PDF

TUMOR
BLOOD
CIRCUL ATION:
Angiogenesis , Vascular
Morphology and Blood
Flow of Experimenta l and
Human Tumors
Editor
Hans-Inge Peterson
Chief Surgeon
Lundby Hospital
and
Research Instructor
Laboratory of Biorheology
Department of Surgery I
Sahlgrenska University Hospital
Goteborg, Sweden
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an informa business
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
Reissued 2019 by CRC Press
© 1979 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an lnforma business
No claim to original U.S. Government works
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have
been made to publish reliable data and information, but the author and publisher cannot assume responsibility
for the validity of all materials or the consequences of their use. The authors and publishers have attempted to
trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if
permission to publish in this form has not been obtained. lf any copyright material has not been acknowledged
please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted,
or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented,
including photocopying, microfilming, and recording, or in any information storage or retrieval system,
without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www. copyright.com
(http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive,
Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration
for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate
system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only
for identification and explanation without intent to infringe.
A Library of Congress record exists under LC control number:
Publisher's Note
The publisher has gone to great lengths to ensure the quality of this reprint but points out that some
imperfections in the original copies may be apparent.
Disclaimer
The publisher has made every effort to trace copyright holders and welcomes correspondence from those they
have been unable to contact.
ISBN 13: 978-0-367-24532-0 (hbk)

ISBN 13: 978-0-429-28302-4 (ebk)
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the
CRC Press Web site at http://www.crcpress.com
PREFACE
Early morphological studies of tumor vascularization have given a fairly detailed

picture of the development and morphology of tumor blood vessels. Later observations
by refined techniques have added further information. Tumor angiogenesis has been
a focus of interest in recent years, and a specific tumor factor responsible for neovas-
cularization of tumor implants by host blood vessels has been isolated.
The literature dealing with the quantification and dynamics of tumor blood flow
has until recently been rather sparse. A lack of uniformity in tumor blood flow distri-
bution, as found in several studies, has been an obstacle to quantitative determinations
of tumor blood flow rate. Several methods of recording tumor blood flow have been
reported, including measurement of venous outflow from tissue-isolated tumor trans-
plants, different isotope and thermodynamic techniques, semiquantitative analysis of
serial microangiograms and plethysmography. Labeled microspheres seem to have
been used in only a few studies.
Much remains to be done towards finding reliable methods for recording tumor
blood flow in the clinic. However, the recording of human tumor blood flow and the
possibility of influencing tumor blood flow by vasoactive drugs or by other procedures
seem to have great importance in tumor diagnosis and therapy. Thus, such procedures
might be better exploited in pharmacoangiography and in perfusion treatment of ma-
lignant tumors with cytostatic drugs based on an improved knowledge of tumor vessel
reactivity. An improved knowledge about the intra-tumor distribution and diffusion
of cytostatic drugs is also of importance to reach optimal concentrations of such drugs
in tumor tissue.
Studies on oxygen diffusion and consumption in tumors might contribute to solve
the problem with radioresistant hypoxic tumor cells.
The editor wishes to express his sincere gratitude to all contributors to this volume.
Their enthusiasm and skill have been an invaluable support. With them he would like
to associate the directors and staff of the CRC Press, Inc., all of whom have spared
no pains in the production of this uniscience publication.
Hans-Inge Peterson
June, 1978
THE EDITOR
Hans-Inge Peterson, M.D., Ph.D., is Chief Surgeon, Department of Surgery,

Lundby Hospital, Gõteborg, and Research Instructor, Laboratory of Biorheology, De-
partment of Surgery I, Sahlgrenska University Hospital, Gõteborg.
Dr. Peterson received his M.D. and Ph.D. degrees in 1960 and 1968, respectively,
from the University of Gõteborg, Sweden.
Current research projects for Dr. Peterson include the influence of vasoactive drugs
and ischemia on tumor blood flow, fibrinolysis in growth and spread of tumor, toco-
pherol and tumor irradiaton, and tissue fibrinolysis in the gastrointestinal tract. Dr.
Peterson has published over 100 papers dealing with tumor biology, fibrinolysis, and
surgery.
CONTRIBUTORS
K. Lennart Appelgren, M.D., Ph.D. Jan Mattsson, M.D.

Assistant Professor Laboratory of Biorheology
Department of Anasthesiology III Department of Surgery I
Sahlgrenska University Hospital Sahlgrenska University Hospital
Gõteborg, Sweden Gòteborg, Sweden
Stig Bengmark, M.D., Ph.D. Eva Peterson-Dahl, M.D.

Professor and Head Department of Surgery
Department of Surgery University Hospital
University Hospital Lund, Sweden
Lund, Sweden
Hans-Inge Peterson, M.D., Ph.D.
Leif Ekelund, M.D., Ph.D. Associate Professor
Associate Professor Laboratory of Biorheology
Department of Radiology Department of Surgery I
University Hospital Sahlgrenska University Hospital
Lund, Sweden Goteborg, Sweden
Per E. Fredlund, M.D., Ph.D. Aleksander S. Popel, Ph.D.

Associate Professor Research Associate Professor
Department of Surgery Departments of Chemical Engineering
University Hospital and Aerospace and Mechanical
Lund, Sweden Engineering
University of Arizona
Joseph F. Gross, Ph.D. Tucson, Arizona
Professor and Head
Department of Chemical Engineering Huibert S. Reinhold, M.D.
University of Arizona Department of Experimental
Tucson, Arizona Radiotherapy
Erasmus University
Rotterdam
Larsolof Hafstrõm, M.D., Ph.D.
and
Associate Professor
Radiobiological Institute TNO
Department of Surgery
Rijswijk, The Netherlands
University Hospital
Lund, Sweden
Peter Vaupel, Dr. Med.
Professor
Bertil Hamberger, M.D., Ph.D. Institute of Physiology
Associate Professor University of Mainz
Department of Histology Mainz, West Germany
Karolinska Institutet
Stockholm, Sweden Bruce A. Warren, M.A., M.B.,
D. Phil.
Per-Ebbe Jònsson, M.D. Professor
Department of Surgery Department of Pathology
University Hospital University of Western Ontario
Lund, Sweden London, Ontario
TABLE OF CONTENTS
Chapter 1
The Vascular Morphology of Tumors 1
Bruce A. Warren
Chapter 2
Tumor Angiogenesis 49
Bruce A. Warren
Chapter 3
Vascular and Extravascular Spaces in Tumors: Tumor Vascular Permeability 77
Hans-Inge Peterson
Chapter 4
Methods of Recording Tumor Blood Flow 87
K. Lennart Appelgren
Chapter 5
Tumor Blood Flow Compared with Normal Tissue Blood Flow 103
Hans-Inge Peterson
Chapter 6
In Vivo Observations of Tumor Blood Flow 115
Huibert S. Reinhold
Chapter 7
Tumor Vessel Innervation and Influence of Vasoactive Drugs on Tumor Blood
Flow 129
Jan Mattsson, Lennart Appelgren, Bertil Hamberger, and Hans-Inge Peterson
Chapter 8
Irradiation and Tumor Blood Flow 137
Jan Mattsson and Hans-Inge Peterson
Chapter 9
Oxygen Supply to Malignant Tumors 143
Peter Vaupel
Chapter 10
Mathematical Models of Transport Phenomena in Normal and Neoplastic
Tissue 169
Joseph F. Gross and Aleksander S. Popel
Chapter 11
Angiography and Pharmacoangiography in Human and Experimental Tumors 185
Leif Ekelund
Chapter 12
Hepatic Dearterialization and Infusion Treatment of Liver Tumors 203
Stig Bengmark, Eva Peterson-Dahl, and Per E. Fredlund
Chapter 13
Regional Perfusion with Anticancer Drugs for Treatment of Malignant Tumors . . . 217
Larsolof Hafstrom and Per-Ebbe Jõnsson
Index 225
Taylor & Francis
http://taylorandfrancis.com
1
Chapter 1
THE VASCULAR MORPHOLOGY OF TUMORS
Bruce A. Warren
TABLE OF CONTENTS
I. Introduction and Basic Concepts 1
II. Historical Review of the Literature to 1966 5

A. The Themes Involved 5
B. Human Tumors 6
C. Animal Tumors 12
III. The Vascular Morphology of Certain Experimental Tumors 20

A. Tumors of Melanin-Forming Tissues 22
B. Sarcomas and Tumors of Subcutaneous Tissues 23
C. Tumors of the Mammary Gland 25
D. The Walker Carcinosarcoma 256 28
E. Hepatomas 29
F. Tumors of the Blood-Vascular System 30
IV. The Organization of the Vascular Architecture in Tumors 31
V. Radiographic Observations of Vascular Morphology in Man and Animals . . .39
VI. Methods of Quantitation of Vascular Morphology 41

A. Network Changes 41
B. Vascular Density Changes and Redundancy Index of Tumor Vessels .. .41
VII. Conclusion 42
Acknowledgment 42
References 43
I. I N T R O D U C T I O N A N D BASIC C O N C E P T S
It is easy to approach the subject of the vascular morphology of tumors in the ex-
pectation that the architecture of the vasculature will vary in regular defined ways from
the norm. However, what is the norm in this sense? The concept of the microcircula-
tory module has been clearly enunciated and expanded by Zweifach,1 Frasher, 2 and
others and elucidated in ultrastructural detail in the fascia lata of the rabbit by
Rhodin. 3 Yet this is, perhaps, the simplest of the physiological microcirculatory units,
being as it is an almost two dimensional structure in thin tissue without structural
2 Tumor Blood Circulation
specialization. Tissues with highly developed functions have more complex vascular
arrangements. Thus, the nephron of the kidney has an artery which breaks up into
multiple capillaries (the glomerular tuft) and reforms to constitute another artery.
Again, the vascular arrangement of the hepatic acini represents a complex arrangement
of both venous and arterial supply to a sinusoidal * 'capillary* ' bed whose channels
unite to form a venous draining system.
Tumors in any of their functions or capacities do not tend to obey simple regular
and defined patterns. Otherwise, the study of tumors might not have generated the
enormous literature that it has to date. The rule which is more often supported is that
each tumor type, and in some cases each tumor, tends to be a law unto itself. It has
to be recognized, therefore, that the vascular morphology of tumors, like other char-
acteristics, has to be studied for each tumor type and that generalizations may be dif-
ficult to make. The very characteristics which define tumors and which divide them
into benign and malignant types are disputed.
What is a tumor?
Willis4 defined a tumor as "an abnormal mass of tissue, the growth of which exceeds
and is uncoordinated with that of the normal tissues and persists in the same excessive
manner after cessation of the stimuli which evoked the change".
A benign tumor is usually slow growing, well encapsulated, and closely resembles
the tissue of origin. It does not give rise to secondary deposits.
In contrast, a malignant tumor may grow rapidly, be poorly encapsulated, and show
marked differences from the tissue of origin; however, its prime characteristics are the
abilities to invade locally and to disseminate and give rise to secondary deposits at sites
distant to the site of origin.
The new growth or tumor is an additional mass of tissue superimposed on or in the
original host tissue. There remains, therefore, that part of the host's vasculature that
is not destroyed by the tumor. As well as this, there is the new formation of vessels or
angiogenesis from neighboring vascular beds.
One of the commonest misconceptions of students considering this topic is that of
suggesting a unitary blood supply to a tumor. This is simply not so with malignant
tumors, except at a very early stage in their development or in a peculiar anatomic
situation. In most instances, malignant tumors induce a veritable Medusa's head of
vessels supplying the growing core of malignant cells. The induction of this growth
results in multiple adhesions, and the new vessels will penetrate natural capsules and
mesothelial surfaces.
A tumor mass is an expansile, growing mass which moves into new host tissue by
commandeering the supply of blood from each of the regions of its growing arcs.
A consideration of the blood supply to tumors may be divided up into two major
subheadings. (1) the vascular morphology of the established tumor, and (2) the earliest
development of blood vessels by the tumor. In one sense, the first encompasses the
latter because as the tumor expands it recruits further blood supply by inducing capil-
lary sprouts from the adjacent microcirculatory networks or takes over the pre-existing
vascular pattern of the organ. The topic of vascular morphology of tumors spans a
number of different aspects of neoplasia. The subject of the early stages of the induc-
tion of vessels into the tumor transplant in vivo or in the small micrometastasis is dealt
with in the next chapter.
Section II of this chapter is a review of the earlier literature to 1966 tracing the
various themes (Table 1) in simple chronological order. It deals first with human ma-
terial (Table 2) and then with experimental tumors (Table 3).
Section III deals with the vascular morphology of experimental tumors including
those of melanin forming tissue, sarcomas and tumors of s.c. tissues, tumors of the
3
TABLE 1
The Main Themes of Papers Relating to the Vascular Morphology of

Tumors
1. Diagnostic use of the vascular patterns in tumors

2. Value of knowledge of vascular morphology in specific tumors
when the delivery of pharmacologically and radiotherapeutically
active drugs is considered
3. Role of the vascular system in the biology of the tumor
During carcinogenesis and the development of the malignant neo-
plastic state
During growth of the tumor
As the factor dictating the distribution of living, dying, and dead
tumor cells
As the anatomical site for the shedding of tumor cell emboli into
the circulation; the importance of this site in the first stage of
the development of metastases
TABLE 2
Selected Papers Dealing with the Vascular Morphology of Tumors in Man in Chronological Order to 19665~23
Principal topic Authors Date Ref.
Capillary network in Virchow 1863 5

stroma of tumors Thiersch 1865 6
demonstrated
Intra-arterial injections of Goldmann 1907 7
bismuth in oil
Vascularity of benign and Thiessen 1936 8
malignant gastric tumors
Arterial pattern in lungs Wright 1938 9
with metastases
Angioarchitecture of the Hardman 1940 10
gliomas
Capillary angioarchitecture Lindgren 1945 11
in tumors
Vascularity of carcinoma de Busscher 1947 12
of stomach
Fluorescence in cutaneous Bierman and co-workers 1951 13,14
lesions 1952 15,16
Arterial pattern of liver
with metastases
Blood supply to liver with Breedis and Young 1954 17
metastases
Vascularity of Delarue and co-workers 1954 18
bronchogenic carcinoma 1956 19
Vascularity of cancers of 1964 20
large intestine
Vascularization of cancers
of alimentary canal
Skin-tumor capillaries Urbach and Graham 1962 21
Review of microcirculation Rubin and Casarett 1966 22
in animal and human 1966 23
tumors and
radiotherapeutic
implications
TABLE 3
Selected Papers Dealing with the Pattern of Tumor-Vessel Morphology and Alterations in Blood-
vessel Pattern in Animals as Tumors Develop, or Following Transplantations: from 1907 to 1965
in Chronological Order7*'
Blood vessels in tumors Goldmann 1907 7

Vascular pattern of tumors Lewis 1927 24
Alterations of vessels Kreyberg 1927 25
during tumor formation
Vital staining of tumors Orr 1937 26
during carcinogenesis
Vascularization of the Ide, Baker, and Warren 1939 28
Brown-Pearce rabbit
epithelioma
Manner of growth of frog Lucke'and Schlumberger 1939 27
carcinoma
Significance of hyperemia Cowdry and Sheldon 1946 29
around tumor transplants
Growth rate of Algire and co-workers 1943 30
transplantable tumors in 1945 31
relation to latent period 1947 32
and heat vascular reaction 1947 33
Growth rate of tumor Lutz and co-workers 1950 34,35
transplants in the hamster
cheek pouch, and effects
on small blood vessels
Transplantation of human Toolan 1951 36
tumors into cortisone- 1953 37
treated animals 1954 38
Heterologous Greene 1952 41
transplantability of
human cancer
Transplantation of human Chute, Sommers, and Warren 1952 39
tumors Patterson, Chute, and Sommers 1954 40
The arterial supply of Braithwaite 1958 42
benzpyrene-induced
tumors in the rat
Localization of fibrinogen Day, Planinsek, and Pressman 1959 43
in transplanted rat tumors
The vascular system of two Waters and Green 1959 44
transplantable mouse
granulosa-cell tumors
Growth of circulation in Kligerman and Henel 1961 45
hepatoma
Stromal response to tumors Gullino, Grantham, and Clark 1962 46
Co-opting of the arterial Gullino and Grantham 1962 47
network in the kidney by
tumor transplant
X-ray photography of Merker and Hurley 1962 49
vessels of human
epidermoid carcinoma
growing in cortisone-
conditioned Swiss mice
Blood flow to transplants Cataland, Cohen, and Sapirstein 1962 50
measured by radioactive
tracers
Access of blood-born dyes Goldacre and Sylven 1962 48
to different tumor regions
Modification of the blood Delarue, Mignot, and Caulet
vessels of the hamster
cheek pouch during the
growth of melanoma
transplants
Arterial supply to Ogilvie and co-workers 1964 52
experimental metastases in
the lungs
5
TABLE 3 (continued)
Selected Papers Dealing with the Pattern of Tumor-Vessel Morphology and Alterations in Blood-Vessel
Pattern in Animals as Tumors Develop, or Following Transplantations: from 1927 to 1965 in Chronological
Order7"56
Observations on the Warner 1964 53

vascular tree of the frog
renal adenocarcinoma in
ocular implants
Vascular relationships of Day 1964 54
tumor and host
Early changes following Witte and Goldenberg 1965 55
transplantation of
epidermal carcinoma onto
the hamster cheek pouch
Vascular patterns in living Goodall, Sanders, and Shubik 1965 56
tumors viewed through an
optical implant in the
hamster cheek pouch
mammary gland, the Walker Carcinosarcoma 256, hepatomas, and tumors of the
blood-vascular system.
Section IV discusses the organization of the vascular architecture in tumors from the
viewpoint of the hierarchy of anatomical structures and categorizes the blood vessels
in tumors into various classes. The tracing of network changes and the changes with
growth can then more easily be discussed and current problems enunciated.
Section V records in chronological order a number of observations made by radio-
logical means concerning the vascular morphology of tumors in man and animals. Al-
though some methods of quantitation of vascular morphology in tumors have been
discussed earlier, in Section VI further recent mathematical concepts regarding network
analysis and vascular density are cited. In the final section, a number of conclusions
regarding the vascular morphology of tumors are listed.
II. HISTORICAL R E V I E W O F T H E L I T E R A T U R E
A. The Themes Involved

On reviewing the literature on this subject, certain broad themes can be seen to
thread their way through the published writings. From the diagnostic viewpoint, the
increased vascularity of the peripheral shell of neoplastic tissue provides a valuable
indication of the occurrence of a neoplasm. Identification of such increased vascularity
and of unusual vascular patterns in organs has been a topic of considerable interest to
radiologists and has proved its value in diagnosis. The pattern of blood vessels in his-
tologic sections is not usually of value in pathological interpretation of sections, except
of course, in tumors of the blood vessels themselves or in those tumors in which this
altered vascularity is a prime feature of the pattern of the cells and tissues of the tumor
itself, e.g., glioblastoma multiforme. From a therapeutic viewpoint, the access of phar-
macologically and radiotherapeutically active agents to tumor cells depends on the
patency and organization of the tumor vascular bed in relation to the cells of the tumor
and the host vascular bed.
The organization and recruitment of the supporting tissues, including the vascular
system, comprises one of the fundamentally important aspects of the biology of neo-
plasia. In many tumors, there is a peculiar mixture of living cells around the vascular
arborizations with sequential layers of dying and dead cells beyond an adequate oxygen
gradient from the blood channels.
The published literature on the subject can be divided into three themes (Table 1):
1. The use of the vascular morphology of tumors in diagnosis

2. The value of an understanding of vascular morphology of specific tumors in the
interpretation of the delivery of active agents
3. The role of the vascular system in the biology of the tumor (e.g., during carcino-
genesis, in angiogenesis — that is, the recruitment of vessels into the service of the
tumor, in the distribution of living, dying, and dead cells within the tumor, in the
response to injury of the tumor, haemostasis within the tumor, and in the release
of blood-born tumor emboli in the malignant tumor)
Because of the wide-ranging nature of this subject each individual paper usually
deals only with a small corner of the general topic. This is usually only an aspect of
the vascular morphology of tumors, and that aspect is only in connection with a small
group of specific tumors, or indeed, with one specific tumor.
The papers, in general, reflect both the technological capabilities and the current
topical problems of their period.
The major approaches to a study of the vascular morphology of tumors have been
predicated on what specifically can be done in man and in animals. In man the ap-
proach has been
1. A general description of the vascularity of human tumors at autopsy and on surg-

ical removal
2. Injection studies of such material
3. Radiological studies of human tumors for diagnosis.
A series of papers in chronological order to 1966 dealing with the vascular morphol-
ogy of tumors in man is listed in Table 2.
In animals, of course, a far wider range of studies is possible, and the problems of
the development of blood vessels during carcinogenesis, the growth rate of tumor
transplants, and the general host response to tumors have been investigated. In Table
3, selected papers in date order are found which deal with these aspects of tumor
growth and development from the viewpoint of the vascular system.
In the following historical review, the papers are divided up into selected important
papers concerning the vascular morphology of tumors and the animal experimental
work up to 1966. Work published more recently than this has been included in subse-
quent divisions of this chapter.
B. Human Tumors
In human material after the initial descriptive phase, injection techniques were used
by Virchow5 and Thiersch6 to demonstrate the distinctive capillary network which sup-
plied the stroma of tumors.
Goldmann, 7 in 1907, described the growth of malignant disease in man and the lower
animals with special reference to the vascular system. He considered that the vasa
vasorum of the arteries and veins played a major part in the development of tumor
metastases, and believed that cancer was spread by arteries and appeared as a form of
periarteritis. However, cancer spread by the veins resulted in a condition of endophle-
bitis carcinomatosa. He exposed the femoral artery and injected an emulsion of bis-
7
muth and oil into the blood vessels in a number of cases of human cancer. After tying
the afferent and efferent vessels of the cancerous organ he dissected them carefully
and exposed them to X-rays. His conclusions were that: (1) there was a disturbance of
the regular distribution of blood vessels by the invading growth, (2) there was a for-
mation of new blood vessels which was most apparent in the zone of proliferation,
which in infiltrating tumors is at the periphery, and (3) as the cancerous growth in-
creased in volume, its centre became necrotic, and the newly formed blood vessels
merely occupied the capsule. Ultimately, the blood vessels seemed to disappear alto-
gether so that at this stage the cancer was not rich in blood vessels, but rather, denuded
of them. The majority of newly formed blood vessels were of small caliber and their
branching was so irregular that big vessels split up into the smallest of their kind without
intermediary types.
Goldmann also injected spontaneous and experimental growths in mice by intracar-
diac puncture and injection of India ink. One common feature was observed in all of
the investigations. As soon as the infiltrative growth of the transplanted cells set in, " a
great commotion" was produced in the surrounding system of blood vessels, and this
was dependent on the wealth of the pre-existing vessels. Hence, in a strongly vascular-
ized tissue such as the mammary gland or muscle, the vascular "irritation" was far
more pronounced than in s.c. tissue which was poor in vessels.
This "irritation" was seen most distinctly in the vessels in front of the growing
tumor. The most striking feature of the "irritation" was the dilatation of the affected
vessel. It curled itself up into spiral coils and sent forth numerous capillary offshoots
towards the invading growth. In carcinoma, the newly formed vessels arranged them-
selves almost entirely in the peripheral area. As the volume of the growth increased
the number of vessels decreased and eventually, especially in cases of complete necro-
sis, the vessels totally disappeared. In sarcoma, the numerous vessels of new formation
were evenly distributed throughout the whole growth, presenting themselves as a deli-
cate, closely woven network even in the interior of the tumor. In chondroma of the
mouse, Goldmann found the capsule to be rich in vessels and in wide bands of connec-
tive tissue. Many of its vessels penetrated the cartilagenous mass and opened out into
large vascular spaces apparently losing all the characteristics of blood vessels. These
vascular spaces disintegrated the cartilage, and numbers of cartilage cells were thus
destroyed. Goldmann quoted Bashford57 as having demonstrated that the stroma of
experimental tumors owes its origin to the innoculated animal, is not derived from the
grafted cell, and that he had observed complete and distinctive differences in the strains
of the cancers. He considered that the structural qualities of the stroma were determined
by the tumor cell. He noted that different forms of cancer varied in the "wealth" of
blood vessels that they possessed. Goldmann argued that the impetus which gave rise
to the proliferation of blood vessels emanated from the invading cell.
On the other hand, he believed that the new formation of blood vessels itself was
dependent upon the general powers of the individual to react, as well as upon the
physiological conditions of the blood supply in the invaded tissues.
Thiessen8 examined the vascularity of benign and malignant lesions of the stomach
in 57 surgically removed gastric specimens. He was concerned with the then-current
vascular theory of causation of gastric ulceration. He developed a vascular-density
index. The cross sectional areas of the lumens of the blood vessels at the edge of the
ulcer and in the normal mucosa were calculated by means of camera lucida drawings
and a planimeter. The difference between the two measurements, expressed as a per-
centage, he called the vascular density. The mean percentage of tissue occupied by
blood vessels in benign ulcers was 3.28% (associated normal areas 1.98%) and in early
carcinomas 5.12% (associated normal areas 0.91%). His conclusion was that this work
did not support the hypothesis that ulcers begin in a region of lowered vascularity.
The index of vascular density increased from the benign ulcers to the definite carcino-
mas. He noted a gradation from normal tissue up through benign ulcers, ulcers with
secondary "cytoplasia", beginning carcinoma, and then down to fully developed car-
cinoma.
Wright,9 in 1938, reported work concerning the arterial pattern in lungs with primary
tumors and those with metastatic deposits. He examined two specimens of broncho-
genic carcinoma. In one, there was a marked stromal reaction in an adenocarcinoma,
and this had produced a bronchial obstruction before any metastases had developed
and had resulted in the patient's death. The vessels of the stroma were all injected
from the pulmonary artery. The vessels of the secondary tumors in the right lung were
all injected from the bronchial artery. Many were of even caliber, but a great number
gave the appearance of twisted rope. The margins showed a clear line of demarcation
between the compressed lung with vessels injected from the pulmonary artery, and the
neoplastic tissue with vessels injected from the bronchial artery. Five specimens of
secondary carcinomata in the lung were examined. In three cases, the tumors were
secondary to carcinoma of the breast, and in one, to a tumor of the stomach. The
tumors developed in two main ways. Firstly, there were discreet rounded masses, pre-
sumably developed from emboli of carcinomatous tissue in the pulmonary artery.
These showed numerous vessels injected from the bronchial arteries, and presented the
same dissociation from vessels injected from the pulmonary artery as the massive sec-
ondary tumors in the case of bronchogenic carcinoma. Secondly, the central area
showed masses of carcinomatous tissue with a fibrovascular stroma arranged not in
relation to the pre-existent alveoli at all, but to the masses or acini of the tumors. In
both these latter zones, the vessels were injected from the bronchial arteries. Where
large masses were present, the branches of the bronchial arteries in the vicinity were
greatly enlarged. Blue gelatin was injected from the bronchial artery. The growth of
fibrous tissue was accompanied by twigs of capillaries into the tissue in the lumen so
that the original pulmonary alveolar pattern became less obvious. In deposits of sec-
ondary sarcoma measuring over 1 cm in diameter, there was the development of blood
vessels. These spread out as fine branching tubes from branches of the bronchial artery
which were apposed to the sides of the tumor. Nowhere did they show any association
with vessels injected from the pulmonary artery. In examination of the specimen of
secondary chorionic carcinoma, there was a solid rind of tissue around the periphery,
and in the center there was liquified brown material. In the right lung which was in-
jected via both systems, there were lakes of gelatin which had escaped mainly from
the pulmonary artery, but in some cases from the bronchial artery.
Wright, in 1938, concluded that, if in the adult lung there develops a tissue which is
usually supplied from the systemic arterial circulation, the vessels which grow with it
are filled by material injected via the bronchial artery. This development of new vessels
from the systemic arteries may be closely linked with the excitation of collagenous
tissue to further development, irrespective of the nature of the causative agent.
Hardman 10 studied material obtained from 23 autopsies and two operation cases. He
classified his material into the malignant gliomata and the relatively benign gliomata
while recognizing that there were transitions of vascular pattern from one to the
other. In the first series of experiments, he injected a suspension of barium sulphate
in hot gelatin into the internal carotid arteries of those who died of intracranial tumor.
The brains were removed, fixed in formalin, sectioned into slices 1 cm thick, and each
slice was radiographed. He was disappointed in this method and finally abandoned it
for a number or reasons. Chief among these was that only the large vessels could be
distinguished, and although the sections might appear to be satisfactory to superficial
examination, the capillary bed was usually incompletely filled. If injections were made
9
under pressure, extravasations were liable to occur in the zone between active and
necrotic tissue. He noted that the tumor vessels were much more fully injected than
the normal tissue and that there was a tendency for the injection mass to pass through
the tumor vasculature into the veins. The region of the tumor was thus shown to be
an area of low resistance. His later specimens were processed using a microchemical
process which consisted of treating sections of 250 jum with benzedrine, sodium nitro-
prusside, and hydrogen peroxide. Treatment with these reagents results in the conver-
sion of hemoglobin to a black precipitate, and all blood vessels that contain hemoglo-
bin are thus stained. He recognized that negative results with this method had to be
treated with some reserve since the delineation of blood vessels by this method de-
pended on the presence of red cells. Hardman divided the vascular pattern in the gliom-
ata into four zones, A to D. In zone A, the capillaries of the surrounding white matter
were poorly stained. At the invasive edge of the tumor, zone B, the vessels stained
more constantly than in zone A, the orderly, normal, uniform network became cha-
otic, and the capillaries were irregularly dilated and occasionally sinusoidal in appear-
ance. In the more malignant tumors, sinusoids were common and capillaries relatively
scarce. In zone C, the sinusoids were largest and fewest towards the necrotic center of
the tumor. Every malignant tumor had a necrotic center, zone D, in which extravasated
blood and thrombosed sinusoids were prominent features. In his material, in the ma-
lignant gliomata the greatest degree of cellular activity and differentiation was seen in
the outer zone. In contrast, the central region of the tumors always showed fibrosis,
hyaline change, and occasionally, cyst formation.
Lindgren, 11 in 1945, wrote an extensive review on the subject of the vascular supply
of tumors with special reference to the capillary angioarchitecture. He considered that
during embryonic development the large blood vessels were formed exclusively by dif-
ferentiation of a primitive continuum of capillaries, during which process the walls of
certain capillaries became thickened by the development of connective tissue and
smooth muscle tissue. Simultaneously with this differentiation of certain capillary ves-
sels, others were subject to atresia. This capacity to remodel the vascular system re-
mained throughout life in normal tissues. He found that most tumors were vascular-
ized by newly formed vessels that pierce the tumor from various directions from the
surrounding tissue of the organ. In malignant tumors, these vessels were generally of
low differentiation or of fetal type, their walls lacking in elastic lamellae. In benign
tumors, the vessels were more highly differentiated and more like those of normal
tissues. In poorly differentiated malignant tumors, the capillary angioarchitecture dif-
fered greatly from that usually found in the corresponding normal tissue, whereas they
were more similar in benign highly differentiated tumors. When a malignant tumor
expanded in a tissue, many preformed vessels of the latter were destroyed, and even-
tually, vessels remained chiefly in the periphery of the tumor. These were most easily
demonstrated in elastin-stained preparations, which he believed could be of diagnostic
value. The amount of capillaries in the tumors was usually greater if the stroma were
loose than if it were fibrous and poor in cells. There were however, exceptions to this
rule. He described the border between living and dead tumor tissue in which there was
regularly a zone of dilated vessels often surrounded by diapedetic hemorrhages. These
originated in connection with intravital stasis phenomena caused by blood-vessel
compression, local fall of the blood pressure, intravascular tumor proliferation, etc.
The capillary angioarchitecture of the metastases on the whole generally agreed with
that of the primary tumor. The tumors seemed to possess a capillary angioarchitecture
which was, to a certain extent, specific. This sometimes allowed classification of the
tumor type, but only in blood-corpuscle-stained preparations. This was especially true
concerning more highly differentiated tumors, and is evident from the illustrations of
Lindgren. Only undifferentiated tumor types showed so uncharacteristic a capillary

angioarchitecture that it was impossible to determine the origin of the tumor by this
method alone. The coarser vessels of malignant tumors were usually devoid of elastic
lamellae, but these were sometimes developed in the vessels of certain benign tumors.
The reason why capillaries grow into the stroma of the tumor was just as un-
known as the cause of the vascularization of normal tissue. Simultaneously with the
formation of new vessels, an extensive destruction of the smaller preformed vessels
that were included within the tumor took place as it grew. However, Lindgren con-
cluded that some of the coarser vessels could sometimes be "taken over" by the tumor,
for some time at least. Lindgren examined 25 mesenchymal and 15 epithelial benign
tumors, 5 mesenchymal and 71 epithelial malignant tumors, and 38 metastases of dif-
ferent localizations. The methods he utilized were
1. The injection into blood vessels of barium sulphate followed by roentgenographic

examination of th^ preparations and histological examination of the larger vessels
and the capillary angioarchitecture of these tumors
2. The capillary angioarchitecture was investigated by selectively staining the eryth-
rocytes within histologic preparations. The blocks were fixed in 10% formalde-
hyde,200 /^m frozen sections were cut, and the remainder processed for normal
paraffin section histology, using various stains.
In thinner sections, the angioarchitecture stood out indistinctly because the vessels
were cut into short segments. The interrelationship of the capillaries could not be stud-
ied. On the other hand, thicker sections did not give sharp images when the vessels
were photographed. He found the most suitable magnification was *25.
The theme of the correlation of the vascular pattern with histological tumor type
was elaborated by de Busscher12 in the specific case of gastric carcinomas. During
examination of the vasculature in carcinomas in this location he found that increase
in small and large diameter arteries was most obvious in cases of adenocarcinoma of
tubulo-papillary type, while in contrast, it was moderated, or even not apparent, in
those cases of colloid carcinoma.
In a series of papers, Bierman and co-workers 1316 described the results of investiga-
tions into the blood supply of tumors in man by fluorescence,13 arteriography, 14 the
increased oxygen content of venous blood draining neoplasms, 15 and skin tempera-
ture. 16 The arterial blood supply to most primary cutaneous tumors was demonstrated
to be greater than that to normal tissues, as judged by the fluorescein appearing in
tumor areas more quickly, remaining longer, and being found in greater concentration
than in the surrounding tissues.13 Necrotic metastatic lesions did not fluoresce as
promptly, nor as brightly, as those of mycosis fungoides or lymphoepithelioma. The
human hepatic arterial circulation14 was demonstrated on 150 occasions in 50 patients
by timed rapid roentgenographic exposures immediately following the injection of ra-
dio-opaque materials through an intra-arterial catheter introduced into the femoral
artery and manipulated into the celiac or hepatic arteries. The hepatic arterial circula-
tion in the uninvolved normal liver exhibited an orderly branching pattern with a pro-
gressive and gradually diminishing caliber of the vessels as they terminated in the par-
enchyma of the liver. No gross anastomoses were observed between the vessels outlined
by this technique. The small vessels in the involved areas originated from dispropor-
tionately large parent arteries in the vicinity, branched abruptly, and formed bizarre
disorderly patterns that were characteristic of a neoplastic mass. The arterial circula-
tion through the normal liver with metastases is apparently more rapid than through
the uninvolved liver. They concluded that increased vascularity of the type described
11
as common in cerebral neoplasms is not a specific characteristic of brain neoplasms,

but is a common characteristic of, probably, the majority of tumors throughout the
entire body, regardless of their location. This has been suggested by the arterial pat-
terns observed in neoplasms in the liver, bone, kidney, and other viscera by arteriog-
raphy. Characteristic abnormalities of the smaller vessels within the tumor areas were
noted. These were sinusoidal dilatations in which the radio-opaque material appeared
to persist longer than in the uninvolved areas. The increased oxygen content of venous
blood draining neoplastic lesions15 indicated that there were considerable arteriovenous
shunting networks, or the lack of oxygen extraction within some neoplasms. These
findings strongly suggested that there was an increased and more rapid blood flow
through these tumors than in comparable normal tissues.
Breedis and Young, in 1954,17 by means of injection experiments examined the blood
supply of neoplasms in the liver. They showed that malignant neoplasms growing in
the liver tend to acquire an exclusively arterial blood supply. The neoplasms tested
were the VX2 carcinoma of rabbits, T-241 sarcoma of mice, a variety of metastatic
carcinomas found at necropsy of human cases, metastases of spontaneous carcinoma
of the kidney of the frog, and primary hepatomas and cholangiomas of rats induced
by p-dimethyl-amino-azobenzene. The blood supply of transplanted rabbit and mouse
tumors was arterial whether innoculations had been made into the hepatic artery or
portal vein or directly into the hepatic parenchyma. Much, if not all, of the failure of
portal blood to supply tumors growing in the liver was due to progressive invasion and
occlusion of portal branches by the tumor cells.
In a series of investigations by Delarue and co-workers, the vascularity of broncho-
genic carcinomas 18 and malignant neoplasms of the alimentary canal19 20 were exam-
ined. In order to study the tumor vasculature, these workers used metal salts which
were opaque to X-rays and which possessed characteristics which would progress at
least to the precapillary level. The general angioarchitecture depended, at least in part,
on the macroscopic location and the regional topography. In localized tumors in the
stomach, a hypervascular network around the zone of the tumor was found, as well
as a variable, segmental dilatation of small and large arteries. In diffuse tumors such
as linitis plástica, a completely different angioarchitecture was recognized. Modifica-
tions of the circulatory bed were not circumscribed or delimited to the neoplastic area
in such cases.
In carcinomas of the colon, Delarue and his co-workers20 recognized several changes
depending on the growth characteristics of the tumors. In purely exophytic growths,
it was difficult in the radiographs to localize the position of the tumor. In other cases
in contrast, the tumor was made more obvious by its circulatory pattern. In some
instances, the tumor was very vascular. The arteries were straight or curved, occasion-
ally long and perforating, and were of greater diameter than normal. They led into a
dense irregular plexus in the tumor. In other cases, the vascularization pattern of the
tissue delimited a diffusely opaque tumor zone. Lastly, in some cases, there appeared
two zones with different vascular densities. In one zone, there was a rich vascular
network while in the other only precapillary vessels were apparent and these corre-
sponded to the extra colic extension.
Urbach and Graham 21 examined the capillaries supplying the skin in a variety of
human premalignant and malignant lesions by means of staining frozen sections of
the lesions by the Gomori technique for demonstrating alkaline phosphatase. In sec-
tions stained in this manner, capillary loops entering the dermal papillae were seen
together with a rich capillary plexus around the cutaneous appendages. They found
that in malignant skin neoplasms this capillary structure was strikingly altered. There
was a significant increase in the number of visible capillaries, and they were elongated,
dilated, and tortuous showing many anastomoses and cross linkages. These altered
vessels surrounded the groups of tumor cells of basal cell carcinomas and extended
beneath them. In 15 basal cell carcinomas, no capillaries were seen within the groups
of neoplastic cells, despite thicknesses sometimes exceeding 300 to 350 /¿m. In sections
of squamous cell carcinomas, however, large numbers of short, widely dilated, anas-
tomosing capillaries were seen in the main masses of the tumors. In malignant mela-
noma, the capillaries were extremely long, sometimes branched, and often arranged
in parallel rows. In both squamous cell carcinoma and in malignant melanoma, the
vessels were associated with very little connective tissue, appearing to be intermingled
with the main mass of the tumor. In the local tissue surrounding both types of tumor,
there were increased numbers of altered capillaries similar to those described in the
vicinity of basal cell carcinomas. Urbach and Graham 21 remarked on the striking cap-
illary abnormalities which they found beneath the cutaneous premalignant lesions of
senile keratosis and Bowen's disease. In these sites, the vessels showed endothelial pro-
liferation, elongation, tortuosity, ectasia, and shunting. These abnormalities began
sharply at the point where epithelial hyperplasia began.
Rubin and Casarett 22 23 approached the subject of the vascular morphology of tu-
mors from the importance of the subject for radiotherapy. They studied by microan-
giographic and histologic techniques the Walker carcinosarcoma and the Murphy lym-
phosarcoma in order to correlate the microcirculation to tumor necrosis. In their first
paper on the subject,22 they reviewed animal and human work with particular attention
to the possible oxygen gradients involved and the patterns of necrosis of tumor cells
which were thereby induced. From their studies they concluded that the regions of
necrosis found in tumors were not due to the absence of capillaries in tumors, and they
emphasized the need for more functional studies. In their second paper, 23 they corre-
lated microangiographic with histologic techniques to demonstrate a "supervasculari-
zation" phenomenon found in irradiated, regressing tumors. This effect was seen more
consistently with tumor regression after fractionated dose schedules than with others.
After single large doses, they found that the vasculature of both tumors and normal
tissues was fragmented despite regression of the tumor mass. They felt that it was
essential to preserve the fractionation schemes because as a result of the combined
effects of regression and supervascularization better oxygenated cell populations were
made available for each subsequent fractional dose of radiation than would otherwise
occur. Because of this work, they indicated that a clinical trial was in progress which
used atmospheric-oxygen breathing by mask so that fractionation dose schedules could
be more easily preserved.
C. Animal Tumors
Goldmann 7 noted that different forms of cancer vary in the number of vessels that
were present and quoted Bashford's observations that there were complete and distinc-
tive differences between the "strains" of tumors. Lewis,24 using a method developed
by the Johns Hopkins School of Anatomy of injecting 3 % India ink into blood vessels,
examined five different types of rat tumor which arose spontaneously in Dr. George
Walker's rat colony (Walker fibrosarcoma, spindle cell sarcoma, round cell sarcoma,
adenofibroma of the breast, and adenocarcinoma). He found that the vascular patterns
were characteristic for each type of tumor. The three different types of sarcoma were
found to differ from each other on macroscopic and microscopic examination. An-
giological observation of the vascular pattern permitted identification. He found that
in each tumor examined there were characteristic growth phenomena, gross and histo-
logical appearance, and vascular pattern.
13
Kreyberg25 and Orr 26 studied the changes whîcii occur in blood vessels during carcin-
ogenesis. Kreyberg25 examined the local alterations of the blood vessels in tar-painted
white mice. He found that tarring of the skin of white mice produced a marked local
hyperemia with increasing transudation. The hyperemic condition lasted as long as the
tarring was continued. The epithelium became hypertrophic and later formed warts
and papillomas. The formation of these benign tumors was intimately connected with
the occurrence of excessively dilated capillaries. At this point, small multiple capillary
thrombi were found, especially in and around the developing tumors. In a tissue which
had been previously furnished with an increased amount of nourishing substances and
which had shown an increased rate of growth, the blood vessels became more or less
obstructed, and accordingly, the supply of blood, and especially of oxygen, corre-
spondingly decreased. In the following period, the manifest malignant tumor devel-
oped. Later alterations of the blood vessels and blood supply have been described by
Itchikawa and Baum in 1924, cited by Kreyberg,25 who noted that the tumor formation
after the tar-painting of the skin of rabbits and mice is always connected with a marked
reaction in the form of hyperemia. In animals that do not respond to the tarring by
tumor formation as in rats and guinea pigs, no such vascular reaction is found. Orr26
followed the results of vital staining with phenol red during the progress of carcinoge-
nesis in mice treated with tar, dibenzanthracene, and benzpyrene. The earliest change
was an increase in the intensity of staining throughout the area treated, the color re-
maining red, but of a considerably deeper shade than that of the normal skin. At a
later stage, foci in the treated skin stained yellow instead of red, and tumors began to
appear at the same time. In some cases, the whole area became orange or mottled with
yellow. The progress of the staining phenomena in the case of each of the three carcin-
ogens was closely associated with the rate of tumor induction. A high proportion of
the tumors first appeared in relation to yellow areas. It is suggested that the yellow
coloration is an indicator of local functional ischemia. Rapid growth of tumors was
in general associated with the disappearance of the yellow color, while its persistence
was frequently accompanied by retarded growth or actual regression of the warts. It
was found that yellow staining might occur around actively healing ulcers.
Ide et al., in 1939,28 wrote the first account of serial observations of the growth of
blood vessels in vivo around and into a tumor transplant within a transparent chamber.
They noted blood vessel proliferation in from 3 to 8 days following transplantation
and remarked on the fact that if blood vessel growth did not occur the transplant failed
to take. There were 56% takes in the rabbit-ear chamber of the Brown-Pearce rabbit
epithelioma, compared with 98% takes with testicular innoculation of the tumor. An-
other method of observing the growth of tumors in vivo which appeared at approxi-
mately the same time was that of intraocular transplantation. The development of slit-
lamp microscopy allowed Lucké and Schlumberger27 to examine the growth of trans-
plants of a carcinoma of the kidney of the leopard frog innoculated into the anterior
chamber of the eye. They used large invasive adenocarcinomas of the kidney obtained
from other frogs of the same species (Rana pipiens). They noted three morphogenetic
patterns. Unimpeded growths assumed a tubulo-papillary arrangement. Where there
was contact with firm, even surfaces, the tumors grew in broad membranes and sec-
ondary tubular processes. Where the tumor was adjacent to loose tissue, invasion of
the tissue occurred, and with supporting stroma, an acinar pattern was produced sim-
ilar to that of the original renal adenocarcinoma. The rate of growth of the implants
typically described a variable lag period, then a period of rapid growth, following
which the growth increments became smaller. The attempts to transplant normal kid-
ney were unsuccessful.
Cowdry and Sheldon, in 1946,29 reported on the significance of hyperemia around

tumor implants. They noted that intense local hyperemia was a constant finding in the
vicinity of transplantable mouse tumors and directed their experiments towards deter-
mining the significance of this phenomenon.
It was found that hyperemia appeared within 18 hr after implantation and was pro-
gressive thereafter, so long as the tumor grew. Implants of homologous adult muscle
tissue failed to produce hyperemia, indicating that hyperemia was not caused merely
by the presence of foreign tissue. Heterologous tumor implants did not produce hyper-
emia, showing that vascular response did not depend upon the fact that the tissue was
neoplastic. Homologous embryonic tissue, which grew for a time in the host, excited
strong hyperemia that faded as the embryonic tissue finally regressed. This suggested
that the hyperemia was due to the presence of proliferating cells. Tumor 1 from Bagg
albino mice, when implanted in C57 mice, grows for a time and then regresses, leaving
the mice resistant to subsequent implants of this tumor. It was found that the initial
implants of tumor 1 during their short growth period produced hyperemia, whereas,
subsequent implants of the same tumor in the same mice did not grow and did not
cause hyperemia. It was concluded from the several experiments that the hyperemia
surrounding transplanted mouse tumors was due to the presence of proliferating cells.
In a series of papers Algire and co-workers, 3033 using a modified form of the rabbit-
ear chamber for the mouse back, studied the vascular reactions of normal and malig-
nant tissues in vivo in a skin flap of the mouse. They examined the growth rate of
transplantable tumors in relation to the latent period of the host vascular reaction. In
the tumors examined, there were two general classes with respect to their growth rate
and vascular development. Among the rapidly growing group, which included sarco-
mas, mammary gland carcinomas, and a malignant epithelial tumor of the skin, there
were elicited new capillary sprouts from the host as early as 2 to 3 days. The surround-
ing host vessels became hyperemic, and numerous leucocytes accumulated about the
implants. The percentage of the vascular tissue rose to approximately 509b within the
tumor and then stabilized at this level. The capillaries of the tumors mentioned had
an average diameter five times greater than those in normal tissues, e.g., striated mus-
cle, and they appeared as enormous sinusoid-like vessels which showed little tendency
to differentiate into arterioles and venules. In striking contrast to the rapidly growing
tumors listed above, that killed the host in 3 to 6 weeks, were the slowly growing
tumors, which resulted in the death of the host in from 3 to 6 months. These included
the Harding-Passey and Cloudman S91 pigmented melanomas, and an amelanotic mel-
anoma derived from the S91. The slow growth of these tumors was correlated with a
more prolonged latent period prior to capillary proliferation, usually 8 days or more.
In addition, vascular levels in these tumors rarely exceeded that of the vessels in the
surrounding s.c. connective tissue and were less than one half that of the rapidly grow-
ing tumors. There was very little leucocytic accumulation about the implants, and vas-
cular hyperemia in the surrounding tissues was lacking. The capillaries formed were
small in diameter, like those of normal striated muscle, and showed considerable dif-
ferentiation into arterioles and venules.
Lutz and co-workers, in 1950,34 35 examined the growth rate of tumor transplants in
the cheek pouch of the hamster. Sarcomas which were induced by the i.v. injection of
methylcholanthrene or 9,10-dimethyl-l,2 benz-anthracene in the hamster were intro-
duced into the cheek-pouch membranes of other hamsters. Following a latent period
of 1 to 3 days during which the transplant became vascularized, growth proceeded
rapidly and exponentially so that an original transplant of 0.5 mm 3 could exceed 100
mm 3 within 2 weeks and 2.3 cc in 6 weeks. The morphology assumed by the growing
transplant was either a sphere or a prolate spheroid which facilitated fairly accurate
determination of growth by measurement with an ocular micrometer.
15
Toolan in a series of studies, 1951, 1953, and 1954, 3638 examined the proliferation
and vascularization of human epithelium and human tumors maintained in condi-
tioned laboratory animals. He minced epithelium and planted it in normal and X-ir-
radiated animals. Three days after implantation, the epithelium was found to have
survived, and the fragments with their small amount of attached dermis lay un vascu-
larized and unincorporated in the host tissue. On the fourth day after implantation,
host capillaries penetrated the collagen and elastic fibres of the surviving human dermis
in the X-irradiated animals. With one exception, the material in the control animals
was never vascularized. The majority of fragments in normal animals were dead by
the seventh day. In contrast, the human epithelium in the X-irradiated hosts was well-
vascularized and actively proliferating on the sixth or seventh day. By the 10th or 14th
day, the fragments of human epithelium had considerably increased their original di-
ameter. Occasionally, large cystic nests of epithelium were seen, between which small
groups of cells had become separated during implantation. At approximately 14 to 16
days after implantation, the tissues in the irradiated animals died quite suddenly. In
all the 18 and 25 day sections, the human epithelium was dead. Toolan, in 1953,37
examined 101 human tumors implanted in cortisone-treated X-irradiated rats. He
found that 90% of these survived and proliferated for 12 to 20 days. In some instances,
they were maintained as long as 30 days. A similar percentage of tumors implanted in
cortisone-treated nonirradiated rats were also positive for growth. None survived in
normal control animals. Toolan, in 1954,38 found that a single s.c. dose of cortisone
injected at the time of tumor implantation was sufficient to ensure growth of some of
the tumor strains in the pouches of nonirradiated hamsters, and he considered that
these animals were ideal for research purposes in this regard.
Further experiments were reported along these lines by Chute et al. 39 and Patterson
et al. 40 They transplanted 33 human tumors onto the cheek pouch of cortisone-treated
hamsters. This resulted in the survival of 7097b of the tumors for periods in excess of
12 days. One epidermoid carcinoma appeared to hold the promise of being perma-
nently transplantable. They considered that the use of cortisone allowed the elimina-
tion of the initial defense of an animal host to a foreign tumor as first described by
Toolan. Spontaneous tumors in humans and heterologous transplants in animals both
excite varying responses indicative of multiple lines of host defense. They considered
that the stromal response determined the adequacy of the blood supply to the tumor
and that an inflammatory exudate and allergic responses might regulate the survival
of the tumor and spread of the neoplasm. Endocrine factors have been shown to be
significant in the growth rate of both human and animal tumors. The nutritional state
of the animal has been known to be related to the survival of transplanted tumors.
They compared their results in the transplantation of heterologous tumors into ham-
sters with the initial attempts to cultivate bacteria. There was a considerable period
between the early stage of the culture of bacteria in which the most hardy varieties
grew on the simplest media and the culturing of the more sensitive types which awaited
the development of more carefully controlled substrates.
Greene, in 1952,41 reviewed his experiences with transplantation experiments from
February 1939 and February 1950. He transplanted tumors from surgical patients at
the New Haven Hospital and used the anterior chamber of the eye of the guinea pig
exclusively as the transplantation site. He found that certain tissues were incapable of
heterologous transfer and that this group included normal tissues, benign tumors, hy-
perplasias, "precancerous" lesions, and inflammatory granulomas. In contrast, em-
bryonic tissues and human cancers survived and grew, although heterotransplantability
in this site was not a property found in all human malignant neoplasms.
16 Turn or Blood Circula tion
Further study of chemically induced cancers was made by Braithwaite, who in 195842
examined the arterial supply of benzpyrene-induced tumors in the rat. The arterial
supply of the benzpyrene tumor occurring in the right flank was studied in eight male
rats. He found that the blood supply of the tumor arose from vessels normally supply-
ing the area of the integument in which the tumor had grown. The important ones
were (1) The subscapular trunk from above, (2) The terminal ramifications of the su-
perficial epigastric and ilio-lumbar arteries from below, and (3) The lateral cutaneous
branches of the intercostal arteries from the medial side. The branches of these vessels
contributed to a superficial plexus lying on the capsule of the tumor and a deeper
plexus in the interstices of the tumor. He noted a rich communication between the
two. In view of the multiple sources of supply and adherence of the tumor to the chest
wall, obliteration of the arterial supply without causing direct trauma to the tumor
was impractical. These findings were in agreement with those of previous authors re-
garding the sources of supply to the tumor and the fact that hemorrhagic zones, as
manifested by leaks of injection material within the substance of the tumor, were a
constant feature. He found that the latter were usually most prominent near the center
of the tumor, this being the site farthest removed from the source of the arterial blood.
In 1959, Day et al. 43 examined the localization of radioiodinated rat fibrinogen in
transplanted rat tumors. This paper was one of the earliest studies concerning the lo-
calization of injected materials in tumors. They found that radioiodinated rat fibrino-
gen localized in three different transplantable rat tumors during the initial inflamma-
tory reaction of the host to the implant and during the rapid-growth stage of the tumor.
The extent of the localization during the inflammation paralleled the amount of the
tissue which became inflamed. The extent of the localization during subsequent tumor
growth was parallel to the rate of tumor growth. In the Murphy-Sturm lymphosarcoma
at its most vigorous state in the their hands (10 gm at 8 days after implantation), 40%
of the i.v.-injected fibrinogen localized in the tumor within 6 hr, as the tumor grew
nearly two more grams. The disappearance of labelled fibrinogen from the tumors was
slower than that of labelled fibrinogen from the blood, but when antifibrin antibodies
were present, the disappearance of labelled fibrinogen from both was markedly has-
tened without a parallel disappearance of antibody.
In 1959, a further report on vascular morphology detailed the vascular system of
two transplantable mouse granulosa tumors by standard techniques (Walters and
Green).44 The morphology of the vascular system of these two tumors of mice was
studied throughout the life of the tumor by means of histologic sections and India-
ink-injected, cleared, whole mounts. Immediately after s.c. transplantation of the neo-
plasm, the capillary bed surrounding the tumor became engorged. During the first 11
days, this capillary bed appeared to become more dense. By 11 days, the tumor itself
became vascularized. The vessels of the tumor parenchyma were always sinusoids.
These sinusoids were endothelial sheaths with relatively small amounts of connective
tissue associated with them.
Kligerman and Henel, in 1961,45 used the Algire mouse-back chamber to examine
the microcirculation of a transplantable tumor, hepatoma 134, in C3H/CRGL mice.
They found that the arterioles supplying the tumor arose from somatic vessels after
the formation of an initial venous capillary bed. The response of these arterioles to
acetylcholine, epinephrine, 94% oxygen, and 6% carbon dioxide mixture remained sim-
ilar to those of somatic vessels of the animal. They noted that some of the vessels of
the capillary bed might be sinusoids.
In 1962, Gullino et al. 46 examined the collagen content of 11 hepatomas, 2 fibrosar-
comas, Walker carcinoma 256, and lymphosarcoma R-2788 in rats and mice. All he-
patomas studied contained more collagen than the normal liver. The amount of colla-
17
gen formed in each tumor remained constant in all the transplant generations studied,
regardless of the site of transplant or the age of the tumor. In all tumors except hepa-
toma 5123, the increase of the collagen content followed in constant proportion the
increase of the tumor mass. In this respect, hepatomas differed from the regenerating
liver. Hepatomas in rats and mice behaved in the same manner. The neoplastic cells
appeared to influence the collagen content in the tumor mass. This paper indicated
that the stromal response to certain tumors with regard to both vascular supply and
collagen content remains constant.
In a further paper in 1962, Gullino and Grantham 46 studied Novikoff hepatoma,
Hepatoma 5123, Hepatoma LCI8, Hepatoma 3683, Ethionine hepatoma, Walker car-
cinoma 256, and Lymphosarcoma R2788. These tumors were grown in a * 'tissue-iso-
lated* ' fashion. This consisted in isolating the organ injected with the tumor in a par-
affin sac so that only one artery and vein connected the tumor with the host. This
technique allowed separation of the vascular response of the tumor from that of the
vascular net of the host. Under the influence of various stimuli, the vessels of the
tumor behaved in a similar fashion to that of the host organs. Vinylite casts were made
of the blood vessels of the tissue-isolated organs which had been replaced by neoplastic
cells. It was found by comparison with casts of the normal kidneys and ovaries that
the main vascular branches which supply the host organ became the main vascular
branches which supply the tumor.
Further work on the aspect of delivery of active agents was conducted by Goldacre
and Sylvén.48 In 1962, they examined the access of the blood-born dyes to various
tumor regions. They changed the color of the systemic blood in the living animal by
the addition of harmless dyes, especially lissamine green. They found that it was pos-
sible to mark out regions of the tumors which could not be readily reached by blood-
born substances. They noted that these types of regions suddenly appeared in tumors
at a critical age. In the transplanted mouse tumors studied, mammary carcinoma, sar-
coma 37, Ehrlich-Sandschritz solid-ascites tumor, this stage was found at about 12
days after transplantation. In the rat Walker carcinosarcoma it was found in 5 to 10
days, depending upon the type of the host. A few days later these zones usually occu-
pied all but a thin, well-vascularized shell of the tumor. The width of this shell varied
from a few millimeters in thickness to as little as 0.1 mm. This region often contained
vessels with red blood in them when the blood in the rest of the body was green,
showing that the vessels were in stasis and their openings blocked. These uncolored
zones contained living cells, as shown by vital staining, tissue culture, and particularly,
by transplantation into new hosts. In 41 transplants from these "white" zones, 23
typical tumors were produced, including all of the tumor types tested. The penetration
of dye into these so-called necrotic zones of tumors which contained living cells was
determined at various time intervals after the blood had been made green, after re-
peated daily injections of the dye, and with injections into the necrotic zone. The move-
ment of interstitial fluid which might have carried the dye into the interior of the tumor
was shown to be retarded or prevented in tumors with necrotic centers, although each
tumor behaved as an individual case in this respect.
Merker and Hurley, in 1962,49 employed a diagnostic procedure using X-ray photog-
raphy together with the injection of thorotrast® as a contrast material, thus combining
different aspects of earlier studies. In this manner, they studied the growth and regres-
sion of a human tumor which was transplanted into cortisone-treated mice. The pro-
cedure allowed for the satisfactory observation of the vasculature of the growing trans-
plant. Following the third day of transplantation, the tumor inocula growing in
cortisone-conditioned mice can readily be distinguished from inflammatory host reac-
tions which develop in mice not so treated. The use of this technique suggested that
the effects of Actinomycin D against transplants were not related to interference with
the development of a satisfactory tumor-bed vasculature.
Cataland et al., in 1962,50 examined the relationship between size and perfusion rate
of transplanted tumors. The blood flow to s.c. transplants of mammary carcinoma
755 and sarcoma 180 was measured by the indirect technique of indicator fractionation
with the use of radioactive rubidium 86. The smallest tumors showed the largest frac-
tional uptake of indicator per gram of tissue. Tumors of increasing size had progres-
sively lower fractional uptake of indicator. They concluded that mass increased more
rapidly than the blood supply in these transplanted tumors. The technique used meas-
ured only functional, rather than total, blood flow. Blood flow through arteriovenous
shunts or other nonexchanging areas was not measured. Nevertheless, the conclusion
remained that the tumors were not well serviced by the circulation, and in fact, the
growth of the fibrovascular stroma of the tumor and the mass of tumor cells might be
discordant.
In an extensive study on the nature of the reaction of the host vasculature to the
presence of a malignant neoplasm, Delarue et al. 51 traced the vascular modification of
the hamster cheek pouch during the course of development and growth of grafts from
a melanoma. Under general anesthetic provided by nembutal, the cheek pouch of a
golden hamster was exteriorized. An incision was made with scissors onto the stretched
pouch, and a small fragment introduced between the two layers.
The fragment measured 1 to 2 mm in diameter and was taken from the side of a
host tumor in a nonnecrotic zone. The fragment was introduced into the end of the
pouch with a thick needle pushed from the opening into the pouch. It was
then pushed further by a rod left in place, and the opening was closed with collodion.
This technique was used on 35 hamsters several times, two passages having been ef-
fected by leaving the first tumors which developed. The development was followed
step by step from the beginning, every day or every second day on one or two animals,
then every 3 to 4 days, and finally, every week or at longer intervals. The examination
under a binocular loupe permitted from the beginning a study of the graft, the vessels,
and the tissue in the pouch. When the tumor reached a large enough size, it became
too opaque, and only the neighboring tissue and peripheral vessels could be observed.
The morphology of the vessels was studied by aortic injection with India ink into the
vascular system of the sacrificed animal. The vessels were studied by thin, cleared,
serial sections. The grafts put into the pouch were followed and showed variable re-
sults. In seven cases, the graft did not take. After 24 or 48 hr, the graft appeared
unchanged and without a peripheral vascular reaction. Over the following days, it
became smaller, deposits of brown or black pigment were present in the neighborhood,
and the pouch was congested. This all disappeared, with deposits of melanin left in
the pouch in some instances. In other cases, following an early inflammatory reaction,
the pouch ulcerated, the graft was eliminated, and the area scarred over. More often,
the graft took and developed (28 hamsters). Observations under a small hand lens
showed the majority of cases went through the following general pattern:
1. From the second day the vascular pattern of the pouch was more accentuated.
This peritumoral pattern was made of a capillary lacework which was regular
and dilated. Sometimes there would be peripheral hemorrhagic suffusions which
could follow injury to the position of the graft. The graft stayed quite translucent
and deposits of pigment could always be seen. The vascular network eventually
became obscure.
2. From the fourth to the sixth day (sometimes only towards the eighth day) the
venous and capillary pattern enlarged, and the tumor appeared vascularized. From
19
this moment, the tumor grew quite regularly in the form of a spherical mass. The
graft also became opaque, and the tumor mass took on a darker tint.
A vascular pedicle reached towards the hilum of the pouch. It was made up of a
very dilated vein four times the normal caliber, which was tortuous and came from
one pole of the tumor. It arose from the confluence of intratumoral vessels which were
difficult to distinguish from sinusoidal peritumoral vessels. The arterial supply to the
tumor was very hard to individualize once this pedicle was constituted. The tumor
grew, generally, quite rapidly to reach the size of a cherry or larger. Necrotic material
was found on the surface and was also within the tumor mass, occurring frequently
with, or followed by, ulceration. The pouch remained, however, very easy to evert,
but optical observation was, because of this, impossible. Usually, the tumor reached
the size of a nut in 30 days. However, in certain cases where the graft had stayed
stationary without peripheral vasodilatation for several days or several weeks with
some reduction in its volume, the growth was slower, and the size of a nut was not
reached within 90 days. The morphology of the vessels was studied in thin, cleared
sections after opacification of the vascular system of the animal with India ink. Within
these slices of the tumors three zones were identified:
1. In the center, there was a zone of avascular necrosis.

2. There was a middle zone which contained viable tumor cells with a vascular retic-
ulum of the capillary type which was richly anastomotic at times. These vessels
were tortuous, irregular, with some saccular dilatations. In section, the vessels
isolated cords of tumor cells in a quadrilateral fashion.
3. A peripheral zone with vessels of large caliber surrounded the tumor. Sometimes
several distended venous canals ran parallel to the convex surface of the tumor,
and numerous branches of the intratumoral vessels contributed to these and ter-
minated within such vessels. Lobules of the tumor appeared oriented by vessels
which contribute to the principal pedicle.
Further away, the vessels of the pouch were normal. From the grafted tumor, there
followed 2 or 3 months later multiple metastases—renal, peritoneal, hepatic, pulmo-
nary, cardiac, and s.c. metastases. One intravascular injection of India ink was given
in these cases, and the disposition of the vascular pattern presented the same appear-
ances as the initial tumors. Delarue and his co-workers came to the following conclu-
sions:
1. The particular morphology of the vessels of the malignant melanoma were the
same in its initial location as in its metastases.
2. The development of the vascular pattern depended upon the vessels of the host.
3. Extension of the tumor appeared to be concurrent with the peripheral vascular
reaction developed around the graft.
Ogilvie and co-workers, in 1964,52 returned to the question raised earlier in the lit-
erature regarding the arterial supply to experimental metastatic tumors in rabbit lungs.
They investigated the developed blood supply to the metastatic tumors by a variety of
injection techniques. A bronchial arterial supply was shown in many specimens in
which the major bronchial arteries were injected and which also showed ink particles
in the vessels of the tumor. At the periphery of the lungs, tumors 3 mm or larger were
injectable via both the bronchial and pulmonary arteries. Tumors in this situation, less
than 3 mm, possessed a blood supply from the pulmonary artery alone. In all prepa-
rations, tumors near the hilus were reached by materials injected via the bronchial
arteries. During in vivo studies during which the circulation to pulmonary tumors in
living rabbits was observed by quartz-rod illumination, India ink injected via the sys-
temic circulation reached the tumor.
Warner, in 1968,53 used the model developed by Lucké and Schlumberger and ex-
amined the living vascular-tree in ocular transplants of frog renal adenocarcinoma. In
all, 49 frogs were inoculated from a single, massive, spontaneously occurring renal
adenocarcinoma. 70% of the frogs showed signs of tumor growth. The growth patterns
of the tumors, as observed at intervals for a 4-month period in living frogs, varied
considerably, as did the vascularization of the tumor tissue. Some tumor transplants
never seemed to be vascularized although there was increasing vascularity of the adja-
cent iris. These tumors usually spontaneously disappeared. Tumors which eventually
eroded the cornea, permitting avulsion of the inner layers of the eye, appeared as
glomeruli complete with vascular tufts and tubules with intervening blood vessels. Both
resembled the vascular bed of the normal frog kidney. The vessels were seen to be
extremely fragile, and microscopic examination of the eye was almost always accom-
panied by some hemorrhage. Between these two extremes of vascularity many sub-
stages were found to exist.
Day, in 1964,54 reviewed the published work on the vascular relationships of tumor
and host. He put forward the hypothesis that certain discrepancies in the literature
could be accommodated by supposing that there were two main types of sources for
the blood supply of tumors. These would be (1) the pre-existing network of the host
tissue recruited by the tumor, and (2) an entirely new set of vessels which resulted from
a fibrovascular response induced in the host by the influence of the presence of the
tumor.
Witte and Goldenberg 55 observed a tumor, originally described by Toolan as
H.Ep.3, a human epidermoid carcinoma, growing in vivo within the hamster cheek
pouch. They found that the growth of the vasculature beneath and into this tumor
conformed with the earlier accounts of the blood-vessel phenomena associated with
transplantation. The first change was a hyperemia of the blood vessels which occurred
about 8 hr after the injection of the suspension. After this, there was spiral-like twisting
of capillary and muscular blood vessels for 12 to 24 hr. At the end of the second day,
there was the establishment of a dense vascular network within the tumor transplant.
The growing tumor further deformed the vessels.
The concept of the rabbit-ear chamber and the availability of the hamster cheek-
pouch membrane were combined by Goodall et al. 56 They described a transparent win-
dow for the observation of the growth of tumor transplants in vivo on the hamster
cheek-pouch membrane. The morphology of the microcirculation in four transplanta-
ble tumors of the hamster was recorded. The two melanoma tumors possessed similar
vascular patterns, while the anaplastic carcinoma of the breast and the hemangioperi-
cytoma showed distinctly different and characteristic networks.
III. T H E V A S C U L A R MORPHOLOGY OF EXPERIMENTAL TUMORS
As can be seen from the above review to 1966, investigators returned with improved
methods and technology to tackle again questions that were originally posed at the
beginning of the century. The principal methods of investigting the vascular morphol-
ogy of experimental tumors, in addition to standard histological sectioning, have been
(1) by examining growing tumor transplants in transparent chambers in the living ani-
mal, (2) by injection techniques, (3) by angiography, and (4) by the clearance of radio-
active tracers. The latter division will be dealt with in detail in a later chapter. One of
the major advances in the study of the reactions of tissues and the growth of tumors
21
has been the development of the transparent-chamber technique. The earliest transpar-
ent chamber was described by Sandison in 1928,58 while he was a student of Dr. E. R.
Clark. The design and construction of most chambers for use in the ears of rabbits
trace their origin from the varieties described by Clark and co-workers in 1930.59 The
aim of the transparent chambers from the time of the development of the rabbit-ear
chamber has been to develop a thin membrane, or to imprison an already formed thin
membrane, between two transparent plates, which are fixed together at the rim. The
thinness of the membrane permits transillumination of the vessels and cells within the
membrane and their observation during life by microscopy. Williams,60 in an extensive
review of microscopic studies in living animals with transparent chamber methods,
considered that this technique reduced the need for interference when determining a
dynamic, vital sequence of the response of tissues to the implantation of potent chem-
icals, tumor and other grafts, parasites, and bacteria. Since this review, there have
been two notable additions involving this technique: (1) the Sanders-Shubik chamber61
for the hamster cheek-pouch membrane, and (2) the "sandwich" chamber system de-
veloped by Reinhold et al.62 for use in rats. Partly because of the availability of a
number of transplantable tumors of the hamster, Sanders and Shubik modified the
transparent chamber for the rabbit's ear to a form which would fit in the hamster
cheek pouch. The chamber consists of upper and lower plates which fit together by
means of two pegs. The upper plate consists of a ring glued to a disc. The ring contains
the recesses which take the pegs from the lower plate. The base, or lower, plate fits
through the animal's mouth to be located in the dilated end of the pouch. The skin
over the back of the animal is incised under anesthesia, and the upper plate fitted over
the pegs of the lower plate. The skin is tied by means of a purse-string suture in the
groove of the flange in the ring of the upper plate. The chamber is transilluminated
by means of a plexiglas rod ("Knisely quartz-rod technique") while the animal is an-
esthetized and mounted on a special stand attached to a microscope. The methodology
of the hamster cheek pouch has been reviewed by Greenblatt et al.63 The round table,
tantalum gauze, bay and moat, large graft, and preformed tissue chambers are all
discussed from the technical aspects, and their individual advantages and disadvan-
tages enumerated in a technical report on chamber techniques of studying the micro-
vasculature by Nims and Irwin.64 Using the hamster cheek-pouch chamber technique,
Greenblatt and co-workers were able to transplant renal65 and cardiac homografts66
onto the pouch membrane and to observe vascularization of these tissues. Donor tissue
for the renal homografts was obtained from hamsters less than 24 hr old, and the
material was minced to 1 to 2 mm fragments. These were transplanted onto the pouch
membrane, and glomerular circulation was established in 100% of grafts from newly
born animals. Grafts derived from adult animals did not show any glomerular circu-
lation. In a similar fashion, donor heart tissue from hamsters less than 24 hr old was
obtained and transplanted. Cardiac grafts from fetal and newborn animals showed
functional activity, i.e., rhythmic contractions, more frequently than those derived
from adult animals.
Injection techniques for the study of vascular morphology have recently been used
by Falk67 and Eddy.68 Falk described two methods for the production of a three-dimen-
sional image of the patterns of vasculature in rat tumors. In both, benzidine is used to
indicate the location of blood. In the first method, tracings onto acetate sheets 200 /¿m
thick are made, placed on squares of glass, and stacked in the appropriate order. In
the second method, the blocks of tissue containing the tumors are halved, stained in
benzidine, and mounted in resin. Similar methods to these have been used for a number
of years for anatomical preparations, but in this particular paper, they are applied to
the study of vascular morphology.
vessels or the complete vasculature of normal and malignant tissues. The method fa-
vored here was injection of red lead oxide or India ink suspended in a heparinized
gelatin-Joy® detergent medium. The vessels perfused with such material can be seen
by high resolution, microradiography, or photomicrography.
Kjartansson69 studied intratumor flow distributions using radioactive tracers and
reviewed this method of approach to the vasculature of tumors.
Stewart et al., 70 in a major work in the A.F.I.P. Fasicle series, list the transplantable
and transmissible tumors of animals and detail their characteristics under the headings
of synonyms, definition, history and description of original tumor, transplantation
studies, and description of current tumor. When considering the vascular morphology
of experimental tumors, one of the main constraints is the nature of the pattern of
growth of the tumor parenchymal cells themselves. This pattern of growth, together
with the size and shape of the cancer cells, are the traits recognized by the pathologist
on histologic section of tumors which permit diagnosis of a specific type. O'Meara 71
considered that there were two forms of growth in carcinomas: (1) maturation growth,
which was a property of normal as well as carcinomatous tissue, and (2) invasive
growth, which was a property only of malignant tissues. He considered that as carci-
nomas grew fibrin was deposited on the cells, and that invasive growth depended upon
fibrin formation by the carcinoma cells. He suggested that the invaded tissues re-
sponded to this fibrin by laying down the stroma of the tumor.
A. Tumors of Melanin-Forming Tissue

Algire30 found that the vascular pattern of a melanoma of the mouse (e.g., Figure
1) contained large afferent and efferent vessels and a rich capillary plexus. Goodall et
FIGURE 1. The edge of a transplanted melanoma growing in the hamster cheek pouch in a transplant
chamber is shown en face. The tumor and adjacent host membrane are transilluminated from below so that
the vessels are obvious. The edge of the tumor (E) contains many draining vessels. (Magnification x 70.)
23
al 56.72 e x a m i n e c i two melanomas of the hamster. Originally described by Fortner 73 as

M. Melanoma No. 1 and 3, the patterns of these two tumors were so similar that it
was difficult to distinguish between them. The distribution of the smallest vessels was
chaotic, and there were leashes of capillaries interlacing in haphazard fashion. A char-
acteristic feature was that the capillaries ran for relatively long distances without
branches and the angles of the branches, when they did occur, were always acute.
Closed geometrical figures were extremely rare, so that the pattern was arboreal rather
than a network. Within the transparent chamber in the hamster cheek pouch at about
8 days after vascularization, a pattern became clearer when the whole growing tumor
was transilluminated. An "organoid" arrangement became apparent which consisted
of relatively large vessels which were situated at the edge of the graft and described
large arcs. These vessels entered and left the graft and communicated with the vessels
of the membrane. The accumulation of a number of such vessels at the growing edge
of the tumors gave the appearance of a "venous capsule". Within this rim, there were
short loops of capillaries which opened toward the center of the graft. These vessels
were associated with the finest capillaries, which at this stage were straight. Transplants
of two varieties of amelanotic melanomas (A-Mel-4B32 and ZGPY1) were examined
in the hamster cheek-pouch chamber. 74 75 The first tumor grew at a rate which covered
the viewing area in 3 to 4 days while the second tumor covered less than 10% of the
viewing area at this time. Although there was this considerable difference in growth
rates, the morphology of the vascular network was so similar as to be indistinguisha-
ble.74 By means of combined in vivo and scanning electron microscopy, the surfaces
of the blood vessels within the transplant and the form of the tumor itself could be
observed.75 There was a considerable variation in the structure of the vessels within
the tumors, which is discussed in a later section of this chapter.
B. Sarcomas and Tumors of Subcutaneous Tissues

Lewis24 described the vascular patterns of a fibrosarcoma, a spindle-cell sarcoma,
and a round-cell sarcoma of rats. The fibrosarcoma possessed a pattern that was sim-
ilar to that of normal s.c. tissue. There was a rich capillary net and large afferent and
efferent vessels. In contrast, the spindle-cell sarcoma formed a viable shell around a
necrotic center. Within the peripheral shell was a rich plexus of fine capillaries, par-
ticularly abundant close to the necrotic center. The capillaries beneath the capsule con-
tained elongated loops. The round-cell sarcoma possessed yet a further vascular pat-
tern. In this type, there developed large necrotic centers and a thin shell of peripheral
living tumor 1 to 2 mm in thickness. The vigorous part of the tumor was limited cen-
trally by the zone of blood vessels. The zone of capillaries and the necrotic center were
separated by two zones in which there were different stages of degeneration. The ves-
sels of this type of sarcoma were coarser than that of the spindle-cell sarcoma. The
capillary plexus was abundantly supplied with large afferent and efferent vessels. There
were only short distances between such vessels and the terminal capillary plexus next
to the degenerating tissue. All of these tumors arose spontaneously in rats of strain P
in Dr. George Walker's rat colony, and the specific organs of origin were the fibrosar-
coma from tissues of the breast , the spindle-cell sarcoma from the liver, and the
round-cell sarcoma from the wall of the vagina.
Falk76 77 investigated the patterns of vasculature in two pairs of related fibrosarco-
mas by staining methods. The first two associated lines were SSBIA and SSBIB. These
arose from a single, spontaneous fibrosarcoma in breast tissue of a rat. Following
transplantation, the two lines were isolated and distinguished by their growth rates,
SSBIA and SSBIB taking 1 Vi and AVi days, respectively, for doubling their volume at
9 mm diameter. The second two tumors were RIB5 and RIB5C. RIB5 occurred as a
result of the s.c. injection of benzopyrene in 1945. RIB5C was derived by culturing the
cells in vitro for 12 months. During this time, plating efficiency rose from 15 to 80%.
Falk draws attention to the effects of constriction by the capsule or pressure by the
pseudocapsule on the vasculature of tumors. He noted that cryostat sections of SSBIA
and RIBs became saucer shaped on thawing, as if the outer portion were constricting
the inner region, whereas sections of SSBIB and RIB5C remained flat. The tumors
SSBIA and RIB 5 can be regarded as tense tumors, i.e., with the tumor substance under
pressure, and SSBIB and RIB 5 C as lax tumors with little buildup of central pressure.
Investigation of the vascular morphology of these tumors revealed that the tense tu-
mors possessed an external blood supply and drainage, while the lax tumors were sup-
plied by an internal system. The organization of the vascular tree of a lax fibrosarcoma
(SSBIB) of the rat commenced with the entry of an artery and vein at the base of the
tumor, with sequential multiple branching more marked in the venous system. The
veins and arteries retained their muscle coats until terminating in very fine vessels. In
RIB 5 C, the only additional morphological feature was that, in addition to the main
vessels at the base of the tumor, there were other vessels which ran obliquely along
part of the circumference before entering the tumor from the side. These particular
vessels, unlike similar vessels in the tense tumors, showed no evidence of areas of
constriction or stretching. While both showed external systems of blood supply, the
two tense tumors, SSBIA and RIB 5 , differed considerably from each other in the de-
tails of their vascular patterns. The outside of tumor SSBIA was covered with veins
possessing a "riverlike" pattern. On growth, these developed a short, broad vessel
which divided within the tumor into 6 to 8 broad branches. Each of these ran in a
circumferential pattern just within the margin of the tumor, and many extended nearly
a quarter of the way around the periphery. A very large number of fine branches
extended; into the tumor from these main branches. They were usually without
branches, and there was no muscle coat. The vessels tended to run in bundles, the
longest extended to more than half the diameter of the tumor. The arteries ran over
the surface of the tumors, running along it for considerable distances, with the for-
mation of occasional hair-pin bends, before passing through the surface and immedi-
ately dividing into very fine branches. They were only able to be separated from the
drainage vessels because of their straightness and a still lesser tendency to divide. They
finally fed into the capillary network. Specific staining revealed a layer of collagen
fibres at the advancing front of SSBIA tumor, and both arteries and veins were in-
vested with this fibrous tissue at the edge of the tumor. Falk's interpretation of this
morphology was that the expanding tumor forced back the bases of the veins while,
at the same time, the increasing circumference of the tumor spread out the first set of
branches. As a result of the increasing radius together with the general expansion, the
final radial branches were compressed and stretched, The attachment of the vessels to
the collagen of the capsule could similarly explain the looping of arteries on the surface
of the tumor. Eventually after increased growth, the vascular pattern of the tumor was
reduced to the vessels of the periphery of the tumor and the capillary network within
it. As a contrast with regard to some features, RIB 5 has no collagen at the outside of
the tumor or attached to the vessel walls. In this tumor, an artery enters the peripheral
rim of the tumor and runs parallel with the tumor edge giving off branches and even-
tually passes out back into the host tissues. The arterial supply was supplemented by
other arteries at the sides of the tumor. Veins appeared deep in the tumor, and where
the artery turned to run parallel to the tumor edge they were markedly constricted.
Within the tumor, the veins were wide. The origin of the veins appeared as a "mop
head" of coarse tributaries. Falk believed that in this type of tumor there existed a
25
pressure, possibly of the dividing cells, which prevented "penetration" by the arteries
and gave rise to constriction of the vein.
Eddy and Casarett 78 traced the development of a malignant neurilemmoma of ham-
ster by transplanting a fragment of the tumor into a hamster cheek-pouch chamber.
They noted the establishment of a tumor capillary net from host venous vessels at the
perimeter of the transplant in 4 to 7 days. With continued remodelling, the vascular
bed assumed a dense "netlike" arrangement of short, profusely anastomosing, thin-
walled endothelial tubes through which blood flowed at a rapid rate. Increase in the
caliber of the capillaries was noted with tumor growth. Focal areas of reduction of
blood flow or general reduction of flow occurred after about 10 days of growth. They
attributed this to pressure effects due to growth of the tumor within the chamber.
Sarcoma 37 and other sarcomas were used by Algire and co-workers 3132 in some of
their studies. They found that with sarcoma 37 there was a rapid development of ves-
sels, and after the tumor attained a vascular level double that of the connective tissue,
the vascular level then stabilized. When a polysaccharide fraction from Serrada mar-
cescens was tested32 in sarcomas, they found that the vascular occlusions and stasis in
normal and neoplastic tissues were associated with edema. Vascular occlusion in sar-
comas was followed after a variable time by the sudden appearance of petechial hem-
orrhages throughout the tumor. An apparently complete necrosis of the tumor oc-
curred, but some peripheral tumor cells survived, elicited a vigorous new vascular
supply, and growth was resumed.
Natadze 79 worked on the regulation of blood flow in mouse sarcoma 180 and Ehrlich
carcinoma. He pointed out that, as well as the fibrovascular response induced by the
tumor, there were vessels, previously formed with differentiated multilayered walls,
which however, might undergo degenerative changes and fibrosis on occasion.
Glazunov80 considered that a poorly regulated network of vessels was formed in
tumors without well-defined arteries, veins, and capillaries. He considered that the new
growth of capillaries took place in the zone of invasion by the tumor and that some
pre-existing vessels of arterial and venous type persisted next to recently induced vas-
cular formations. One of his illustrations shows a benzanthracene-induced sarcoma of
the soft tissues of the leg of a rat. A radiograph of the injected specimen showed that
the tumor was supplied not only from the femoral artery, but from arteries branching
off from above the aorta.
C. Tumors of the Mammary Gland

From his injection studies of the Walker adenofibroma of the breast in rats, Lewis24
described the highly specialized vascular tree in this tumor (e.g., Figures 3 and 4). It
had large afferent and efferent vessels which broke up into fine vessels which supplied
the lobes, lobules, and acini. He recognized the unit capillary plexuses which supplied
the acini. Algire and co-workers31 examined the growth of transplants from mammary
gland carcinomas originating in C3H mice. New capillary sprouts appeared at about
the third day, and by the fifth day the cell clump was surrounded by capillaries. By
the seventh day the vascular level within the transplant was about twice that of the
surrounding connective tissue. Capillary sprouts arose from the underlying muscle cap-
illaries and from vessels in the nerve trunk at some distance from the transplant.
Within the transplant there developed a sinusoid-like plexus of vessels which retained
their capillary structure. No evidence of differentiation into arterioles or venules was
detected. The vascular bed stabilized at double the level of the connective tissue. The
growth rate was rapid and constant until the 12th day when a ring of leukocytes was
noted around the transplant. All the vessels of the tumor, except for the main artery,
which was the only preformed vessel, had walls consisting of only a single layer of
endothelium.
Transplants of mammary adenocarcinoma in the hamster developed a distinctive vas-
cular pattern. 56 72 The smallest capillaries formed a reticular pattern of closed geometr-
ical figures of irregular outline. Capillary junctions or branches were deltoid or pyram-
idal. Roughly spheroidal regions were delineated by the vascular pattern at this level
(See Figures 2 and 3). Sinusoidal and saccular distortions and other malformations were
common in this tumor (Figure 4). The larger pre- and postcapillary vessels formed wide
loops, frequently of paired vessels with the direction of blood flowing in opposing
directions. The rate of blood flow was faster in the mammary carcinoma than in the
melanomas and hemangiopericytoma types of hamster tumors when these were com-
pared.56
Kruuv et al.81 examined blood flow and oxygenation in transplanted, isologous
C3HBA mammary tumors. They emphasized that the fine vessels in tumors are devoid
of contractile elements and neural junctions and that, when pharmacologically active
agents which affect the circulation are given, the importance of the relative resistance
of the tumor vascular bed to the vascular bed of the surrounding tissues comes to the
fore. An increase, induced by such agents, in the flow in surrounding tissues, because
of this effect, may cause a decrease of flow within the tumor. The tumor-bearing ani-
mals were given chlorpromazine, isoproterenol, or allowed to inhale amyl nitrite in air,
or to inhale 100% oxygen at one atmosphere. The drugs all diminished tumor blood
flow and oxygen tension. They concluded that administration of these drugs during
radiotherapy of tumors was unlikely to be of value.
Tannock, 82 by means of a combination of histological stains and autoradiographic
technique, studied population kinetics in the C3H mouse mammary tumor. He found
FIGURE 2. Histological section of mammary adenocarcinoma of the mouse to show the fibrovascular
stroma (FV) dividing the lobules of the tumor. (Magnification x 530.)
27
FIGURE 3. En face view of a growing mouse mammary adenocarcinoma in vivo in the hamster cheek
pouch. Although there is some blurring of the details of the vessels because of the thickness of the prepa-
ration, wide arcs of small vessels around the lobules of the tumor can be distinguished (at A and B).
Hamster cheek pouch chamber preparation. (Magnification x 55.)
FIGURE 4. Observation of vessels in vivo within a hamster mammary adenocarcinoma revealed straight
arterioles (Al, A2) and very irregular vessels with saccular dilatations (S). Hamster cheek pouch chamber
preparation. (Magnification x 70.)
that mitotic index, labelling index, and mean grain count per labelled cell decreased
with the distance of carcinoma cells from a capillary. He noted migration of cells from
locations near blood vessels where there was rapid proliferation to regions near nec-
rotic changes where proliferation was low. The turnover time for the carcinoma cells
was about 22 hr. In contrast, endothelial cells showed a turnover time of 50 to 60 hr
and fibroblasts 70 to 80 hr.
D. The Walker Carcinosarcoma 256

The vascular pattern in the Walker 256 tumor comprises a branching system of slen-
der angulated arteries which drain into a profuse venous plexus.83 Kido84 examined by
angiography the vascular pattern which was generated when approximately 10,000 cells
of Walker's carcinosarcoma 256 were injected into liver lobes of rats. The changes
found were divided into changes at an early stage (1 to 5 days, 18 rats), intermediate
stage (6 to 7 days, 17 rats), and advanced stages (8 to 13 days, 53 rats) of tumor growth.
The early stage—No tumor formation was seen macroscopically in the rats in the
first three days. After four days a tumor between 2 and 8 mm in diameter was found
in 10 animals. The main vessels which fed the tumor revealed a peculiar dilatation and
tortuosity in some animals and were only noted with tumors greater than 4 mm in
diameter.
The intermediate stage—The tumor size at this stage ranged from 4 to 14 mm in
diameter. The distinctive angiographic features were (1) a remarkable increase in new
vessels (71%) with irregular calibers, (2) interruption in new vessels (5997b), (3) a tend-
ency for the tumor to be lobulated, and (4) a tendency to have vessels encircling its
surface. These encircling vessels branched to form several smaller branches which cre-
ated a coarse network. Anastomoses between such vessels were present in 47% of the
tumors.
The advanced stage—As enlargement of the tumors occurred there were alterations
in vessel shape and tumor infiltration. Remarkable and irregular vascularization was
always present. Widely anastomosing vessels formed a complete network which corre-
sponded to the extent of the tumor mass. As spread occurred, small new vessels were
noted outside the main tumor mass, and the demarcation between tumor and normal
tissue became obscured. Partially and completely avascular areas were sometimes noted
in some animals, and considerable ascites occured in 13 rats in this terminal stage.
Oikawa et al.85 studied by serial microangiography and radioactive ,33 Xe, the growth
and immune rejection of Walker 256 tumor implanted in rat tails. There were two
tumor groups. In group A, the tumor grew rapidly, resulted in a two- to sixfold increase
in blood flow, and caused death in 10 days. In group B, the tumors grew in a slower
fashion, and the blood flow increased only two to four times. At 6 to 8 days after
implantation, the tumors diminished in size, the blood flow decreased, and an extensive
lymphocytic infiltration occurred. At 21 days, the tumor was not discernible.
These events were followed by angiography. In group A at 8 to 10 days after trans-
plantation, there was an obvious tumor circulation with much neovascularization and
profuse small veins, which were dilated. No arteriovenous shunting was recognized.
The animals died about 10 days from transplantation. In group B at 6 to 8 days, the
tumor vessels were abundant and showed no notable differences from the tumors in
group A. By 10 days, however, the vascular pattern changed in parallel with diminished
tumor size. Thinning of the vascular pattern so that it became very slender and "ghost-
like" was noted. By 17 days, the vasculature present remained tortuous. By 21 days,
the angiographic appearances were consistent with anatomical normality.
In my own studies86 of the microcirculation of Walker 256 carcinoma growing in
29
hamster cheek-pouch chambers, extravasation of red cells at the advancing edge was
linked to the occurrence of capillary sprouts. By 4 days after transplantation, a capillary
network had formed in the transplant. These vessels were most obvious at the periphery
of the tumor. The pressure on the growing tumor obviously influenced the macroscopic
pattern of vessels. When pressure was released by raising the top plate, many vessels
filled that had previously been empty within the central mass of the transplant. Vessels
at the leading edge of the tumor contained intracellular endothelial fenestrations and
saccular endothelial processes. These fenestrations were not evenly distributed and were
closed by a diaphragm that had a thickness equal to the plasma membrane of the cell.
In width, they were from 500 to 700 A.
Van den Brenk and co-workers87 described the microvascular changes induced by
Walker 256 and YP388 tumor cells. They found that the free blood present in ascitic
and solid forms of these tumors was due to leakage of blood from the microvasculature
as the result of the formation of capillary sprouts. They found that the formation of
capillary sprouts was largely confined to a peripheral shell of actively growing tumor,
and that within this active zone extravasated blood persisted. The central part of the
tumor eventually becomes apparently avascular because there is failure of the remo-
delling process, and capillaries degenerate. Some capillaries may remain closed, possi-
bly due to the tumor pressure as described above. 86
E. Hepatomas
Guillino and Grantham 47 in their work on the exchange of fluids between host and
tumor used a number of hepatomas of rats including the Novikoff hepatoma, Hepa-
toma 5123, Hepatoma LC18, Hepatoma 3683, and Ethionine Hepatoma. Transplan-
tation of the tumors into various organs resulted in the infiltration of those organs by
the tumor cells and the stretching of the larger vessels with a resulting increase in
length. The combination of cell growth and stretching of pre-existing vessels was sim-
ilar to the appearances seen in liver regeneration. They found that the difference be-
tween the vasculature of a regenerating liver and a hepatoma consisted in the appear-
ance of a great proliferation of vessels, mainly at the periphery of the tumor.
Kligerman and Henel 45 experimented with a transplantable hepatoma 134 in C3H/
CREL mice in the Algire chamber. Tumor fragments were placed next to one of the
cutaneous nerves. Usually by the third to fifth day, capillaries invaded the tumor trans-
plant, and these were supplied and drained by venules from the host tissue. Not until
the venous circulation was established did evidence of arteriolar supply appear, and
this had its origin from existing somatic vessels. The venous and arterial connections
usually were derived from the respective vessels of the nerve. At no time was differen-
tiation of the tumor capillaries into arterioles or venules observed.
Yamaura and Sato 88 examined in detail the vascular morphology of the growth of a
rat ascites hepatoma AH109A in a transparent chamber. A small fragment of the tu-
mor (approximately 0.2 mm 3 ) was placed on the tissue during insertion of the chamber.
These workers divided the changes into four parts. In the first stage within 4 days of
transplantation, the capillaries became irregularly widened at branches and bends and
began to form an irregular net. The changes of the second stage were seen 8 to 10 days
after transplantation. The new sinusoidal capillaries developed homogeneously over
the whole area since all the tissue surface was occupied by tumor cells. Venous blood
flowed in light and dark colored stripes, and the color of the bloodstream altered
greatly from one location to another. No changes were noted in the arteries. In the
third stage, 14 to 16 days after transplantation, there were changes in the arterial sys-
tem, especially in arterioles of less than 100 /¿m in diameter. The walls of these vessels
became thin and ragged. The lumens of arteries widened at branch regions with capil-
laries, and a zigzag pattern developed. The color of the arterial bloodstream became
mottled. The sinusoidal capillaries which branched out from such vessels formed an
alveolar reticular pattern. The venous vessels (structurally they may not strictly be
"veins") varied in diameter dramatically. There were variations in lumen diameter from
200 to 50 ^m. The velocity and direction of the flowing blood was easily changeable
by a little movement or by respiration of the animal. In the fourth stage, up to 24 to
26 days after transplantation, accumulations of necrotic tumor cells made their appear-
ance, mainly in the areas of the capillary network. Arteries measuring more than 30
^m showed parallel clear zones of about 100 yun on each side. Histology of this tissue
indicated that these zones contained viable tumor cells and that, external to the zones,
necrotic cells were found. Capillaries in these darker zones contained nearly normal
blood at first, but eventually, severe hemorrhage occurred. With this, the blood flow
stopped, and the tissue became completely necrotic. Attempted capillary regrowth into
the necrotic area was obscured, but the ability to mount a new wave of capillary sprouts
was severely limited in the third and fourth stages. A further interesting phenomenon
concerning the effect of the hepatoma tumor cells on the capillary network was detected
by Yamaura and Sato. Although the initial neovascularization process of the tumor
transplants was the same as that seen in normal tissues with sprouts and << comblike ,,
arrangement of capillaries, this initial pattern was altered under the influence of the
tumor cells to sinusoidal capillary networks.
F. Tumors of the Blood-Vascular System

Transplants of a hemangiopericytoma (IZ-1) of the hamster when observed in the
hamster cheek-pouch chamber 56 72 possessed a simple, regular, geometrical pattern in
which the smallest capillaries described enclosed polygonal figures, often five sided
(Figure 5). In some transplants, there was a suggestion that in this particular tumor
some contribution to the vascular tree might have been made by the tumor itself. This
would be concordant with the peculiar nature of this tumor, i.e., derived itself from
blood vessels. This tumor showed a far more regular pattern to its vascular morphol-
ogy than hamster melanomas. 88 Following the lag period of 3 to 5 days after transplan-
tation, there appeared scattered groups of red cells in the graft, and often hemorrhage
at its edge. Within 5 to 8 days, the tumor fragment contained vessels, and these drained
in an arboreal fashion89 90 towards a central vein. The arterial supply was not easily
seen in life, but on examination of fixed specimens it was found to arise from directly
beneath the tumor. The capillaries possessed intact endothelial walls and a distinct
basement membrane, outside which were fine strands of collagen fibres. Unlike the
growth of melanoma transplants in which the advancing edge occurred as a large arc
scarcely distinguishable from the main mass of tumor, growth of the hemangiopericy-
toma occurred by the division of the main mass of tumor into lobules, with further
growth of the individual lobules. The blood flow from a growing segment of the tumor
altered its direction out toward the new principal draining vessel and away from the
old "stem" vein.90 This watershed between old and new drainag 2 areas constituted the
boundary between the older tumor unit and the new one (Figure 5). Subsequent induc-
tion of further drainage vessels resulted in a repetition of the process. The overall value
for the capillary volume of this tumor, using a morphometric method, was 21 % of
the total tumor mass. 90 Comparable figures are 12 and 14% of the parenchymatous
tissue for the capillaries in the placenta and lung, respectively.90
31
FIGURE 5. En face in vivo view of a hemangiopericytoma growing in the hamster cheek-pouch chamber.
The edges of the tumor are made visible by its microcirculation. A watershed area (W) is present between
the main mass of the tumor (M) and the new lobule that is forming (L). (Magnification x 80.)
IV. ORGANIZATION OF THE VASCULAR ARCHITECTURE IN

TUMORS
August Krogh,91 in his landmark treatise on the anatomy and physiology of capillar-
ies, addresses in the first chapter the question of the distribution and number of capil-
laries in selected organs. He illustrates the great variation in the organization of the
microcirculation in different tissues, such as in striated muscle, in subpapillary arterial
plexus of the skin, in small intestinal villi, and in the rete mirabile of the eel. He found
an average number of 40.4 ± 5 capillaries in an area of 0.03 mm2 in five different
transverse sections of the gastrocnemius muscle of the horse, thus showing a "remark-
able regularity of distribution" within a specific tissue. However, there is a dichotomy
in the classification of blood vessels due to differences in the physiological and the
anatomical approach, which becomes obvious when the organization of the microcir-
culation in tumors is considered (Figure 6).
The physiological approach is that "the assignment of a name to a specific vessel
should be determined by its position and function in the vascular system".92 The ana-
tomic approach is based on the recognition of the specific structural morphology of
the wall of the vessel together with the size of the lumen as being identical with the
appearances of known segments of the vascular tree.93,94 No problems arise in the
"model" case where blood passes successively through large arteries, small arteries,
arterioles, capillaries, venules, small veins, and large veins. The physiological route is
recognized, and the anatomical structures characteristic of these vessels known, at spe-
cific points in the microcirculation in the "model" case or "simple" route between
arterial and venous system.95 There are a number of routes from the arterial, high-
32 Turn or Blood Circula don
FIGURE 6. Diagram to show the relationships between various classes of vessels within a growing tumor
on the hamster cheek pouch membrane (PM). The situation is closely analogous to the growth and devel-
opment of seedling metastases on pleural and peritoneal surfaces in man. The tumor transplant (T) is grow-
ing on the pouch membrane (PM), which has squamous epithelium (S) on its buccal side and muscle fibers
(F) within it. The numbers throughout the diagram refer to the classification of blood vessels in neoplasms
listed in Table 4. Arteries and arterioles (A) (class 1 vessels) run in the pouch membrane beneath the tumor
and are unaffected by it. Within the tumor there are mature capillaries with intact basement membranes
surrounded by tumor (class 2 vessels). At the growing edge of the tumor there are capillary sprouts (class 3
vessels). In the substance of the tumor, there may be sinusoidal vessels with interrupted endothelial lining
(class 4 vessels) as well as blood channels without endothelial lining (class 5 vessels). At the edge of the
tumor transplant, there are giant capillaries (class 6 vessels), which run as marginal vessels (M) describing
arcs of various lengths around the tumor, and which communicate with the venous system of the pouch
membrane (class 8 vessels). Capillaries with fenestrated endothelium (class 7 vessels) and arteriovenous an-
astomoses (class 9 vessels) are not illustrated. A reactive zone (R) occurs at the edge of the transplant and
consists of leukocytes and macrophages.
pressure side of the circulation to the venous, low-pressure side, and such routes are
in parallel. The main categories of these routes according to Burton 95 are
1. A simple route where a single capillary bed is perfused.

2. Two capillary beds are present in series, e.g., in the kidney where the glomerular
tuft is in series with the tubular capillaries.
3. More than two capillary beds are traversed, e.g., the capillary beds of the spleen,
which are in parallel with the mesentery and in series with the hepatic sinusoids.
4. The pulmonary circulation.
5. The bronchial circulation, which constitutes a route from one chamber of the left
heart to another.
As well as this variation in routing between the arterial and venous systems, there
are marked differences in the location of the "control points". In the type 1 listed
above, a single set of control points is found, whereas in 2 and 3, there are multiple
33
sets.95 All this refers to the situation in the normal mammalian host. When a tumor
microcirculation is superimposed upon different host tissues, the "position and func-
tion" of specific segments of vessels becomes difficult to determine. In order to com-
pare and discuss different combinations of hierarchical order in the circulatory system
of tumors, it is useful to have a morphological classification of the vessels found within
tumors (Table 4).
Class 1 : arteries and arterioles (Figure 7)—Willis96 has concluded from wide experi-
ence and considerable review of the literature that "the walls of arteries exhibit striking
immunity from neoplastic invasion". He cites studies in which all categories of vessel
in the arterial system from the aorta to the arteriole have been observed to persist
TABLE 4
The Varieties and Classification of Blood Vessels in the Sub-

stance of Neoplasms
Class 1 Arteries and arterioles

Class 2 Capillaries with basement membrane
Class 3 Capillary sprouts
Class 4 Sinusoidal vessels — interrupted endothelial lining
Class 5 Blood channels without endothelial lining
Class 6 Giant capillaries
Class 7 Capillaries with fenestrated endothelium
Class 8 Venules and veins
Class 9 Arterio-venous anastomoses
FIGURE 7. A normal artery (AR) is present in the center of a mass of infiltrative tumor. The tumor is a
transplantable melanoma of the hamster growing on the hamster cheek pouch. There is no involvement of
the arterial vessel wall. (Magnification x 330.)
within, sometimes massively, infiltrating growths. He considered it "exceedingly rare"

to observe malignant invasion of arterioles. He traced arterioles through dense infiltra-
tive growth that had destroyed all semblance of normal structure. In reviewing the
literature cited above concerning experimental tumors, the arteries and arterioles are
reported to persist within the new growth, despite infiltrative growth up to the walls
of the vessels. The persistence of normal structure allows possible vasomotion of the
arterial walls within tumors. The blood flow in capillaries in thyroid grafts in a trans-
parent chamber varied periodically due to rhythmic changes in the caliber of arter-
ioles.97 Vasomotion in the arterial supply to a tumor (mouse rhabdomyosarcoma
BA1112) has been reported. 6298 They observed periodic fluctuations in tumor perfu-
sion which appeared modulated by (1) arteriolar vasomotion and (2) the effect of res-
piration acting on the venous circulation.
Class 2: capillaries with basement membranes—The more highly differentiated tu-
mors possess capillaries which have intact and complete endothelial linings with normal
basement membranes and with well formed cell junctions between the endothelial cells.
The capillaries are supported by fine bundles of collagen.90
Class 3: capillary sprouts—In cross sections of growing tumors, there are regions in
which the host capillary bed is reacting to the presence of the tumor by putting forth
capillary sprouts. This is discussed further in the next chapter.
Class 4: sinusoidal vessels; vessels with interrupted endothelial lining—This type of
vessel is present in regions of poorly differentiated adenocarcinomas. In a series of six
human renal adenocarcinomas, 99 many of the intrinsic vessels of the tumors were sin-
usoidal in type. The vessels had nonparallel walls consisting of endothelial cells and
occasional pericytes. Some of the endothelial cells on scanning electron microscopy
appeared damaged and were covered by fibrin (Figure 8). Numerous extra vascular
deposits of fibrin were noted.
Class 5: blood channels without endothelial lining (Figure 9)—Adhesion between
tumor cells within a neoplasm can be quite tenuous, and the form of the circulation
can be such that blood percolates around and between tumor-cell cords. This form of
circulation occurs in the melanomas and some of the sarcomas. When the blood supply
to small melanoma transplants was traced from its inception, eventually channels were
found within the tumor substance that possessed only a partial investment of endoth-
elial cells.100 (Figure 9A and 9B)
Class 6: giant capillaries (Figure 10)—At the edge of many tumors, a collection of
wide-bored vessels containing venous blood is frequently found and has been referred
to as the "venous capsule" of the tumor. On histological examination, however, al-
though the size of the vessels would warrant the term "venule", often being in excess
of 50 ¿¿m in diameter (e.g., in melanomas of the hamster 101 ), the actual vessel wall is
composed only of endothelial cells with some fibrous supporting tissue. These giant
capillaries are frequently quite tortuous and usually describe arcs around the expanding
mass of the tumor.
The giant capillaries have been identified as a location for the migration of tumor cells
from the extravascular space to the intravascular compartment. 102103
Class 7: capillaries with fenestrated endothelium— Capillaries with intracellular fe-
nestrations are found in many endocrine organs. The presence of intracellular fenestra-
tions provides the distinguishing feature for one of the major divisions of capillaries
enunciated by Bennett et al. 104 The occurrence of intracellular fenestrations in capillar-
ies of tumors has been noted in the Walker 256 carcinoma, 86 in transplants of human
renal carcinoma in the " n u d e " mouse, 105 and in a variety of brain tumors. 106 It has
been shown107 that the escape of the larger marker particles, such as carbon, ferritin,
and mercuric chloride, occurs in considerable quantity from fenestrated and nonfenes-
35
FIGURE 8. (A) Scanning electron micrograph of a sinusoid (S) in a partially necrotic area of a human
renal adenocarcinoma. Fibrin (F) is attached to one region of the sinusoid. (Magnification x 1200.) (From
Warren, B. A., in Platelets: A Multidisciplinary Approach, de Gaetano, G. and Garattini, S., Eds., Raven
Press, New York, 1978, 427. With permission.) (B) Higher power view to show the fibrin strands (F) covering
one wall of the sinusoid. (Magnification x 4100.)
36
Tumor Blood Circulation
FIGUR E 9. (A) and (B) Within a malignant melanom a of th e hamster, channels ar e present which are not bordered by endothelial
cells and which are continuous with sinusoidal vessels partially invested with endothelium. Red cells (RC) ar e present in direct contact
with malignant melanom a cells (M). (Malignification of A and B x 3000.)
37
FIGURE 10. (A) A giant capillary (GC) (class 6) is present at the edge of a malignant melanoma infiltrating
the hamster cheek-pouch membrane. An artery (A) (class 1) is uninvolved and is surrounded by tumor cells
. (Hematoxylin-eosin stained section; magnification x 140.) (B) A higher power view of the wall of a giant
capillary to show that it consists of a single layer of endothelial cells (E) supported by fibrous tissue (F).
Tumor cells (T) are in close proximity to the outer surface of this vessel. (Hematoxylin-eosin stained section
x 1400.)
trated vessels in the intestinal mucosa of normal rats and mice. Large aggregates of
the particles form beneath the junctions between vascular endothelial cells. The leakage
may be due to a temporary opening of the intracellular junctions, and it was suggested
by Hurley and McCallum107 that leaks of this type might account for the escape of
plasma protein into the intestinal mucosa, submaxillary gland, and other normal tis-
sues.
Class 8: venules and veins—The venules and veins draining a tumor usually show
remarkable degrees of tortuosity, saccular dilatations or "ectasias", and variations in
velocity of blood flow. There may be such a range of changes in the oxygen content
of the draining blood that stripes of different colors within the veins are seen.
Class 9: arterio-venous anastomoses—These structures occur in the normal micro-
circulation where metarterioles or arterioles flow suddenly into a venule or small
vein.108 A fusiform dilatation is formed on the venous side of the siphon, and the
velocity of blood flow slows in conformity with that of the venous side of the circula-
tion. The arterial side of the structure has a stellate lumen.
This type of classification allows the morphological definition of all the vessels
within a histological section. The actual hierarchial organization may vary from tumor
to tumor. Thus, the arterial-venous route through the microcirculation of a tumor
(e.g., a melanoma) can be written in the following manner: arteriole (class 1) — capil-
lary with basement membrane (class 2) — sinusoid (class 4) — blood channel (class 5)
— giant capillary (class 6) — venule (class 8). Certain controversial points can be more
clearly enunciated and discussed using this system, e.g., what is the nature of the deri-
vation of the capillary bed in tumors? Is it a "portal" system, i.e., are the capillary
networks in tumors derived from the venous side in some tumors a priori [i.e., venule
(class 8) — capillary (class 2) — sinusoid (class 4) — capillary (class 2) to return to
venule (class 8)]? The actual "module" of the tumor circulation must, of course, start
from the basis of the module of the microcirculation of the host organ. The possible
variations that might occur are considerable. The region of the tumor, that is, whether
it is part of a growing arc, an intermediate zone, or a central necrotic zone, would
influence the nature of the routing from the arterial side to the venous side.
Liebow109 reviewed the changes that induce alteration in vascular patterns and noted
that the venous collaterals developed to a larger size more quickly than arterial.
Hellmann and his group 1 1 0 1 1 3 examined the antitumor activity of a series of bisdi-
ketopiperazines and gave the term ICRF159 (standing for Imperial Cancer Research
Fund agent number 159) to a cytostatic agent 1,2. bis (3,5-dioxopiperazine-l-yl) pro-
pane. The mode of action was considered to be prevention of the entry of cells into
mitosis and arrest of dividing cells in prophase and early metaphase. At a dose level
which did not influence the growth rate of the primary transplant of Lewis lung carci-
noma, treatment with ICRF159 completely inhibited the development of metastases.
This prevention of metastatic development was attributed to the effect of ICRF159 on
the blood vessels at the invading margins of the primary tumor. The vascularity of the
tumor margin was greatly increased. This difference between the blood-vessel pattern
between treated and control groups was attributed either to an effect of the agent on
the "fundamental growth pattern of the tumor" or an effect secondary to an effect
on tumor growth. It was suggested that the slowing of tumor growth allowed matura-
tion of the tumor blood vessels to occur. If the apparent structural pattern of each
tumor is characteristic for the histological type of that tumor, then the replicating
module of the tumor probably contains a constant number of each of the above classes
of vessels. It is apparent that the numbers of vessels in each class will play a profound
part in determining the nature of the permeability of the tumor micro vasculature. The
distribution of i.v.-administered, labeled, rat fibrinogen was studied in a 20-methyl-
39
cholanthrene-induced sarcoma of the rat by Peterson et al. 114 They concluded that the
uptake of fibrinogen in the sarcoma was not an active process, but due to a passive
leakage of the substance owing to the high permeability of the vessel walls in the tumor.
V. R A D I O G R A P H I C O B S E R V A T I O N S O N V A S C U L A R M O R P H O L O G Y
O F T U M O R S IN M A N A N D A N I M A L S
One of the earliest commentaries on tumor vascularity was that of Dibbelt.115 Using
radiography, he contrasted the vascular supply of fibromas with fibrosarcomas. He
noted a congruence between the nature of the development of the vascular tree and
the differentiation of the tumor. Thus, fibrosarcomas contained vessels which ap-
peared similar to undifferentiated embryonic vessels, while fibromas showed vessels
with normally differentiated wall structures.
De Saunders116 reviewed the contribution of microradiography to the study of the
living circulation with the use of microangiograms.
The infusion of barium into arteriole beds of tumors allows radioopaque angiogra-
phy.117 Modifications of the original technique were made by Bellman118 and Lagergren
etal. 119
Margulis et al. 120 published a report based on the angiograms of 171 tumor-bearing
adult mice. The tumors studied were transplanted squamous cell carcinoma, sponta-
neous and transplanted carcinoma of the breast, hepatoma, lymphosarcoma, sarcoma
37, and rhabdomyosarcoma. The number of tumors examined in each category ranged
between 13 and 46. Transplants of the squamous cell carcinoma tended to assume a
rounded shape, and when large, to become cystic. The tumors which were themselves
poorly vascularized were circled with a coarse network of large vessels. In the mice
with hepatomas, the vessels showed a tendency to curve around parts of the tumor
substance and, hence, to indicate a lobular pattern. Saccular structures within the tu-
mors were noted. Transplanted adenocarcinomas of the breast were more vascular
than squamous-cell carcinoma, but less vascular than hepatomas. Margulis et al. in
their paper describe the vessels of adenocarcinomas of the breast as tending to form
lobules, forming multiple tufted areas throughout the tumors. Occasionally, there were
small vessels of variable character densely arranged in a parallel fashion with "brush-
like" forms. Most of the spontaneous adenocarcinomas of the breast in C3H mice
showed the same vascularity present in the transplanted tumors. Infiltration was sug-
gested by a blurred demarcation between normal and tumor tissue. In lymphosarco-
mas, most vessels were smaller than in other neoplasms and tended to run parallel with
the surface of the body of the animal. The next order of branches arose and ran toward
the surface. In observations of transplants of sarcoma 37, there were similar appear-
ances in that the vessels below the tumor ran parallel to the surface. Numerous ill-
defined "lakes" were scattered throughout the neoplasms. They described a "cande-
labra" arrangement in which vessels arranged perpendicular to the surface led into
large superficial channels. Numerous, irregular vessels with lakes were found in rhab-
domyosarcomas when these were studied with thorotrast®. When micropaque® was
injected on the systemic arterial side, a completely different angiographic pattern ap-
peared, and there were no filled veins or "lakes" present. They suggested that the
question of arteriovenous communications should be investigated further.
Margulis in 1964121 reviewed the progress of the artériographie study of tumors from
the initial procedure with poor and irritating contrast media to the advent of organic,
iodinated contrast media and improved methods of percutaneous catheterization. He
cautions that, although many types of neoplasms have been studied, the methods var-
ied greatly, and the literature on the subject is replete with sweeping statements on the
basis of only one or two cases. Margulis concluded that, while some benign lesions
could possess very vascular patterns that might mimic those of carcinoma, close scru-
tiny at high magnification of the angiogram would not detect the tell-tale changes in
the fine vessels only found in malignant lesions. The shape of the lumen, direction of
the many vessels, and the presence of lakes were the features which established the
diagnosis of a malignant tumor.
Milne in 1967122 using arteriography and microarteriography, returned to the ques-
tion of the blood supply of primary and metastatic pulmonary neoplasms earlier dis-
cussed by Wright in 1938. He examined 14 cases of pulmonary metastasis from various
primary neoplasms and 18 cases of primary bronchogenic carcinomas. His studies
showed that, while the circulation is mainly bronchial in type, some tumors may be
supplied by both the bronchial and pulmonary circulations. The pulmonary metastases
that were investigated by him could be supplied by both the bronchial or pulmonary
circulation separately or together. Although the number of primary tumors examined
was small, there were consistent differences in the arterial patterns of squamous cell,
anaplastic, and adenocarcinoma of the lung. In the squamous cell carcinomas, hyper-
trophy of the bronchial arterial tree with increased numbers of fine vessels was noted,
some of which were quite tortuous. A feature was large broncho-pulmonary anasto-
moses at the periphery of the tumors. In comparison, anaplastic carcinomas were less
vascular, especially towards the central part of the tumor, which was frequently nec-
rotic. Fine, perforating branches arising from the bronchial artery supplied the medial
edge, while the lateral edge was likewise endowed by similar vessels, although from
the pulmonary arteries. In both the above types of carcinoma, the more peripheral the
situation of the lesion, the greater the pulmonary component of its vasculature. The
one adenocarcinoma examined contained increased numbers of bronchial arterial
branches, which were bizarre, tortuous vessels associated with "lakes" of the contrast
medium.
In an investigation of vascular patterns in primary and secondary pulmonary tumors
in dogs, Jonas and Carrington 123 used in vivo angiography in one dog, and vinylite
corrosion cast, and gelatin injection techniques. They concluded that bronchial artery
proliferation occurred after the destruction by the tumor of alveolar septa and pul-
monary parenchyma. If the septa remained intact, the pulmonary arteries supplied the
tumor as though in a cancerous pneumonia. Anastomoses between the bronchial and
pulmonary arterial systems and the venous drainage of tumors were demonstrated.
Shivas and Gillespie124 addressed a similar problem in the liver, another organ with
a dual afferent blood supply, this time an artery and vein. They reviewed the earlier
studies relating to work concerning the blood supply to tumors by angiography, vinyl
and gelatin casts, and India ink injection. They concluded from their own studies on
the Brown-Pearce carcinoma in rabbits and the literature that a growing metastatic
tumor in the liver draws its circulation preferentially from the hepatic artery.
Herman et al. 125 examined by microangiography and histology the popliteal lymph
node in 49 rabbits 3 to 55 days after transplantation of V2 carcinoma to the hind paw.
At the end of about 4 weeks, metastatic deposits of tumor cells were established and
were surrounded by numerous plasma cells. The site of the earliest lymph node metas-
tasis was adjacent to the subcapsular sinus. Microangiography revealed displacement
of the cortical vessels by the tumor deposit. No significant increase in vascularity was
noted at the tumor-lymphoid tissue interface. The medullary cords adjacent to the
metastasis, however, were increased in vascularity at 14 to 21 days. At the next stage
of 22 to 40 days, metastatic involvement was increased, and there were displaced cor-
tical vessels at the tumor-lymphoid tissue interface. Metastatic deposits were found to
41
be essentially avascular. The medullary cords did not show evidence at this stage of
hypercellularity.
VI. M E T H O D S O F Q U A N T I T A T I O N O F V A S C U L A R M O R P H O L O G Y
OF TUMORS
Good starting points in considering this topic are the reviews of the methods of
measurement of the collateral circulation by Liebow in 1963109 and Shivas and Gillespie
in 1969.124 References to the various methods for the production of vinyl and gelatin
casts are given. Angiography is dealt with in a later chapter in this book. I would like
to confine my comments here to the analysis of the anatomy of the vascular network
of tumors. Two basic elements of the change in the vessels in relation to tumors can
be considered.
A. Network changes
The first is a change in the surrounding network of vessels of the host and the pe-
culiar network of vessels of different classes ( vide supra) that lie within the tumor.
Questions concerning the mathematical analysis of dynamic alterations in networks
have been addressed by geographers, and there have been published mathematical
analyses of network alterations by Haggett and Chorley126 and Haggett. 127 The classi-
fication of the vessels in the tumors described above could be used as a base to com-
mence analysis using these mathematical tools. As soon as it is recognized that the
vascular morphology of tumors differs in a radical fashion from that of normal tissue
in the same way that a tumor cell pattern differs from a normal tissue pattern, then
appropriate mathematical analysis and discussion can be commenced.
B. Vascular Density Changes and Redundancy Index of Tumor Vessels

Algire and co-workers 31 were early practitioners of what are now known as stereo-
logic techniques. Parameters, such as the relative volume and contents of capillaries
with reference to total volume can be discussed and comparison of peripheral and
central fields of tumors can be made 90 with the techniques of Freere and Weibel.128
Various stereological techniques for the estimation of the frequency of occurrence
of three-dimensional objects from two-dimensional images are detailed in the text by
Underwood. 129 More recent information regarding mathematical techniques which can
be applied to this problem is available, 130 131 for example, in the analysis of the heter-
ogeneous composition of central nervous system (Eins and Wolff), and the use of
morphometry, stereometry, and stereology as applied to cancer development in the
large intestine of man (Bokelmann and Vogtle).
With a tumor growing between two plates in a transparent chamber, provided that
the space between the plates remains constant, the photographs showing the extension
of the vascular pattern can be used to plot the daily growth of the tumor. If photo-
graphs are taken at a constant magnification, then the weight of the photographic
paper of the viewing area of the chamber will remain constant. The weight of the
tumor image on the photographic paper will represent the size of the tumor. When
the size of the tumor is expressed as a percentage of the area of the viewing region of
the chamber, increments in this percentage will present the growth of the tumor. Thus,
if x = the area of the tumor on the photograph at day 4 following transplantation,
and y = the area of the tumor on the photograph at day 5 following transplantation,
then the increment from day 4 to day 5 is y - x; z = the area of the viewing area of
the transparent chamber (constant throughout the experiment). The area of z is calcu-
lated by weighing the cut-out area of z on standard photographic paper (let this weigh
M mg) and a 1 x 1 cm square of paper (let this weigh P mg). The area of z will then
be f cm2 at the magnification of the photograph.
In a similar fashion, the areas of x and y can be arrived at. The percentage increment
between day 4 and 5 for this tumor would then be
Changes in the dimensions of the vessels, their form, and tortuosity (simple dilata-
tion for the arterial tree, redundancies of form for the venous system) can be calculated
from daily serial photography. The *'redundancy index" of veins and tortuous vessels
can be calculated in the following way: the "redundancy index" is defined as the per-
centage increase of the tortuous segment over the length of a straight vessel between
the ends of the segment, i.e., with a tortuous vessel a specific segment, e.g., between
the points S and T, is measured first as a straight line. Let this be V cm. The route
blood takes in the vessel between S and T around all the curves is next measured with
a map measurer. Let this by C cm. The Redundancy Index is then
It is a measure of the meandering of a tortuous vessel, and indicates the increase in

percentage length over a straight line that the curves in the vessel have achieved.
VII. C O N C L U S I O N S
Tumors vary from one to the other with regard to every detail including the organi-
zation of their vascular morphology.
The arterial system of the host tissue persists in almost all tumors.
The principal changes in the vasculature of small tumors is seen in the transforma-
tion of the capillaries and veins. The latter become dilated, tortuous, and exhibit re-
dundant curves.
Vessels in histological sections of tumors do not conform in toto to the standard
morphology or to the standard artery to capillary bed to vein-network pattern.
A classification of the morphology of tumor vessels to include the aberrant vascular
structures, such as sinusoids and channels through which blood percolates and which
have limited or no endothelial investment is suggested so that different types of net-
work patterns can be described.
The form of the capillary beds of tumors is dictated by the growth pattern of the
neoplastic cells, e.g., adenocarcinomas have a lobular basket-work type of capillary
pattern that encloses the acini of the tumor; poorly differentiated tumors do not have
such constraints.
The vascular morphology for a specific tumor is characteristic for that tumor, but
may not be unique to that tumor.
In small tumors, there is extreme variability in the red cell route through the tumor
on a day to day basis.
The variations in the tumor vascular pattern accommodates the expansile growth of
the tumor. The method of this accommodation varies from tumor to tumor.
ACKNOWLEDGMENT
My own studies reported here were supported by the National Cancer Institute of
Canada. Some of the work was performed at the Eppley Institute for Research in
43
Cancer, University of Nebraska Medical Center, Omaha, during a period of sabbatical

leave. I am grateful for able technical assistance from Mr. R. Feldman, W. Chauvin,
and Mrs. J. Denisovs.
REFERENCES
1. Zweifach, B. W., Functional Behavior of the Microcirculation, Charles C Thomas, Springfield, 111.,
1961.
2. Frasher, W. G. and Wayland, H., A repeating modular organization of the microcirculation of cat
mesentery, Microvasc. Res.,4, 62, 1972.
3. Rhodin, J. A. G., The ultrastructure of mammalian arterioles and precapillary sphincters, / . Ultras-
truct. Res., 18, 181, 1967.
4. Willis, R. A., Pathology of Tumors, 3rd éd., Butterworths, London, 1960.
5. Virchow, R., Die Krankhaften Geschwulste, August Hirschwald, Berlin, 1863; cited by Rogers, W.
etal., Surg. Clin. N. Amer.,47, 1473, 1967.
6. Thiersch, C., Der Epithelialkrebs namentlich der Haut mit Atlas, Leipzig, 1865, as cited in Rogers,
W., Edlich, R. F., Lewis, D. V., and Aust, J. B., Surg. Clin. North Am., 47, 1473, 1967.
7. Goldmann, E., Growth of malignant disease in man and the lower animals with special reference to
vascular system, Proc. R. Soc. Med., 1,1, 1907.
8. Thiessen, N. W., The vascularity of benign and malignant lesions of the stomach, Surg. Gynecol.
Obstet.,2, 149, 1936.
9. Wright, R. D., The blood supply of abnormal tissues in the lung, J. Pathol. Bacteriol., 47, 489,
1938.
10. Hardman, J., The angioarchitecture of the gliomata, Brain, 63, 91, 1940.
11. Lindgren, A. G. H., The vascular supply of tumours with special references to the capillary angioar-
chitecture, Acta Pathol. Microbiol. Scand., 22, 493, 1945.
12. de Busscher, G., La vascularisation macroscopique et microscopique du cancer de l'estomac, Acta
Gastro-Enterol. Belg., 10, 481, 1947; as cited in Delarue, J., Abelanet, R., Baril, A. M., Lavrent,
M.,andGalian,P., Bull. Cancer, 51, 25, 1964.
13. Bierman, H. R., Kelly, K. H., Dod, K. S., and Byron, R. L., Jr., Studies on the blood supply of
tumors in man. I. Fluorescence of cutaneous lesions, / . Natl. Cancer Inst., 11, 877, 1951.
14. Bierman, H. R., Byron, R. L., Jr., Kelly, K. H., and Grady, A., Studies on the blood supply of
tumors in man. III. Vascular patterns of the liver by hepatic arteriography in vivo, / . Natl. Cancer
/nsr.,12, 107, 1951.
15. Bierman, H. R., Kelly, K. H., and Singer, C., Studies on the blood supply of tumors in man. IV.
The increased oxygen content of venous blood draining neoplasms, J. Natl. Cancer Inst., 12, 701,
1952.
16. Bierman, H. R., Gilfillan, R. S., Kelly, K. H., Kuzma, O. T., and Noble, M., Studies on the blood
supply of tumors in man. V. Skin temperature of superficial neoplastic lesions, J. Natl. Cancer Inst.,
13, 1,1952.
17. Breedis, C. and Young, G., The blood supply to neoplasms in the liver, Am. J. Pathol, 30, 969,
1954.
18. Delarue, J., Mignot, J., Paillas, J., and Sors, Ch., Etude sur la vascularisation des cancers bronc-
hiques, C. R. Seances Soc. Biol. Paris, 148, 846, 1954.
19. Delarue, J., Mignot, J., and Bulliard, A., Etude sur la vascularisation des cancers du gros intestin,
C. R. Seances Soc. Biol. Paris, 150, 1104, 1956.
20. Delarue, J., Abelanet, R., Baril, A. M. Laurent M., and Galian, P., Etude de la vascularisation des
cancers epitheliaux du tube digestif, Bull. Cancer, 51, 25, 1964.
21. Urbach, F. and Graham, J. H., Anatomy of human skin tumour capillaries, Nature (London), 194,
652,1962.
22. Rubin, . and Casarett, G., Microcirculation of tumors. I. Anatomy, function and necrosis, Clin.
Radiol., 17,220, 1966.
23. Rubin, R. and Casarett, G., Microcirculation of tumors. II. The supervascularized state of irradiated
regressing tumors, Clin. Radiol., 17, 346, 1966.
24. Lewis, W. H., The vascular pattern of tumors, Johns Hopkins Hosp. Bull., 41, 156, 1927.
25. Kreyberg, L., On local alterations of the blood vessels of tar-painted white mice, Br. J. Exp. Pathol.,
8,465, 1927.
26. Orr, J. W., The results of vital staining with phenol red during the progress of carcinogenesis in mice
treated with tar, dibenzanthracene and benzpyrene, J. Pathol. Bacteriol., 44, 19, 1937.
27. Lucké, B. and Schlumberger, H., The manner of growth of frog carcinoma studied by direct micro-
scopic examination of living intra ocular transplants, / . Exp. Med., 70, 257, 1939.
28. Ide, A. G., Baker, N. H., and Warren, S. L., Vascularization of the Brown-Pearce rabbit epithelioma
transplant as seen in the transparent ear chamber, Am. J. Roentgenol., 42, 891, 1939.
29. Cowdry, D. R. and Sheldon, W. F., The significance of hyperaemia around tumor transplants, Am.
J. Pathol., 22, 821, 1946.
30. Algire, G. H., Microscopic studies of the early growth of a transplantable melanoma of the mouse,
using the transparent chamber technique, / . Natl. Cancer Inst., 4, 1, 1943.
31. Algire, G. H., Chalkley, H. W., Legallais, F. Y., and Park, H. D., Vascular reactions of normal
and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic
transplants, J. Natl. Cancer Inst., 6, 73, 1945.
32. Algire, G. H., Legallais, F. Y., and Park, H. D., Vascular reactions of normal and malignant tissues
in vivo. II. The vascular reaction of normal and neoplastic tissues of mice to a bacterial polysacchar-
ide from Serratia Marcescens (Bacillus prodigius) culture filtrates, J. Natl. Cancer Inst., 8, 53, 1947.
33. Algire, G. H. and Legallais, F. Y., Growth rate of transplantable tumor in relation to latent period
and host vascular reaction, Cancer Res., 7, 724, 1947.
34. Lutz, B. R., Fulton, G. P., Patt, D. I., and Handler, A. H., The growth rate of tumor transplants
in the cheek pouch of the hamster (Mesocricetus auratus), Cancer Res., 10, 231, 1950.
35. Lutz, B. R., Patt, D. I., Handler, A. H., and Stevens, D. F., Serial sarcoma transplantation in the
hamster cheek pouch and the effects of advanced neoplasia on the small blood vessels, Anat. Rec,
108,545, 1950.
36. Toolan, H. W., Proliferation and vascularization of adult human epithelium in subcutaneous tissues
of x-irradiated heterologous hosts, Proc. Soc. Exp. Biol. Med., 78, 540, 1951.
37. Toolan, H. W., Growth of human tumors in cortisone treated laboratory animals: the possibility of
obtaining permanently transplantable human tumors, Cancer Res., 13, 389, 1953.
38. Toolan, H. W., Transplantable human neoplasms maintained in laboratory animals, H. S. No. 1,
H.Ep. No. 1, H.Ep. No. 2, H.Ep. No. 3, and H. Emb. Rh. No. 1, Cancer Res., 14, 660, 1954.
39. Chute, R. N., Sommers, S. C., and Warren, S., Heterotransplantation of human cancer. II. Hamster
cheek pouch, Cancer Res., 12, 912, 1952.
40. Patterson, W. B., Chute, R. N., and Sommers, S. C , Transplantation of human tumors into corti-
sone treated hamsters, Cancer Res., 14, 656, 1954.
41. Greene, H. S. N., The significance of the heterologous transplantability of human cancer, Cancer
(Philadelphia), 5, 24, 1952.
42. Braithwaite, J. L., The arterial supply of benzpyrene induced tumors in the rat, Br. J. Cancer, 12,
75,1958.
43. Day, E. D., Planinsek, J. A., and Pressman, D., Localization of radio iodinated rat fibrinogen in
transplanted rat tumors, J. Natl. Cancer Inst., 23, 799, 1959.
44. Waters, H. G. and Green, J. A., The vascular system of two transplantable mouse granulosa cell
tumors, Cancer Res., 19, 326, 1959.
45. Kligerman, M. M. and Henel, D. K., Some aspects of the microcirculation of a transplantable exper-
imental tumor, Radiology, Id, 810, 1961.
46. Gullino, P. M., Grantham, F. H., and Clark, S. H., The collagen content of transplanted tumors,
Cancer Res., 22, 1031, 1962.
47. Gullino, P. M. and Grantham, F. H., Studies on the exchange of fluids between host and tumor.
III. Regulation of blood flow in hepatomas and other rat tumors, / . Natl. Cancer Inst., 28, 211,
1962.
48. Goldacre, R. J. and Sylvén, B., On the access of blood borne dyes to various tumor regions, Br. J.
Cancer, 16, 306, 1962.
49. Merker, P. C. and Hurley, J., A study of human epidermoid carcinoma (H. Ep. 3) growing in corti-
sone conditioned Swiss mice. IV. X-ray diagnostic procedures, Cancer Res., 22, 646, 1962.
50. Cataland, S., Cohen, C , and Sapirstein, L. A., Relationship between size and perfusion rate of
transplanted tumors, J. Natl. Cancer Inst., 29, 389, 1962.
51. Delarue, J., Mignot, J. and Caulet, T., Modifications vasculaires de la poche jugale du hamster doré
au dours du développement de greffes d'une tumeur mélanique, C. R. Seances Soc. Biol. Paris, 157,
69, 1963.
52. Ogilvie, R. W., Blanding, J. D., Jr., Wood, M. L., and Knisley, W. H., The arterial supply to
experimental metastatic VX2 and XY tumors in rabbit lungs, Cancer Res., 24, 1418, 1964.
45
53. Warner, L., Frog renal adenocarcinoma: living vascular tree in ocular transplants, Anat. Rec, 148,
425,1964.
54. Day, E. D., Vascular relationships of tumor and host, Prog. Exp. Tumor Res.,4, 57, 1964.
55. Witte, S. and Goldenberg, D. M., Vitalmikroskopische Beobachtungen über die frilhen Gefâ'ssreak-
tionen eines menschlichen Transplantattumors, Z. GesamteExp. Med., 139, 633, 1965.
56. Goodall, C. M., Sanders, A. G., and Shubik, P., Studies of vascular patterns in living tumors with
a transparent chamber inserted in hamster cheek pouch, / . Natl. Cancer Inst., 35, 497, 1965.
57. Bashford, E. F,, The zoological distribution, the limitations in the transmissibility and the compa-
raaive histological and cytological characteristics of malignant new growths, Imp. Cancer Res. Fund
Sci.Rep.,1,3, 1904
58. Sandison, J. C. The transparent chamber of the rabbit's ear giving a complete description of im-
proved technique of construction and introduction and general account of growth and behaviour of
living cells and tissues as seen with the microscope, Am. J. Anat.41, 447, 1928.
59. Clark, E. R., Kirby-Smith, H. T., Rex, R. C , and Williams, R. G., Recent modifications in the
method of studying living cells and tissues in transparent chambers inserted in the rabbit's ear, Anat.
Record, 47, 187, 1930.
60. Williams, R. G., Microscopic studies in living mammals with transparent chamber methods, Int.
Rev. Cytol., 3, 359, 1954.
61. Sanders, A. G. and Shubik, P., A transparent window for use in the Syrian hamster, Isr. J. Exp.
Med., 11, 118, 1964.
62. Reinhold, H. S., Blochiwiecz, B., and Blok, A., Oxygenation and reoxygenation in "sandwich"
tumors, Bib!. Anat., 15, 270, 1977.
63. Greenblatt, M., Choudari, K. V. R., Sanders, A. G., and Shubik, P., Mammalian microcirculation
in the living animal: méthodologie considerations, Microvasc. Res., 1, 420, 1969.
64. Nims, J. C. and Irwin, J. W., Technical report: chamber techniques to study the microvasculature,
Microvasc. Res., 5, 105, 1973.
65. Greenblatt, M., Choudari, K. V. R., Sanders, A. G., and Shubik, P., Mammalian microcirculation
in the living animal: microcirculation of renal homograft transplants in hamsters, Am. J. Pathol.,
56,317,1969.
66. Greenblatt, M., Kaufman, J., and Kommineni, V. R. C , Functioning heart homografts in hamsters,
Transplantation, 11, 50, 1971.
67. Falk, P., Two methods for three-dimensional examination of the pattern of vasculature in tumors,
Microvasc. Res., 15, 83, 1978.
68. Eddy, H. A., Microangiographic techniques in the study of normal and tumor tissue vascular systems,
Microvasc. Res., 11, 391, 1976.
69. Kjartansson, I., Tumor circulation: an experimental study in the rat with a comparison of different
methods for estimation of tumor blood flow, Acta Chir. Scand. Suppl.471, 1976.
70. Stewart, H. L., Snell, K. C , Dunham, L. J., and Schleyen, S. M., Transplantable and transmissible
tumors of animals, Atlas of Tumor Pathology, Section 12, Fasicle 40, Armed Forces Institute of
Pathology, Washington, 1959.
71. O'Meara, R. A. Q., The growth pattern of carcinomas, Arch. De Vecchi Anat. Pathol. Med. Clin.,
31,365, 1960.
72. Goodall, C. M., Feldman, R., Sanders, A. G., and Shubik, P., Vascular patterns of four transplant-
able tumors in the hamster (Mesocricetus auratus), Angiology, 16, 622, 1965.
73. Fortner, J. G., Mahy, A. G., and Schrodt, G. R., Transplantable tumors of the Syrian (golden)
hamster. I. Tumors of the alimentary tract, endocrine glands and melanomas, Cancer Res., 21(b),
161, 1961.
74. Warren, B. A., Shubik, P., Wilson, R., Garcia, H., and Feldman, R., The microcirculation in two
transplantable melanomas of the hamster. I. In vivo observations in transparent chambers, Cancer
Lett., 4, 109, 1978.
transplantable melanomas of the hamster. II. Scanning electron microscopy, Cancer Lett., 4, 117,
1978.
76. Falk, P., The angioarchitecture of rat tumors, Bibl. Anat., 15, 245, 1977.
77. Falk, P., Patterns of vasculature in two pairs of related fibrosarcomas in the rat and their relation
to tumor responses to single large doses of radiation, Eur. J. Cancer, 14, 237, 1978.
78. Eddy, H. A. and Casarett, G. W., Development of the vascular system in the hamster malignant
neurilemmoma, Microvasc. Res.,6, 63, 1973.
79. Natadze, T. G., Regulation of blood circulation in malignant tumors, Probl. Oncol. (USSR), 5(12),
14,1959.
80. Glazunov, M. F., The General Morphology of Tumors in Cancer, Petrov, N. N., Ed., Pergamon
Press, Oxford, 1962, chap. 4.
81. Kruuv, J. A., Inch, W. R., and McCredie, J. A., Blood flow and oxygenation of tumors in mice,
Cancer (Philadelphia), 20, 60, 1967.
82. Tannock, I. F., Population kinetics of carcinoma cells, capillary endothelial cells, and fibroblasts in
a transplanted mouse mammary tumor, Cancer Res., 30, 2470, 1970.
83. Milne, E. N. C , Margulis, A. R., Noonan, C. D., and Stoughton, J. T., Histologic type-specific
patterns in rat tumors, Cancer (Philadelphia), 20, 1635, 1967.
84. Kido, C , Hepatic angiography of experimental transplantable tumor, Invest. Radiol., 5, 341, 1970.
85. Oikawa, M., Milne, E. N. C , Whitmore, E., Gilday, D., and Oliver, C , A morphological and
quantitative study of tumor blood flow, Cancer (Philadelphia), 35, 385, 1975.
86. Warren, B. A., The ultrastructure of the microcirculation of the advancing edge of Walker 256 car-
cinoma, Microvasc. Res.,2, 443, 1970.
87. Van den Brenk, H. A. S., Crowe, M., Kelly, M., and Stone, M. G., The significance of free blood
in liquid and solid tumors, Br. J. Exp. Pathol., 58, 147, 1977.
88. Yamaura, H. and Sato, H., Experimental studies on angiogenesis in AH 109A ascites tumor tissue
transplanted to a transparent chamber in rats, in Chemotherapy of Cancer. Dissemination and Me-
tastasis, Garattini, S. and Franchi, G., Eds., Raven Press, New York, 1973, 149.
89. Warren, B. A., In vivo and electron microscopic study of vessels in two transplantable tumors in the
hamster, Bibl. Anat., 1, 412, 1967.
90. Warren B. A., In vivo and electron microscopic study of vessels in a haemangiopericytoma of the
hamster, Angiologica,5, 230, 1968.
91. Krogh, A., The Anatomy and Physiology of Capillaries, Yale University Press, New Haven, Conn.,
1922.
92. Wiedeman, M. P., Dimensions of blood vessels from distributing artery to collecting vein, Circ. Res.,
12,375, 1963.
93. Bloom, W. and Fawcett, D. W., Blood vascular system, in A Textbook of Histology, 9th éd., W. B.
Saunders, Philadelphia, 1968, Chap. 13.
94. Windle, W. F., Blood vessels and lymphatic vessels, in Textbook of Histology, 5th éd., McGraw-
Hill, New York, 1976, Chap. 11.
95. Burton, A. C , Arrangements of the many vessels, in Physiology and Biophysics of the Circulation,
Year Book Medical Publishing, Chicago, 1965, chap. 6.
96. Willis, R. A., The Spread of Tumors in the Human Body, Butterworths, London, 1973, 15.
97. Merwin, R. M. and Wollman, S. H., Transparent chamber studies of vessels, circulation and follicles
in thyroid grafts in unanesthetized mice, J. Natl. Cancer Inst., 34, 415, 1965.
98. Intaglietta, M., Myers, R. R., Gross, J. F., and Reinhold, H. S., Dynamics of microvascular flow
in implanted mouse mammary tumors, Bibl. Anat., 15, 273, 1977.
99. Warren, B. A. and Chauvin, W. J., Transmission and scanning electron microscopy of renal adeno-
carcinomas, Ann. R. Coll. Phys. Surg. Can., 10, 74.
100. Warren, B. A. and Shubik, P., The growth of the blood supply to melanoma transplants in the
hamster cheek pouch chamber, Lab. Invest., 15, 464, 1966.
101. Warren, B. A., Shubik, P., and Feldman, R., Changes in the microcirculation of melanomas with
growth, Proc. Microscop. Soc. Can., 4, 34, 1977.
102. Warren, B. A., Shubik, P., and Chauvin, W. J., The transmural passage of tumor cells across vessel
walls, Ann. R. Coll. PhysandSurg. Can., 11, 66, 1978.
103. Warren, B. A., Shubik, P., and Feldman, R., Metastasis via the blood stream: the method of intra-
vasation of tumor cells in a transplantable melanoma of the hamster, Cancer Lett., in Press, 1978.
104. Bennett, H. S., Luft, J. H., and Hampton, J. C , Morphological classification of vertebrate blood
capillaries, Am. J. Physiol., 196, 381, 1959.
105. Groscurth, P. and Kistler, G., Human renal cell carcinoma in the " N u d e " mouse: long-term obser-
vations, Beitr. Pathol., 160, 337, 1977.
106. Hirano, A. and Matsui, T., Vascular structures in brain tumors Hum. Pathol., 6, 611, 1975.
107. Hurley, J. V. and McCallum, N. E. W., The degree and functional significance of the escape of
marker particles from small blood vessels with fenestrated endothelium, / . Pathol., 113, 183, 1974.
108. Cliff, W. J., Vessels as functional units, in Blood Vessels, Harrison, R. J., McMinn, R. M. H., and
Treherne, J. E., Eds., Cambridge University Press, Cambridge, 1976, Chap. 7.
109. Liebow, A: A., Situations which lead to changes in vascular patterns in Handbook of Physiology,
Section 2, Vol. 2, Hamilton, W. F., Ed., American Physiological Society, Washington, D.C., 1963,
chap.37.
110. Creigton, A. M., Hellmann, K., and Whitecross, S., Antitumor activity in a series of bisdiketo-
piperazines, Nature (London), 222, 384, 1969.
111. Hellmann, K., Newton, K. A., Whitmore, D. N., Hanham, I. W. F., and Bond, J. V., Preliminary
clinical assessment of ICRF 159 in acute leukaemia and lymphosarcoma, Br. Med. J., 1, 822, 1969.
47
112. Hellmann, K. and Field, E. O., Effect of ICRF 159 on the mammalian cell cycle: significance for its
use in cancer chemotherapy, J. Natl. Cancer Inst., A4, 539, 1970.
113. Salsburg, A. J., Burrage, K., and Hellmann, K., Inhibition of metastatic spread by ICRF 159 selective
deletion of a malignant characteristic, Br. Med. J., 4, 344, 1970.
114. Peterson, H-L, Appelgren, K. L., and Rosengren, B. H. O., Experimental studies on the mechanisms
of fibrinogen uptake in a rat tumor., Eur. J. Cancer, 8, 677, 1972.
115. Dibbelt, W., Über die blutgefôsse der tumore Arb. Pathol. Anat. Bacteriol., 8, 114, 1912.
116. De, C. H. and Saunders, R. L., The contribution of microscopes to the study of living circulation:
radiographic techniques, J. R. Microscop. Soc, 83, 55, 1964.
117. Bohatyrtschuk, F., Die Fragen der MikrorontgenSgraphie, Fortschr. Geb. Rdntgenstr., 65, 253, 1942.
118. Bellman, S., Microangiography, Acta Radiol. Suppl., 102, 1973.
119. Lagergren, C , Lindbom, A. and Soderberg, S., Vascularization of fibromatous and fibrosarcoma-
tous tumors, Acta Radiol., 53, 1, 1970.
120. Margulis, A. R., Carlsson, E., and McAlister, W. H., Angiography of malignant tumors in mice,
Acta Radiol., 56, 179, 1961.
121. Margulis, A. R., Arteriography of tumors: Difficulties in interpretation and the need for magnifica-
tion, Radiol. Clin. North Am., 2(3), 543, 1964.
122. Milne, E.N. C , Circulation of primary and metastatic pulmonary neoplasms: a postmortem microan-
giographic study, Am. J. Reontgenol., 100(3), 603, 1967.
123. Jonas, A. M. and Carrington, C. B., Vascular patterns in primary and secondary pulmonary tumors
in the dog, Am. J. Pathol, 56, 79, 1969.
124. Shivas, A. A. and Gillespie, W. J., The vascularization of Brown-Pearce carcinoma implanted in
rabbit liver, Br. J. Cancer, 23, 638, 1969.
125. Herman, P. G., Kim, C.-S., de Sousa, M. A. B., and M ell ins, H. Z., Microcirculation of the lymph
node with metastases, Am. J. Pathol., 85, 333, 1976.
126. Haggett, P. and Chorley, R. J., Network Analysis in Geography, Edward Arnold, London, 1969.
127. Haggett, P., Geography: A Modern Synthesis, Harper & Row, New York, 1972, chap. 14 and 15.
128. Freere, R. H. and Weibel, E. R., Stereologic techniques in microscopy, J. R. Microscop. Soc, 87,
25, 1967.
129. Underwood, E. E., Quantitative Stereology, Addison-Wesley Publishing, Reading, Mass., 1970.
130. Elias, H., Bokelmann, D., and Vbgtle, R., Morphometry, stereometry and stereology applied to
cancer development in the large intestine of man, in 4th Int. Congr. Stereology, Underwood, E. E.,
de Wit, R., and Moose, G. A., Eds., National Bureau of Standards Special Publication 431, Wash-
ington, D.C., 1976,321.
131. Eins, S. and Wolff, J. R., Analysis of the heterogeneous composition of central nervous tissue, in
4th Int. Congr. Stereology, Underwood, E. E., de Wit, R., and Moose, G. A., Eds., National Bureau
of Standards Special Publication 431, Washington, D.C., 1976, 331.
Taylor & Francis
49
Chapter 2
TUMOR ANGIOGENESIS
Bruce A. Warren
TABLE OF CONTENTS
I. Introduction 49
II. General Aspects of Angiogenesis 50
III. Fetal Angiogenesis 51
IV. Factors from Tumor Tissue 56

A. Chemical Mediators and Angiogenesis: Tumor Angiogenesis Factor or
Factors 56
B. Nerve Growth Factor (NGF) of Levi-Montalcini 58
C. Fibroblast-Growth Factor of Gospodarowicz 59
V. Angiogenesis Induced by Specific Tumors and Cells 60

A. Brain Tumors 60
B. Skin Tumors and Neoplasia of Cervix Uteri 60
C. Breast Carcinoma 61
D. Neovascularization of the Eye 62
E. Various Cells and Angiogenesis 62
VI. The Morphology of Tumor Angiogenesis 64
Acknowledgment 71
References 72
I. I N T R O D U C T I O N
Tumor angiogenesis is the formation of capillary sprouts induced by a group of

tumor cells with eventual development of a microcirculatory network within the mass.
Its closest normal analogue is the production of capillary sprouts in granulation tissue
during repair of tissues. The phenomena of regeneration of limbs by amphibia and
the subtle processes of fetal angiogenesis may provide examples which bridge the gap
between the normal processes of wound repair and granulation-tissue formation and
tumor angiogenesis. There are two principal types of tumor angiogenesis in terms of
the time frame of the events following implantation of metastatic seedlings on surfaces
and in organs. The first, or primary angiogenesis is the initial vascularization of the
mass of multiplying tumor cells, and is regarded as an essential prerequisite for the
survival and further growth of a metastatic deposit. The second is the continuing or
secondary angiogenesis, and is the phenomenon which occurs in waves at the periphery
of a growing mass of tumor, and is essential for the accretion of new microcirculatory
territories into the service of the expanding and infiltrating tumor. A full understand-
ing of tumor angiogenesis would presuppose knowledge of the normal constraints on
remodelling of the microcirculation, how these are released or modified, if there is a
"factor" released during induction of granulation tissue, the determination of differ-
entiation of the tissues and the maintenance of differentiation, the nature of the inva-
sion of fetal tissues by the vascular system, and the return to a "quiescent state'' after
a period of growth both in the healing of a wound and on maturation of the vessels
of a fetal part. The subject of tumor angiogenesis impinges on many fundamental
aspects of dynamic changes in fetal growth and development. Clues as to its nature
may be present in these phenomena, which are kin to tumor angiogenesis. The funda-
mental position as to whether the tumor cells drive the vasculature or the vasculature
* Controls' ' the tumor cells must be addressed. If the primary angiogenesis was halted,
would this prevent further growth of the tumor mass? If secondary angiogenesis was
halted, would this prevent increments in the mass of the tumor? Can certain tumors
develop nonconventional vascular systems without endothelium which subserve the
usual purposes (albeit less efficiently than normal)? Are the mechanisms of evocation
of the initial primary angiogenic response of the vascular bed and the secondary angi-
ogenic response similar or entirely different in the stimulus required or in the type of
vessel affected? How does the induction of new blood vessels by tumors fit into the
general biological framework of the reaction of mammals to injurious agents, to the
marvel of regeneration, and to fetal development?
II. G E N E R A L A S P E C T S O F A N G I O G E N E S I S
Capillary growth, constituting as it does one of the main elements of granulation

tissue (Figure 1), is probably one of the commonest responses of tissues following
injury. The restitution of tissues and the replacement of dead or necrotic tissue by less
specialized tissue takes place after the invasion of the part by capillaries. There is an
intimate relationship between the fibrous supporting structures, basement membrane
of the capillary, and the endothelial cells lining the lumen of the capillary. The endoth-
elial cells of mature capillaries are anchored to the basement membrane by hemides-
mosomes. The basement membrane is in turn supported by fine bundles of collagen
fibers and an investment of pericytes.
For capillary growth, the supporting framework of the basement membrane and
collagen fibers is altered. Capillary sprout formation is not dependent solely upon
endothelial cell division. Mitotic division within a capillary sprout occurs proximal to
the growing tip. The cells in the sprout are not anchored to the basement membrane,
but appear to slide freely over one another. Indeed, the basement membrane of capil-
laries is poorly formed or absent in the region of the capillary sprouts. Capillaries were
extensively studied by the Clarks 1 in the rabbit-ear chamber. Fine-pointed capillary
sprouts arising from pre-existing capillary vessels in the rabbit-ear chamber were de-
scribed by Clark et al. 1 Balloon or saccular types of sprouts were found in the rabbit-
ear chamber by Abell2 and Williams. 3 Cliff4 combined studies by light and electron
microscopy on the repair tissue in rabbit-ear chambers. He identified proliferating ves-
sels in vivo and found both the tapering or fine-pointed type of sprouts and the sac-
cular type. A collapsed or potential lumen was present in the tapering type. Endothelial
cells of these capillary sprouts differed from normal endothelium of mature vessels in
a number of ways. The cells were plump, they possessed a plentiful, ribosome-studded,
endoplasmic reticulum, and there were numerous mitochondria. In some instances,
the cytoplasm of these cells could be seen to be divided into a fibrillary outer region
51
FIGURE 1. In vivo micrograph of the vascular pattern of granulation tissue (G) next to a
necrotic area (N) in a hamster cheek pouch chamber. (Magnification x 120.)
with an inner region with much fewer fibers. These newly formed vessels were more
fragile than mature vessels, and leukocytes were found to adhere to the newly formed
endothelium. Tapering sprouts possessed many overlapping endothelial cells and had
only a rudimentary lumen, in contrast to the well-developed lumen of the saccular type
of sprout. A similar division of capillary sprouts is possible on examination of the
vasoproliferation response called forth by the presence of a tumor transplant (Figures
2 and 5). Cliff4 suggested that closely apposed endothelial cells first formed from an
existing vessel, and that after the formation of the cord of cells, there was the progres-
sive development of a lumen by a splitting mechanism down the center of the cord of
cells. Basement membrane was formed early in the process of new vessel formation.
The basement membrane was incomplete around some of the tapering sprouts and al-
ways complete around the saccular types of sprouts.
Ill. FETAL ANGIOGENESIS
Loeb, in 1893,5 demonstrated that in the embryo of the fish, Fundulus, in which
the effective action of the heart had been prevented by the addition of 1.5% potassium
FIGURE 2. Macroscopic photographs of the vessels in a hamster cheek pouch membrane within a cham-
ber. A fragment of cheek melanoma tumor (T) has been placed on the surface of the membrane. The blush
of fine vessels which represents an early phase of vascularization of the implant is seen. (A) Three days after
transplantation. (Magnification x 8.) (B) The same chamber 1 day later. The vascular pattern in the tumor
implant is better developed. The veins of the surrounding pouch membrane are more tortuous. Small hem-
orrhages (H) from induced capillary sprouts dot the membrane in some areas. (Magnification x 7.75.)
53
chloride to the sea water in the hatchery, new vessels developed in relation to the de-
veloping tissues. There was no effective blood pressure and no circulation of blood.
Therefore, he considered that the vessels developed in response to what would now be
conceived as an evocation of vascular growth by the growing tissues. Loeb noted that
in such vessels growing without being subjected to normal blood pressure the diameters
of the lumen were irregular and the wall thickness was uneven. Therefore, although
vessel formation might occur under such circumstances, the development of normal
pattern and tissue differentiation was dependent upon other factors.
In a consideration of the development of peripheral vessels, Arey6 considered that
there were general principles of development which pertained to the following subjects:
(1) the source of the angioblast, (2) the origin of embryonic vessels, (3) the formation
of temporary capillary plexuses which were the forerunners of definitive vascular sys-
tems, (4) the emergence of larger pre-emptive channels within such provisional net-
works, (5) histogenetic advances in which additional tissue coats were laid down on
certain channels, and (6) casual development. Arey considered that the primary tissue
of blood vessels was endothelium, and that all else was auxilliary tissue. The first blood
islands or earliest vascular primordia are clusters of cells arising on the yolk sac. These
are at first compact masses between the splanchnic mesoderm and endoderm. Separa-
tion into peripheral cells (primitive endothelium) and more centrally located cells (pri-
mitive blood cells) soon occurs, and a plexus of vessels is formed. Progressive vascu-
larization is observed. Although this tissue was originally considered to be the sole
source of all blood vessels (the "angioblast theory"), later workers, 7 8 took the position
that mesenchyme throughout the embryo was able to differentiate into endothelium
as needed, and eventually the concept of the local origin of blood vessel endothelium
within the embryo was accepted. 6 Once the initial circulation of the embryo is formed,
the system is extended only by sprouting and subsequent transformation.
Haar and Ackerman, 9 by using phase and electron microscopy in their studies of
vasculogenesis in the mouse embryo, classified a number of morphologic features con-
cerned with the early development of vascular channels. In the mouse, they found that
at 7 days of gestation localized proliferations of mesoderm produced angioblastic cords
in which the cells were attached to each other by tight junctions and desmosomes. By
8!/2 days, lumina appeared within the cords. The several distinct, but interrelated, fac-
tors which occurred at the same time and involved both lumenization and vasculoge-
nesis included:
1. Dissociation of cells of the angioblastic cord as a result of release of the adhesion

forces initially imposed by the presence of tight junctions. The cells of the angiob-
last cords and blood islands then rounded up.
2. The primitive endothelial cells elongated while the primitive erythroblasts of the
blood islands remained fixed, so that differential growth between these two now
distinct elements came to pass.
3. The visceral basement membrane fragmented and was lost. This discontinuity of
the visceral basement membrane allowed differential growth and movement of the
endothelial cells as well as ready passage of fluid into the angioblastic cords and
their contents.
4. Wide intercellular channels developed between adjacent endothelial cells. By 9
days of gestation, the primitive erythroblasts were noted free in the vitelline ves-
sels.
There was an early controversy as to whether there was a direct outgrowth of definitive
vessels which took up the standard pattern of the organ or whether there was an initial
plexus in which competition for supremacy and survival occurred.
The work of Thoma 10 and Evans 11 indicated the necessary existence of provisional
capillary networks wherever a vessel was to appear and the selection of a definitive
channel within it, with only a few exceptions (e.g., dorsal segmental branches of the
aorta). The series of stages are represented, first, by a provisional temporary plexus
and, next, by the appearance of a preferred channel through the plexus. Subsequent
developments of the network consist of concomitant enlargement of the (now) major
pathway of the preferred route together with atrophy of the disused components of
the network. In the embryo, 6 capillaries invade tissues from primary vascular areas
into regions not yet vascular. The timing of the vascularization process is distinctive,
and some tissues such as cartilage and cornea became permanently avascular.
Clark12 made direct observations on the tail of the frog tadpole and described how
the patterns of the vessels change with time. The influences that govern the final stable
pattern of the vasculature are considered by Arey6 to be heredity (only in major arteries
and veins), heart beat, blood flow, volume of blood flowing, and its pressure. Thoma 10
concluded from studies on the yolk sac of the chick that the development of arteries
and veins were consequent upon a functional adaptation to the demands of conduction
for the flowing blood in the area. Arey6 cites Thoma's laws10 as follows:
1. Increase or decrease in the size of the lumen of a vessel (or what is the same thing, the increase or
decrease in the surface of the vessel wall) depends upon the rate of the blood flow. (In practice, the
surface ceases to grow when the blood current acquires a definite rate; the vessel increases in size when
this rate is exceeded, becomes smaller when the blood stream is slowed, and disappears when it is
finally arrested.)
2. Increase or decrease in the length of a vessel is governed by the tension exerted on the vessel wall in a
longitudinal direction by tissues and organs outside the vessel.
3. Increase or decrease in the thickness of the vessel wall is dependent upon the blood pressure. Further,
the tension of the wall is dependent on the diameter of the vessel and on its blood pressure.
4. Increase of blood pressure in capillary areas leads to the formation of new capillaries.
The metabolism of the organ involved was identified as the ultimate governor of the
pattern and size of the capillary network. Variations in the size of capillaries second-
arily influences factors which change the size of arteries and veins. The different types
of alterations in vessels that can be observed in the process of the production of the
stable adult pattern of vessels from the fetal distribution are listed in Table 1.
Rhodin, 13 in a study of angiogenesis in the human fetus, noted that vascular growth
occurred through endothelial sprouting. Microangiogenesis was examined in 4-month-
old human fetuses in skeletal muscles, lungs, and kidneys. After formation of the
endothelial sprout, a thin basement membrane is laid down around the bud conjointly
by undifferentiated mesenchymal cells and the endothelium. Lumenization of the
sprout occurs simultaneously with mesenchymal cells settling on the surface of the
endothelial cell cord. The mesenchymal cells differentiate to form pericytes. An incom-
plete basement membrane forms between the pericyte and the endothelial cell. As the
capillaries lengthen and branch in the tissues, the pericytes become less numerous per
unit length of capillary. In the hamster cheek-pouch chamber (for details see Chapter
1), contracting cardiac homografts 14 and glomeruli with circulating blood 15 have been
observed in vivo. The function of the grafted tissues was related to the age of the
donor. Cardiac grafts from adult animals showed no contractile activity. Those derived
from the atria contracted if the donors were 6 days old or less, in the majority of
preparations. In the renal grafts, no glomerular circulation was evident in 55 grafts
from adult donors, in contrast to the 21 grafts from newborn animals in which glo-
merular circulation was always present, albeit in some instances abnormal. In the car-
diac homograft experiments, vascularization of the transplanted fragment occurred
within 24 to 36 hr. The graft rapidly became dark red, and was surrounded by a cir-
55
TABLE 1
Fetal Angiogenesis: Table Showing Different Types of Vascular Development6
Type of development that

gives rise to final vessel form Examples
Selection of preferred route Commonest manner of origin of vessels, may further participate in
from capillary network secondary changes with remolding of anatomical course
Direct sprouting as tubal Segmental spinal arteries, not preceded by provisional capillary plexus
structures
Parallel fusion: the merger Basilar artery, portions of umbilical veins, merger of those ventral
of earlier paired vessels branches of aorta which form coeliac and mesenteric arteries
Shifts in level of origin Final position of vessels are far removed from original site, e.g., coeliac
and mesenteric arteries
Longitudinal annexation Blood supply of posterior region of lower limb
Cross anastomosis Left innominate vein, left common iliac vein
Combining of paired vessels: Portal vein (comprises left vitelline vessel, portion of right vessel, plus a
compound definitive vessel middle anastomatic union)
Linkage at tips Vertebral arteries (longitudinal linkage of six dorsal branches of aorta)
Survival of original segments Primitive sciatic artery only represented in adult by survival of some of
its segments
Replacements Inferior petrosal sinus replaces a degenerated segment of primary head
vein
Secondary root of origin Hypogastric arteries
Secondary side branches The branches of an artery may arise from a different plexus than that
from which the main vessel was derived, e.g., main aortic branches
Portal system development Venous trunks become invaded by developing organs, hepatic and
hypophyseal systems, temporary renal portal system
Progressive reduction in Original series of ventral aortic branches which supplied yolk sac
numbers reduced to three vessels (coeliac and 2 mesenteric arteries) supplying
main segments of GI tract
Disappearance and atrophy Abandonment of fetal plan of circulation results in certain obsolete fetal
of fetal vessels vessels which survive as fibrosed relics, e.g., left umbilical vein as
round ligament of liver
cular area of hemorrhage. The previously empty vessels of the graft filled with red
cells without movement. Endothelial tubes proliferated from pre-existing homograft
vasculature and radiated out towards the host microcirculation. These endothelial
tubes connected with the host venules and venous capillaries. Retrograde flow from
the venous system into the graft next appeared. By the third or fourth postoperative
day, arterialization of the blood flow within the graft was apparent. At this time, the
graft changed in color from dark red to a pink color. Contractions of the graft were
not present prior to vascularization. Commonly, two or three contracting foci were
present in the atrial grafts, each beating rhythmically at different rates, usually between
80 and 130 beats/min. In the ventricular grafts, contractile foci were frequently nu-
merous, but of small amplitude generally, at rates of between 30 and 60 beats/min.
The homografts of newborn kidney tissue in the hamster cheek-pouch membrane
underwent a similar series of changes, but with a different time schedule. The graft
became dark red in color within 12 to 24 hours of implantation. If two fragments were
present in the same chamber, the initial vascularization occurred with the larger one.
Establishment of continuity of the vasculature of the graft with the host tissue was
achieved by capillary buds from the graft. After 24 to 36 hr the graft changed color,
becoming lighter in appearance, and arterioles were seen at this stage connected with
the vascular plexus of the pouch membrane. In grafts from newborn animals, glomer-
ular circulation appeared with the onset of this stage (the intermediate stage). Approx-
imately 30 to 50% of the glomeruli were abnormal.
This capacity of fetal or newborn tissue to stimulate angiogenesis has been directly
compared with transplanted neoplastic tissue by Huseby et al.16 They found that one-
day-old mouse testes stimulated angiogenesis when transplanted into castrated young
adult male animals "in certain respects more dramatically" than expiants of compa-
rable size from a transplantable mouse mammary adenocarcinoma. In the testicular
grafts, new vessels rapidly extended throughout the interstitium of the expiants, and
this effect was evident as early as 3 days after introduction of the tissue. The fetal
characteristics of some neoplasms have been repeatedly emphasized over many years
(for review see Uriel17), and this provides another facet of similarity.
IV. F A C T O R S F R O M T U M O R TISSUE
A. Chemical Mediators and Angiogenesis: Tumor Angiogenesis Factor or Factors

Chemical mediators that might induce a vasoproliferative response in the host tissues
have been suggested (for review regarding the development of collateral circulation,
see Liebow18), and indeed, observation of the ingrowth of capillaries into tumor trans-
plants suggests the existence of such substances.19-20 Greenblatt and Shubik21 separated
the host tissue (in this case hamster cheek-pouch membrane) from a tumor transplant
(melanoma) by means of a barrier membrane (Millipore® filter 25 pm thick with a
pore size of 0.45 ± 0.2 jum). Diffusion of a chemical mediator was possible through
the filter which at the same time prevented contact between the tumor and the host
vasculature. A vasoproliferative effect was consistently observed, and they concluded
that this evidence strongly pointed to the existence of a humoral factor which was
active in tumor angiogenesis. A similar transfilter stimulation of vasoproliferation in
the hamster cheek pouch was achieved through a microporous filter membrane with
dose size 0.5 \xm using a transplantable choriocarcinoma by Ehrmann and Knoth. 22
Folkman and his associates have published a considerable number of papers 2350 on
the subject of a diffusible factor from tumors, putatively responsible for angiogenesis,
including the theoretical implications of the existence of such a substance. 24 2 7 3 6 3 9 In
the initial studies by Folkman et al., 23 a method of creating a "dorsal air sac" in the
57
rat by aseptically injecting 30 mi of air, which dissected into a plane of areolar tissue
on the dorsal surface of the rat, was used. Tumor cells within millipore filter chambers
or solutions in millipore tubing were assayed for their effects on the vascular bed by
being placed in the air sac. After 48 hr, the degree of vasoproliferation was graded,
the tube was removed, and sections taken for histological examination. This type of
assay has now been replaced by assay techniques using the rabbit cornea (Gimbrone
et al.34) and the chorio-allantoic membrane of the developing chick (Ausprunk et al. 40 ).
It has become apparent that diffusion of the factor can occur over distances greater
than that of the millipore filter (25 ¡urn) (Folkman 39 ). Studies by Folkman's group for
example, found diffusion of the effect up to 1 to 3 mm in the dorsal air sac of the rat
(Cavallo et al. 26 ), up to 2.5 mm in the rabbit cornea (Gimbrone et al. 34 ), and up to 5
mm in the anterior chamber (Gimbrone et al. 31 ). However, it is difficult to make gen-
eral statements from these observations in artificial sites since it appears that the net
effect of angiogenesis depends not only on exciting factors from the tumor transplant,
which for a given mass of proliferating tumor may be constant, but also upon inhibi-
tory influences. For example, cartilage expiants inhibit tumor-induced angiogenesis in
the anterior chamber of the rabbit eye (Brem and Folkman 38 ). The transmission of the
effect of tumor angiogenesis thus appears to be dependent, at least partially, upon the
nature of the local tissues. Eisenstein et al. 51 found that cartilage extracts inhibit the
growth of endothelial cells in culture more than those of the dermis, and these workers
suggested that this type of influence may bear upon the question of the resistance of
certain tissues to invasion. For a review of the work of this group, the reader is referred
to the paper by Folkman and Cotran. 45 Great interest currently centers upon the puri-
fication methods for tumor angiogenesis factor (TAF) and the bioassay procedure. It
is, naturally, essential to have a quantitative bioassay procedure available for this agent
since it is produced in such small quantities (1 to 3 ¡¿g of protein containing TAF
activity are released from 1 x 107 cells when roller bottles are used). 45 TAF activity
been found in a wide variety of tumor lines, including cultures of mouse tumors
(BALB/c SVT2), B-16 melanoma, a meningioma, a glioblastoma, and SVW126. The
initial procedure using the dorsal air sac of the rat has been discontinued because it
requires so much protein containing TAF activity (up to 500 jug). In the 1976 paper, 45
the current bioassay procedures for TAF are described as using the chorio-allantoic
membrane of the chick embryo (CAM) and the rabbit cornea. A 1 mm piece of Milli-
pore® filter is soaked with the test substance, or the test material is implanted as a
lyophilized crystal. After 48 to 72 hr, the CAM is examined under a low-power ster-
eoscope and the vascular reaction graded according to its intensity on a scale of one
to five by comparison with a wall chart. With the rabbit cornea, 1 mm pieces of tissue
are implanted into a pocket 1.5 x 2.0 mm in the cornea which is 2.0 mm or less from
the limbal edge. TAF fractions are added to this pocket, and the effect with regard to
the stimulation of new vessels observed. The density and rate of growth of the vessels
are used to determine the activity. These assays are recognized as "essentially non-
quantitative" and "subject to biological variation". 45
The work of Phillips et al. 52 and Fenselau and Mello53 supports the concept of a
tumor angiogenesis factor (TAF) present in human and animal tumors. Phillips and
co-workers52 prepared extracts from Walker 256 carcinoma, spontaneous rat mam-
mary adenocarcinoma, Wilms' tumor, human neuroblastoma, and human heman-
gioma. Four fractions were harvested from the material after chromatography of the
extracts on Sephadex G-100®. Growth of new capillaries in the s.c. fascia of rats re-
sulted after injection of two of the fractions. Similar extracts of rat liver or human
kidney did not produce a proliferative response. The endothelium of the positive test
material contained many mitotic figures. The development of a quantitative in vitro
assay for TAF using short-term primary cultures from adult rat brain white matter
was attempted. The fractions which were active in vivo failed to stimulate endothelial
proliferation in vitro in their system.
Fenselau and Mello53 produced short-term cultures from fetal bovine heart and
aorta, which in their hands consistently displayed increased growth rates when crude
tumor cell homogenates from Walker 256 carcinoma were added to the culture media.
The material derived from the tumor acted on both confluent and sparse endothelial
cell cultures. They found that the tumor-derived cultures were not absolutely necessary
for the in vitro growth of fetal bovine endothelial cells, provided that 5 to 20% of fetal
bovine serum was present in the medium. They concluded that the effects in their system
were attributable to macromolecules in the tumor homogenates and not to viral con-
taminants, because of the appearances of the cell cultures. The stimulated endothelial
cells grew in monolayers as before, persisted in their original morphological appear-
ance, and when the homogenates were removed from the medium, resumed a slower
growth rate. These features were unlike those that would be expected of virally trans-
formed cells. Homogenates from various adult tissues were ineffective, while homoge-
nates of embryonic tissues and cultured cells showed growth-promoting effects similar
to those of the tumor homogenate. This is generally consistent with the results of trans-
plantation in vivo of tumor grafts, embryonic tissue grafts, and adult tissue grafts, and
their subsequent angiogenic capabilities.54 An examination of vascularization induced
in the cheek pouch by tumor and nontumor substance was made when these substances
were enclosed in microporous filters which prohibited cellular contact with the test
substance or tissue. Fresh amelanotic and dry amelanotic melanoma tissue, gelatin,
embryo, adult connective tissue, fresh and dry liver, fibrinogen, fibrin, dry pouch mem-
brane, hamster embryo, and glycogen were tested. There were both quantitative and
qualitative differences in the vasoproliferative response produced in the underlying
pouch membrane. The glucocorticoid, 6a methyl prednisolone (MPS), decreased and
sometimes totally inhibited new vessel formation. Dry tumor tissue induced angioge-
nesis in seven of eight membranes 3 days after implantation. Only two of seven cham-
bers of MPS-treated animals showed a reaction 10 days after transplantation of tumor
tissue. MPS had a suppressive effect on capillary new growth in chambers with im-
plants. This is an interesting effect in view of the work discussed later in this chapter
(V.D.).
B. Nerve Growth Factor (NGF) of Levi-Montalcini

Levi-Montalcini55 in 1965 reviewed her work on nerve growth factor (NGF) and epi-
dermal growth factor (EGF). Although the second half of the last century saw the
recognition of the action of hormones and, to a lesser extent, other "growth factors",
more rigorous analysis and investigations of their action on their target-cell populations
has awaited relatively recent times. Hormones have held center stage with only rudi-
mentary investigation of other growth-promoting agents. Bueker56 implanted mouse
adenocarcinoma, fowl Rous sarcoma, and mouse sarcoma 180 into the body wall of 3-
day embryos. This was an approach to certain neuroembryological situations in that
he wished to test the capacity of nerve fibers to invade homogeneous tissues rather than
the heterogeneous tissues which are found in the developing limb. The differentiation
of primary, sensory, and motor systems in chick embryos is mostly under extrinsic-
factor control. Transplantations in amphibia in previous experiments had shown the
ability of nerve fibers to invade heteroplastic structures. The results of his transplanta-
tions were that the mouse adenocarcinoma failed to grow, the fowl Rous sarcoma
caused extensive hemorrhage, and mouse sarcoma 180 grew vigorously and was invaded
by nerve fibers of the host. Levi-Montalcini57 investigated this phenomenon further and
59
found that sympathetic ganglia of embryos transplanted with sarcomas 180 or 37 are
enlarged even if they are not involved with innervation of the implanted tumor. A
remote effect was proved by transplantation not into the body of the embryo, but onto
the chorio-allantoic membrane of 4 to 6 day chick embryos so that the tumor shared
the circulation with the embryo, but did not have direct contact with it. The affected
ganglia were hypertrophic and hyperplastic and produced nerve fibers far in advance
and in excess of ganglia of control embryos. These fibers entered in large bundles into
most of the embryonic viscera, as well as forcing their way into the lumens of large
and small blood vessels. Further investigation of the source of origin and the nature of
the nerve growth factor (NGF) followed,58 and an assay method using a hanging drop
was developed. In an attempt to further purify this agent, Cohen used a crude snake
extract as a source of phosphodiesterase. It was found that the venom itself was a
potent source of the agent. On the hypothesis that similar glands in other animals, e.g.,
the mouse salivary gland, might harbour NGF, these were assayed for activity. The
mouse salivary glands were found to be a third and more potent source than the other
two.59 The equivalent growth stimulation effects were similar with 15,000 /¿g of tumor
homogenate, 6 fig of snake venom, and 1.5 /¿g of mouse submaxillary salivary glands.
Salivary NGF is a protein particle which is heat labile, destroyed by acid, resistant to
alkali, and is not dialyzable. The biological activity was destroyed on incubation with
proteolytic enzymes, and the salivary factor was antigenic. Injection of the antiserum
drastically decreased the sympathetic cell population in newborn animals. From their
studies, it was concluded that NGF acted in very low concentration, was specific in its
effects, was normally present, and was essential to the subsistence of sympathetic nerve
cells during an early phase of differentiation.
A further growth factor, epidermal growth factor,55 was eventually isolated because
side effects on the skin were noted with crude NGF. When daily injections of EGF
were given in a dose of 0.5 mg/1.5 g of body weight to newborn mice for 6 days,
marked alteration in epidermal growth was noted. The skin thickened, underwent early
keratinization, and the height of the epitelia lining the oral cavity, esophagus, and stom-
ach mucosa increased. Levi-Montalcini55 distinguished growth factors from hormones
by the following features. The primary effect of growth factors is to increase growth
activity rather than functional activity. Growth factors are important early in life during
growth and differentiation of the target cells. Levi-Montalcini55 speculated that similar
control mechanisms to those of NGF might exist for all cell types and might function
as a primitive integration system. A relationship of metabolites to the growth factors
might exist. Such substances, released by poorly organized cells not present as defined
organs, might be utilized by other cells as growth factors. The ability to respond to
NGF persists even in neuronal-HeLa heterokaryons.60 Cells from dorsal root ganglia
of 7-day chick embryos can be fused with HeLa cells by inactivated Sendai virus. Nerve
growth factor resulted in the growth of long processes from the neuronal-HeLa heter-
okaryons.
C. Fibroblast Growth Factor of Gospodorowicz

Serum is a complex mixture, and for the proliferation of animal cells in tissue culture
it is necessary to supply as yet unidentified macromolecules which are present in
serum.61 Gospodorowicz made the following assumptions: (1) growth promoting fac-
tors may be synthesized in organs other than blood, and if those organs could be
identified purification would be simpler than with blood, and (2) two sources which
appeared likely were (a) the pituitary gland, since stimulation of cell growth in vitro
had been reported, and (b) the brain, since a neurotrophic substance was necessary
for blastema formation in the amputated limbs of vertebrates capable of regeneration.
Using both pituitary and brain, a growth factor was isolated which was named fibro-
blast growth factor (FGF). Although the initial studies demonstrated the effect of this
agent on fibroblasts, the range of its target cells has been extended in further work to
Yl adrenal cell line, bovine adrenal cells, myoblasts, glial cells, chondrocytes, vascular
smooth muscle, and endothelial cells derived from the vascular bed. From the range
of cells sensitive to this mitogenic agent, it became apparent that it was effective on
mesoderm-derived cells. In contrast, it was not effective on epithelial cells, anterior
pituitary cells, liver, or pancreas (i.e., cells of other germ layers such as ectoderm or
endoderm). FGF was found to be nonmitogenic for avian fibroblasts and myoblasts.
FGF is similar to the growth promoting factor found in platelet extracts which is con-
cerned with endothelial turnover. Endothelial cells from fetal bovine aortas maintained
in the presence of 10% plasma divided every 72 to 92 hr, while in 10% serum they
divided every 48 hr. When FGF was added to plasma, the division time was reduced
to 24 hr. When FGF was added to serum, the division time remained at 24 hr, but the
final cell density was higher. Fetal bovine endothelial cells, whether from umbilical
cord or aorta, proliferate poorly in the absence of FGF and show altered morphology.
The effect of FGF on morphology may be indirect. Since FGF increases cell density,
this population density alone may be a determining factor with regard to the morphol-
ogy of the cells.
Recently, Gospodorowicz and Thakral 62 have examined the factors controlling
changes in the vascular pattern of the ovary. By using the rabbit cornea assay system,
they found that the follicles did not induce neovascularization while the corpora lu-
teum did. They suggested that at the stage of the corpus luteum an agent was secreted
which induced new vessel formation.
V. A N G I O G E N E S I S IN S P E C I F I C O R G A N S A N D T U M O R S
A. Brain Tumors
Intense new vessel formation is associated with rapidly growing tumors and is a poor
prognostic sign.63 If human and experimental gliomas are transplanted to the rabbit
cornea, intense neovascularization is induced. This does not occur with control tissues.
If a fragment of tumor which is active in the cornea in inducing angiogenesis is trans-
planted into the vitreous humor, the tumor does not induce vascularization, but re-
mains "dormant". 6 3 Using a system involving a human endothelial cell culture derived
from postpartum umbilical veins, Kelly et al. examined the growth-promoting proper-
ties of media from cultures of human and experimental central nervous system tu-
mors. 64 As an indicator of endothelial proliferation, they used a determination of the
3
H thymidine uptake with autoradiography and represented this as the thymidine la-
belling index (Tl). This is the proportion of 3 H thymidine-labelled endothelial cells to
the total number of cells counted. They tested the following human central nervous
system tumors (which are ranked from tumors with the highest labeling index to the
lowest with the Tl in parentheses): angioblastic meningioma (98.3%), neuroblastoma
(78.7%), ependymoma (40%), syncytial meningioma (38.2%), acoustic neurinoma (2
tumors, 34.5% and 32%) and ependymoma (30.1%). Experimental tumors tested were
C6 (Pfeiffer, 96.6%), C6 (JDV, 83.3%), and C6 (CCL107, 28.7%). The control media
induced labelling of only 2.5 (endothelia alone) to 4.5% (endothelia with fibroblast-
conditioned media). They concluded that there was an endothelial growth promoting
factor produced by these tumor cells.
B. Skin Tumors
Wolf and his associates,65"68 in a number of papers, have described angiogenic factor
or factors associated with the epidermis, 65 skin tumors, 66 s.c. lymphoma, 67 and mela-
61
noma. 68 The complex interplay between the epidermis and dermis has been observed
clinically, and there is the implication that alternations in the epidermis are capable of
affecting the dermal blood vessels. Wolf and Harrison 65 used the cheek-pouch chamber
system to study the effects of implants of epidermis. They noted a vasoproliferative
response of the underlying pouch membrane in situations where the implant was placed
directly on the pouch membrane, and when it was separated from it by a microporous
filter. There was no significant new vessel growth in chambers in which the following
were implanted: dermis, polythene, dialysis membrane, or when the epidermis had
been inactivated by heat treatment. In contrast to preparations from the dermis, epi-
dermal homogenates, Millipore® filtrates, and dialysates induced a vasoproliferative
response in the pouch membrane. The activity persisted in the aqueous, but not the
organic phase after ethyl acetate extraction, was inactivated by heating, and was re-
moved by trichloracetic acid precipitation. It was not affected by storage at 4°C for
up to 7 days. These investigators considered that there existed in epidermis a specific,
heat-labile, diffusible, nondialyzable protein which they named "epidermal angiogenic
factor". A variety of human tumors, and vascular and other cutaneous lesions were
next examined for angiogenic activity.66 A filterable and diffusible angiogenic factor
was extracted from a number of types of skin tumor (e.g., melanoma, basal cell epi-
thelioma, squamous cell carcinoma, and lymphoma). The activity of extracts from
these tumors induced a distinctive pattern of sequential vasodilatation, tortuosity, and
new vessel formation in the hamster cheek pouch membrane. A group of lesions,
which included pyogenic granuloma, vascular histiocytoma, and Kaposi sarcoma, was
productive of extracts which incited dramatic hyperemia and ectasia. New vessel for-
mation was not induced by extracts from nevoid normal cutaneous components, with
the notable exception of epidermis or control materials. Direction stimulation of en-
dothelial mitosis resulted from the released angiogenic factor, and these authors con-
sidered that it might be essential for survival of "nutritionally ravenous" tumor cells.
Investigation of the activity of fragments of human lymphoma and human melanoma
showed that these tumors were active both when implanted directly on the hamster
pouch membrane or when separated from it by microporous filters.67 68 Combined
colposcopic and histochemical studies of the cervix uteri69 have indicated that there
are alterations in the vascular pattern of the tissues as lesions in the area progress from
early dysplasia to carcinoma in situ. In noninvasive cervical lesions, restructuring of
the terminal vascular network of the columnar epithelium occurs by compression of
the capillaries because of epithelial proliferation. New-vessel formation is noted in
those cases of carcinoma in situ which will advance to invasive carcinoma. The devel-
opment of horizontal vessels characterizes this process and may result from the effects
of an angiogenic factor.
C. Angiogenesis in Neoplastic and Preneoplastic Lesions of the Breast

Certain strains of mice (C3H, C3H-A, C3H-AvyfB) have a high incidence of mam-
mary tumors. This biological model was used by Gimbrone and Gullino to test for the
timing of the ability to induce new vessel formation during the progression of mam-
mary tissue from the normal state, through a preneoplastic stage, to mammary carci-
noma. 70 The capacity to induce angiogenesis was compared in normal, preneoplastic,
and mouse mammary tumors by implantation on the iris of rabbits. 70 71 Neovasculari-
zation of the iris was studied by in vivo slit-lamp stereomicroscopy and fluorescein
angiography, injection of colloidal carbon into the vasculature, and histological ex-
amination. After 48 to 72 hr, 90% of the mammary tumor implants induced a vaso-
proliferative response, and this was without regard to histological type or to the pres-
ence or absence of mammary tumor virus. The immediate postoperative inflammation
was reduced by corticosteroid treatment, but did not abolish further vasoproliferation.
Necrotic tumor fragments did not induce any angiogenesis. When normal tissues from
resting mammary glands were tested in the same system, only 6% elicited any vasopro-
liferation. Hormone-stimulated mammary tissues from pregnant and lactating mice,
when tested, only showed transient activity in their capacity to develop new blood
vessels, and even that was lost during postweaning involution. Thirty percent of im-
plants from premalignant hyperplastic alveolar nodules induced vessel growth similar
to that of tumors. In two lines, Dl and D2, the capacity of the hyperplastic alveolar
nodules to induce angiogenesis was parallel with the incidence of mammary tumors
(low and high, respectively). From their work, the authors concluded that the capacity
to induce angiogenesis was acquired during progression of the mouse mammary tissues
to neoplasia.70 71 Further work by members of the same group tested whether the in-
duction of angiogenesis by fragments of hyperplastic human breast tissue could indi-
cate a high risk for the future development of mammary carcinoma. 72 73 The next
study72-73 entailed testing 947 mammary fragments obtained from surgical biopsy spec-
imens from 10 patients with mammary carcinoma and 32 patients with nonneoplastic
lesions, including fibrocystic disease, stromal fibrosis, lipoma, fibroadenoma, and gy-
necomastia. The fragments were implanted into the iris of female rabbits. Angiogenesis
occurred with fragments of neoplastic tissue, but was absent in other fragments which
consisted of fat or stroma. Less than 2% of the nonmalignant lesions induced angioge-
nesis.72 Boiled tumor fragments in 49 preparations did not induce angiogenesis.72 Fifty
fragments from hyperplastic lobules were identified, and of these, 28% induced angi-
ogenesis. They concluded that the capacity to induce growth of new vessels was a
characteristic of the human mammary carcinoma as well as that of the mouse, in a
similar way to that shown earlier. They speculated that the angiogenesis assay might
be able to separate patients with hyperplastic lobules into those of high (positive angi-
ogenic response) and low (low or absent angiogenic response) risk of developing mam-
mary neoplasia.
D. Neovascularization of the Eye

The subject of ocular neovascularization has particular interest in a discussion of
tumor angiogenesis, since, as can be seen above, assay procedures involving the cornea
and the iris have been used for testing the capacity of tissue fragments to induce angi-
ogeneis. Henkind, 74 in a review of ocular neovascularization, considered that the eye
had many (and specialized) vascular channels which differed in both anatomic and
physiologic senses, depending upon the special requirements of the tissues which they
served. The blood vessels in the mature eye, although in reality labile structures, give
the superficial impression of stability because of the balanced interplay between biol-
ogic forces. Disruption of this balanced, biologic system releases the vascular endothe-
lium to embark on a course of proliferation and new vessel formation. Henkind con-
sidered that there were at least two antivasculogenic agents: (1) an agent within the
cornea which prevented normal tissues entering it, and (2) another in the vitreous. New
vessels from the retina rarely enter the vitreous. Whether the vii reous antivasculogenic
agent is continuously produced, or what its chemical nature ij, is unknown. Ocular
neovascularization, according to Henkind, is a dynamic process and requires both an
initiating stimulus and a sustaining stimulus or the neovascularization involutes. The
known vasculogenic factors include inflammation and its products, a hypoxic retina-
diffusible product, the tumor angiogenic factor, and possibly an ageing factor.
E. Various Cells and Angiogenesis

Sidky and Auerbach, 75 in 1975, reported a method for the quantitative assay of the
graft versus host reaction by using as an indicator in the skin lymphocyte-induced
63
angiogenesis. If homologous spleen cells were injected, there were few, if any, extra
blood vessels developed. When immunocompetent lymphocytes were injected into the
skin of irradiated unimmunized mice, the scar region became surrounded by an intri-
cate network of vessels. These workers enumerated distinct vessels by counting every
discreet vascular branching. A number value was, hence, assigned to the vascular reti-
culation induced by the foreign lymphocytes. They considered that this number bore
a direct relationship to the number of cells injected, immunological state of compe-
tence of the injected cells, and the degree of histoincompatability between recipient
and donor. The assay was termed the lymphocyte-induced angiogenesis (LIA) assay.
The reaction was found to be dose dependent within a range of 2 x 105 to 4 x 106 cells.
In this range, the number of vessels induced correlated with the number of immuno-
competent cells injected. Cells from spleen, lymph node, and hydrocortisone-resistant
thymocytes are effective in this range, and bone marrow and thymus cells are not.
Spleen cells from nude mice did not induce LIA, and mitomycin C and irradiated
lymphocytes, although capable of imitating the response, could not maintain it.
As a result of the observation of marked endothelial proliferation in delayed hyper-
sensibility reactions in the skin of guinea pigs at the time of maximum mononuclear
infiltration, Polverini et al. 76 investigated whether the activated macrophage was in-
volved in vascular proliferation. "Activated" macrophages were derived from guinea
pigs or mice that had previously been injected with paraffin oil or thioglycollate. Neo-
vascularization was tested in the cornea of the guinea pig eye by introducing cells or
media into the stroma and observing the formation of vascular sprouts from the lim-
bus. It was found that macrophages, activated in vivo and in vitro, induced vascular
proliferation in the guinea pig cornea. A dense brush work of new capillary plexuses
and loops grew from the adjacent limbal plexus towards the depot of test material
when a positive neo vascular response was observed. The reaction was not associated
with inflammation and was in a completely syngeneic system.
Fromer and Klintworth 77 78 79 have provided data to support the hypothesis that the
polymorphonuclear leukocytes were directly implicated in corneal vascularization and
produced one or more factors which induced directional vascular growth. Corneal ex-
plants in the hamster cheek-pouch chamber were invaded only following leukocytic
infiltration. 77 In all the models studied, which included exposure of the cornea to nox-
ious agents and intracorneal injection of antigens in sensitized animals, corneal vascu-
larization was part of the reparative phase of inflammation. With regard to vasculari-
zation, three phases were noted: (1) an early phase of leukocytic infiltration prior to
vascular invasion, (2) a phase when both leukocytes and vessels were present in the
cornea, and (3) a phase when blood vessels remained in the cornea in the absence of
leukocytes. The lag period prior to vascular invasion was directly related to the degree
of leukocytic infiltration. Early and considerable leukocytic invasion was matched by
a rapid and extensive vasoproliferative response. Similarly, a lessened polymorph in-
vasion resulted in a delay in vascularization. Leukocytic infiltration and neovasculari-
zation occurred in the same sites within the cornea. In the next series of experiments,78
leukocytic depletion in weanling Fischer albino rats was attempted by total body irra-
diation (ranging from 1100 to 2100 rd). Animals that received 1500 rd or more became
severely leukopenic within 4 days. Silver nitrate cauterization of the cornea in these
rats caused neither leukocytic infiltration nor cornea neovascularization. At lower doses
of irradiation when the circulating leukocytes were not totally eliminated, both leuko-
cytic invasion and vasoproliferation of the cornea resulted from this injury. In nonir-
radiated rats after silver nitrate cauterization, there was, by 2 or 3 days, a vasoprolifer-
ative response in the cornea. If corticosteroid was administered immediately after the
cauterization, there was inhibition of leukocytic invasion and the following vascular
response. If it was administered 1 day after cauterization, the response both of leuko-
cytic infiltration and new-vessel formation were both dampened. A comparison of the
vasoproliferative abilities of polymorphonuclear leukocytes and lymphocytes was made
by Fromer and Klintworth78 using the model of corneal vascularization. Polymorphon-
uclear leukocytes, lymphocytes, and components of leukocytes were instilled into the
cornea. Polymorphonuclear leukocytes (produced from glycogen-induced peritoneal
exudates) resulted in a corneal vascular response. Lymphocytes from thymus, spleen,
and nodes did not induce new-vessel formation in this system. Similar polymorphonu-
clear leukocytes were next suspended in isotonic saline, ultrasonified, and centrifuged
at 101,952 g for 1 hr. Using rats which were leukopenic due to irradiation, the nonse-
dimentable supernatant resulted in vascular new growth into the cornea. In contrast,
there was no activity in the sediment.
Fromer and Klintworth7779 considered that these experiments implicated the poly-
morphonuclear leukocyte in the induction of ingrowth of vessels into the cornea, pos-
sibly by the release of chemical mediators.
Sholley et al.80 also examined the effects of leukocyte depletion on corneal vascular-
ization in adult rats. Combined treatment of radiation (800 rd) and repeated injections
of antineutrophil serum resulted in a reduction of the vascular ingrowth following
cauterization with silver nitrate. There was a reduction of vascular length to 67% of
control at 3 days and 33% at 4 days after cauterization. In their animals despite the
absence of neutrophils, neovascularization occurred in all corneas. These authors con-
cede, however, that complete inhibition of neutrophilic infiltration significantly re-
duces corneal neovascularization. The relationship of the various cells and their prod-
ucts to the induction of angiogenesis is currently under investigation. If polymorph
leukocytes and their products are directly involved in the angiogenesis associated with
inflammation, then this has a great bearing on tumor angiogenesis, since there is usu-
ally a low-grade inflammation in the lag period from implantation to initial vasculari-
zation. Hypersensitivity reactions could help induce angiogenesis by way of the inflam-
matory response and the immigration of leukocytes into the area.
VI. THE MORPHOLOGY OF TUMOR ANGIOGENESIS
The accompanying figures illustrate the major features of the morphology of tumor
angiogenesis as seen in a tumor transplant in the hamster cheek-pouch chamber. This
is an animal model of the situation in man when a seedling metastatic tumor deposit
implants on a thin membrane, such as pleura or peritoneum, and induces a vasoproli-
ferative response eventually sufficient to supply nutrition for further growth. Figure
2A and 2B shows the changes with time as the thin "spread-out" form of a melanoma
creeps across the pouch membrane. Capillary sprouts indicate their presence because
of their fragile nature and the great tendency for pinpoint hemorrhages to occur
around them. If there are enough capillary sprouts, then a confluent small area of
hemorrhage appears. The rate of growth in different arcs of the tumors is not constant;
first one region will show the fastest advance, and then the other (Figure 3). In the
melanoma tumors, the growth is by expansion outward between the plates of the pouch
chamber. Some early movement frequently occurs, and this is attributed to a relatively
narrow fixation base of capillaries from the host into the tumor. Later, fixity is
achieved by more widespread invasion of the tumor by host capillaries. There are great
changes in vascular patterns that occur with growth of the tumor, both within the tumor
and within the pouch membrane as far as the capillary and venous systems are con-
cerned. The arterial arcades within the pouch membrane, however, remain constant in
pattern (Figure 4). On transillumination of the pouch membrane, the transplanted tu-
mor fragment may first shrink a little. After a lag period of 3 to 5 days, small collections
65
FIGURE 3. Composite diagram of the growth of a malignant mel-

anoma on a pouch membrane showing its extent at 2,3,5,7,8,9, and
10 days after transplantation. By superimposing the regions of the peg
holes (P), the extent of the tumor was traced on transparent film at
each of these times. An exact register was possible by locating the
circles of the peg holes (P) in each film. The resulting combined out-
line is shown here and indicates the extent and location of the growing
areas during the period 2 to 10 days following transplantation. Some
movement on the pouch membrane occurred in the period to 5 days.
Thereafter, expansion occurred around the circumference of the tu-
mor, though some regions showed greater growth than others. (Mag-
nification x 5.5.)
of red cells within capillary sprouts are seen (Figure 5A). These form a fine network
(Figure 5B) which has an irregular lumen and contains static columns of compressed
red cells. Within a day, these static columns start to flow, and this signals the formation
of the first capillary network of the tumor implant. Tumor growth in the hamster cheek-
pouch chamber is in the form of a flattened disc whose surfaces form a thin edge on
the areolor surface of the membrane (Figure 6). This is the edge which grows across
the observation area and is outlined in Figure 3. In the capillary sprouts, the endothelial
cells are remarkable labile (Figure 7) and tend to seek a way between bands of collagen.
When collagen is exposed to the lumen, platelets attach to the exposed bands. Red cells
and fibrin deposits are plentiful around the capillary sprouts. Mitotic division may not
necessarily occur in the capillary sprout, but may occur back in the parent vessel81
(Figure 8). The cells may then slide over one another to extend the capillary sprout.
Both saccular sprouts and tapering sprouts were observed to be induced by tumor im-
plants (Figures 9 and 10). Saccular sprouts appear to be derived most frequently from
large-bore vessels, such as venules, rather than from capillary networks per se. The
capillary nets, in contrast, appeared to produce more capillary sprouts of tapering type.
FIGURE 4. Tracings on transparent film of the extent of

a melanoma tumor transplant as it grows in a Hamster
cheek-pouch chamber. The pattern of the adjacent arteries
is shown and remains constant as the tumor grows. The
letters indicate the number of days from transplantation:
A,2; B,3; C,5; D,7; E,8; F,9; G,10 days.
67
FIGURE 5. (A) This photomicrograph shows a transilluminated area of pouch membrane bearing a mela-
noma at 3 days after transplantation. The underlying vessels are blurred because of the inflammatory exu-
date, the presence of red cells, and capillary sprouts. (Magnification x 185.) (B) A slightly later stage in the
development of the vessels in the transplant. Fat cells (F) are present near the transplant of melanoma tumor
tissue. The capillary sprouts have joined to form a fine network (N) which contains static red cells. Three
days after transplantation. (Magnification x 120.)
68
FIGURE 6. Scanning electron micrograph of the edge of a melanoma tu-

mor transplant (T) on the hamster cheek-pouch membrane. This transplant
had been growing in a hamster cheek-pouch chamber for 7 days. The muscle
fibers (MF) of the pouch membrane and the squamous epithelium of the
buccal surface of the pouch (S) are shown. A host vessel (V) is present at the
edge of the tumor, in the pouch membrane. (Magnification x 170.)
FIGURE 7. (A) and (B) low and high power, respectively, transmission electron micro-
graphs of saccular capillary sprouts adjacent to a tumor implant in the hamster cheek-
pouch membrane. Red cells are present in the lumen of the vessel (L) which connects with
the outpocketing of the wall formed by rather complex arrangements of the endothelial
cells (E). In (B), platelets (P) from the lumen of the saccular sprout are in contact with a
bundle of collagen fibers (C). There are fibrin strands (F) in the extravascular tissue just
beyond the break in the vessel wall. (Magnification (A) x 4400, (B) x 9200.)
FIGURE 7B. FIGURE 7C. A further view of an "open" capillary sprout containing red cells in its
lumen. This sprout has passed through a gap between two bands of collagen CI and
C2. Fibrin (F) is present in the extravascular tissue (Magnification x 9200.)
69
70
Tumor Blood Circula tion
FIGURE 8. (A) An endothelial cell is shown in mitosis (M). This cell is part of an established vessel wall, with formed cell junction with the neighboring endothelial cell. (Mag-
nification X 3600). (B) Some cells were seen in mitosis which were outside the rim of endothelial cells forming the lining of the vessel. Such a cell is present here (P). These
were presumed to be pericytes, though conceivably if there was endothelial overlapping, they could be endothelial in origin. (Magnification X 3600).
71
FIGURE 9. The tapering capillary sprout in tumor angiogenesis. A variety

of profiles (1 to 5) are shown which were drawn from electron micrographs
of capillary sprouts passing into tumors. Some possessed bulbous ends (1)
containing red cells (RC) which were closely packed and beyond a tapering
mass of endothelial cells. The lumen (L) at the tip did not always contain
red cells (2). Cross sections of "closed" vessels with potential lumens were
noted (3). Extravasation of red cells from the sprouts was common (4).
"Young" capillaries with pericytes were noted in areas of angiogenesis (5).
ACKNOWLEDGMENT
The support of a grant from the National Cancer Institute of Canada is gratefully
acknowledged together with the able technical assistance of Mr. R. Feldman and Mr.
W. J. Chauvin. Some of the work was performed at the Eppley Institute for Research
in Cancer, University of Nebraska Medical Center, Omaha, Nebraska.
FIGURE 10. The saccular capillary sprouts. Two profiles of

saccular capillary sprouts from regions of tumor growth are
shown. Groups of platelets (P) were often noted, as well as red
cells (RC), both within the lumen and in an extravascular po-
sition. Endothelial cells (E) sometimes showed thin prolonga-
tion of their cytoplasm to enclose the lumen of the sac. The
nuclei of the endothelial cells (N) occasionally were in mitosis
(M) within the saccule. Leukocytes (WBC) could sometimes be
seen passing through the wall of the capillary.
REFERENCES
1. Clark, E. R., Clark, E. L., and Abell, R. G., Cytological studies on the new growth of blood capil-
laries in the living animal, Anat. Rec, 55, (Suppl.) 50, 1933.
2. Abell, R. G., The permeability of blood capillary sprouts and newly formed blood capillaries as
compared to that of older blood capillaries, Am. J. Physiol., 147, 237, 1946.
3. Williams, R. G., Experiments on the growth of blood vessels in thin tissue and in small autographs,
Anat. Rec, 133, 465, 1959.
4. Cliff, W. J., Observations on healing tissue: a combined light and electron microscopic investigation,
Philos. Trans. R. Soc. London Ser. B, 246, 305, 1963.
73
5. Loeb, J., Über die Entwicklung von Fischembryonen ohne Kreislauf, Pfluegers Arch. Gesamte Phys-
iol. Menschen Tiere, 54, 525, 1893.
6. Arey, L. B., The development of peripheral blood vessels, in The Peripheral Blood Vessels, Orbison,
J. L. and Smith, D. E., Eds., Williams & Wilkins, Baltimore, 1963, chap. 1.
7. Huntington, G. S., The development of the mammalian jugular lymph sac, Am. J. Anat., 16, 259,
1914.
8. McClure, C. F. W., The endothelium problem, Anat. Rec,22, 219, 1921.
9. Haar, J. L. and Ackerman, G. A., A phase and electron microscopic study of vasculogenesis and
erythropoiesis in the yolk sac of the mouse, Anat. Rec, 170, 199, 1971.
10. Thoma, R., Untersuchungen über die Histogenèse und Histomechanik des Gefassystems, F. Enke,
Stuttgart; as cited in Arey, L. B., The development of peripheral blood vessels, in The Peripheral
Blood Vessels, Orbison, J. L. and Smith, D. E., Eds., Williams & Wilkins, Baltimore, 1963, chap.
1.
11. Evans, H. M., On the development of the aorta, cordinal and umbilical veins and other blood vessels
of embryos from capillaries, Anat. Rec.,3,498, 1909.
12. Clark, E. R., Studies on the growth of blood vessels in the tail of the frog larva by observation and
experiment in the living animal, Am. J. Anat.,23, 37, 1918.
13. Rhodin, J. A. G., Microangiogenesis in the human fetus, Anat. Rec, 151, 404, 1965.
14. Greenblatt, M., Kaufman J., and Kommineni, V. R. C , Functioning heart homografts in hamsters,
Transplantation, 11, 50, 1971.
15. Greenblatt, M., Choudin, K. V. R., Sanders, A. G., and Shubik, P., Mammalian microcirculation
in the living animal: microcirculation of renal homograft transplants in hamsters, Am. J. Pathol.,
56,317, 1969.
16. Huseby, R. A., Currie, C , Lagerborg, V. A., and Garb, S., Angiogenesis about and within grafts
of normal testicular tissue: a comparison with transplanted neoplastic tissue, Microvasc. Res., 10,
396,1975.
17. Uriel, J., Fetal characteristics of cancer, in Cancer, Vol. 3, Becker, F. M., Ed., Plenum Press, New
York, 1975, chap. 2 and 21.
18. Liebow, A. A., Situations which lead to changes in vascular patterns, in Handbook of Physiology,
Section 2, Vol. 2, Hamilton, W. F., Ed., American Physiological Society, Washington, D.C., 1963,
1251.
19. Warren, B. A. and Shubik, P., The growth of the blood supply to melanoma transplants in the
hamster cheek pouch, Lab. Invest., 15, 464, 1966.
20. Warren, B. A., The ultrastructure of capillary sprouts induced by melanoma transplants in the golden
hamster, / . R. Microscop. Soc, 86, 177, 1966.
21. Greenblatt, M. and Shubik, P., Tumor angiogenesis:transfilter diffusion studies in the hamster by
the transparent chamber technique, J. Natl. Cancer Inst., 41,111, 1968.
22. Ehrmann, R. L. and Knoth, M., Choriocarcinoma transfilter stimulation of vasoproliferation in the
hamster cheek pouch studied by light and electron microscopy, / . Natl. Cancer Inst., 41, 1329, 1968.
23. Folkman, J., Merler, E., Abernathy, C , and Williams, G., Isolation of a tumor factor responsible
for angiogenesis, J. Exp. Med., 133, 275, 1971.
24. Folkman, J., Tumor angiogenesis: therapeutic implications, N. Engl. J. Med., 285 (21), 1182, 1971.
25. Folkman, J. and Gimbrone, M. A., Jr., Perfusion of the thyroid, Acta Endocrinol. (Copenhagen),
Suppl. 157,237, 1971.
26. Cavallo, T., Sade, R., Folkman, J., and Cotran, R. S., Tumor angiogenesis, J. Cell Biol., 54, 408,
1972.
27. Folkman, J., Anti-angiogenesis: a new concept for therapy of solid tumors, Ann. Surg., 175, 409,
1972.
28. Brem, S., Cotran, R., and Folkman, J., Tumor angiogenesis: a quantitative method for histologic
grading, J. Natl. Cancer Inst., 48, 347, 1972.
29. Tuan, D., Smith, S., Folkman, J., and Merler, E., Isolation of the nonhistone proteins of rat Walker
Carcinoma 256. Their association with tumor angiogenesis, Biochemistry, 12, 3159, 1973.
30. Cavallo, T., Sade, R., Folkman, J., and Cotran, R., Ultrastructural autoradiographic studies of the
early vasoproliferative response in tumor angiogenesis, Am. J. Pathol., 70, 345, 1973.
31. Gimbrone, M. A., Jr., Leapman, S. B., Cotran, R. S., and Folkman, J., Tumor angiogenesis: iris
neovascularization at a distance from experimental intraocular tumors, / . Natl. Cancer Inst., 20,
219, 1973.
32. Folkman, J., Tumor angiogenesis factor, Cancer Res., 34, 2109, 1974.
33. Brem, S. S., Cotran, R. S., and Folkman, J., Angiogenesis in brain tumors: a quantitative histologic
study, Surg. Forum, 25, 462, 1974.
34. Gimbrone, M. A., Jr., Cotran, R. S., Leapman, S. B., and Folkman, J., Tumor growth and neovas-
cularization: an experimental model using rabbit cornea, J. Natl. Cancer Inst., 52, 413, 1974.
35. Folkman, J., Tumor angiogenesis, Adv. Cancer Res., 19, 331, 1974.
36. Folkman, J., Tumor angiogenesis: role in regulation of tumor growth, in Macromolecules Regulating
Growth and Development, Hay, E. D., King, T. J., and Papaconstantinou, J., Eds., Academic Press,
New York, 1974,43.
37. Folkman, J. and Klagsbrun, M., Tumor angiogenesis: effect on tumor growth and immunity, in
Fundamental Aspects of Neoplasia, Gottlieb, A. A., Plescia, O. J., and Bishop, D. H. L., Eds.,
Springer-Verlag, New York, 1975, 401.
38. Brem, H. and Folkman, J., Inhibition of tumor angiogenesis mediated by cartilage, J. Ex. Med.,
141,427,1975.
39. Folkman, J., Tumor angiogenesis, a possible control point in tumor growth, Ann. Intern. Med., 82,
96,1975.
40. Ausprunk, D. H., Knighton, D. R., and Folkman, J., Vascularization of normal and neoplastic
tissues grafted to the chick chorioallantois, Am. J. Pathol., 79, 597, 1975.
41. Auerback, R., Arensman, R., Kubai, L., and Folkman, J., Tumor induced angiogenesis lack of
inhibition by irradiation, Int. J. Cancer, 15, 241, 1975.
42. Folkman, J., Tumor angiogenesis, in Cancer: A Comprehensive Treatise, Vol. 3, Becker, F. F., Ed.,
Plenum Press, New York, 1975, 355.
43. Kessler, D. A., Langer, R. S., Pless, N. A., and Folkman, J., Mast cells and tumor angiogenesis,
Int. J. Cancer, 18, 703, 1976.
44. Folkman, J., The vascularization of tumors, Sci. Am., 234, 59, 1976.
45. Folkman, J. and Cotran, R., Relation of vascular proliferation to tumor growth, Int. Rev. Exp.
Pathol., 16, 207, 1976.
46. Klagsbrun, M., Knighton, D., and Folkman, J., Tumor angiogenesis activity in cells grown in tissue
culture, Cancer Res., 36, 110, 1976.
47. Brem, S., Brem, H., Folkman, J., Finkelstein, D., and Patz, A., Prolonged tumor dormancy by
prevention of neovascularization in the vitreous, Cancer Res., 36, 2807,1976.
48. Ausprunk, D. H. and Folkman, J., Migration and proliferation of endothelial cells in preformed
and newly formed blood vessels during tumor angiogenesis, Microvasc. Res., 14, 53, 1977.
49. Finkelstein, D., Brem, S., Patz, A., Folkman, J., Miller, S., and Ho-Chen, C , Experimental retinal
neovascularization induced by intravitreal tumors, Am. J. Opthalmol., 83, 660, 1977.
50. Ausprunk, D. A., Falterman, K., and Folkman, J., The sequence of events in the regression of
corneal capillaries, Lab. Invest.,38, 284, 1978.
51. Eisenstein, R., Kuettner, K. E., Neapolitan, C , Soble, L. W., and Sorgente, N., The resistance of
certain tissues to invasion. III. Cartilage extracts inhibit the growth of fibroblasts and endothelial
cells in culture, Am. J. Pathol., 81, 337, 1975.
52. Phillips, P., Steward, J. K., and Kumar, S., Tumor angiogenesis factor (TAF) in human and animal
tumors, Int. J. Cancer, 17, 549, 1976.
53. Fenselau, A. and Mello, R. J., Growth stimulation of cultured endothelial cells by tumor cell homog-
enates, Cancer Res., 36, 3269, 1976.
54. Shubik, P., Feldman, R., Garcia, H., and Warren, B. A., Vascularization induced in the cheek pouch
of the Syrian hamster by tumor and non-tumor substances, J. Natl. Cancer Inst., 57, 769, 1976.
55. Levi-Montalcini, R., Biological aspects of specific growth promoting factors, Proc. R. Soc. Med.,
58,357,1965.
56. Bueker, E. D., Implantation of tumors in the hind limb field of the embryonic chick and the devel-
opmental response of the lumbosacral nervous system, Anat. Rec, 102, 369, 1948.
57. Levi-Montalcini, R., Effects of mouse tumor transplantation on the nervous system, Ann. N.Y.
Acad. Sci., 55,380, 1952.
58. Levi-Montalcini, R., Morphological and metabolic effects of the nerve growth factor, Arch. Biol.,
76, 387,1965.
59. Cohen, S., Purification and metabolic effects of a nerve growth promoting protein from snake
venom, / . Biol. Chem.,234, 1129, 1959.
60. Di Zerega, G. and Morrow, J., The effect of nerve growth factor on dispersed neuronal HeLa het-
erokaryons, Exp. Neurol., 28, 206, 1970.
61. Gospodarowicz, D., Humoral control of cell proliferations: the role of fibroblast growth factor in
regeneration, angiogenesis, wound healing and neoplastic growth, in Membranes and Neoplasia: New
Approaches and Strategies, Marchesi, V. T., Ed., Alan R. Liss, New York, 1976, 1.
62. Gospodarowicz, D. and Thakral, K., Production of a corpus luteum angiogenic factor responsible
for proliferation of capillaries and neovascularization of the corpus luteum, Proc. Natl. Acad. Sci.,
75,847,1978.
63. Brem, S., The role of vascular proliferation in the growth of brain tumors, Clin. Neurosurg., 23,
440,1976.
75
64. Kelly, P. J., Suddith, R. L., Hutchison, H. I., Werbach, K., B., and Haber, B., Endothelial growth
factor present in tissue culture of CNS tumors, J. Neurosurg., 44, 342, 1976.
65. Wolf, J. E., Jr. and Harrison, R. G., Demonstration and characterization of an epidermal angiogenic
factor, J. Invest. Dermatol.,61, 130, 1973.
66. Wolf, J. E., Jr. and Hubler, W. R., Jr., Tumor angiogenic factor and human skin tumors, Arch.
Dermatol., 3,321, 1975.
67. Wolf, J. E., Jr. and Hubler, W. R., Jr., Tumor angiogenic factor associated with subcutaneous
lymphoma, Br. J. Dermatol.,92, 273, 1975.
68. Hubler, W. R., Jr. and Wolf, J. E., Jr., Melanoma tumor angiogenesis and human neoplasia, Cancer
(Philadelphia), 38, 187, 1976.
69. Stafl, A. and Mattingly, R. F., Angiogenesis of cervical neoplasia, Am. J. Obstet. Gynecol., 121,
845, 1975.
70. Gimbrone, M. A., Jr. and Gullino, P. M., Neovascularization induced by intraocular xenografts of
normal, preneoplastic and neoplastic mouse mammary tissues, J. Natl. Cancer Inst., 56, 305, 1976.
71. Gimbrone, M. A., Jr. and Gullino, P. M., Angiogenic capacity of preneoplastic lesions of the murine
mammary gland as a marker of neoplastic transformation, Cancer Res., 36, 2611, 1976.
72. Brem, S. and Gullino, P. M., Angiogenesis produced by malignant versus benign tumors of the
human breast, Fed. Proc. Fed. Am. Soc. Exp. Biol, 35, 568, 1976.
73. Brem, S., Jensen, H. M., and Gullino, P. M., Angiogenesis as a marker of preneoplastic lesions of
the human breast, Cancer (Philadelphia), 41, 239, 1978.
74. Henkind, P., Ocular neovascularization, Am. J. of Ophthalmol., 85, 287, 1978.
75. Sidky, Y. A. and Auerbach, R., Lymphocyte induced angiogenesis, a quantitative and sensitive assay
of the graft vs. host reaction, J. Exp. Med., 141, 1084, 1975.
76. Polverini, P. J., Cotran, R. S., Gimbrone, M. A., Jr., and Uranue, E. R., Activated macrophages
induce vascular proliferation, Nature (London), 269, 804, 1977.
77. Fromer, C. H. and Klintworth, G. K., An evaluation of the role of leucocytes in the pathogenesis of
experimentally induced corneal vascularization. I. Comparison of experimental models of corneal
vascularization, Am. J. Pathol.,79, 537, 1975.
experimentally induced corneal vascularization. II. Studies on the effect of leucocytic elimination on
corneal vascularization, Am. J. Pathol.,81, 531, 1975.
experimentally induced corneal vascularization. III. Studies related to the vasoproliferative capability
of polymorphonuclear leucocytes and lymphocytes, Am. J. Pathol., 82, 157, 1976.
80. Sholley, M. M., Gimbrone, M. A., Jr., and Cotran, R. S., The effect of leucocytes depletion on
corneal neovascularization, Lab. Invest.,38, 32, 1978.
81. Warren, B. A., Greenblatt, M., and Kommineni, V. R. C , Tumor angiogenesis: ultrastructure of
endothelial cells in mitosis, Br. J. Exp. Pathol., 53, 216, 1972.
Taylor & Francis
77
Chapter 3
V A S C U L A R A N D E X T R A V A S C U L A R S P A C E S IN T U M O R S : T U M O R
VASCULAR PERMEABILITY
Hans-Inge Peterson
TABLE OF CONTENTS
I. Introduction 77
II. Vascular and Extravascular Spaces in Tumors 78

A. Vascular Space 78
B. Extravascular Space 79
C. Vascular and Extravascular Spaces Recorded by an Isotope Technique
in Two Transplantable Rat Tumors 79
D. Summary 81
III. Tumor Vascular Permeability 81

A. Studies with Different Techniques 81
B. Capillary Permeability Recorded by an Isotope Technique in Two Trans-
plantable Rat Tumors 82
C. Summary 83
Acknowledgment 84
References 84
Morphological observations of an extensive vascularization of malignant tumors

have initiated studies on vascular and extravascular spaces of tumors. These parame-
ters, as well as tumor blood flow, will probably have some importance for the distri-
bution of drugs and isotopes in tumor diagnosis and therapy.
Owing to methodological difficulties, experimental investigations on tumor vascular
space have mostly been based on the blood content of tumors after tumor dissection,
and they will most probably not show the functional vascular volume, as some authors
propose. A progressive development of central necroses in growing tumors might af-
fect the results, if these are mainly based on the vital parts of a tumor or on tumor
specimens which include necrotic areas.
The extravascular tumor space, especially interstitial, has been measured in some
human and experimental tumors. In all these studies, a markedly large interstitial space
has been found.
Tumor capillary permeability has been studied in human brain tumors on the basis
of the appearance of brain edema with such tumors. Morphological observations of
specific-tumor vascular properties which make possible the transcapillary transport of
large molecules have also been supported by observations in experimental studies of a

high tumor-capillary permeability for such molecules. The transport of large molecules
over the tumor capillary wall seems to be limited by a passive plasma-tissue equilibra-
tion. However, the finding of a large interstitial space and a high tumor-capillary
permeability can be taken as a challenge in our efforts to reach optimal concentrations
of active drugs in tumor tissue.
II. VASCULAR AND EXTRA VASCULAR SPACES IN TUMORS

A. Vascular Space
In rapidly growing mouse tumors observed through a transparent chamber, the
blood vessels were found to reach a volume of 40 to 50% of total space within 5 to 8
days. The tumors elicited a production of new capillary endothelium from the host.
Coincident with the establishment of a blood supply, the capillaries took on an irreg-
ular pattern, sinusoids developed, and little differentiation into arterioles and venules
occurred.1
Gullino and Grantham found in studies of several transplantable rat-liver tumors a
vascular space which was from 4.5 to 12.4% of tumor weight, with marked variations
in different parts of tumors. 2 The vascular space of host liver was 18.6%. They used
dextran 500 in determining vascular and interstitial spaces in tumors. However, it is
not clear if the rats studied were dextran resistant or if anaphylactoid reactions might
have interfered with the results. 3 The vascular space observed in rat tumors increased
linearly with tumor growth in small tumors, but no prediction was possible for larger
tumors. The plasma vascular volume of Walker 256 carcinoma recorded with dextran
was 10.6% of tumor weight,2 but the RBC volume was 4.0%, as recorded with 51Cr-
labeled erythrocytes. 4
In studies of a mouse mammary carcinoma by microangiography and morphometric
analyses, the vascular volume, as a percentage of total viable tumor volume, was found
to remain stable during tumor growth at 17%. 5 Necrotic tumor volume increased pro-
gressively during tumor growth. The average vessel diameter increased, and the capil-
lary-like vessels in small tumors gave way gradually to sinusoid-type vessels. The av-
erage vessel length and surface area per mm 3 of viable tumor tissue decreased during
growth. A transient decrease of vascular volume was found after fractional irradiation
with 500 rd, while a more lasting depression was found after a single exposure of 1500
to 3000 rd. Capillary morphometries showed a decreased vascular space in growing
DS-rat carcinosarcomas. This was especially distinct during the first period of tumor
growth. 6
The "functional" vascular volume in A / J mice transplanted with a neuroblastoma
was determined with 51Cr-labeled red blood cells.7 One hr after i.v. injection of the
labeled red cells, tumors were excised, avoiding obvious blood loss from tumor tissue,
and blood samples were drawn by cardiac puncture. The vascular volume in this tumor
was found to be about 0.27 m i / g (dry). No significant changes were found during
growth. X-ray irradiation of an SCL carcinoma in the same strain of animals increased
the vascular volume 1 day after 1000 to 2000 rd, the volume being thereafter de-
creased.8 Calculations of vascularity indicated that the vascular volume per gram of
tumor increased two and three times over control volume after irradiation with 1000
and 2000 rd, respectively.
In humans, vascularization has been found to be most extensive in brain tumors
(glioblastoma), slightly less in carcinomas, and least in sarcomas. 9 After tumors have
reached a certain diameter (1 to 3 cm), vascular compression might occur, followed
by the development of central necroses. When the blood flow has stopped, capillary
79
endothelial cells probably die rapidly. 10 It has been suggested that there is a critical
depth in most tumors beyond which blood flow stops, owing to endothelial damage
caused by external compression. 11 Angiographically hypo vascularized human renal
carcinomas were also found to have more frequent, and large, necrotic areas when
examined histopathologically. 12 The reduction of vascular surface area with increasing
tumor size has also been explained by a difference in turnover times between endothe-
lial and tumor cells.13 Hypoxia, anoxia, and glucose depletion in the growing tumor
caused by the absence of a sufficient neovascularization and a general rarefaction of
the terminal vascular bed might explain the development of necrotic areas in large
tumors. 14
B. Extravascular Space
The extravascular space includes the interstitial space (extracellular space — plasma
space), tumor cell volume, and tissue stroma. Direct measurement by electron micro-
scope of the extracellular space in human gliomas and meningiomas showed a large
extracellular space in gliomas (20 to 40%). 15 The extracellular space in meningiomas
amounted to 13 to 15%. That in normal brain tissue was 6 to 7%. A large extracellular
space might be expected to influence the localization of drugs and isotopes in tumor,
but in this study no correlation was found between the size of the extracellular space
and the degree of isotope uptake ( 203 Hg-chlormerodrin).
A large extracellular space (38%) was also found in DS carcinosarcoma of the rat
as measured by the correlation of total tissue chloride to blood chloride content. 16 In
studies of several transplantable rat tumors, the extracellular space was determined by
24
NaCl, 36 NaCl, and D-[1-14C] mannitol, and the vascular space was determined by
dextran 500. The interstitial water space was found to be large, between 30 and 60%
of the tumor water. 17
C. Vascular and Extravascular Spaces Recorded by an Isotope Technique in Two

Transplantable Rat Tumors
We have studied the vascular and extravascular spaces in two transplantable rat
tumors, a 20-methylcholanthrene-induced sarcoma and a benzpyrene-induced sar-
coma. 1819 Both tumors were transplanted i.m. into one hindleg of inbred Lister rats,
and the studies were performed 2 weeks after the tumor transplantation, with a mean
tumor diameter of 10 mm.
All animals were given 0.25 juCi 59Fe-chloride (59Fe) i.v. 10 days before the experi-
ments for labeling of red blood cells. Human albumin labeled with 125I (125I-RISA) was
injected i.v. into a tail vein in a volume of 1.5 m i , containing 5 \xC\. The extracellular
space in tumors was determined by i.v. injection of 51 Cr-EDTA (5 p*Ci), which is a
monovalent extracellular anion with a molecular weight of 400 and with capillary ex-
change properties similar to those of sucrose. 51 Cr-EDTA is known not to enter red
blood cells, and its strictly extracellular distribution was verified in tumors in in vitro
studies. 1819
Isotope activities, in cpm/mi blood and cpm/g muscle or macroscopically vital tu-
mor tissue, were recorded at different intervals after isotope injection and calculated.
Hematocrit was used to calculate the activity per mi plasma (125I, 51Cr) and per mi
red blood cells (59Fe). The data obtained were further calculated as activity ratios be-
tween tissue and plasma or red blood cell activity in mi plasma or red blood cells per
g tissue.
Animals were sacrificed by bleeding under ether anesthesia. This means that the red
blood cell and plasma activity in muscle and macroscopically vital tumor reflects the
residual plasma and red blood cell volume in tissue after bleeding. This procedure was
designed to restrict variations due to differences in blood loss from tissue at dissection,
and it, thus, does not reflect any attempt to reproduce the "functional" vascular vol-
ume.
The activity ratio of 59Fe gives an estimation of the residual red cell volume per g
tissue (RCV). The activity ratio of 51Cr-EDTA, after equilibration between plasma and
tissue to a comparatively stable value, gives an estimation of the extracellular volume
(ECV), if equal concentrations of a tracer in plasma and interstitial fluid are assumed,
and the tracer distribution is extracellular. The activity ratio of 125I after complete
mixing of 125I-RISA in plasma, but before any considerable extravasation has oc-
curred, gives an estimate of the volume of residual plasma (PV) in the tissue. RCV
equals volume in m i / g tissue of red cells. ECV and PV are extracellular and plasma
volume in mi/g tissue.
The results for the two tumors and for normal muscle are presented in Figure 1.
From this figure,it is evident that both tumors had a noticeably large ECV (0.35 to
0.50 mi/g) as compared with muscle (0.13 mi/g). Owing to the rapid renal excretion
of 51Cr-EDTA, observations of ECV were based on plasma versus tissue activity ratios
during the first 50 min after injction of the isotope.
After local X-ray irradiation of the 20-methylcholanthree-induced sarcoma, a
marked increase (from 0.49 ± 0.04 to 0.56 ± 0.06) of the ECV was found when studied
by the same isotope technique (Figure 2). Based on an approximate density of tumor
to 1 g/mi, the cellular volume (CV), which will mainly be tumor cells with a small
volume due to stroma and blood vessel walls, could be calculated as 1.0 - RCV —
ECV. The cellular volume in tumors was thus reduced by irradiation from approxi-
mately 0.49 ± 0.04 to 0.42 ± 0.06 7 days after local irradiation.
In all these experiments, the residual RCV and PV in macroscopically vital tumor
tissue remained at stable values. This might be looked upon as a control of the experi-
mental model studied.
FIGURE 1. Plasma to tissue ratios in tumors and normal muscle of the rat for 51Cr-EDTA (circles) and
for ,25I-RISA (arrows) from which are estimated in ml/% tissue the ECV and the PV, respectively. RBC to
tissue ratios of 59Fe (points) indicate the residual RCV in m l / g tissue after exsanguination of the animals.
All data are based on mean values from 5 animals. (From Appelgren, L., Peterson, H.-I., and Rosengren,
B., Bib!. Anat, 12, 504,1973. With permission.)
81
FIGURE 2. Tissue to plasma ratios in control and irradiated tumor 7 days after tumor irradiation. Ratios
for 51Cr-EDTA (circles) are 10, 20, and 30 min after i.v. injection of this isotope. ECV is estimated from
the calculated mean value of these ratios. Ratios for 125I-RISA (arrows) are 10, 20, 30, 60, and 360 min
after i.v. injection. PV based on early ratios for this isotope. Ratios for 59Fe (points) indicate residual blood
volume in tissue after exsanguination of animals. All separate data are based on mean values from 5 animals.
CV, which is mainly the volume of tumor cells with a small volume of cells in stroma and vessel walls,
could be calculated from these values. CV = 1.0 - RCV - ECV. (From Peterson, H.-L, Appelgren, L.,
Kjartansson, I., and Selander, D., Z. Krebsforsch., 87,17,1976. With permission.)
D. Summary
The vascular volume in vital parts of tumors seems to remain rather stable during
growth. However, central necroses develop during growth, probably owing to the
compression of vessels by increasing tumor cell masses or to a more rapid growth of
the tumor cell mass compared with the vascular endothelial cell proliferation. A mark-
edly large interstitial space has been observed in both experimental and human tumors.
III. TUMOR VASCULAR PERMEABILITY
A. Studies with Different Techniques

Morphological studies of blood vessels in different human brain tumors showed
alterations which might increase the trans vascular transport of different materials.
Thus, fenestration, widened intercellular junctions, increase in pinocytotic vesicles,
and infolding of the luminal surface were observed. These alterations were connected
with the appearance of brain edema associated with brain tumors. 20
In low-grade human astrocytomas and glioblastomas, similar observations were
made, including increased overall vessel diameter and a greater number of endothelial
cells, with a corresponding increase in the number of intercellular junctions. 21 It was
suggested that the vascular pattern observed in brain tumors could be interpreted as
an active, hyperplastic response.
In studies of the advancing edge of Walker 256 rat carcinoma by light and electron
microscopy, the early microvasculature showed intracellular fenestrations and saccular
endothelial processes.22 The cytoplasm of newly formed capillaries was occasionally
humped into high ridges, and the fenestrations separating the ridges had a width of
from 500 to 700 A. They were closed by a diaphragm with a thickness equal to that of
the plasma membrane of the cell.
The vasculature of a rat fibrosarcoma showed irregular channels lined by plump
endothelium with mainly pentalaminar junctions. 23 An increased vascular permeability
was found after histamine challenge, accompanied by the formation of endothelial
gaps in the irregular vascular channels. The majority of the vascular channels, it was
suggested, had a structural and functional relationship to small venules.
An increased permeability of tumor vessels might be by defects in vascular endothe-
lium due to poor cellular differentiation, break of vascular walls by invading tumor
cells, release of endogenous serotonin from platelets, or response to peptides derived
from necrotic tumor. 24
An extravasation of albumin was found in C3H-mouse Gardner lympho-sarcoma. 25
This extravasation was increased by local irradiation of tumors. The increased albumin
space in tumors after irradiation was found not to be dependent on increased vascular
volume as determined by 59Fe labeled red cells. However, a slight increase of the vas-
cular volume was also found after irradiation in some animals. Furthermore, irradiated
normal muscle showed an increased permeability for albumin. The albumin space after
extravascular equilibration in irradiated tumor was increased by 200% and in irradi-
ated muscle by 80%.
The intratumoral concentration of 125I-albumin in tumor-bearing mice increased
during the first 24 hr after albumin administration to values four to five times higher
than those obtained in normal organs. 26 The albumin was found to have an essentially
extracellular distribution, but in some tumor areas in the proximity of vessels and
necrotic areas an intracellular uptake of albumin was suggested.
An increased vascular permeability, studied by Evans blue, was found by histamine
in solitary liver tumors in rats, which were compared with the surrounding normal
tissue.27
Observations of increased capillary extravasation prompted experimental studies to
determine effective combinations of Adriamycin and irradiation in a hamster carci-
noma. 28 An increased therapeutic effect of the combined treatment was observed in a
phase of increased capillary extravasation after irradiation.
B. Capillary Permeability Recorded by an Isotope Technique in Two Transplantable

Rat Tumors
We have studied the capillary permeability in two transplantable rat tumors in inbred
Lister rats, a 20-methylcholanthrene-induced sarcoma and a benzpyrene-induced sar-
coma, by labeled proteins with different molecular size.29 These studies were prompted
primarily by earlier observations of a localization of fibrinogen in tumor tissue, which,
it was suggested, was the result of an active binding of fibrinogen as fibrin in tumors
with a high content of tissue thromboplastin (for a concentrated review see Peterson 30 ).
However, in an experimental study in the rat in which the uptake of labeled fibrinogen
in tumor and normal tissues with a high content of tissue thromboplastin (lung, brain)
was compared, we were unable to confirm that there was an active binding of labeled
fibrinogen to normal tissues with high thromboplastic activity.31 This suggested that
another property of tumor, e.g., a high capillary permeability, might explain the local-
ization of labeled fibrinogen in tumor tissue that had been observed.
83
We studied the distribution in tumors and various normal tissues of the rat of 125I-
labeled human albumin (125I-RISA) with a molecular weight of 69,000, of 125I- or 131I-
labeled human immunoglobulin G (IgG) with a molecular weight of 150,000, of 125I-
labeled rat fibrinogen with a molecular weight of 340,000, and of 125I-labeled human
immunoglobulin M (IgM) with a molecular weight of 900,000.29
The estimation of capillary permeability in transplantable rat tumors was compared
with various normal organs, among them organs, such as intestine, with a known high
capillary permeability for substances within this molecular weight area. The technique
used was based on the identification of a period of penetration of labeled proteins into
interstitial space when the protein was well mixed in the plasma volume of the tissue,
but had not penetrated to any extensive amount into the tissue. The increase of the
activity ratio during this period (generally from ti = 1 hr to t2 = 8 hr after i.v. injection
of labeled protein) was used for the calculation of capillary permeability in the follow-
ing way:
PS, the capillary permeability, or more correctly the permeability-surface-area prod-

uct, will then be given as the activity increase in percent of plasma activity/mi plasma/
hr/g tissue.
The permeability in percent of plasma activity/ml/g tissue and hr was then, e.g.,
for 12/-RISA, 1.5 to 1.7% for tumors, 0.8% for small intestine, and 0.5% for muscle.
The corresponding values for IgG were 1.2 to 1.9% for tumors, 0.4% for small intes-
tine, and 0.14% for muscle. The data for IgM were probably disturbed by small pro-
tein molecules included in the preparation.
A concentrated review of the results for tumor, muscle, and intestine is given in
Figure 3, in which capillary permeability data for all proteins studied are correlated to
earlier observations on muscle and intestine. Diffusion of large protein molecules ov-
erthe capillary wall takes place mainly through the large pore system and by pinocyto-
sis. It is obvious from this figure that there is a passive-diffusion transport of labeled
protein molecules over the tumor capillary wall, and that the permeability of the tumor
capillary wall for large protein molecules is high compared with intestine, which is
itself known to have a relatively high capillary permeability.
In an experimental study, the irradiation effect of heavily 131I-labeled albumin in-
jected i.v. into rats with a sarcoma transplant to the stomach was investigated.32 A
temporary arrest of tumor growth was observed with large isotope doses, but, owing
probably to the passive transport of albumin over the tumor capillary wall, a concen-
tration of labeled albumin sufficient for a more marked therapeutic effect was never
reached.
C. Summary
Most experimental data confirm that there is a high permeability of the tumor cap-
illary wall for large protein molecules. This is probably explained by morphological
changes in tumor vessels as observed in electron microscopy studies. A high tumor-
capillary permeability might be of interest in the localization of active drugs and iso-
topes in tumor diagnosis and treatment. However, it is also evident that the transport
of large molecules over the tumor capillary wall is based on a passive diffusion, and
concentrations of active drugs sufficient for a therapeutic effect are difficult to reach.
FIGURE 3. Capillary permeability or permeability-surface area product (PS, percent of plasma concen-
tration/mi/g tissue/hr, or mi/hr/100 g tissue) at different molecular weights. Crosshatched area = skeletal
muscle; light grey area = GI tract. Values of PS are given for tumor tissue (TB = benzpyrene-induced
sarcoma; TM = 20-methylcholanthrene-induced sarcoma), small intestine (I), and skeletal muscle (M). Val-
ues for albumin (mol. wt. 69,000), IgG (mol. wt. 150,000), fibrinogen (mol. wt. 340,000), and IgM (mol.
wt. 900,000). For comparison, the approximate position of 51Cr-EDTA (mol. wt. 400) has been indicated in
the diagram. The diagonal broken lines indicate the expected reduction of free diffusion with increasing
molecular weight (diffusion rate ~ 1 \f mol. wt.). (From Peterson, H.-L, Appelgren, L., Lundborg, G.,
and Rosengren, B., Bibl Anat., 12, 511,1973. With permission.)
ACKNOWLEDGMENT
All experimental tumor studies performed at our laboratory were supported by

grants from the Swedish Cancer Society.
REFERENCES
1. Algire, G. H., Vascular reactions of normal and malignant tissues in vivo. VII. Observations on
vascular reactions in destructions of tumor homografts, /. Natl. Cancer Inst., 15, 483, 1954.
2. Gullino, P. M. and Grantham, F. H., The vascular space of growing tumors, Cancer Res., 24, 1727,
1964.
85
3. Ivarsson, L., Appelgren, L., and Rudenstam, C.-M., Plasma volume after dextran infusion in rats
sensitive and non-sensitive to dextran, Eur. Surg. Res., 7, 315, 1975.
4. Song, C. W. and Levitt, S. H., Quantitative study of vascularity in Walker carcinoma 256, Cancer
Kes.,31,587, 1971.
5. Hilmas, D. E. and Gillette, E. L., Tumor microvasculature following fractioned X-irradiation, Ra-
diology, 116,165,1975.
6. Vaupel, P., Braunbeck, W., Schultz, V., Günther, H., and Thews, G., Critical 0 2 and glucose supply
and microcirculation in tumor tissue, Bibl. Anat., 12, 527, 1972.
7. Song, C. W., Sung, J. H., Clement, J. J., and Levitt, S. H., Vascular changes in neuroblastoma of
mice following X-irradiation, Cancer Res., 34, 2344, 1974.
8. Clement, J. J., Song, C. W., and Levitt, S. H., Changes in functional vascularity and cell number
following X-irradiation of a murine carcinoma, Int. J. Radiât. Oncol. Biol. Phys., 1, 671, 1976.
9. Brem, S., Cotran, R. S., and Folkman, J., Tumor angiogenesis: a quantitative method for histologic
grading, / . Natl. Cancer Inst., 48, 347, 1972.
10. Mervin, R. and Algire, G. H., The role of graft and host vessels in the vascularization of grafts of
normal and neoplastic tissue, J. Natl. Cancer Inst., 17, 23, 1956.
11. Folkman, J., Tumor angiogenesis, Adv. Cancer Res., 19, 331, 1974.
12. Ekelund, L., Jonsson, N., and Lunderquist, A., Tumor vessels. Angiographic-histopathologic cor-
relation, Radiologe, 17, 95, 1977.
14. Vaupel, P., Hypoxia in neoplastic tissue, Microvasc. Res., 13, 399, 1977.
15. Bakay, L., The extracellular space in brain tumours. I. Morphological considerations, Brain, 93,
693, 1970.
16. Rauen, H. M., Norpoth, K., and van Husen, N., Extracellularen Raum und Blutraum von DS-Car-
cinosarkom auf der Chorioallantoismembran des HUhnerembryos, Naturwissenschaften, 54, 540,
1967.
17. Gullino, P. M., Grantham, F. H., and Smith, S. H., The interstitial water space of tumors, Cancer
Res.,25, 727, 1965.
18. Appelgren, L., Peterson, H.-L, and Rosengren, B., Vascular and extravascular spaces in two trans-
plantable tumors of the rat, Bibl. Anat., 12, 504, 1973.
19. Peterson, H.-L, Appelgren, L., Kjartansson, I., and Selander, D., Vascular and extravascular spaces
in a transplantable rat tumor after local X-ray irradiation, Z. Krebsforsch., 87, 17, 1976.
20. Hirano, A. and Matsui, T., Vascular structures in brain tumors, Hum. Pathol., 6, 611, 1975.
21. Waggener, J. D. and Beggs, J. L., Vasculature of neural neoplasms, Adv. Neurol., 15, 27, 1976.
22. Warren, B. A., The ultrastructure of the microcirculation at the advancing edge of Walker 256 car-
cinoma, Microvasc. Res., 2, 443, 1970.
23. Papadimitriou, J. M. and Woods, A. E., Structural and functional characteristics of the microcir-
culation in neoplasms, / . Pathol., 116, 65, 1975.
24. Underwood, J. C. E. and Carr, I., The ultrastructure and permeability characteristics of the blood
vessels of a transplantable rat sarcoma, J. Pathol., 107, 157, 1972.
25. Potchen, E. J., Kinzie, J., Curtis, Ch., Siegel, B., and Studer, R. K., Effect of irradiation on tumor
microvascular permeability to macromolecules, Cancer (Philadelphia), 30, 693, 1972.
26. Cerottini, J.-C. and Isliker, H., Transport d'agents cytostatiques par les protéines plasmatiques, Eur.
J. Cancer,3, 111, 1967.
27. Ackermann, N. B., Differences in vascular permeability between normal and tumor vessels produced
by vasoactive agents, Surg. Forum, 24, 105, 1973.
28. Eddy, H. A., Effectiveness of radiation/Adriamycin combinations as influenced by changes in tumor
microvascular function, Radiât. Res.,70, 662, 1977.
29. Peterson, H.-L, Appelgren, L., Lundborg, G., and Rosengren, B., Capillary permeability of two
transplantable rat tumours as compared with various normal organs of the rat, Bibl. Anat., 12, 511,
1973.
30. Peterson, H.-L, Fibrinolysis and antifibrinolytic drugs in the growth and spread of tumours, Cancer
Treat. Rev.,4, 213, 1977.
31. Peterson, H.-L, Appelgren, K. L., and Rosengren, B. H. O., Experimental studies on the mechanisms
of fibrinogen uptake in a rat tumour, Eur. J. Cancer, 8, 677, 1972.
32. Peterson, H.-L, Appelgren, L., Javelin, L., and Rosengren, B., Influence of intravenously injected
13,
I-labeled albumin on the growth rate of a sarcoma transplanted into the rat stomach, Eur. J.
Cancer, 13, 1357, 1977.
Taylor & Francis
87
Chapter 4
METHODS OF RECORDING TUMOR BLOOD FLOW
Lennart K. Appelgren
TABLE OF CONTENTS
I. Introduction 87
A. Flow Concepts 87
B. Flow Measuring Principles 88
II. Direct Volume Flow Measurements 88

A. Venous Outflow 88
B. Arterial Inflow 89
C. Plethysmography 89
D. Comments 89
III. Radioactive Tracers in the Study of Tumor Circulation 90

A. Choice of Tracer Substances 90
B. The Tracer Analysis with the Tissue as a "Black Box" 91
C. From Tracer Theory to Tracer Practice 92
D. Outline of Different Radioactive Tracer Methods 93
1. Radioactive Microspheres 93
2. Distribution of 86Rb+ and Radioiodinated Antipyrine According
to Sapirstein 94
3. Distribution of Radioactive Antipyrine According to Kety 95
133
4. Xenon Used According to the Height Over Area Method . . . .96
5. Heat Clearance Methods 96
6. Local Radioactive Tracer Tissue-Clearance Methods 97
133
a. Xenon and 85Krypton by Local Tissue Clearance 97
b. Simultaneous Local Tissue Clearances of More Than One
Radioactive Isotope 98
IV. Summary and Conclusions 99
References 99
I. INTRODUCTION
A. Flow Concepts
A discussion of various methods of measurement of blood flow through a tissue
may start from some concepts of flow, and there are several.
Flow is the volume flow of plasma, red cells, or whole blood through the arterial
vessels to the whole tumor and through venous vessels from the tumor. It is also the
volume flow distributed to several parallel areas of the tumor, the flow being perhaps
higher in peripheral and well-vascularized tumor tissue, lower in central parts on the
edge of necrosis. Furthermore, volume flow includes the different movements of blood
and blood elements in serial sections of the tumor vascular bed from the arterioles
branching to the immature, sinusoidlike capillaries and venules of the tumor. Quanti-
fication of the distribution of flow between several parallel regions, or of flow patterns
in serial sections of the tumor vascular bed, may be achieved with different grades of
spatial resolution, and therefore, with different grades of disparity compared with the
average for the whole tumor.
Flow is also the material flow oí various substances through the tumor to and from
the cells: oxygen and nutrients, metabolites and waste products, electrolytes and pro-
teins, and in addition, drugs and drug metabolites. This material flow is partly convec-
tive because of the movement of blood through the vasculature and the movement of
fluid within the large tumor interstial space, in many tumors "loose" owing to a low
glycosaminoglycan content. 1 It is partly diffusive through the vascular wall and
through the tissue for most substances by routes and with rates of diffusion which are
different for lipid-soluble and water-soluble substances. As a last step, there may be a
specific or nonspecific passage of the cell wall. Some substances remain exclusively
extracellular.
B. Flow Measuring Principles

The aim of a flow measurement on a tumor may be a recording of the total volume
flow of the tumor, e.g., in mi per min, of the mean flow of the total tumor, e.g., in
mi/min/unit volume or weight of tumor, or it may be a recording of the distribution
of volume flow within the tumor, expressed as the fractions of the tumor having certain
ranges of flow. There are direct volume-flow recording methods, and there are tracer-
flow methods to study tumor circulation. Tracer-flow methods generally lead to results
that might be transposed to volume flows or volume-flow distributions. The flows of
material are by themselves basic functional variables of the material balance of the
tumor; data on tracer flows might help to quantify this balance without necessarily
being translated to a volume-flow scale.
In addition, a dynamic panorama of the microcirculation might be achieved by mor-
phological methods, such as serial microangiography 2 and microscopy in vivo. 3 4 These
methods are outside the scope of the present chapter, however.
The present chapter will deal with the measurement of tumor blood flow with an
emphasis on experimental work, although it is appreciated that much clinical work is
going on, sometimes with less choice of methods or of optimal conditions for methods.
Figure 1 gives a summary sketch of some principles for flow measurements, also taking
into account the spatial resolution of the methods.
II. D I R E C T V O L U M E F L O W M E A S U R E M E N T S
A. Venous Outflow
To measure the total-volume blood flow, the tumor has to be isolated to a certain
extent within the body. Gullino and Grantham 5 substantiated this by implanting tumor
tissue into the kidney or ovary, mobilized and isolated with its vascular pedicle within
a paraffin envelope, into a pouch of the subcutaneous tissue of the rat. By growth,
the tumor now more or less replaced the normal tissue, giving a complete and encap-
sulated tumor preparation for circulatory studies. The vein is catheterized and venous
outflow measured, here, by a peristaltic pump device, one of several possible venous-
outflow methods. Gullino and Grantham's experiments were pioneer work in the field
of quantification of tumor circulation and should be read in extenso as a classic within
this field. In the same study, these authors compared venous outflow data with data
from uptake of 86Rb+ or 42K+, according to Sapirstein, a method described under Sec-
tion III. D. 2 of this chapter. Their method has recently been applied by Vaupel6 to
the study of pressure-flow relationships in tumors.
89
FIGURE 1. Survey of some principles of measurement of total tumor blood flow, local tumor blood
flow, and blood flow distribution. The lines join together the methods with the volume of the tumor where
the blood flow is measured. The tumor is illustrated by a Spalteholtz injection specimen of a rat hepatoma
grown in skeletal muscle of the thigh of the rat.
B. Arterial Inflow
To the author's knowledge, tumor blood flow has not been described as measured
by an electromagnetic flow-meter placed on an artery leading to a tumor. The require-
ments, however, are the same — the artery should be going exclusively to the tumor
without contamination of surrounding tissue.
C. Plethysmography
Plethysmography can be looked upon as an arterial inflow method. The venous
outflow is stopped momentarily and the tumor volume increase by arterial inflow is
registered for a few seconds. The procedure is assumed not to change the circulatory
state of the tumor. The volume-change registration places certain demands on the lo-
calization of the tumor, the two present possibilties being the s.c. tissue of the paws
or the tail of small animals. 7 9 Plethysmography has been applied to the measurement
of tumor blood flow in a rat 20-methylcholanthrene-induced sarcoma implanted into
the dorsum of the hindpaw of the rat 10 (see Figure 2, upper panel). Venous occlusion
was achieved by small rubber cuffs which could be rapidly inflated above venous pres-
sure by opening a line to a pressure reservoir. The paw were placed in thermostat-
controlled, water-filled, plexiglass cylinders. The change of the paws volume was meas-
ured by the precalibrated pressure change of a small air bubble in the cylinder. The
volume of the tumor and the tumor blood flow were calculated as the differences of
volume and change of volume per time unit between the tumor-bearing and the normal
hindpaw. The toes were temporarily ligated to diminish the flow of normal tissue
within the cylinders. This difference calculation did not account for differences in paw
flow due to inflammatory and edematous changes in the skin adjacent to the tumor
or to the displacement of the skin by the tumor.
D. Comments
Distribution of volume flow within parallel sections of the tumor vascular bed may
be measured by quantitative microangiographic 2 or microscopic methods, 3 4 or indi-
FIGURE 2. Overall picture of three techniques of tumor blood-flow quantification as described in Chapter
4, The central field is a Spalteholtz injection specimen of a MCH-H tumor implanted into skeletal muscle
of the thigh of the rat. Upper panel. Plethysmography of the hindpaw of the rat with and without tumor.
See Section II. C for details. Lower left panel. Distribution of radioactive tracer according to Sapirstein.
See Section III. D. 2 for details. Lower right panel. Local tissue clearance of two isotopes after local injection
into a tumor transplanted into skeletal muscle of the thigh of the rat. See Section III. D. 6. b for details.
(From Appelgren, K. L., Kjartansson, I., and Peterson, H.-I., Bibl. Anat., 15, 255, 1977. With permission.)
rectly, by radioactive tracer methods to be described below. Flow within serial sections
from the arterial to the venous side can at present only be quantitatively described by
the same morphological methods. However, it is felt that much of classical macro-
scopic analysis11 of neurogenic, hormonal, pharmacological, and local regulation of
peripheral circulation — expressed as vascular resistance, pre- vs. postcapillary resist-
ance, capillary pressure and capillary fluid balance, capillary filtration coefficient, etc.
— can in fact be done with the Gullino-Grantham preparation or by the plethysmo-
graphic method, and a beginning has been made. 610
III. RADIOACTIVE TRACERS IN THE STUDY OF TUMOR

CIRCULATION
A. Choice of Tracer Substances

Material exchange between blood and tissue is a compound effect of volume flow,
flow distribution, and of diffusion through the tissues. Material flow is studied by
means of tracer substances, often radioactive for convenience of measurement, but
colored substances, fluorescent substances, or heat are useful alternatives. It should
91
be remembered that tracer flows are not identical to flows of nutrients and metabolites,
but obviously, they might give important information concerning these.
The radioactive isotope may be bound to microspheres or to high-molecular-weight
substances, like proteins, with different distribution volumes within the tumor vessels.
The distribution volume for the microspheres is the arterial end of the vascular space,
and, for high molecular substances, it is the vascular space, at least during the first
few passages of the tracer through the tissue.
The radioactive isotope may be part of molecules or ions of lower molecular weight.
Useful lipid-soluble substances are radioactively labeled antipyrene (131I, 125I, 14C) and
the radioactive inert gases 133xenon and 85 krypton. These have practically no diffusion
restriction at the capillary wall and a large distribution volume, including the intracell-
ular space within the tissue. The lipid-containing tissue to water-partition coefficient
is more sensitive to the lipid concentration for 133Xe than for 85Kr and labeled antipyr-
ene.
Useful water-soluble substances are the 51 chromium-EDTA ion, which is extracellu-
lar, the 24Na and 131iodide ions, which are practically extracellular, and the 86rubidium
and 42K ions, which behave as if the potassium pool within the cell were a sink, giving
in practice a large distribution volume. Water-soluble substances have a diffusion re-
striction at the capillary wall and may be called barrier-limited within certain blood-
flow ranges.
B. The Tracer Analysis with the Tissue as a "Black Box"

Of all the tracers mentioned above, except labeled microspheres, a known bolus may
be injected intra-arterially, assuming mixing with arterial blood, and the variation of
the venous concentration may be followed (and eventually corrected for recirculation).
After Fick (the steady-state input-output analysis), and Stewart and Hamilton (the
bolus method), a material balance for this "dye dilution" maneuver will give:
(1)
The same data also define the mean transit time t for passage of an intra-arterially
injected bolus through the tissue to the venous end:
(2)
which, in turn, is related to the distribution volume VD of the tracer within the tumor
and to the flow, Q, by (Meyer and Zierler12):
(3)
External measurement of residual radioactivity in the tumor after intra-arterial injec-

tion gives flow as (Zierler,13 Lassen, 14 Bassingthwaighte and Yipintsoi15):
(4)
Note that so far no model assumption has been made concerning the "black box",
the tissue. However, if such assumptions are made, there might be information to be
extracted from the shape of the venous concentration-time curves. The exploration of
such information is better done by simultaneous injection of multiple tracers with dif-
ferent transport characteristics in the same bolus injection and comparison of their
venous concentration-time curves.
This short summary gives the essence of the use of the tracer method for circulatory
studies, but it is appreciated that the reader has to go to the references for more details
and that the original theory is not easily grasped. Virtually no studies exist for tumors
along these lines. However, there are more practical than theoretical reasons for that.
As pointed out earlier, a well-defined artery and vein of an isolated tumor are only
available under certain experimental conditions. In addition, owing to the relatively
slow and heterogeneous circulation of tumors, long tails of the venous concentration-
time curves are to be expected. These, in turn, make it difficult to correct for recircu-
lation. One may have to work with a tumor perfused without recirculation or with a
large venous reservoir. Besides, measurements and calculations involve a considerable
amount of work, and it is wise to plan a study along these lines with automatic analysis
and computerized calculations. 15
Readers who want to read more about the tracer method may consult reference
works such as Welch et al. 16 A review which reconciles a complicated field and sums
up some key references is that by Bassingthwaighte and Yipintsoi.15 Winbury 17 has
written a review with many practical aspects and with references to the application of
radioactive tracers for the study of peripheral circulation of practically every tissue of
the body.
C. From Tracer Theory to Tracer Practice

When tracer methods have been used to study tumor circulation, it has most often
been done by adapting some simplifying assumption and only part of the full scheme
outlined under Section III. B. Figure 3 shows schematically where to label the tumor
tissue with the tracer (v) and where to measure the activities or concentrations (•). The
situations discussed under Section III. B respond to the combinations:
1. T 1 bolus arterial labeling with • 2 serial venous sampling or • 4 external moni-

toring of residual activity
Some combinations which will be discussed later are
1. T 1 left ventricle arterial blood labeling with • 3 tissue sampling (III. D. 1.).
2. Bolus i.v. injection with small extraction in the lung giving T 1 arterial labeling
with • 3 tissue sampling (III. D. 2.).
3. Bolus i.v. injection with small extraction in the lung giving T 1 arterial labeling
with • 2 arterial and • 3 tissue sampling (III. D. 3.).
4. T 1 bolus arterial labeling with • 4 external monitoring of residual tissue activity
(III.D.4.).
5. T 2 constant heat flow labeling with • 3 tissue temperature measurement (III. D.
5.).
6. T 1 bolus heat labeling with • 3 tissue temperature measurement (III. D. 5.).
7. T 2 local tissue labeling with • 4 external registration of residual tissue activity
(III. D. 6.).
93
FIGURE 3. Schematic sketch of different possibilities of labeling a

tumor (A, filled triangles) and of measuring the activity or the concen-
tration ( # , filled circles) of tracers for the study of tumor circulation
by different tracer methods.
D. Outline of Different Tracer Methods

1. Radioactive Microspheres
The choice of radioactive microspheres as blood-flow indicators 1819 greatly simpli-
fies the tracer method by giving a simple measuring principle; the size of the spheres
is chosen so that they are trapped in the arterioles and capillaries of the first vascular
bed downstream from the injection site. This is where perfect mixing with blood should
have been achieved. This mixing generally requires injection into the left atrium or
ventricle by a permanent indwelling catheter (PE 10 in the rat, if introduced into the
ventricle through the aortic orifice, according to Svedman20) or through cardiac punc-
ture (Wetterlin et al. 21 ). Recirculation is no great problem. Several consecutive injec-
tions may be made with different labeling isotopes on the spheres. If properly mixed,
the spheres follow the distribution of cardiac output to different organs or within or-
gans, with the obvious exception of organs with a postcapillary vascular bed. The
relative distribution of flow is fairly easily transposed to an absolute flow scale by the
artificial reference-organ method; 22 a suitable artery is cannulized, and a reference
sample collected through use of a calibrated syringe to draw blood at a constant vol-
ume rate by means of a withdrawal pump during the microsphere injection period.
The calculation of cardiac output and flow of different organs from measured activi-
ties, including total injected activity, organ weight, and reference-syringe blood volume
and sampling time, is straightforward.
The microsphere method seems temptingly simple; however, few studies have been
made in experimental tumors. Blanchard et al., 23 in a study made while 3M
microspheres were still at the introductory stage, injected spheres into the hepatic ar-
tery and the portal vein and sampled tumor tissue and normal tissue from the liver.
The method applied in this way might be valid for demonstrating gross differences in
vascular supply of tumors vs. normal hepatic tissue. A word of caution is necessary
here, however; arteries or veins are not perfect mixing chambers, which invalidate them
for use for more exact circulatory studies with the microsphere method.
Rogers24 correlated the uptake of microspheres in tumors of the hamster and the rat
with uptake of 131I-antipyrine and lissamine green and found a positive correlation.
One recent contribution to the use of microspheres in the study of V2 carcinoma cir-
culation of the rabbit has been made by Rankin and Phernetton.25
However, the main work on tumor circulation with the microsphere method seems
yet to be done. The reader who wishes to take up the method will be wise to consult
Buckberg et al.26 for measuring statistics of the microsphere method, and Svedman20
and Warren and Ledingham27 for several practical questions. It should be remembered
that, when striving to achieve reproducibility and good spatial resolution in a tissue
with such a relatively low and variable blood flow as that in most tumors, the micro-
sphere method will require a good solution of the mixing problem and a balance of
the total dose of microspheres between, on the low side, few microspheres and poor
measuring statistics in the samples, and on the high dose side, effects on general hemo-
dynamics, central nervous system, and heart, with no guarantee of an undisturbed
tumor circulation.
2. Distribution of 86Rb+ and Radioiodinated Antipyrine According to Sapirstein

This is a method akin to the microsphere method which is more easily used in prac-
tice, but depends upon less rigid theoretical foundations through the introduction of
some unproven assumptions and approximations. The method was devised by Sapir-
stein28 for determination of the fractional distribution according to blood-flow fraction
of cardiac output of 86Rb ion or 42K ion in most organs except the brain, and of ra-
dioiodinated antipyrine in brain, skin, and carcass, after an i.v. bolus injection of a
known amount of radioactive tracer. The tracers, which are taken up in a very small
amount in the lung, are mixed in the right and left ventricle, are transported to the
tissues in proportion to their blood flows as fractions of cardiac output, and are ex-
tracted by the tissues, not necessarily to 100%. The nonextracted part is recirculated
and handled in the same way. This cycle continues. These tracers are characterized by
a large distribution volume and a reasonable extraction fraction within most tissues.
Therefore, within a period of 40 sec to several min, a pseudostable state is found with
a constant level of the tracers in many organs in proportion to their fraction of the
blood flow of total cardiac output. This pseudostable state slowly redistributes to a
state where the isotopes are distributed more in accordance with their true whole-body
equilibrium distribution. At the same time, excretion and metabolism may have started
(Sapirstein and Moses29).
The animal is killed 40 to 80 sec after the i.v. injection, the tissue samples dissected,
weighed, and their radioactivity measured along with a reference sample in order to
calculate the total injected amount of radioactivity. With the given assumptions, blood
flow is expressed as a percentage of cardiac output to tissue-weight unit. The same
bolus of 86Rb can be used for measurement of cardiac output by the Stewart-Hamilton
method from the arterial concentration-time curve (Sapirstein,28 Wetterlin and Bjõrk-
man34), as described in principle under Section III. B above. Absolute values of blood
flow to weight unit can then be calculated.
This method was used first in tumors by Gullino and Grantham,5 who established
the pseudostable period and compared 86Rb+ and 42K+ uptake (assuming a standard
cardiac output in the rat) with their venous-outflow blood-flow values. Sapirstein and
co-workers30 also applied their method to a tumor-circulation study. Controls of the
plateau have been made in tumors for 86Rb by Takács et al.31 and by Kjartansson et
al.32 The latter authors32-33 used the method for the study of intratumoral blood-flow
distribution. They showed that the uptakes of 86Rb+ and 125I-antipyrine, injected in the
same bolus, were independent of tumor sample size between 10 and 100 mg. Good
correlations between uptakes of 86Rb+ and 125I-antipyrine in the same samples were
found in two rat tumors (see Figure 4). The general set up of the experiment is shown
in Figure 2, lower left panel.
95
FIGURE 4. Correlation of the tumor-tissue uptake of "Rubidium

ion and of I25I-antipyrine. Data are expressed as percentages of in-
jected dose/g of tumor tissue. Single data, denoted 1, refer to mea-
surements of 86Rb- and I25I-activities in the same tumor samples after
injection of both tracers in the same bolus. Correlation coefficient, r
= 0.9556. The tumor is a rat hepatoma transplanted into skeletal
muscle of the thigh of the rat. (From Kjartansson, I., Acta Chir.
Scand. Suppl., 471, 1976. With permission.)
The correlation found validates 86Rb+ and 125I-antipyrine against each other. 86Rb
ion is recommended, however, because of its shelf stability; iodinated antipyrine is not
safely stable in saline for more than hours, in water for more than days. The intracell-
ular-sink uptake of 86Rb ion is also preferable, although the extracted amount of tracer
in one passage of the tumor might be higher for antipyrine than for rubidium ion.
86
Rb ion seems better suited for cardiac output determination than iodinated antipyrine
because of a slight uptake of the latter in the lung (Goldman and Sapirstein35).
More work seem necessary to validate the Sapirstein method against direct volume-
flow methods. Validation of 86Rb for determination of intratumoral blood-flow distri-
bution has to be done, e.g., against serial microangiography.2
3. Distribution of Radioactive Antipyrine According to Kety

Kety and co-workers36 devised a method, not to be confused with the local tissue-
clearance method, the latter being sometimes called the Kety-Schmidt method. Kety's
method was suggested for measurement of local blood flow in heterogeneously per-
fused regions. The method has recently been applied by Allen et al.37 to the measure-
ment of heterogeneity of blood flow in CNS tumors compared to the normal tissues
within cerebral cortex, striatum, subcortical white matter, spinal cord, and nerve roots.
This, so far, single work is mentioned because of the abundance of data and the good
spatial resolution (down to 10 mg of tissue) achieved with this method, but also because
the method gives an interesting example of combination of labeling, sampling, and
measuring.
Arterial labeling with 14C-antipyrine given i.v. is combined with serial arterial sam-
pling and a final tissue-radioactivity sampling when the animal is killed after 20 sec
(cf., Figure 3). Radioactivities were originally estimated by Kety and co-workers by
densitometry of autoradiographs of blood and tissue, valuable when a morphological
identification is wanted as well. Autoradiography may be replaced by measurement of
,4
C-activity of blood and tissue samples with a liquid scintillation counter (Goldman
and Sapirstein35). The calculations of flow/unit tissue volume depend on the loading
of the tissue during a short time with a changing, but recorded, arterial concentration
to a final tissue concentration during such a short time that venous washout is negligi-
ble. The tissue to blood partition coefficient for antipyrine, close to 1, is needed in
the original method. The reader is referred to Goldman and Sapirstein's paper35 for
comments and for a practical simplified approach, giving a measuring principle with
similarities both to Kety's original method and to the Sapirstein method described
under III. D. 2.
133
4. Xenon Used According to the Height Over Area Method
As described under Section III. B, blood flow to tissue volume can be measured by
external monitoring of residual activity in the tissue after intra-arterial bolus injection
by the height over area method without the need to know the tissue to blood partition
coefficient for 133Xe for the particular tissue. The method requires access to an arterial
inflow, and the radioactive gamma-detector is collimated onto the tissue of interest.
The calculation will give the mean flow/unit volume of this tissue, regardless of
whether the tissue is homogeneously perfused or not. The measured tissue is not nec-
essarily the total tissue labeled by the arterial injection. Counter-current vein to artery
diffusion (see Figure 3, arrows marked 5) outside the detector field will tend to give
an underestimation of flow values. 133Xe is used because of its easily collimated low-
energy gamma-energy radiation and its low recirculation due to efficient washout in
the lungs, in turn due to low solubility in blood. Correction might be made for recir-
culation by injecting the same activity amount outside the detector field.
The correct method is to follow the activity-time curve down to background values;
this is tedious and a 10 min registration approximation may be sufficient, so that
(5)
The constant is a correction factor which might be determined by going through the
whole procedure or by determining flow by a separate method as well. It is not far
from 1 and is not changed much for a certain vascular bed if changes in the circulation
are not found to be to drastic. To correct a misunderstanding, it should be mentioned
that this constant is not the tissue-blood partition coefficient for 133Xe.
This method has been used by Oikawa and co-workers38 39 for quantification of
blood flow in tumors implanted into the tail of the rat; serial microangiographies were
used for comparison. By using indwelling arterial catheters, the tumor circulation
could be followed for several days in the same animals.
5. Heat Clearance Methods

Heat is an ideal tracer with a unit tissue to blood partition coefficient and no recir-
culation. Its main drawbacks are the effect of induced temperature changes on circu-
lation and the difficulty of finding suitable places for heat labeling and for temperature
registration in the tissue. It has been applied to the study of tumor circulation in var-
ious ways, from attempts to discuss tumor blood flow in terms of temperature differ-
ences from surrounding tissue to more exact tracer methods. M0ller and Boj sen40 com-
pared s.c. tumor temperature with skin temperature, estimating blood flow in the
97
tumor with the local 133Xe clearance technique (see Section III. D. 6. a below). They
used the atraumatic tissue labeling technique according to Sejrsen;41 no relation be-
tween tumor blood flow and temperature was found. Johnson42 used a steady-state
heat-clearance method measuring the temperature of superficial tumor placed in close
contact with a heat sink or source of known temperature. Straw et al.43 used a local
heat clearance technique, with arterial injection of a bolus of cold saline and local
measuring of equilibration of temperature at various sites of the tumor with 23-gauge-
needle thermistors. While Johnson's method is theoretically clear and mainly limited
by the experimental setup, the local heat-clearance technique is in many ways subject
to the same problems as the radioactive local tissue-clearance technique. On the one
hand, the tissue is labeled intra-arterially, and the temperature is measured locally. On
the other, the tissue is labeled locally, and the residual activity at the injection site is
followed by external gamma-counting (cf. Figure 3).
6. Local Radioactive Tracer Tissue-Clearance Methods

a. 133Xenon and 85Krypton by Local Tissue Clearance
The local tissue-clearance technique builds on the assumption that by local injection
part of the tissue is labeled homogeneously with the tracer. The tracer assumes equilib-
rium with the venous blood passing from the injection site, the tissue-blood partition
coefficient being A (lambda). The residual activity at the injection site is registered,
and this activity is plotted in a semilogarithmic scale against time. The fractional dis-
appearance rate of the tracer activity (A) can be calculated as:
(6)
where In2 is the natural logarithm of 2, and t 1/2 is the time for any slope of the curve
to go down to half its value. The initial part of the curve analyzed in this way should
then give the flow/unit weight of tissue
(7)
as a mean value for the labeled part. Generally, k is not constant, but tends to diminish
with time. This is an indication that the best-perfused parts of the labeled tissue have
a more rapid washout. Observe that bolus arterial labeling of the tissue is not the same
as local injection labeling. If a mean value of flow/unit weight by this labeling route
is wanted for the whole tumor, prolonged labeling by arterial infusion should be used,
and the initial slope after stop of infusion used for calculation of flow. However, the
method described under Section III. D. 4 is preferable.
The reader should consult Kety's classical paper44 concerning this method; a valuable
discussion concerning the local labeling technique versus arterial labeling is given by
Lassen.14
Partition coefficients of tumor to blood have been determined by Kallman et al.,45
Song et al., 46 and for various brain tumors by O'Brien and Veall.47
Consider Figure 3. It is evident that in many experimental and in most clinical situ-
ations the only possible labeling-measuring combination is just the local tissue-clear-
ance technique with local tissue labeling (T 2) and external registration of radioactivity
( • 4) during washout. If this method is chosen, one has to be prepared to meet diffi-
culties due to the injection artifact of the needle, to the impossibility of registering the
curve from close after the injection, with the consequent problem of deciding which is
the proper part of the curve to analyze. These difficulties are due, finally, to an un-
known partition coefficient (most often related to an unknown lipid content of the
FIGURE 5. Diagrams of simultaneously registered fractional disappearance rates of 13'iodide (k,) and
l33
xenon (kXe) in untreated (upper left panel) and X-irradiated MCH-H tumors in the thigh of rats (upper
right panel, crosses). The lower diagrams give the distribution as numbers of observations within the squares
of the upper diagrams, illustrating the scattering of the observations. (From Kjartansson, I., Appelgren, K.
L., Peterson, H.-I., Rosengren, B., Rudenstam, C.-M., and Lewis, D. H., Bibl. Anat., 12, 519, 1973. With
permission.)
tumor and an unknown hematocrit of the blood). The local injection technique will
also give a large variation of values from different injections owing to the heterogeneity
of blood flow in many tumors (Kjartansson et al.48). See Figure 5.
In sum, the easiest method applicable for estimation of tumor circulation will give
the most variable values, which may, in part, be due to defective injections or to erro-
neous analysis of the registered curve. At the same time, it may be the only method
available for repeated measurements in the tumor or the only clinically available
method in the operation ward. The author suggests that data obtained by this method
should be handled with caution and that, if possible, some other method be used for
comparison, e.g., the height over area method (Section III. D. 4).
b. Simultaneous Local Tissue Clearances of More Than One Radioactive Isotope

Kety44 introduced a factor to correct for nonequilibrium between tissue and venous
blood for the tracer during washout, but generally assumed this factor to be close to
1. It is, therefore, of interest to compare one lipid-soluble isotope, 133Xe, and one
water-soluble isotope, 131I, with respect to washout rates after local injection of a mix-
ture of the two isotopes. Fractional disappearance rates of the two tracers taken from
Kjartansson et al.48 have been plotted in the diagrams of Figure 5 where points along
the diagonal lines would mean equilibration for both tracers. Now in fact, values for
the barrier-limited substance iodide seem almost independent of the xenon values.
99
They illustrate that the capillary-diffusion capacity of the capillary bed for diffusive
transport is the rate-limiting step for washout of iodide. This is opposite to xenon,
where the blood flow or perfusion is the rate-limiting step for washout. The reader is
referred to the original work and to Lassen and Trap-Jensen49 for a fuller discussion.
The lower panels of Figure 5 amply illustrate that both washout limited by perfusion
and that limited by diffusion are subject to large variations in various parts of the
tumors, when studied by the local tissue-clearance method.
The setup of the double-isotope local clearance method is shown in Figure 2, lower
right panel.
IV. SUMMARY A N D CONCLUSIONS
A survey of different methods for quantification of tumor blood flow has been
given. These build either directly on volume-flow registrations or indirectly on tracer-
transport methods. It has been argued that the more unambiguous methods with re-
gard to theoretical ground principles require access to vessels leading to and/or from
the tumor and a tumor reasonably well separated from surrounding tissue. The simple
local tissue-clearance method is hazardous to use, owing to several practical difficulties
and to the heterogeneous character of the tumor blood flow. If it is applied, measure-
ment should be based on many injections in different parts of the tumor.
There are many circumstances in which different methods might supplement each
other, e.g., a tracer method and a morphologically directed method,38 or several meth-
ods might confirm blood flow heterogeneity within a tumor.48 There is a definite need
for investigations where more than one method is applied simultaneously in order to
validate different methods for quantification of tumor circulation. Thus, measurement
of tumor circulation is still in a period of development and evaluation of methods,
and few of the methods reviewed here are yet at a stage where they can be applied for
the detailed study of tumor circulatory physiology.
REFERENCES
1. Swabb, E. A., Wei, J., and Gullino, P. M., Diffusion and convection in normal and neoplastic
tissues, Cancer Res., 34, 2814, 1974.
2. Reinhold, H. S., Improved microcirculation in irradiated tumours, Eur. J. Cancer, 7, 273, 1971.
in implanted mouse mammary tumors, Bibl. Anat., 15, 273, 1977.
4. Warner, N. E., Puffer, H. W., and Schaeffer, L. D., Observations of the microcirculation in spon-
taneous mammary tumors in C3H mice, Bibl. Anat., 13,311, 1975.
5. Gullino, P. M. and Grantham, F. H., Studies on the exchange of fluids between host and tumor. II.
The blood flow of hepatomas and other tumors in rats and mice, J. Natl. Cancer Inst., 27, 1465,
1961.
6. Vaupel, P., Interrelationship between mean arterial blood pressure, blood flow, and vascular resist-
ance in solid tumor tissue of DS-carcinosarcoma, Experentia, 31, 587, 1975.
7. Maistrello, I., Spazzoli, G., and Matscher, R., Measurement of blood flow in the rat tail by strain-
gauge plethysmography, / . Appl. Physiol., 22, 826, 1967.
8. Clarke, E. W. and Jamison, J. P., The measurement of the flow through the hindpaw of a mouse
by venous occlusion plethysmography, Proc. Physiol. Soc. Surg., 71, 148, 1972.
9. Weaver, J. P. A. and Evans, A., A plethysmograph with an air capacitance recording system, / .
Appl. Physiol.,23, 591, 1967.
10. Kjartansson, I. E., Appelgren, L. K., Ivarsson, L., Peterson, H.-L, and Sivertsson, R., Total blood
flow in a 20-methylcholanthrene induced rat sarcoma determined by plethysmography. Effect of
aging and of a single dose of X-ray irradiation., Acta Chir. Scand. SuppL, 471, 45, 1976.
11. M el lander, S. and Johansson, B., Control of resistance, exchange and capacitance functions in the
peripheral circulation, Pharmacol. Rev.,20, 117, 1968.
12. Meyer, P. and Zierler, K. L., On the theory of the indicator-dilution method for measurement of
blood flow and volume, / . Appl. Physiol., 6, 731, 1954.
13. Zierler, K. L., Equations for measuring blood flow by external monitoring of radioisotopes, Circ.
Res., 16, 309, 1965.
14. Lassen, N. A., On the theory of the local clearance method for measurement of blood flow including
a discussion of its application to various tissues, Acta Med. Scand. SuppL, 47'1, 136, 1967.
15. Bassingthwaighte, J. B. and Yipintsoi, T., Organ blood flow, wash-in, washout, and clearance of
nutrients and metabolites, Mayo Clin. Proc, 49, 248, 1974.
16. Welch, T. J. C , Potchen, E. J., and Welch, M. J., Fundamentals of the Tracer Method, W. B.
Saunders, Philadelphia, 1972.
17. Winbury, M. D., Use of radioactive tracers in the study of nutritional circulation, in International
Encyclopedia of Pharmacology and Therapeutics, Section 78, Vol. 1, Pergamon Press, Oxford, 1971,
chap.12.
18. Rudolph, A. M. and Heymann, M. A., The circulation of the fetus in utero. Methods for studying
distribution of blood flow, cardiac output and organ blood flow, Circ. Res.,21, 163, 1967.
19. Wagner, H. N., Rhodes, B. A., Sasaki, Y., and Ryan, J. P., Studies of the circulation with radioac-
tive microspheres, Invest. Radiol.,4, 374, 1969.
20. Svedman, P., Distribution of cardiac output in small laboratory animals determined with a micro-
sphere method, Thesis, Department of Plastic Surgery, University Lund, Sweden, 1977.
21. Wetterlin, S., Aronsen, K. F., Bjorkman, I., and Ahlgren, I., Studies on methods for determination
of the distribution of cardiac output in the mouse, Scand. J. Clin. Lab. Invest., in press.
22. Bartrum, J. J., Berkowitz, D. M., and Hollenberg, N. K., A simple radioactive microsphere method
for measuring regional blood flow and cardiac output, Invest. Radiol., 9, 126, 1974.
23. Blanchard, R. J. W., Grotenhuis, I., La Fave, J. W., and Perry, J. F., Blood supply to hepatic V 2
carcinoma implants as measured by radioactive microspheres, Proc. Soc. Exp. Biol. Med., 118, 465,
1965.
24. Rogers, W., Tissue blood flow in transplantable tumors of the mouse and the hamster, Diss. Abstr.
B, 28,5185,1968.
25. Rankin, J. H. G. and Phernetton, T., Effect of prostaglandin E2 on blood flow to the V 2 carcinoma,
Fed. Proc. Fed. Am. Soc. Exp. Biol., 35, 297, 1976.
26. Buckberg, G. D., Luck, J. C , Payne, D. B., Hoffman, J. I. E., Archie, J. P., and Fixler, D. E.,
Some sources of error in measuring regional blood flow with radioactive microspheres, / . Appl.
Physiol., 31, 598, 1971.
27. Warren, D. J. and Ledingham, J. G. G., Measurement of cardiac output distribution using
microspheres. Some practical and theoretical considerations, Cardiovasc. Res., 8, 570, 1974.
28. Sapirstein, L. A., Regional blood flow by fractional distribution of indicators, Am. J. Physiol., 193,
161, 1958.
29. Sapirstein, L. A. and Moses, L. E., Cerebral and cephalic blood flow in man: basic considerations
of the indicator-fractionation technique, in Dynamic Clinic Studies with Radioisotopes, Report U.S.
Atomic Energy Commission, Washington, D.C., 1964, 135.
30. Cataland, S., Cohen, C , and Sapirstein, L. A., Relationship between size and perfusion of trans-
planted tumors, J. Natl. Cancer Inst., 29, 389, 1962.
31. Takács, L., Debreczeni, L. A., and Farasang, C , Circulation in rats with Guérin carcinoma, / . Appl.
Physiol. ,38,696, 1975.
32. Kjartansson, I., Appelgren, L. K., and Peterson, H.-L, Intratumor flow distribution studied by
86
Rubidium-ion and I2S I-antipyrine. I. Methodological aspects with a comparison of two different
tracers, Acta Chir Scand. Suppl., 471, 20, 1976.
33. Kjartansson, I., Appelgren, K. L., and Peterson, H.-L, Intratumor flow distribution studied by
86
Rubidium-ion and 125I-antipyrine. II. Influence of tumor growth and age, Acta Chir Scand. SuppL,
471,30, 1976.
34. Wetterlin, S. and Bjorkman, I., Determination of cardiac output in the mouse, Thesis, Department
of Surgery, University Lund, Sweden, 1977.
35. Goldman, H. and Sapirstein, L. A., Brain blood flow in the conscious and anesthetized rat, Am. J.
Physiol., 224, 122, 1973.
36. Kety, S. S., Theory of blood-tissue exchange and its application to measurement of blood flow, in
Methods in Medical Research, Vol. 8, Year Book Medical Publishing, N.Y., 1960, 223.
101
37. Allen, N., Goldman, H., Gordon, W. A., and Clendenon, N. R., Topographic blood flow in exper-
imental nervous system tumors and surrounding tissues, Trans. Am. Neurol. Assoc, 100, 157, 1975.
38. Oikawa, M. and Milne, E. N. C , A morphological and quantitative study of tumor blood flow
during growth and immune rejection, Tohoku J. Exp. Med., 112, 325, 1974.
39. Oikawa, M., Milne, E. N. C , Whitmore, E., Gilday, D., and Oliver, C.,A morphological and quan-
titative study of tumor blood flow. I. During growth and immune rejection, Cancer (Philadelphia),
35,385,1975.
40. Miller, U. and Bojsen, J., Temperature and blood flow measurements in and around 7,12-dimethyl
(a) anthracene-induced tumors and Walker 256 carcinosarcomas in rats, Cancer Res., 35, 3116, 1975.
41. Sejrsen, P., Atraumatic local labelling of skin by inert gas: epicutaneous application of Xenon 133,
J. Appl. Physiol., 24, 570, 1968.
42. Johnson, R., A thermodynamic method for investigation of radiation induced changes in the micro-
circulation of human tumors, Int. J. Radiât. Oncol. Biol. Phys., 1, 659, 1976.
43. Straw, J. A., Hart, M. M., Klubes, P., Zaharko, D. S., and Dedrick, R. L., Distribution of antican-
cer agents in spontaneous animal tumors. I. Regional blood flow and methotrexate distribution in
canine lymphosarcoma, J. Natl. Cancer Inst., 52, 1327, 1974.
44. Kety, S. S., Theory and applications of the exchange of inert gas at the lungs and tissues, Pharmacol.
Rev., 3 , 1 , 1951.
45. Kallman, R. F., de Nardo, G. L., and Stasch, M. J., Blood flow in irradiated mouse sarcoma as
determined by the clearance of Xenon-133, Cancer Res., 32, 483, 1972.
46. Song, C. W., Payne, J. T., and Levitt, S. H., Vascularity and blood flow in x-irradiated Walker
carcinoma 256 of rats, Radiology, 194, 693, 1972.
47. O'Brien, M. D. and Veall, N., Partition coefficients between various brain tumors and blood for
I33
Xe, Phys. Med. Biol., 19, 472, 1974.
48. Kjartansson, I., Appelgren, K. L., Peterson, H.-L, Rosengren, B., Rudenstram, C.-M., and Lewis,
D. H., Capillary flow, exchange and flow distribution in transplantable rat tumors, Bibl. Anat., 12,
519,1973.
49. Lassen, N. A. and Trap-Jensen, J., Theoretical considerations on measurement of capillary diffusion
capacity in skeletal muscle by the local clearance method, Scand. J. Clin. Lab. Invest., 21, 108, 1968.
50. Appelgren, L. K., Kjartansson, I., and Peterson, H.-L, Quantification of tumor blood flow and
blood flow distribution, Bibl. Anat., 15, 255, 1977.
Taylor & Francis
103
Chapter 5
TUMOR BLOOD FLOW C O M P A R E D WITH NORMAL TISSUE BLOOD

FLOW
Hans-Inge Peterson
TABLE OF CONTENTS
I. Introduction 103
II. Total Tumor Blood Flow 104
III. Local Tumor Blood Flow 107
IV. Intratumor Blood-Flow Distribution 108
V. Summary 112
Acknowledgment 113
References 113
I. INTRODUCTION
Ribbert postulated in 1904 that tumors have a vascular supply superior to that of
normal tissues.1 This postulation, which was based on morphological observations,
was generally accepted for over half a century. In 1961, presenting their experimental
technique for recording the venous outflow from ''tissue-isolated" transplanted tu-
mors, Gullino and Grantham2 summarized the situation: "to our knowledge the eval-
uation of the blood supply to a tumor is an unsolved problem."
The main obstacles to studying tumor blood flow have been due to methodological
difficulties. When isotope techniques were introduced, it was suggested that these
problems were now solved. The local tissue clearance by 133Xe seemed to be ideal for
clinical use. However, by this method the blood flow of only a very limited tumor area
is examined, and several experimental studies have verified the wide lack of homogene-
ity in tumor blood-flow distribution. Furthermore, large variations in blood-tissue par-
tition coefficients for 133Xe have been found in different tumors, and these might influ-
ence the flow data recorded.
Methods used in experimental studies for recording tumor blood flow have included
venous outflow from isolated tissue transplants, intratumor isotope distribution, serial
microangiography, and plethysmography. By these different techniques and by the
local isotope clearance technique, different aspects of tumor blood flow have been
studied, namely total tumor blood flow, local tumor blood flow, and intratumor
blood-flow distribution.
As will be apparent from the different studies to be reviewed in this chapter, Rib-
bert's postulation does not seem to have any strong support from recent tumor blood-
flow studies. On the contrary, a comparatively low blood-flow rate has been found in
many tumors, and this flow rate decreases rapidly with tumor growth.
There still remain many problems to be solved, especially in finding methods for
recording human tumor blood flow. However, it must be remembered that this re-
search field has only been open for study for a fairly short time, and that considerable
methodological difficulties have been met with. A thorough knowledge of tumor blood
flow will be of the utmost importance in cancer diagnosis and treatment, based on the
influence of tumor blood supply and distribution and also on the localization of diag-
nostic substances and of active antitumor substances (see Chapters 10 to 13).
II. T O T A L T U M O R B L O O D F L O W
Wartnaby et al. 3 used the bromsulphonphtalein uptake to measure blood flow

through a large hepatic metastasis in a patient by passing a catheter into the vein and
draining the tumor. Such a method is only applicable when the venous blood drains
the tumor tissue exclusively.
In experimental studies, Gullino and Grantham 2 introduced the ingenious technique
of transplanting tumor to isolated organs in rats and mice, such as kidneys and ovaries.
By the destructive growth of the tumor, the normal tissue of the transplantation area
will be eliminated or left as very small residues. By cannulating the vein draining the
transplanted organ, it will be possible to record the total tumor blood flow.
The authors found that in ovarian implants larger than 4.0 g no residues of normal
tissue could be found, while in large renal tumors small residues of normal kidney
tissue were still present. This means that transplantation to an isolated ovary might be
the method of choice in these studies. The average value of total blood flow in tumors
grown in isolated tissue was 0.14 m i / m i n / g . This value was found for a hepatoma
transplanted to isolated kidney, and the blood supply of this hepatoma was calculated
to be roughly 20 times less than blood supply to normal liver. The tumor blood flow
in kidney transplants decreased with increasing tumor growth, and in tumors with a
weight of 12 g, the total blood flow was between 0.09 and 0.12 m i / m i n / g . Gullino
and Grantham also compared their venous outflow studies by registration of tumor
blood flow, using the uptake of 42K and 86Rb in tumor and normal tissues. By this
technique, they found a tumor flow in ovarian implants of about 0.15 m i / m i n / g ,
compared to 0.14 mi with the venous outflow technique. This flow was calculated to
be about 50 times less than the average normal ovarian blood flow. The comparatively
low tumor blood flow values found in these studies might be explained by the technique
used. Thus, it is known that the tumor blood flow decreases with increasing tumor
growth in parallel with the development of central tumor necroses. At a time when
most normal tissue has been destroyed by the growing tumor, this tumor might be in
a late phase of growth with a decreased blood-flow rate. In a later study, a method
was used by which the whole efferent blood of chemically induced rat mammary car-
cinomas was recorded. 4 In tumors weighing 2.0 to 3.5 g, the blood flow was 0.03 to
0.13 m i / m i n / g .
A linear dependence of total tumor blood flow on mean arterial blood pressure was
found in studies on a DS-ascites-carcinoma transplanted and grown tissue-isolated in
rat kidney through use of the venous outflow technique. 5 No autoregulation of tumor
blood flow as in normal kidney was seen. An increased vascular resistance was found
during tumor growth. This was explained by the general reduction of the vascular
space and rarefaction of the terminal vascular bed. With a mean arterial blood pressure
105
of 120 mmHg, the recorded blood flow in a tumor with a weight of 2.5 g was 0.32
mi/min/g, and in a tumor with a weight of 8.2 g, it was 0.065 mi/min/g.
The fractional uptake of i.v. injected 86Rb in tumors as a fraction of cardiac output
has been used in several experimental studies. The blood flow in hamster and mouse
tumors was studied by the uptake in large biopsies or whole tumors of 131I-antipyrine,
radioactive microspheres, and lissamine green.6 The mean blood flow of a hamster
melanoma with a weight of 0.5 g remained approximately 0.6 mi/min/g, regardless
of the site of implantation. In most tumors, the average recorded blood flow decreased
10 to 100 times with growth. This was correlated with an enlarging volume of central
necroses. The mean tissue blood flow was 2 to 12 times higher in peripheral tumor
compared with central, more necrotic, tumor areas. The tumor blood flow was well
correlated to tumor histopathology.
In a study on Guérin carcinoma in rats by tumor uptake of 86Rb, no significant
difference in tumor blood flow was found between small and large tumors.7 Thus,
tumors with a mean weight of 1.21 g showed a flow of 20.8 ± 1.5 mi/min/100 g, and
tumors with a mean weight of 56.6 g showed a flow of 19.6 ± 1.2 mi/min/100 g. This
observation indicates that different results on tumor blood flow in experimental studies
might be obtained, according to the tumor studied. In tumors with an early develop-
ment of central necroses, the tumor blood flow will decrease rapidly with increasing
growth. Furthermore, the technique used and the areas of tumor studied will influence
the results. The blood flow of peripheral nonnecrotic parts of tumor will probably
remain comparatively stable in spite of increasing tumor volume.
Thus, the fractional uptake of 86Rb in a mouse carcinoma and a mouse sarcoma
was studied without any attempt to exclude necrotic tumor tissue.8 In this study, tu-
mors of increasing size showed progressively lower uptake of indicator, and it was
concluded that the tumor mass increased more rapidly than the blood supply. The
average tissue was calculated to receive 5% of the cardiac output, while the corre-
sponding values in sarcoma and carcinoma were 0.5 to 1.5% and 1.0 to 2.0%, respec-
tively.
Observations of a low blood-flow rate in human brain tumors by the Xe-clearance
technique led to studies on various nerve and brain tumors in the rat by 14C-antipyrine
uptake.9 Tumors were induced in rats by transplacental exposure to ethylnitrosurea.
Brain and tumor samples as small as 10 mg were studied, and determinations of cardiac
output in animals were performed. In these studies, the cerebral cortex flow was 1.14
± 0.06 mi/min/g, while the blood flow in different nerve or brain tumors varied from
0.44 to 0.79 mi/min/g. Small tumors showed greater flow than large tumors or central
growth zones. Well-differentiated tumors had also a considerably larger flow rate than
anaplastic tumors. A lower blood-flow rate was found in cerebral cortex adjacent to
tumor.
The total tumor blood flow in a 20-methylcholanthrene-induced sarcoma trans-
planted into the hindpaw of inbred Lister rats was studied by a plethysmographic tech-
nique worked out and thoroughly described by Kjartansson at our laboratory.10 The
resting and postischemic flows in tumor-bearing and contralateral normal hindpaws
were recorded separately under ether anesthesia. In later studies (to be published by
J. Mattsson, see Chapter 7), the technique was further developed for simultaneous
registration of the blood-flow rate in both hindpaws with two plethysmographs.
In all animals studied, the paw volume, resting flow, and postischemic flow were
recorded for the nontumor-bearing leg. These values were then subtracted from the
corresponding values for the tumor-bearing leg to get an estimate of the tumor volume
and the tumor blood flow/min. From this, the blood flow/min and the blood flow/
100 mi tumor tissue was calculated.
The resting flow rate for the tumor-bearing paw minus that for the nontumor-bear-
ing paw was found to be 58 ± 28 mi/min/100 mi tissue on day 3 of the experiment,

which means 21 days after implantation, and 22 ± 13 mi/min/100 mi tissue on day 7
after the experiment. The corresponding blood-flow values after 10 min of ischemia
were 74 ± 37 and 30 ± 12. This means a decreased tumor blood-flow rate in tumors
with increasing volume and age. It is also evident that small tumors reacted with a
large postischemic increase of blood-flow rate compared with large tumors. The data
from separate animals are given in Figure 1.
It is obvious that, with this tumor and transplantation area and with this technique,
a high tumor blood-flow rate was found compared to many other experimental tumors
and other techniques studied. On the other hand, the changes of tumor blood-flow
rate with growth of tumors were identical to most other observations.
The temperature in the plethysmograph chamber was kept at 31.5°C during the ex-
periment to minimize the skin flow, and the animal body temperature was kept at
37.5°C. Despite this, a comparatively high blood flow was found in the normal hind-
paw which was similar to that found in normal rat tail. 11 This might be explained by a
FIGURE 1. Resting (black circles) and postischemic (white circles)

tumor blood flow in mi/min and per 100 mi tissue recorded by pleth-
ysmography. Corresponding tumor-flow values are connected with
vertical lines (resting and postischemic flow values) and with broken
straight lines with an arrow in the direction from day 3 to day 7 of
the experiment. The A-A curve indicates the upper range for postis-
chemic flow, and the B-B curve the upper range for resting flow.
(From Kjartansson, I., Acta Chir. Scand. SuppL, 471, 1976. With
permission.)
107
high s.c. blood flow. S.c. transplantation of tumor into the paw might induce an in-
flammatory reaction which will further increase this blood flow. By the plethysmo-
graphical technique, the blood flow of tumor and of an unknown portion of s.c. paw
tissue is studied. This means that a high blood flow in s.c. tissue surrounding the tumor
might influence the estimated tumor blood-flow values.
III. LOCAL TUMOR BLOOD FLOW
The main technique used in these studies has been local isotope clearance of
133
xenon. By this technique, the isotope is injected locally into tumors, and the isotope
washout from tumor tissue is studied. This necessarily means that the tumor blood
flow studied is limited to a rather small tumor area around the injection site, and it is,
thus, the local tumor blood that is shown.
In tumor surgery, the local isotope-clearance technique was introduced by Gelin et
al.,12 who studied liver blood flow in man during abdominal surgery, and the technique
was used to investigate the effect of hepatic artery ligation on liver-tumor blood flow.
It was also used in a recent study to measure the blood flow of primary liver carcinoma
by Plengvanit et al.13 They found a normal hepatic blood flow of 29 ± 10 mi/min/
100 g tissue. The blood flow in carcinomatous nodules was noticeably low, 12 ± 6 m i /
min/g tissue.
In a study on human superficial tumors, including lymphomas, anaplastic carcino-
mas, and various differentiated malignant tumors, the same isotope technique was
used.14 In this study, 133Xe was injected into central parts of tumor nodules, and dif-
ferent flows were registered in different types of tumors. Thus, the blood flow in lym-
phomas was 38 ± 14 mi/min/100 g, in anaplastic carcinomas 11 ± 5 mi/min/100 g,
and in differentiated malignant tumors 14 ± 9 mi/min/100 g. The authors pointed
out that the reproducibility of the method in their study by repeated injections was ±
2 mi/min/100 g. However, the local blood flow was studied, and this might change if
the isotope is injected into necrotic parts of a tumor. Furthermore, as stressed in this
study, the coefficient of 133Xe partition between tumor nodule and blood depends
strongly on the fat content of the tissue and slightly on the hemocrit. No significant
correlation was found between the tumor size and the local tumor blood flow.
It is evident from a study by O'Brien and Veall15 that changes in partition coefficient
between different tumors might influence the results obtained by the local isotope-
injection technique. In this study, the coefficient of 133Xe partition between various
human brain tumors and blood was investigated, and coefficients from 0.70 to 1.37
were observed.
The local 133Xe clearance technique has also been studied in several experimental
tumors. Robert et al.16 studied a methylcholanthrene-induced C3H mouse sarcoma and
compared the blood flow of this tumor to that of normal mouse muscle and skin. They
found a blood flow in muscle of 0.11 mi/min/g and in skin (or more correctly s.c.
tissue) 0.08 mi/min/g. The blood flow in small tumors (4 to 20 mm diameter) was
0.03 to 0.22 mi/min/g and in larger tumors (20 to 42 mm diameter) 0.01 to 0.08 m i /
min/g. This means that the blood flow in small s.c. transplanted tumors was roughly
equal to that of normal muscle and s.c. tissue.
In studies on a KHT sarcoma in syngeneic mice, a rather high partition coefficient
for 133Xe was found, 0.891, compared to the coefficient of 0.72 used in some other
studies.17 This might also have influenced the blood-flow data recorded, which in un-
anesthetized animals were found to be comparatively high, 21.2 ± 0.8 mi/min/100 g.
However, another parameter of importance for blood-flow rate was found in this
study.
In animals anesthetized with sodium pentobarbital i.p., the tumor blood flow was
significantly decreased to 13.8 ± 1 . 2 mi/min/100 g. There was also a marked decrease

with increasing tumor volume over 560 mm 3 . Thus, tumors with a volume of 561 to
1120 mm 3 had a blood flow of 13.7 ± 1.5 mi/min/100 g and tumors with a volume
of 1121 to 2240 mm 3 , a blood flow of 6.7 ± 0.9 mi/min/100 g.
The 133Xe washout technique was studied after intra-arterial injection into the fem-
oral artery in studies on the blood flow of tumors transplanted into the rat tail.11
Animals received transplants of a Walker 256 carcinoma. In one group of animals, a
rapid tumor growth was observed, and animals died 10 days after transplantation. The
normal tail blood flow was found to be 0.21 m i / m i n / g with a SEM of 0.17. In animals
with rapidly growing tumors, the mean blood flow to the tumor with an unknown part
of tail tissue increased six times up to day 10. In another group of animals, tumors
grew up to 8 days after transplantation, but decreased in volume afterwards, and were
totally rejected. In these tumor-transplanted tails, the blood flow increased four times
up to day 8, but parallel with the decrease in tumor volume afterwards, the blood flow
decreased significantly. As in the previous study, sodium pentobarbital anesthesia was
found to decrease the tail blood flow significantly, and therefore, animals were anes-
thetized with ketamine hydrochloride instead. Microangiographic studies during tumor
rejection showed marked tortuosity of feeding vessels and a "ghostlike" fading out
of tumor vessels which was unlike the appearance of necroses.
The local tissue-clearance technique using 133Xe and 24Na ions simultaneously was
applied to the study of tumor blood flow in two transplantable mouse tumors. 18 The
results showed a wide spread of flow values between tumors as estimated by 133Xe
clearance, but a relatively uniform diffusional transport as expressed by the 24Na clear-
ance. Similar conclusions of nonhomogeneous blood flow in tumors have been drawn
in other studies.19 21 This will, of necessity, be a main obstacle in recording tumor
blood flow by local isotope technique and will be further discussed later in this chapter.
By a thermodynamic method, high superficial blood flow was found in human
breast carcinomas compared with normal breast tissue.22
IV. I N T R A T U M O R B L O O D F L O W D I S T R I B U T I O N
The distribution of lissamine green within tumors was early studied by Goldacre and
Sylven23 and by Owen,24 and gave an indication of the tumor blood-flow distribution
within the tumor. However, this method will not give any quantitative information
about it.
In in vivo studies by serial microangiograms, Reinhold19 found a nonhomogeneous
blood-flow distribution (see the chapter dealing with this method). Similar observa-
tions were made in studies at our laboratory, in which, primarily, a very wide distri-
bution of tumor blood flow was recorded by the local 133Xe clearance technique. 20 21
This lack of homogeneity was also verified in studies of the intratumor distribution of
intra-aortally injected 125I-antipyrine.
The intratumor distribution of i.v. injected 86Rubidium and 125I-antipyrine was fur-
ther investigated by Kjartansson. 10 He studied two rat tumors, a 20-methylcholan-
threne-induced sarcoma and a hepatoma transplanted into one hindleg of animals.
After isotope injection, a stable plateau level of externally recorded isotope activity
was reached in tumor in 20 sec, remaining stable for several min. This means that
during this period the uptake and release of isotope balance each other, despite recir-
culation. According to Sapirstein, 25 the fractional uptake of isotope in tumor at a
stable level will reflect the fractional distribution of cardiac output in the tumor.
Animals were sacrificed 45 sec after isotope injection, the aorta and lower caval vein
were clamped, and tumors and normal muscle were rapidly dissected and frozen. From
these tissues, multiple frozen biopsies were taken for isotope-activity measurements.
109
Tumor biopsies between 10 and 80 mg were studied, 20 to 50 biopsies being taken

from each tumor. Down to the level of 10 mg, the biopsy size did not seem to influence
the recorded activity. By the technique used, both isotopes were found to have an
identical intratumor distribution, despite different transport characteristics.
The recorded isotope activities in tumor and muscle specimens were calculated in
% of injected dose/g tissue, since no equipment for registration of cardiac output in
small animals was available. A wide distribution of isotope uptake in both tumors was
found. This changed with increasing tumor age. There was a decrease of the fast flow
population and an increase of the slow flow population, indicating a decreased tumor
blood flow with increasing tumor age. This is illustrated in Figures 2 and 3, in which
FIGURE 2. (A) and (B) Distribution of ,25 I-antipyrine and "Rubidium ions, given in °/o of i.v.-injected
dose/g tumor tissue on different days of the experiments. Data from separate animals and lumped data.
MCH-H = 20-methylcholanthrene-induced sarcoma; Hep-H = hepatoma. Note the similar distribution of
both isotopes. Central tumor specimens indicated as crossed staples. No difference in distribution can be
seen between peripheral and central samples. (From Kjartansson, I., Acta Chir. Scand. SuppL, 471, 1976.
With permission.)
110
FIGURE 3. Conceptual diagrams with smoothed isotope-distri-

bution curves in tumor specimens on different days of the experi-
ment. Data from Figure 2(A) and (B). The decrease of a fast-flow
population and the increase of a slow-flow population with in-
creasing tumor age is seen in both tumors. (From Kjartansson, I.,
Acta Chir. Scand., Suppl., All, 1976. With permission.)
Ill
individual and lumped data from both tumors at different days of the experiment are
given. The data in Figure 2 were used as a basis for the smoothed isotope-distribution
curves of Figure 3 in which the decrease of the fast tumor-flow population with increas-
ing tumor age is more easily observed. The distribution of isotope was similar in central
and peripheral tumor samples, as seen in Figure 2.
A rather wide distribution of isotope uptake was also found in normal muscle, as
presented in Figure 4. However, the intramuscle flow distribution was unchanged dur-
ing the experimental period and might also be regarded as a control of the experimental
model.
FIGURE 4. Distribution of 125I-antipyrine given in % of i.v.-in-

jected dose/g tumor and muscle tissue on different days of the exper-
iment. Tumor, hepatoma, (white bars) and normal muscle (black
bars). (From Kjartansson, I., Acta Chir. Scand. SuppL, 471, 1976.
With permission.)
V. SUMMARY
Some blood-flow data recorded from experimental and human malignant tumors
are summarized in Table 1.
A wide distribution of tumor blood flow has been observed in several experimental
studies. This might be of importance when studying tumor blood flow by local isotope-
clearance technique, which will reflect the actual blood flow in small tumor areas.
Some authors have found a rather good reproducibility with this technique, while oth-
ers have found a large spread of locally recorded tumor blood flow. The 133Xe-clear-
ance technique is easily adapted to clinical use, but since a wide lack of homogeneity
in tumor blood flow distribution has been observed, the data obtained must be consid-
ered very carefully. The large variations in blood - tissue partition coefficients for
various tumors must also be observed.
TABLE 1
Some Blood Flow Data from Experimental and Human Tumors Recorded by Different Techniques—Blood
Flow Data from Normal Tissues are Included
Results
Tumors Methods of blood flow determination (mi/min/g) Ref.
Rat hepatomas Venous outflow 0.14 2

Rat carcinoma Venous outflow Small tumors 5
0.32; large tu
mors, 0.07
133
Mouse sarcoma Xe clearance small tumors, 16
0.03 — 0.22;
large tumors,
<0.01— 0.08;
muscle, 0.11;
s.c. tissue, 0.08
Mouse sarcoma 133
Xe clearance 0.04—0.19 18
Mouse mammary carcinoma 0.01—0.17
Mouse sarcoma l33
Xe clearance Small tumors, 17
0.14; large tu-
mors, 0.07
,33
Rat sarcoma Xe clearance 0.04—0.21 10
133
Human liver carcinoma Xe clearance Tumor, 0 . 1 2 ; 13
liver, 0.29
133
Human lymphoma Xe clearance 0.38 14
Human anaplastic carcinoma 0.11
Human diff. malignant tumors 0.14
131
Hamster melanoma Uptake of I-antipyrine 0.60 6
Rat carcinoma Uptake of 86
Rb Small and large 7
tumors, resp.
0.21—0.20
M
Rat nerve and brain tumors Uptake of C-antipyrine Cerebral cortex, 9
1.14 tumors,
0.44—0.79
Rat sarcoma Plethysmography 0.22—0.58 m i / 10
min/mi tissue
Skeletal muscle Several different methods, see original Rest., 0.02—0.05 26
reference max. 0.40—0.60
Liver Rest., 0.5 max.
3.0
Skin Rest., 0.03—0.10
max. 0.4
113
Intratumor distribution of 125I-antipyrine might be a technique which could be used

in the clinic and which, combined with multiple tumor biopsies and with cardiac output
registration, could be expected to give a broader estimation of tumor blood flow. The
ideal situation, with a single vein draining a tumor, will seldom be met with in tumor
surgery and will probably remain chiefly a technique for experimental studies. The
same restriction will also apply to the recording of tumor blood flow by plethysmog-
raphy.
Tumor blood flow is not only characterized by a nonhomogeneous flow distribution,
but also, with few exceptions, by a low flow rate as compared with the flow rate of
the normal tissue of origin. Furthermore, the blood flow rate in most tumors decreases
rapidly with increasing tumor growth. This decrease of tumor blood flow runs parallel
with the development of central necroses in growing tumors.
ACKNOWLEDGMENT

REFERENCES
1. Ribbert, H., Uber das Gefáss-system und die Heilbarkeit der Geschwülste, Dtsch.Med. Wochenschr.,
30,801, 1904.
1961.
3. Wartnaby, K. M., Bouchier, I. A. D., Pope, C. E., II, and Sherlock, S., Hepatic blood flow in
patients with tumours of the liver, Gastroenterology, 44, 733, 1963.
4. Gullino, P. M., Extracellular compartments of solid tumors, in: Cancer. A comprehensive treatise,
Vol. 3. Becker, F., Ed., Plenum Press, New York, 1975, chap. 12.
5. Vaupel, P., Interrelationship between mean arterial blood pressure, blood flow and vascular resist-
ance in solid tumor tissue of DS-carcino-sarcoma, Experientia, 31, 587, 1975.
6. Rogers, W., Tissue blood flow in transplantable tumors of the mouse and hamster, Diss. Abstr. Int.
B, 28,5185, 1968.
7. Takács, L., Debreczeni, L. A., and Far sang, Cs., Circulation in rats with Guérin carcinoma, Appl.
Physiol., 38, 696, 1975.
transplanted tumors, / . Natl. Cancer Inst., 29, 389, 1962.
9. Allen, N., Goldman, H., Gordon, W. A., and Clendenon, N. R., Topographic blood flow in exper-
imental nervous system tumors and surrounding tissues, Trans. Am. Neurol. Assoc, 100, 157, 1975.
10. Kjartansson, I., Tumour circulation. An experimental study in the rat with a comparison of different
methods for estimation of tumour blood flow, Acta Chir. Scand. Suppl., All, 1976.
11. Oikawa, M. and Milne, E. N. C , A morphological and quantitative study of tumor blood flow
during growth and immune rejection, Tohoku J. Exp. Med., 112, 325, 1974.
12. Gelin, L.-E., Lewis, D. H., and Nilsson, L., Liver blood flow in man during abdominal surgery,
Acta Hepato-Splenol., 15, 13, 1968.
13. Plengvanit, U., Suwanik, R., Chearanai, O., Intrasupt, S., Sutayavanich, S., Kalayasiri, C , and
Viranuvatti, V., Regional hepatic blood flow studied by intrahepatic injection of 133Xenon in normals
and in patients with primary carcinoma of the liver, with particular reference to the effect of hepatic
artery ligation, Aust. N.Z.J. Med., 1, 44, 1972.
14. MgntylS, M., Kuikka, J., and Rekonen, A., Regional blood flow in human tumours with special
reference to the effect of radiotherapy, Br. J. Radiol., 49, 335, 1976.
15. O'Brien, M. D. and Veall, N., Partition coefficients between various brain tumours and blood for
,33
Xe, Phys. Med. Biol., 19, 472, 1974.
16. Robert, J., Martin, J., and Burg, C , Evolution de la vascularisation d'une tumeur isologue solide
de la souris au cours de sa croissance, Strahlenterapie, 133, 621, 1967.
18. Peterson, H.-L, Appelgren, K. L., Rudenstam, C.-M., and Lewis, D. H., Studies on the circulation
of experimental tumours. I. Effect of induced fibrinolysis and antifibrinolysis on capillary blood
flow and the capillary transport function of two experimental tumours in the mouse, Eur. J. Cancer,
5,91, 1969.
20. Kjartansson, I. E., Lewis, D. H., Peterson, H.-L, Rosengren, B., and Rudenstam, C.-M., Effect of
X-irradiation on blood circulation in two experimental tumours, Microvasc. Res.,3, 438, 1971.
21. Kjartansson, I., Appelgren, L., Peterson, H.-L, Rosengren, B., Rudenstam, C.-M., and Lewis, D.
H., Capillary blood flow, transport and flow distribution in two transplantable rat tumours, Bibl.
A/iar.,12, 519, 1973.
22. Johnson, R., A thermodynamic method for investigation of radiation induced changes in the micro-
circulation of human tumors, Int. J. Radiât. Oncol. Biol. Phys., 1, 659, 1976.
23. Goldacre, R. J. and Sylven, B., A rapid method of studying tumour blood supply using systemic
dyes, Nature (London), 184, 63, 1959.
24. Owen, L. N., A rapid method for studying tumour blood supply using lissamine green, Nature (Lon-
don), 187,795, 1960.
25. Sapirstein, L. A., Regional blood flow by fractional distribution of indicators, Am. J. Physiol., 193,
161, 1958.
26. Mellander, S. and Johansson, B., Control of resistance, exchange and capacitance functions in the
peripheral circulation, Pharmacol. Rev.,20, 117, 1968.
115
Chapter 6
IN VIVO OBSERVATIONS OF TUMOR BLOOD FLOW
Huibert S. Reinhold
TABLE OF CONTENTS
I. Introduction 115
II. Methodological Considerations 116
III. Observations of Tumor Vascular Growth 118
IV. Observations of Tumor Blood-Flow Rate 121
V. Future Aspects 126
References 127
I. INTRODUCTION
The rate of tissue blood flow is inevitably related to the microcirculatory bed. The
planning of an investigation of the vascularity of tumors is strongly influenced by the
investigator's philosophy as to how tumor growth should be restricted, or tumors
should be cured.
Obviously, if the proliferation rate of tumor cells is restricted by their vascular sup-
ply, i.e., the supply of nutrients and the removal of catabolites, one may find a very
efficient growth-limiting factor in substances that specifically inhibit tumor angioge-
nesis (Folkman1, and Warren, see Chapter 2). On the other hand, if one wants to treat
tumors with radiation or chemotherapy, the vascular supply must be optimal. In the
case of radiation therapy, the cells must have access to oxygen in order to have an
optimal radiation sensitivity. With chemotherapy, the drug of choice must, of course,
have access to the malignant cells. An optimal therapeutic effect cannot be expected
unless all tumor cells are being supplied with the required oxygen or drug, and in
consequence, the observed therapeutic effect will be strongly related to the efficiency
of the vascular supply.
Many studies, encompassing measurements of physiological parameters like tumor
blood flow and permeability of tumor blood vessels have been performed, and form,
in fact, the main essence of this volume. In addition, a great number of morphological
investigations have been performed (see Chapter 1). The consensus of these investiga-
tions is that the vascular morphology and the blood supply of tumors differ very much
from related normal structures and that there is in all cases a strong indication that
the vascular supply is very heterogeneous. This implies that the previously mentioned
therapeutic factors, i.e., the distribution of oxygen and drugs, will be heterogeneous.
This, in turn, implies that one may find therapy-responsive areas adjacent to therapy-
resistant areas, which is one of the reasons why systems have been developed to study
the dynamics of tumor vascular supply at a microscopic level.
II. METHODOLOGICAL CONSIDERATIONS
Algire was the first to construct a usable observation chamber, based on the ideas
of Sandison and Clark, in the 1920s. They constructed a small windowlike device that
could be fastened in the rabbit ear and which allowed the observation of the flow of
blood cells in the blood vessels. Algire recognized the value of such a method and
designed a similar chamber, but now for the mouse.2 Rather than taking the ear with
its supportive core of cartilage as the site for the window, Algire positioned the window
in a skinfold on the back of the mouse. In order to protect the window, which is in
essence a small-size coverslip attached to the skin only, Algire introduced an external
splint. This splint consisted of two similarly sized sheets of plastic, one for each side.
The sheets were bolted together through the skinfold. The latter is stretched and is
kept in place by sewing it to the external rim of the splint. Many useful studies have
been performed on tumors of the mouse. Adaptation of the afore-mentioned systems
was performed by Goodall et al., 3 Warren et al., 45 and Eddy6 for use in the cheek
pouch of the hamster. This cheek pouch consists mainly of a mucous membrane, and
a light-transmitting rod can be inserted into it for transillumination. By replacing the
skin covering of the cheek pouch with a suitable "window" system, the thin mucous
membrane becomes directly visible. Tumor fragments can be inserted, and the growth
of the tumor and development of its vascular system can be followed. While the sys-
tems mentioned in this paragraph allow the observation of the development of the
vascular system, one interfering factor should be emphasized. It concerns the fact that,
once the tiny piece of tumor tissue that was inserted has "established" and starts grow-
ing, its thickness increases with increasing tumor diameter, and within a few days the
smaller capillaries in the tumor center become blurred. Here it should be noted that
the trick of all such microcirculation studies lies in the fact that the optical transpar-
ency of homogenous, living tissue is sufficient for microscopic examination to a depth
of 0.1 to 0.2 mm. If the tissue thickness exceeds this value, visual observation is seri-
ously handicapped.
This limitation of the possibility of optical observation is the reason why, for the
mouse and rat skin, Yamaura7"9 and the present author1*-11 have designed systems for
constraining tumors in "observation systems" to a "sheetlike" growth pattern. Figure
1 serves as an example of the way such preparations are made. For the mouse, this
was accomplished by separating a "nerve cord" in the subcutis containing blood ves-
sels and letting the tumor grow in a space where thickness is restricted by a mica sepa-
rator.10 In the rat, two systems are presently in use. The one designed by Yamaura is
based on an external frame which is rigid enough to keep the front window and back
disc separated at about 50 /nm to 100 /^m (Figure 1). The other one employs a large
s.c. implanted sheet of mica to which a window system is fastened. Both systems are
quite similar in the sense that they use a thin layer of s.c. tissue in which the microcir-
culation is carefully left intact during the various surgical procedures. This serves for
a pre-existing vascular "tumor bed" (Figure 2). The advantages of using such thick-
ness-restricted chambers are, among other things, that the flow in virtually all blood
vessels can be studied and that a number of physiological determinations can be made.
Moreover, they approximate a cross section through the equatorial plate as closely as
is possible. In this way, it is possible to distinguish differences in vascular structure,
for example, in vivo in "peripheral" versus "central" areas. One word of caution,
however. Although the structure of a tumor, as judged from observations and/or pho-
117
FIGURE 1. Typical example of the sequence of procedures for the installation of

a thickness-restricted chamber. (From Yamaura, H., Suzuki, M., and Sato, H.,
Gann, 62, 177, 1971. With permission.)
tographs, may give the impression of being a sheetlike two-dimensional structure, the
thickness of such tumors in reality ranges between 50 /urn and 200 jum. This means that
not all vessels will run parallel with the plane of the window. This may also have
implications when one attempts to infer diffusion rates etc. from two-dimensional
analyses of images. The previously mentioned methods or in vivo observation of tumor
blood flow require highly refined methods of preparation, transplantation, and meas-
urement. Vascular architecture as such can be very adequately studied in fixed tissue
by means of cleared thick sections (e.g., see Hilmas and Gilette12) or with reconstruc-
tion methods from serial sections.13 Obviously, the fragile and laborious in vivo prep-
arations are therefore only indicated if by the use of them certain dynamic processes
can be analyzed. Examples of these are the factors involved in tumor growth, the
velocity of the erythrocytes in the capillaries, and the topical distribution of blood flow
and/or oxygenation.
118 Tumor Blood Circula tion
FIGURE 2. A "sandwich" tumor based on a modification of the

Algire "access-type" chamber. (From Reinhold, H. S., Eur. J. Can-
cer, 7, 273, 1971. With permission.)
III. O B S E R V A T I O N S O F T U M O R V A S C U L A R G R O W T H
All methods, in effect, first encompass the preparation of a vascular bed, and after
that, the implantation of a small piece of tumor. An excellent description of the se-
quence of events after inoculation and transplantation can be found in Chapter 2.
With regard to the in vivo observations, it should be noted that nonviable tumor has
an opaque appearance, while viable tumor is relatively translucent. The transplanted
piece of tumor invariably appears opaque, and therefore, the majority of the tumor
cells must be nonviable. After a few days, vascular growth begins in venous capillaries
of the draining bed. In ensuing days, a capillary bed develops which becomes supplied
from the arterial side. The tumor tissue in the direct vicinity of the circulating capillar-
ies becomes clear, indicating the presence, and growth, of viable tumor cells. For a
very good description of this effect see Kligerman.14
Once the capillaries are being supplied with arterial blood, the central, translucent,
viable tumor area begins to expand, and generally, the opaque areas are rapidly re-
placed by translucent tumor tissue. In addition, during the following few days, the
tumor, which has now been definitely established, grows with an average increase of
about 0.5 to 1 mm/day. Of course, this growth rate depends upon the type of tumor,
but probably is also partially dependent upon the site of transplantation. Most impor-
tant, however, is the temperature at which the tumor is maintained. With regard to
the latter, it should be recognized that a tumor, growing in a situation in which one,
or even two of the surfaces enclosing it consist of glass "windows", has a temperature
much below the core temperature of the host animals. When the temperature of the
tumor drops below about 32°C, its growth rate becomes very low. Therefore, the hosts
carrying "observation windows" have to be kept at elevated temperatures, e.g., envi-
ronmental temperatures of 35 to 36°C. The same applies, of course, for physiological
assay determinations.
If one follows the growth of a tumor in an observation chamber, especially in a
chamber of the "flat" type, one becomes aware of the peculiar pattern of the vascular
development. Humans probably tend to assume erroneously that the growth of a tu-
mor proceeds from a small nucleus in a similar way to the growth of a city. The latter
can be seen as an expanding structure of consuming units (houses or people) where
expansion is accompanied by an extension of the supply lines (streets). Once a part is
established, it remains unchanged, and the growth of a city takes place via apposition.
119
The development of the vascular system of a tumor, however, proceeds along entirely
different lines in the first place. The tumor probably captures the pre-existing vessels
of the organ in which the tumor is located. Arteries become wider and veins become
dilated, with a curved course. In contrast to the way we are used to seeing a town
developing, a growing tumor, with its large number of growing foci, pushes large parts
of the vascular system aside. Moreover, new vessels appear at one site, while, at other
sites, vessels disappear. This means that, for a given area, the vascular supply system
can have an entirely different appearance from one day to another. In our "sandwich
tumors", we gained the strong impression that increased tissue pressure is instrumental
in the visual disappearance of vessels, while many of the "new" vessels simply develop
from pre-existing collapsed capillaries or tissue interspaces. Our experience confirms
the observation by Eddy and Casarett 6 that the blood flow in a large number of capil-
laries in the tumor can be restored by releasing the pressure. Tissue pressure in this
instance is the result of tumor cell proliferation, and pressure release can be obtained
by lifting of the window. In fact, this mechanism points to the "tense" tumor concept
ofFalk 15 .
When a tumor continues its growth, its center may become necrotic. In the type of
observation chambers that make use of spherical tumors, one can usually just observe
that the center becomes blurred. In the "flat" type of observation chambers, devel-
opment of necrosis is more easily recognized as there is an obvious difference in ap-
pearance between the translucent, viable, tumor tissue and the opaque necrotic tissue
(Figure 3). It should, however, be noted that it requires a high level of technical skill
to produce "flat" tumors with a necrotic center in a reproducible manner. The reason
for this is that for most tumors a tumor size of about 6 mm is required before central
necrosis develops. The time elapsed after inoculation is in the order of 3 weeks, while
the chamber is about one month old. Over this period the fragile, 50 to 200 ¿mi-thick
"chamber" and its supportive skin structure have "aged" considerably and are subject
FIGURE 3. Central necrosis in an experimental rat rhabdomyosarcoma growing in a "sandwich" tumor.

to greatly increased risks of tearing, fibrosis, and infection. Once such a necrosis de-
velops, it is almost invariably secondary to a decreased microcirculatory flow in the
center of the tumor. In some tumor types, the necrotic part is surrounded by a rim
with dilated sinusoids, indicating pressure effects (Figure 3).
In this context, the observation by Falk15 should be mentioned. Falk divided the
tumors he investigated in two types, the "tense" tumors and the "lax" tumors. This
very useful subdivision is based on whether or not the central part bulges up (slightly)
when tumors are cut in two parts. While the "tense" tumors have a circulatory system
that supplies the tumor tissue from the periphery into the center, the circulation of the
"lax" tumor seems to have its vascular system originating in the central part. The
tumor RIBs, as described by Falk, shows what one might expect from a "tense" tumor.
Deep in the tumor tissue venous sinusoids were apparent, and tumors exceeding 7 mm
in diameter showed central necrosis due to venous infarction. It should be noted here
that for the "observation" type of preparations 7 mm is a large diameter indeed. The
previously mentioned mechanism of causing a central necrosis through increased tissue
pressure and sinusoidal pressure in developing sinusoids which encompass an area
without capillary flow, has been definitely observed in "window" preparations, but
only in relatively large tumors.
The widening of capillaries in the vicinity of tumor transplants14 or in the tissue
surrounding the growing tumors15 is well known. In effect, this characteristic is even
being used for diagnostic purposes. With the aid of long-distance illumination and
magnification systems, the so-called colposcope, Kolstad and Bergsj016 not only de-
scribed the various patterns of tumor vascularity in the cervix uteri of patients, but in
addition, performed quantitative determinations on the distances between capillaries.
Their findings can be summarized as follows: the more malignant the tissue, the more
abnormal the blood vessels (on the surface) of the tumor. The distance between the
capillaries was larger in tumors than in the normal cervix epithelium. When the inter-
capillary distance exceeded the 300 /um, a high proportion of the tumors were malig-
nant.17 Bergsjo recognized two types of tumor; one with predominantly "branching"
vessels, and one with complete loops during radiation treatment. This indicates that
there may very well be a correlation between tumor vascularity (oxygenation!) and
response to radiation treatment.
Histological preparations, "whole mount" preparations, or even observations in
"window" systems do not indicate velocity or direction of flow. Falk, however, has
inferred from his laborious reconstruction system the direction of flow in a tumor
bed.15
Another example is given in Figure 4. The "window" system used here is the mouse-
sandwich system, depicted in Figure 2. The tumor is in this instance the mouse mam-
mary tumor C3HBA, inoculated in a nerve-cord. It is obvious that, even in such a
small tumor, there is an almost complete lack of a systemic pattern of circulation. The
rim consists almost completely of venous sinusoids, but in the center parts a number
of sinusoids are also visible. The main arterial supply vessel (coming from the left in
this diagram) is widened, but one of its major connecting tumor vessels also has a
sinusoidal appearance. It is in these vessels that two different types of flow can be
observed, as so clearly described by Falk.15 In addition to the direct connection with
intratumoral wide vessels, the artery gives off a large number of fine branches, some
of which run with a very high flow rate directly into a "venous" system (double ar-
rows). The main conclusion one can draw from this picture is that the general pattern
of circulation is plainly chaotic and that, on this basis only, one can expect serious
heterogeneity in the supply of oxygen and nutrients in tumors. This is even more im-
portant, of course, once a tumor grows to sizes over 10 mm, rather than in the 2 mm
tumor shown in Figure 4.
121
FIGURE 4. Diagram of the microcirculation of a mouse mammary carcinoma growing in a ''sand-

wich' ' tumor. Note the shunts (double arrows) and the many wide sinusoidal vessels. Picture derived
from photographs, with subsequent registration of the in vivo blood flow. (From Reinhold, H. S., Eur.
J. Cancer,!, 273, 1971. With permission.)
One should realize that the efficiency of the supply of nutrients and oxygen must
keep pace with the metabolic level at which they are consumed.18 If the supply rate
falls short, this will invariably result in foci of tumor cells which are deficient of some
of the metabolites.19 The same holds for the removal, or accumulation, of catabo-
lites.20
IV. OBSERVATIONS OF TUMOR BLOOD-FLOW RATE
In this context, it is of obvious interest to analyze the velocity and flow-distribution

pattern in tumor blood vessels. Two approaches to this problem have been made until
now, time analysis studies on microangiograms and the use of dynamic high-resolution
image-analysis techniques for the measurement of flow rate in tumor capillaries.2122
Angiographic methods used in tumor microcirculation studies are similar to the flu-
orescent angiographic techniques used in opthalmology.23 X-ray angiography with a
radio-opaque contrast is frequently used for diagnostic purposes. This method is, how-
ever, not suitable for dynamic microcirculation studies. The reason is that the resolu-
tion of the X-ray films is rather poor at the "micro" level and that the focal spot of
an X-ray tube is relatively large. This means that for analysis at the microscopic level
optical systems are generally preferred. In principle, there is the choice between light-
absorbing and light-emitting dyes. In practice for repeated "in vivo" determinations,
the customary microscopic systems are unsuitable. Light-absorbing substances gener-
ally consist of colloidal suspensions, e.g., carbon particles. A considerable concentra-
tion of such material is required to obtain sufficient differential contrast between the
tissue parenchyma on the one hand and the blood stream which is marked with the
pigment on the other. The pigments most commonly in use are mostly nontoxic vari-
eties of India ink. Other types of (industrial) colloidal carbon suspensions, or copper-
containing organic paint pigments can, however, sometimes be employed. A disadvan-
tage common to all of them is the lack of ability of the body to adequately excrete
pigments. Rather then being excreted, they are removed from the blood stream by
various types of phagocytic cells. This causes an accumulation in these cell types. In
the case of experimental tumors, the pigment tends to penetrate in the semiviable rim
of cells that is located between the necrotic parts and the viable tumor tissue. In this
way, a picture emerges that has been described by Goldacre and Sylvén.24
These effects can, however, be completely avoided when one uses water-soluble dyes
that do not bind in the body. Fluorescent dyes are generally preferred because they
combine the advantages of a high contrast rate with a high (light)' yield at very low
concentrations. This means that for ophthalmological examinations fluorescent an-
giography is generally used. 23 Fluorescein does not bind to proteins, and therefore, the
bolus of the fluorescein solution, used for angiography, is readily excreted. Moreover,
the dye has a low toxicity, which explains its extensive use in ophthalmology.
Similar studies of the flow patterns in the mesentery of experimental animals have
been performed by Witte. 25 He observed a discrete network of extravasation, i.e., the
dye left the vessels in a well-defined pattern.
If one wants to investigate some of the complex permeability functions of the micro-
vascular system, the use of a dye that binds to macromolecules is indicated. Evans
blue is such a dye, and it has been used to examine radiation-induced increased vascular
permeability in frozen sections.26 However, for investigations with serial angiography,
nonbinding dyes which can be used in high concentrations are indicated. An example
of a serial microangiogram in a mammary tumor in the mouse is presented in Figure
5. The tumor is the same as the one depicted in Figure 4, and it is, therefore, possible
to compare the characteristics of the microcirculatory flow with the angioarchitecture.
The dye used in this instance is the water-soluble, nontoxic dye "pyranin" which can
be injected i.v. with a solution concentration as high as 10%.
The dye is miscible with the plasma, and there may be differences in velocity between
erythrocytes and the plasma. It therefore seems better in this type of investigation to
speak about the flow rate of the blood plasma in the blood vessels rather than about
the velocity of the blood as such. From this serial microangiogram, one notices that
there is a very high rate of influx through a widened arterial-type vessel during the
first half sec. At about 1 sec, the majority of the vessels have not yet become fluores-
cent, while at the same time the fluorescent plasma has already reached some of the
draining veins. At 1.5 sec, most of the larger tumor vessels are visible, while the shunts
(indicated with double arrows in Figure 4) have become very obvious. At 2 and 3 sec,
there is a further filling of the vessel network, with some extravasation.
For the purpose of studying the response of the tumor microcirculatory system to
irradiation, a series of microangiograms of the type depicted in Figure 5 have been
quantified.10 The possibilities for quantification of such pictures is restricted by the
fact that the fluorescence intensity as it appears on the film cannot be considered as
an objective measure of the amount of dye present. While the quantitative-intensity
approach is feasible in principle, in practice there are too many unknown variables.
Examples of these are fluctuations in the UV-light intensity, reflections at the coverslip
and/or interfaces, and most of all, the extremely steep contrast and high film sensitiv-
ity, together with unknown variables in the processing procedures. Therefore, the pre-
viously mentioned angiographic films were quantified by visual means, i.e., selected
frames at about 0.5 sec intervals were projected on paper, and the progress of the dye
in the vessels was outlined by pencil. Using different colors for different time intervals
facilitated the subsequent analysis. The quantitative analysis consisted of following the
tracings with a simple map measurer. In this way, it was found that the velocity of
the blood plasma decreased while traversing the tumor (Figure 6). It was estimated
FIGURE 5. Serial microangiogram from the tumor shown in Figure 4. Note the sites of arterial inflow, shunting, extravasation, and the heterogeneity in the distribution of the
dye.
123
FIGURE 6. Quantification of a series of microangiograms. Note that the velocity of the plasma de-
creases while traversing the tumor. (From Reinhold, H. S., Eur. J. Cancer, 7, 273, 1971. With permis-
sion.)
that the mean flow rate during the first sec of its passage was of the order of 650 /¿m/
sec, and during the next few seconds about 300 /um/sec. Considerable differences in
flow rate were encountered, even in adjacent capillaries.
These values are comparable with the ones obtained by Endrich on the RBC velocity
in an experimental rhabdomyosarcoma by means of videoimage analysis. 21222728 En-
drich derived values for RBC velocity on the arterial side ranging from 0.94 mm/sec
to 0.44 mm/sec, depending on the site of the tumor. For capillaries, these values were
between 0.30 mm/sec and 0.09 mm/sec, while the venous side also presented flow rates
ranging from 0.09 mm/sec to 0.36 mm/sec. Taking this data into account, it seems
that the velocity of the blood in single vessels in a tumor can show differences of a
factor of 10. This is assuming that the vessels of interest show consistency in their flow
pattern. Some doubts have arisen recently about the latter. This point will be discussed
later. Endrich et al. have, on the basis of the methods developed by Intaglietta and
Zweifach,29 determined the perfusion rates for various areas in the tumor. The perfu-
sion rate was shown to differ for the various sites in the tumor tissue, but also de-
pended upon the tumor size and the period of time the tumor tissue had been growing.
The perfusion rate of RBC per unit tissue was found to vary between almost zero
values to values of 40 mi/min/100 g. There was a strong tendency for a lower perfu-
125
sion rate in seminecrotic areas compared with areas of "established microcirculation".

These determinations show unambiguously that it is impossible to assign a single value
to a tumor for its blood perfusion rate. One, rather, has to take into account the fact
that the perfusion rate shows tremendous differences between the various areas. The
centrally located, seminecrotic areas have the lowest perfusion rate. This heterogeneity
in tumor circulation is also readily observed in Figure 5, although the size of this ex-
ample did not exceed 2 mm diameter. It has been known for a long time that, due to
a combination of intra- and extravascular pressures and differences in osmotic pres-
sures, fluid may leave the capillary network on the arterial side of a capillary network
and is reabsorbed on the venous side.30
This mechanism has been studied most extensively by means of either fluid-balance
(isogravimetric) methods in isolated limbs,31 or with radioisotopes (see Chapter 3).
With regard to the extravascular fluid distribution in tumors, Figure 5 is of interest.
Here one will note that in some areas in the tumor tissue there is a very rapid extrava-
sation, while in others the extravasation rate is very low and resembles a physical dif-
fusion process. The extravasation velocity of the fluorescent dye, as depicted in Figure
5, was found in a large number of measurements to range between 25 ¿¿m/sec to values
lower than 1 jLtm/sec.10 There can be little doubt that the intensity of the extravascular
fluid transport depends to a large extent on differences in intravascular pressure in the
various parts of the microcirculatory network, with possibly some additional factors
involved that may restrict diffusion. Such factors may be found in the permeability
characteristics of the vessel wall, but the composition of the extravascular (interstitial)
spaces will also be of influence. Moreover, one should consider the possibility that the
rate of interstitial fluid transport may be impaired when the interstitial tissue pressure
is increased. The latter will be rather high due to the proliferative properties of tumor
cells, causing intratumoral expansion. For a mathematical treatise of the factors in-
volved, see Chapter 10.
Some of the factors that tend to escape the attention of investigators are fluctuations
of blood flow, and tissue perfusion over time. This occurs because neither angio-
graphic methods nor the classical type of determinations of tissue blood flow have a
high enough resolving power to recognize heterogeneities in time as well as in place.
In fact, only during recent years have methods been developed that are sufficiently
sensitive to demonstrate time-dependent fluctuations.32,33 Fluctuations in the perfusion
rate of capillaries have been demonstrated,34 but fluctuations in the oxygenation of
the cortex cerebri of the cat32 have also been demonstrated. This seems to indicate that
one should not see a microcirculatory bed as a system in which every part is continu-
ously and equally perfused, but rather as a dynamic structure in which the supply to
the various parts is subjected to continuous adaptation.
In tumor microcirculation studies, fluctuations in the blood supply have also been
encountered. Intaglietta et al.28 encountered two types of periodic changes in flow
(Figure 7). One had a cycle time of 2 to 3 min, which was interpreted as secondary to
vasomotor activity, and there was a very short one caused by the respiratory activity.
In investigations of NAD(H) fluorescence as an indicator for the oxygenation of tumor
tissue, fluctuations were sometimes also observed.11 It should be noted, however, that
in the latter case the frequency of occurrence of such fluctuations was rather low, and
was found in somewhere around 10% of the sites investigated. It is, therefore, pres-
ently rather difficult to estimate its importance for the oxygenation status of tumor
tissue, which is so important for its sensitivity to radiation. With regard to the latter,
the observation by Zanelli35 should be noted. He found that the circulation (i.e., the
fraction of the cardiac output) of several mouse tumors increased when the animals
were anesthetized. This might be interpreted as a potential increase in radiosensitivity.
FIGURE 7. Record of RBC velocity in a 18 \xm arteriole supplying the microvasculature of a "sandwich"
tumor. Two types of periodic changes in flow are discernible. The first is a long period of 2 to 3 min duration
which is associated with vasomotor activity. A second type of fluctuation of 4 to 5 seconds duration is
superimposed on the former, and is due to the respiratory cycle. Smaller perturbations in the flow tracing
are probably due to electrical and hydraulic noise. (From Intaglietta, M., Myers, R. R., Gross, J. F., and
Reinhold, H. S., Bibl. Anat., 15, 273, 1977. With permission.)
Also, the findings by Hornsey 36 that the skin of the mouse is more sensitive during
anesthesia may have the same background. The function of vasoregulatory mecha-
nisms or periods with vasoconstriction may indeed occur less often during anesthesia.
A good treatise on this subject is given by P. C. Johnson. 34 Various substances, like
oxygen or metabolites, are considered to have roles as vasoconstrictive stimuli. That
cooling can also act as an additional vasoconstrictive stimulus has recently been shown
byR. J. R.Johnson. 3 7
V. F U T U R E A S P E C T S
Obviously, our main interest in the microcirculation in tumors will always be di-
rected to unravelling those factors that are of importance for efficient tumor treat-
ment. For many years, this interest has been focused on the mechanisms involved in
the distribution of oxygen (see Chapter 9). Future possibilities in this field encompass
the promising fluorescent reagents, such as pyrene butyric acid,38 for use in the in vivo
determination of tissue oxygenation. More recently, much attention has been paid to
the distribution of chemotherapeutic agents (see Chapter 10). V ;ry recently, a renewed
interest in the distribution of heat in tumors has been shown. This is because of the
quite sudden revival of interest in the application of heat as a therapeutic modality for
tumors. 39 4 0 4 1 4 2 One of the many ideas behind this is that the center of a tumor will
build up a relatively high temperature during heating with electromagnetic energy.
Based on this philosophy, it might be possible to develop therapeutic methods which,
rather than compensating for, may be aimed at exploiting insufficiencies of the tumor
microcirculation.
127
REFERENCES
1. Folkman, J., Merler, E., Abernathy, C , and Williams, G., Isolation of a tumor factor responsible
for angiogenesis, J. Exp. Med., 133, 275, 1971.
2. Algire, G. H., An adaptation of the transparent chamber technique to the mouse, / . Natl. Cancer
Inst. A, 1, 1943.
3. Goodall, C. M., Sanders, A. G., and Shubik, P., Studies of vascular patterns in living tumors with
a transparent chamber inserted in hamster cheek pouch, J. Natl. Cancer Inst., 35, 497, 1965.
transplantable melanomas of the hamster. I. In vivo observations in transparent chambers, Cancer
Lett., A, 109, 1978.
transplantable melanomas of the hamster. II. Scanning electron microscopy, Cancer Lett., 4, 117,
1978.
6. Eddy, H. A. and Casarett, G. W., Development of the vascular system in the hamster malignant
neurilemmoma, Microvasc. Res.,6, 63, 1973.
7. Yamaura, H. and Sato, H., Quantitative studies on the developing vascular system of rat hepatoma,
J. Natl. Cancer Inst., 53, 1229, 1974.
8. Yamaura, H., Suzuki, M., and Sato, H., Transparent chamber in the rat skin for studies on micro-
circulation in cancer tissue, Gann, 62, 177, 1971.
9. Yamaura, H. and Sato, H., Experimental studies on angiogenesis in AH 109A ascites tumor tissue
transplanted to a transparent chamber in rats, in Chemotherapy of Cancer Dissemination and Metas-
tasis, Garattini, S. and Franci, G., Eds., Raven Press, New York, 1973.
10. Reinhold, H. S., Improved microcirculation in irradiated tumours, Eur. J. Cancer,!, 273, 1971.
11. Reinhold, H. S., Blachiwiecz, B., and Blok, A., Oxygenation and reoxygenation in "sandwich"
tumours, Bibl. Anat., 15, 270, 1977.
12. Hilmas, D. E. and Gillette, E. L., Microvasculature of C3H/BÍ mouse mammary tumors after X-
irradiation, Radiât. Res., 61, 128, 1975.
13. Hilmas, D. E. and Gillette, E. L., Tumor microvasculature following fractionated X-irradiation,
Radiology, 116, 165, 1975.
imental tumor, Radiology, 76, 810, 1961.
to tumour responses to single large doses of radiation, Eur. J. Cancer, 14, 237, 1978.
16. Bergsjo, P., Radiation-induced early changes in size and vascularity of cervical carcinoma. A colpo-
photographic and clinical study, Acta Radiol. Suppl.,214, 1968.
17. Kolstadt, P., The development of the vascular bed in tumours as seen in squamous cell carcinoma
of the cervix uteri, Br. J. Radiol., 38, 216, 1965.
18. Thomlinson, R. H. and Gray, L. H., The histological structure of some human lung cancers and the
possible implications for radiotherapy, Br. J. Cancer, 9, 539, 1955.
19. Boag, J. W., Oxygen diffusion in tumour capillary networks, Bibl. Anat., 15, 266, 1977.
20. Tannock, I. F., Oxygen diffusion and the distribution of cellular radiosensitivity in tumours, Br. J.
Radiol., 45, 515, 1972.
21. Intaglietta, M. and Tompkins, W. R., One-line microvascular blood cell flow velocity measurements
by a simplified correlation technique, Microvasc. Res., 4, 217, 1972.
22. Intaglietta, M., Silverman, N. R., and Tompkins, W. R., Capillary flow velocity measurements in
vivo and in situ by television method, Microvasc. Res., 10, 165, 1975.
23. Wessing, A., Fluoreszenzangiographie der Retina (Lehrbuch und Atlas), Georg Thieme Verlag, Stutt-
gart, 1968.
24. Goldacre, R. J. and Sylvén, B., On the access of blood-borne dyes to various tumour regions, Br. J.
Cancer, 16, 306, 1962.
25. Witte, S., Flow pattern pertaining to vascular permeability as observed by fluorescence vital micros-
copy, in 4th Int. Congr. Rheology, John Wiley & Sons, New York, 1965, 451.
26. Lundborg, G. and Schildt, B., Microvascular permeability*in irradiated rabbits, Acta Radiol., 10,
311, 1971.
27. Endrich, B., Reinhold, H. S., Gross, J. F., and Intaglietta, M., Tissue perfusion inhomogeneity
during early tumor growth, / . Natl. Cancer Inst., Submitted for publication, 1978.
in implanted mouse mammary tumours, Bibl. Anat., 15, 273, 1977.
29. Intaglietta, M. and Zweifach, B. W., Microcirculatory basis of fluid exchange, Adv. Biol. Med.
Phys.,15, 111, 1974.
30. Starling, E. H., in Principles of human physiology, Churchill, London, 1949, 687.
31. Landis, E. M. and Pappenheimer, J. R., Exchange of substances through the capillary walls, in
Handbook of Physiology, Section 2, Vol. 2, Hamilton, W. F., Ed., American Physiological Society,
Washington, D.C., 1963, 961.
32. Leniger-Follert, E., Lubbers, D. W., and Wrabetz, W., Regulation of local tissue P 0 2 of the brain
cortex at different arterial 0 2 pressures, Pfluegers Arch., 359, 81, 1975.
33. Johnson, P. C. and Wayland, H., Regulation of blood flow in single capillaries, Am. J. Physiol.,
212, 1405,1967.
34. Johnson, P. C , Landis award lecture. The myogenic response and the microcirculation, Microvasc.
Res., 13, 1977.
35. Zanelli, G. D. and Lucas, P. B., Effect of stress on blood perfusion and vascular space in trans-
planted mouse tumours, Br. J. Radiol.,49, 382, 1976.
36. Hornsey, S., Myers, R., and Andreozzi, U., Differences in the effects of anaesthesia on hypoxia in
normal tissues, Int. J. Radiât. Biol.,32, 609, 1977.
37. Johnson, R., Fowler, J. F., and Zanelli, G. D., Changes in mouse blood pressure, tumour blood
flow, and core and tumour temperatures following nembutal or urethane anesthesia, Radiology, 118,
697,1976.
38. Mitnick, M. H. and Jobsis, F. F., Pyrenebutyric acid as an optical oxygen probe in the intact cerebral
cortex, / . Appl. Physiol.,41, 593, 1976.
39. Dietzel, F., Mbglichkeiten und Grenzen der Hyperthermie-Behandlung maligner Tumoren aus gegen-
wiártiger Sicht. Roentgen Bl., 28, 185, 1975.
40. Cater, D. B., Silver, I. A., and Watkinson, D. A., Combined therapy with 220 kV roentgen and 10
cm microwave heating in rat hepatoma, Acta Radiol. Ther. Phys. Biol.,2, 321, 1964.
41. Miller, R. C , The potential of localized heating as an adjunct to radiation therapy, Radiology, 116,
433,1975.
42. Robinson, J. E., Wizenberg, M. J., and McCready, A., Radiation and hyperthermal response of
normal tissue in situ, Radiology, 113, 195, 1974.
129
Chapter 7
T U M O R VESSEL I N N E R V A T I O N A N D I N F L U E N C E O F V A S O A C T I V E
DRUGS ON TUMOR BLOOD FLOW
Jan Mattsson, Lennart Appelgren, Bertil Hamberger, and Hans-Inge Peterson
TABLE OF CONTENTS
I. Introduction 129
II. Innervation of Tumor Vessels and Host Vessels Related to Tumors 129
III. Influence of Vasoactive Drugs on Tumor Blood Flow 132
IV. Influence of Anesthesia on Tumor Blood Flow 133
V. Arteriovenous Shunting in Tumors 134
VI. Summary 134
Acknowledgment 134
References 134
The principal change of the vascular bed in malignant tumors during growth is a
change from a fine caliber, fairly uniform capillary bed in small tumors to dilated
sinusoidlike vessels in larger tumors. During the growth of tumors, some vessels be-
come stretched, other tortuous, and new vessels appear as others are occluded by the
growing tumor-cell mass. The morphology of the vessel wall in different tumors seems
to vary with age and histologic type of tumor, and the degree of arteriovenous shunting
also varies between different tumors. From this, one might expect a wide distribution
of the tumor blood-flow rate in various tumors. An influence of vasoactive drugs on
tumor blood flow might be dependent upon the incorporation of normal contractile
vessels in the growing tumor mass and upon the degree of connection between tumor
vessels and normal vessels in the transplantation area.
II. I N N E R V A T I O N O F T U M O R VESSELS A N D H O S T VESSELS

RELATED TO TUMORS
Gullino 1 stated that the network of vessels in tumors is constituted both by newly
formed and by already existing vessels, and that host vessels surviving in the tumor
usually retain, at least in part, the contractile and nervous apparatus which makes
them responsive to physiological stimuli. Intaglietta et al. 2 studied a rat rhabdomy-
osarcoma by the Algire chamber technique and found that arteriolar vessels were in-
corporated in the tumor mass without apparent structural changes. Falk 3 examined
the angioarchitecture of different rat tumors by microangiography. In one fibrosar-
coma, both veins and arteries retained a muscular wall until the branching became
very fine. In two other tumors, the intratumor blood vessels had no obvious muscle
layer.
We have studied the vascular bed of two transplantable rat tumors, a hepatoma and
a 20-methylcholanthrene-induced sarcoma, by a microangiographic technique, perfus-
ing the tumors with a suspension of fluorescent particles for about 15 sec before sac-
rificing the animals. 4 With this technique, a large amount of tumor vessels were visu-
alized. The adrenergic innervation of the vessels in tumors transplanted i.m. or into
the anterior chamber of the eye was studied by a histochemical technique for visualiz-
ation of catecholamines. In the borderline between muscle and infiltrating tumor, mus-
cle vessels, mainly arterioles, with adrenergic innervation were found. However, no
blood vessels with adrenergic innervation could be followed into the tumor tissue. Sim-
ilar observations were made in tumors transplanted into the anterior chamber of the
rat eye (Figure 1). In a human renal adenocarcinoma, no adrenergic innervation was
FIGURE 1. Fluorescence microphotograph of a rat hepatoma transplanted into the

anterior chamber of the eye (courtesy of Dr. Lars Olson). The tissue was treated for
visualization of catecholamines. Blood vessels with adrenergic nerves (-*) are seen, but
no nerves are found in small blood vessels in the tumor tissue. (Original magnification
x 190.)
131
found in the main part of the tumor. 22 However, large arteries infiltrated by the tumor
had a scarce adrenergic innervation. Occasionally, nerve terminals could be seen close
to these arteries which also innervated small tumor vessels (Figure 2).
The absence of adrenergic innervation of tumor vessels does not exclude the possi-
bility that these vessels have alpha receptors and might still be influenced by vasoactive
drugs. The tumor blood flow might also be modified through arterioles in the border-
line between tumor and normal tissue. In a study of a rat rhabdomyosarcoma, Intag-
lietta et al. 2 found that the number of arteries and arterioles in the whole tumor mass
was low, but arterioles with a diameter of 20 to 40 \xm showed a strong vasomotor
activity. This was found to modulate the blood flow to the tumor.
FIGURE 2. Fluorescence microphotograph of a human renal adenocarcinoma treated for

visualization of catecholamines. Very few nerve terminals are seen in the tumor, probably
innervating small tumor blood vessels (-*) and located close to arteries. Auto fluorescent gran-
ules are found in certain tumor cells (-*-*). (Original magnification x 190.)
III. INFLUENCE OF VASOACTIVE DRUGS ON TUMOR BLOOD FLOW
Systematically administered vasoactive drugs can indirectly modify the tumor blood
flow by affecting the systemic blood pressure.5 However, as the arteriolar supply to
the tumor is based on normal vessels, one might expect vasoactive drugs to modify
the tumor blood flow in the same way as in normal tissues. This was, in fact, found
by Kligerman and Henel6 in studies of a transplantable mouse carcinoma by the Algire
chamber technique. The response of the arterioles in tumor tissue to acetylcholine,
epinephrine, and a mixture of 94°/o oxygen and 6% carbon dioxide was similar to that
in normal tissue. The authors concluded that some vessels making up the tumor capil-
lary bed might be sinusoids, but that in all instances the flow through these is subject
to the control of the arteriolar blood supply.
A marked fall in the total tumor blood flow was found after i.v. injection of epi-
nephrine as studied by the venous-outflow recording technique from tumors grown
tissue isolated.7 Cater et al.8 found i.v. injection of norepinephrine and epinephrine
to induce a rapid, but temporary, fall in tumor blood flow of rat hepatomas as indi-
cated by changes of the recorded tumor oxygen tension. They also found that 5-hy-
droxytryptamine, in a dose that had little or no effect on the muscle blood circulation,
temporarily arrested the blood flow in tumors. They, therefore, suggested that tumor
vessels might be extremely sensitive to circulating amines.
Rankin et al.9 studied the effect of prostaglandin E2 and epinephrine on the blood
flow of a V2 rabbit carcinoma. Prostaglandin E2 was found to increase the resistance
of the tumor vascular bed about three times; other organs showed some degree of
vasodilation, with the exception of the heart and the G.I. tract. Norepinephrine in-
creased the resistance of the tumor vascular bed about 13 times. The authors concluded
that the vasculature of the V2 carcinoma might be very sensitive to norepinephrine.
Ackermann et al.10 studied a Walker rat carcinoma implanted into the liver. The
tumor vasculature was examined by a microangiographic technique using silicone rub-
ber. Most notably, it was found that epinephrine, bradykinin, and glucagon all ap-
peared to open up capillarylike vessels into the central parts of tumors. At the same
time normal vessels in the transplantation area reacted with contraction or dilation,
depending upon the agent given.
Wickersham et al.11 investigated the response of the tumor micro vasculature to epi-
nephrine, norepinephrine, propranolol, and isoproteronol. The tumor microcircula-
tion was examined by direct inspection of the most prominent tertiary arterioles and
venules. In experimental mammary tumors, blood vessels showed a progressive loss of
the ability to respond to vasoactive drugs as the tumor enlarged. This loss of respon-
siveness was more pronounced in arterioles than in venules and was more conspicuous
following topical rather than i.v. administration of drugs.
We have studied the influence of i.v. injected norepinephrine on the intratumor
blood-flow distribution in a rat 20-methylcholanthrene-induced sarcoma and a rat hep-
atoma.12 The intratissue blood-flow distribution in tumor and normal muscle was stud-
ied by an isotope technique recently described by Kjartansson.13 Norepinephrine was
found to change the distribution of tumor blood flow values significantly towards low
flow values; the same effect was found in normal muscle (Figure 3). The effect of
norepinephrine on the tumor blood-flow rate could, thus, either be due to a direct
effect on tumor blood vessels, or to an indirect effect on normal vessels in the trans-
plantation area (muscle).
In an attempt to differentiate between the reactions of the tumor blood vessels and
the surrounding normal vessels to vasoactive drugs, we have used in preliminary studies
(to be published) a modification of the plethysmographic technique described by Kjar-
133
FIGURE 3. Intratissue distribution of i.v. injected 86Rb, in °/o of given dose/g tumor (hepa-
toma) or muscle, recorded in multiple tissue biopsies. Note the change to low activity values in
both tumor and muscle after administration of norepinephrine (noradrenaline). (From Mattsson,
J., Appelgren, L., Karlsson, L., and Peterson, H.-I., Eur. J. Cancer, 14, 761, 1978. With permis-
sion.)
tansson.13 A 20-methylcholanthrene-induced rat sarcoma was transplanted into one

hindpaw of inbred Lister rats. Simultaneous measurements of the total blood flow in
both hindpaws were made under ether anesthesia. The total tumor blood flow was
calculated as the blood flow in the tumor-bearing hindpaw minus the blood flow in
the normal hindpaw. A wide distribution of resting blood flows in tumor estimated
by this technique was found. Intravenous administration of norepinephrine induced
comparatively small changes in the tumor blood flow. This was contrary to the consid-
erable decrease of flow in the normal paw. The mean arterial blood pressure in animals
was slightly elevated by norepinephrine.
By the same plethysmography technique, we have also studied the total tumor blood
flow after ischemia for 10 min, induced simultaneously in both hindpaws. A large
postischemic increase in blood flow was found in the normal hindpaw, but the calcu-
lated tumor blood flow was mostly decreased. This is contrary to the findings of Kjar-
tansson.13 He calculated the tumor blood flow, however, as the difference between the
mean flow in the normal hindpaw of a group of animals and the blood flow in the
tumor-bearing hindpaw. By this technique, an increase in tumor blood flow was found
after ischemia.
The actual findings might support the view of tumor blood vessels as comparatively
inert vessels.
IV. INFLUENCE OF ANESTHESIA ON TUMOR BLOOD FLOW
In experimental tumor blood-flow studies, the influence of anesthesia on the flow

recording has been observed. However, the reports about this influence have been
contradictory.
The blood-flow rate of tumors seems to be very sensitive to changes in systemic
blood pressure which might be induced by anesthesia. 514 In a study of a Walker rat
carcinoma, Oikawa et al. 15 primarily used sodium pentobarbital for anesthesia, but
found a very wide range of recorded tumor flow values from day to day. They later
changed to ketamine hydrochloride for anesthesia, which was found to have a lesser
influence on the recorded values. However, both an increased16 and a decreased17 tu-
mor blood-flow rate have been found after sodium pentobarbital anesthesia. From
this, it might be considered that different tumors would react differently to the same
anesthetic agent, which is somewhat confusing. This stresses the importance of contin-
uous blood-pressure registration during the recording of tumor blood flow in anesthe-
tized animals. For a discussion about the influence of anesthetics on tumor blood flow,
see also Zanelli and Fowler. 18
V. A R T E R I O V E N O U S S H U N T I N G IN T U M O R S
An arteriolar supply of tumors originates from existing normal vessels in the tumor
transplantation area. The tumor capillary bed has been found to be characterized by
shunts which by-pass large sections of it. 619 Reinhold19 noted the apparently random
way in which blood vessels and blood flow crisscross a tumor. The blood flow in
sinusoids, estimated by direct observation, was sometimes slow and sometimes fast,
especially where shunts emptied into sinusoids.
Gbthlin20 found arteriovenous shunting in human renal carcinomas, and B'licheler
and Bolt21 reported arteriovenous connections as a typical finding in human pancreatic
carcinomas. On the contrary, Oikawa et al. 15 did not find any arteriovenous shunting
in a Walker rat carcinoma.
VI. S U M M A R Y
It must be emphasized that great caution is needed in comparing the results of dif-
ferent studies using different techniques for tumor blood-flow recording. Large meth-
odological difficulties are met with in studies on the effect of vasoactive drugs on
tumor blood flow, including the differentiation between an effect on tumor blood
vessels and that on afferent normal vessels in the host tissue. Most tumor vessels are
probably inert to vasoactive drugs, but in some tumors normal reactive vessels might
be incorporated in the growing tumor-cell mass. (Also see Chapter 11).
ACKNOWLEDGMENT

REFERENCES
1. Gullino, P. M., Extracellular compartments of solid tumors, in Cancer, A Comprehensive Treatise,

Vol. 3, Becker, F. F., Ed., Plenum Press, New York, 1975, 327.
in implanted mouse mammary tumours, Bibl. Anat., 15, 273, 1977.
135
3. Falk, P., The angio-architecture of rat tumours, Bibl. Anat., 15, 245, 1977.
4. Mattsson, J., Appelgren, L., Hamberger, B., and Peterson, H.-L, Adrenergic innervation of tumour
bloodvessels, Cancer Lett., 3, 347, 1977.
5. Algire, G. H., Legallais, F. Y., and Anderson, B. F., Vascular reactions of normal and malignant
tissues in vivo. VI. The role of hypotension in the action of components of podophyllin on trans-
planted sarcomas, J. Natl. Cancer Inst., 14, 879, 1954.
imental tumor, Radiology, 76, 810, 1961.
1961.
8. Cater, D. B., Grigson, C. M. B., and Watkinsson, D. A., Changes of oxygen tension in tumours
induced by vasocontrictor and vasodilator drugs, Acta Radiol., 58, 401, 1962.
9. Rankin, J. H. G. and Phernetton, T., Effects of prostaglandin E2 on blood flow to the V2 carcinoma,
Fed. Proc. Fed. Am. Soc. Exp. Biol., 35, 297, 1976.
10. Ackermann, N. B. and Hechmer, P. A., Effects of pharmacological agents on the microcirculation
of tumor implanted in the liver, Bibl. Anat., 15, 301, 1977.
11. Wickersham, J. K. Barret, W. P., Furukawa, S. B., Puffer, W. H., and Warner, N. E., An evalua-
tion of the response of the microvasculature in tumors in C 3 H mice to vasoactive drugs, Bibl.
Anat., 15, 291, 1977.
12. Mattsson, J., Appelgren, L., Karlsson, L., and Peterson, H.-L, Influence of vasoactive drugs and
ischaemia on intra-tumour blood flow distribution, Eur. J. Cancer, 14, 761, 1978.
13. Kjartansson, I., Tumour Circulation, Acta Chir. Scand. Suppl. 471, 1976.
14. Vaupel, P., Interrelationship between mean arterial blood pressure, blood flow and vascular resist-
ance in solid tumor tissue of DS-carcino-sarcoma, Experientia, 31, 587, 1975.
15. Oikawa, M., Milne, E. N. C , Whitmore, E., Gilday, D., and Oliver, Ch., A morphological and
quantitative study of tumor blood flow during growth and immune rejection, Cancer, (Philadelphia),
35,385, 1975.
16. Zanelli, G. D., Lucas, P. B., and Fowler, J. F., The effects of anaestetics on blood perfusion in
transplanted mouse tumours, Br. J. Cancer, 32, 380, 1975.
17. Johnsson, R., Fowler, J. F., and Zanelli, G. D., Changes in mouse blood pressure, tumor blood
flow, and core and tumor temperatures following Nembutal or Urethane anesthesia, Radiology, 118,
697,1976.
18. Zanelli, G. D. and Fowler, J. F., Anaesthetics in the study of the microcirculation of tumours: pitfalls
and uses, Bibl. Anat., 15, 249, 1977.
20. Obthlin, J., Arteriovenous shunting in carcinomas evaluated by a dye dilution technique, Scand. J.
Urol. Nephrol., 11, 159, 1977.
21. Biicheler, E., Bolt, L, Frommholdt, H., and Kaufer, C , Die angiographische Diagnostik der Pan-
creas Tumoren and der Pancreatitis, Fortschr. Geb. Roentgenstr., 115, 726, 1971.
22. Hamberger, B., unpublished data, 1978.
Taylor & Francis
137
Chapter 8
IRRADIATION A N D TUMOR BLOOD FLOW
Jan Mattsson and Hans-Inge Peterson
TABLE OF CONTENTS
I. Introduction 137
II. Influence of Single-Dose Irradiation on Tumor Blood Flow . . . 137
III. Influence of Fractionated Irradiation on Tumor Blood Flow ... 139
IV. Summary 140
Acknowledgment 140
References 140
I. INTRODUCTION
Several external physical factors might influence tumor blood flow, e.g., hypother-
mia, hyperthermia, and ionizing radiation. Among these the effect of irradiation on
tumor blood flow has been most extensively studied.
Tumors probably contain about 15% hypoxic cells, and these cells are assumed to
be radioresistant. Irradiation of tumors with moderate doses is followed, at least tem-
porarily, by an increased tumor oxygenation, and also, by an increased tumor blood
flow rate. A threshold dose of irradiation for the initiation of reoxygenation of hypoxic
tumor cells has been suggested.1 Reoxygenation of hypoxic radioresistant tumor cells
after irradiation has been one basis for treatment by fractionated irradiation in the
clinic (see also Chapters 6 and 9).
The effect of ionizing radiation on tumors is generally attributed both to a direct
action on the tumor cells and to an indirect action mediated by the tumor's blood
vessel and stroma. The resistance of tumor blood vessels to irradiation compared with
tumor cells has been discussed. Normal capillary endothelium has been reported to
have a great capacity for repair after irradiation.2 3
Studies on the influence of single-dose irradiation and of fractionated irradiation
on tumor blood flow will be briefly reviewed in this chapter.
II. INFLUENCE OF SINGLE-DOSE IRRADIATION ON TUMOR BLOOD

FLOW
Song and Levitt4 studied a Walker rat carcinoma after local irradiation with single
doses of 200 to 6000 rd. The tumor vascular volume measured by 51Cr-labeled red cells
was not influenced by a dose of 200 rd, but after irradiation with doses from 500 rd,
a decrease in the volume was observed up to 12 days after irradiation. They concluded
that a decreased vascular volume in tumors after irradiation is not explained by a
transient vascular constriction, but by irreversible circulatory failure due to vascular

occlusion and stasis.
In a study on a mouse neuroblastoma, Song et al. 5 found an increased tumor vas-
cular volume 1 day after irradiation with 500 rd and a gradually decreased vascular
volume thereafter. X-irradiation with 1000 to 2000 rd reduced the tumor vascular vol-
ume gradually. Extravasation of 125I-labeled human albumin in tumor increased soon
after irradiation and then decreased. These observations were also assumed to be ex-
plained by an irradiation-induced damage to tumor vessels.
After irradiation of a SCL mouse carcinoma with 1000 or 2000 rd, the vascular
volume/g tumor measured by 51Cr-labeled red cells was temporarily increased, and
later decreased.6 The number of living tumor cells/g tumor, estimated from single-cell
preparations, decreased to a minimum three days after irradiation. The authors con-
cluded that a postirradiation reoxygenation of hypoxic cells might be due to a greater
decrease in oxygen-consuming cells than the corresponding decrease in functional vas-
cularity.
McAlister et al. 7 examined the vascular changes in five different experimental tumors
after irradiation in doses from 400 to 3200 rd by an angiographic technique. They
found that after irradiation, tumor vessels became less numerous, more tortuous and
irregular, diminished in size, tapered sharply, and ended abruptly. They also noted
that tumors that failed to regress after irradiation, or that showed a continuous
growth, had the same postirradiation pattern. The timing and doses for vascular
changes varied according to the type of tumor.
Rubin and Casarett 8 studied a Walker rat carcinoma by microangiography and
found that some days after irradiation with a single dose of 1500 rd the tumor vascular
bed was characterized by fragmentation and occlusion of fine vessels.
The proliferative capacity of cells is known to be closely correlated to the degree of
vascularity in solid tumors. 8 Thus, Song and Levitt4 concluded that a retarded growth
rate in tumors after irradiation is at least partly due to vascular damage.
In accordance with this are the results of studies on tumor blood flow in a mouse
rhabdomyosarcoma by the 133Xe clearance technique, in which a marked reduction in
tumor blood flow was found 1 to 9 days after irradiation with 2000 rd. 9
Contrary to this, Kallman et al. 10 found an increased tumor blood flow up to 7 days
after single irradiation in doses from 1000 to 4000 rd. With larger irradiation doses,
the blood flow increase tended to occur later. They suggested that an altered balance
between tumor-cell parenchyma and vascular stroma could explain their results. By a
thermodynamic technique, an increased blood flow was also found in mouse tumors
after irradiation with 1500 rd, and the increased tumor blood flow was associated with
an increased rate of tumor-cell loss. 11
Hilmas and Gilette 1213 observed that the morphological character of tumor vessels
changed towards that seen in small tumors after irradiation. A dose of 500 to 1000 rd
opened up nonfunctioning vessels, while a larger dose from 3000 to 4000 rd appeared
to damage the tumor vascular endothelium severely.
Mervin et al. 14 studied a transplantable mouse mammary carcinoma after single ir-
radiation in doses of 2000 to 5000 rd. One day after irradiation, they found a slow
blood-flow rate in some tumors and a very fast blood-flow rate in others. Two to four
days after irradiation, a generally slow blood flow was found in all tumors. Tumor
vessels became narrow early after irradiation. Later, vessels were observed in areas
where no vessels were found at the time of irradiation.
At our laboratory, Kjartansson studied the influence of local X-ray irradiation in a
dose of 3000 rd on the blood-flow rate of two transplantable rat tumors, a 20-methyl-
cholanthrene-induced sarcoma and a hepatoma. 15 Examining the intratumor blood-
flow distribution by isotope technique (see Chapter V), he found in irradiated tumors
139
a retarding of the progressively decreased tumor blood flow with increasing tumor age.
By plethysmography, a significant increase of the tumor blood flow per unit tumor
volume was observed (Figure 1).
III. I N F L U E N C E O F F R A C T I O N A T E D I R R A D I A T I O N ON T U M O R
BLOOD FLOW
A mouse mammary carcinoma was irradiated with six daily doses of 500 rd. 16 The
tumor volume did not change significantly during irradiation. Three days after irradia-
tion, tumors began to grow again. At this time, vascular changes attributed to the
filling of previously nonfunctional vessels were observed by morphometric methods.
FIGURE 1. Resting (black circles) and postischemic (white

circles) tumor blood flow registered by plethysmography in
mi/min and mi/100 ml tissue. Corresponding tumor flow val-
ues are connected with vertical lines (resting and postischemic
flow values) and with broken straight lines with an arrow in
the direction of day 3 to day 7 of the experiment. The A-A
curve indicates the upper range for postischemic-flow values in
control tumors, and the B-B curve indicates the upper range
for resting-flow values in control tumors. Note the postirradia-
tion-flow values compared to control tumor-flow values, indi-
cating a significantly increased tumor blood flow after irradia-
tion (see also Chapter 5, Figure 1 ). (From Kjartansson, I.,
Acta Chir. Scand. Supp/., 471, 1976. With permission.)
Reinhold17 found an increased blood flow rate showing as an increased vascular

density after fractionated irradiation of tumors with daily doses of 576 rd. The effect
was most marked 7 days after the initiation of irradiation, when tumors showed a
regression of volume beyond the original volume. He concluded that at least part of
the observed effect was due to tumor-cell decay.
Rubin and Casarett 8 irradiated two rat tumors with 500 rd daily to a total dose of
1500 rd and found a marked regression of tumors. An increased vascularization of
tumors was found four days after irradiation. The authors suggested that this phenom-
enon was explained by a loss of tumor parenchymal cells and a better filling of already
existing vessels. More pronounced vascular damage was found after a single-irradia-
tion dose of 1500 rd than after the fractionated irradiation.
Mántylã et al. 18 studied the blood flow of various human malignant tumors by the
133
Xe clearance technique. Tumors were irradiated, receiving five doses a week with a
total weekly dose of 1100 to 3000 rd. A significant increase of tumor blood flow was
found in anaplastic carcinomas one week after irradiation. The blood flow of lympho-
mas and differentiated carcinomas was not affected. Later, a decreased blood flow
was found in all groups of tumors.
IV. S U M M A R Y
Single doses of irradiation seem to have a different influence on various tumors.

After low doses of irradiation, an increased tumor blood flow has been observed in
many tumors. After large doses of irradiation, it is mostly a decreased blood-flow rate
coincident with damage of the vascular bed that has been found. Changes of the tumor
vascular bed towards that of small tumors could be seen after irradiation.
Fractionated irradiation does not seem to damage the tumor vascular endothelium
so much as a large single-dose irradiation. In human tumors, the influence of fraction-
ated irradiation on tumor blood flow seems to be correlated to the histopathological
type of the tumor. Fractionated irradiation, which mainly damages tumor cells without
influencing the tumor vascular bed, increases the tumor blood flow.
ACKNOWLEDGMENT

REFERENCES
1. Reinhold, H. S., Blachiwiecz, B., and Blok, A., Oxygenation and reoxygenation in "sandwich"
tumours, Bibl. Anat., 15, 270, 1977.
2. Anon., Current Topics, The influence of radiation on blood vessels and circulation, Radiât. Res. Q.,
10(1 and 2), 1974.
3. Guette, E. L., Meurer, G. D., and Severin, G. A., Endothelial repair of radiation damage following
beta irradiation, Radiology, 116, 175, 1975.
4. Song, C. W. and Levitt, S. H., Vascular changes in Walker 256 carcinoma of rats following X-
irradiation, Radiology, 100, 397, 1971.
5. Song, C. W., Sung, J. H., Clement, J. J., and Levitt, S. H., Vascular changes in neuroblastoma of
mice following X-irradiation, Cancer Res., 34, 2344, 1974.
141
6. Clement, J. J., Song, C. W., and Levitt, S. H., Changes in functional vascularity and cell number
following X-irradiation of a murine carcinoma, Int. J. Radiât. Oncol. Biol. Phys., 1, 671, 1976.
7. McAlister, W. H. and Margulis, A. R., Angiography of malignant tumors in mice following irradia-
tion, Radiology,SI, 664, 1963.
8. Rubin, P. and Casarett, G., Microcirculation of tumors. II. The supervascularized state of irradiated
regressing tumors, Clin. Radiol., 17, 346, 1966.
9. Robert, J., Martin, J., and Burg, C , Action des rayons X sur la vascularisation spécifique de tumeurs
isologues de la Souris et du Rat, C. R. Seances Soc. Biol. Paris, 161, 867, 1967.
11. Johnsson, R. J. R., The effect of temperature and radiation on tumor blood flow, Radiât. Res., 59,
269, 1974.
12. Hilmas, D. E. and Gilette, E. L., Microvasculature of C3H/BÍ mouse mammary tumors after X-
irradiation, Radiât. Res.,61, 128, 1975.
13. Hilmas, D. E. and Gilette, E. L., Morphometric analyses of the microvasculature of tumors during
growth and after X-irradiation, Cancer, (Philadelphia), 33, 103, 1974.
14. Mervin, R., Algire, G. H., and Kaplan, H. S., Transparent chamber observations of the response of
a transplantable mouse mammary tumor to local Roentgen irradiation, J. Natl. Cancer Inst., 11,
593, 1950.
15. Kjartansson, I., Tumour blood circulation, Acta Chir. Scand. Suppl., 471, 1976.
16. Hilmas, D. E. and Gilette, E. L., Tumor microvasculature following fractionated X-irradiation, Ra-
diology, 116,165,1975.
18. Mantyla, M., Kuikka, J., and Rekonen, A., Regional blood flow in human tumours with special
reference to the effect of radiotherapy, Br. J. Radiol., 49, 335, 1976.
Taylor & Francis
143
Chapter 9
OXYGEN SUPPLY TO MALIGNANT TUMORS
Peter Vaupel
TABLE OF CONTENTS
I. Introduction 144
II. Restriction of Convective and Diffusive Transport within Malignant Tumors

during Growth 144
A. Rarefaction of the Terminal Vascular Bed in Tumor Tissue 144
B. Reduced and Heterogenous Blood Flow in Malignant Tumors 146
III. Oxygen Diffusivity in Neoplastic Tissue 147
IV. Oxygen Consumption of Solid Tumor Tissue 148

A. Respiration Rate of Tumors, a Function of 0 2 Delivery 148
B. Heterogeneity of 0 2 Consumption by Malignant Tumors 149
C. Oxygen Consumption of Tumors During Reduction of Available
Oxygen 150
D. Oxygen Consumption of Tumor Tissue during Abundance of
Oxygen 151
E. In Vivo Oxygen Consumption of Tumors during Hyperthermia 151
V. Theoretical Analysis of the 0 2 Supply in the Intercapillary Region of Tumor
Tissue 152
A. Computation of the Critical Supply Radii for 0 2 in Tumor Tissue . . . 154
B. p 0 2 Distribution within a Tissue Cylinder and its Respective Tumor Cap-
illary 156
VI. Measurement of the p 0 2 Distribution within Neoplastic Tissue 157

A. p 0 2 Distribution in Tumor Tissue during Normoxia 158
B. Dynamic Behavior of p 0 2 Values in Tumor Tissues during Respiratory
Hyperoxia 159
VII. Intracapillary H b 0 2 Saturation in Malignant Tumors 160
VIII. Interrelationships between 0 2 Consumption and Glucose Uptake by Malignant

Tumors 162
A. In Vivo Investigations on the Pasteur Effect in Solid Tumor Tissues or
Ascites Cells 162
B. In Vivo Observations on the Crabtree Effect in Solid Tumors 163
IX. 0 2 Deficiency, a Limiting Factor for Cell Proliferation and Radiosensitivity in

Malignant Tumors 164
References 164
I. INTRODUCTION
The present knowledge of respiratory gas exchange in tumor tissue is based primarily
upon investigations conducted on tissue slices, cell cultures, and tissue homogenates
under in vitro conditions, the experimental approach relying at first upon manometric
techniques.
Until a few years ago, the direct measurement of respiratory gas exchange in malig-
nant tumors under in vivo conditions was not possible because either the amount of
blood flow could not be determined or the respiratory gas parameters for the tumor-
venous blood could not be defined: exactly due to the *'parasitic" blood supply of
tumors. Thus, the earlier conclusions concerning the arterio-tumorvenous partial pres-
sure differences or concentration differences of respiratory gases were only a rough
approximation.
It was the development of a suitable tumor model in kidneys, ovaries, or testes of
rodents by Gullino and Grantham 1 that allowed direct measurements. Following stand-
ard physiological procedures, tumor blood flow as well as the uptake or release of
respiratory gases and various metabolic parameters under in vivo conditions could thus
be evaluated.
Since 1967 oxygen (0 2 ) supply to solid tumor tissue has been very carefully investi-
gated using "tissue-isolated" preparations of implanted tumors. 2 3 4 In this model,
the tumor mass is connected to the host by only a single artery and vein, permitting
the determination of the essential parameters for the supply conditions in neoplastic
tissue and the collection of the relevant data on arterial and tumorvenous blood. Al-
terations of these parameters under variable 0 2 supply conditions can also be deter-
mined.
The advantage of investigations on "tissue-isolated" implantation tumors is that all
important supply parameters for the tumor tissue can be measured. The experimental
findings together with theoretical considerations can thus present an exact description
of the real in situ situation at hand. It became evident that in vitro findings could be
applied to in vivo conditions only when the important steady state and nonsteady state
in vivo conditions were taken into account. Furthermore, it could be shown that con-
clusions which were valid for implantation tumors could be applied without large re-
servations to solid, spontaneous tumors. 4 5
Investigations on this type of tumor and on primary carcinomas have shown that
during tumor growth, the 0 2 uptake as well as the glucose consumption of cancer cells
are largely dependent on the supply conditions for these substrates, whereby changes
in the convective transport of the nutrients in the vascular compartment and changes
in diffusive transport from the tumor capillary to the respiring cells were primarily
responsible for the growth pattern of malignant tumors.
II. RESTRICTION OF CONVECTIVE A N D DIFFUSIVE TRANSPORT

WITHIN MALIGNANT TUMORS DURING GROWTH
The major cause for the changes in 0 2 supply conditions during tumor growth is
the lack of a sufficient vascularization, since endothelial cells are unable to initiate an
adequate neovascularization. The basic modulations of the vascular space in neoplastic
tissue are presented first, since the deterioration of the 0 2 supply conditions during
tumor growth necessarily results from the rarefaction of the terminal vascular bed.
A. Rarefaction of the Terminal Vascular Bed in Tumor Tissue

A major cause for the changes of the supply conditions in tumor tissue can be seen
145
in its reduced vascularization. According to Gullino and Grantham, 6 the main vascular
branches which supply the host organ become the main vascular branches which supply
the tumor. The newly formed nutritive vessels which are incorporated into the growing
tumor mass are stretched to supply a mass of tissue many times greater than the origi-
nal tissue. These vessels proliferate at a slower rate than the rapidly proliferating cancer
cells. Therefore, the vascular network is less dense and reduced to such a degree that
a general rarefaction of the vascular bed occurs. The reduction of vascular space as
tumor size increases has been attributed by Tannock 7 to a difference in the turnover
times between endothelial (50 to 60 hr) and neoplastic cells (22 hr). Neovascularization
thus lags behind the increase in the number of neoplastic cells.
Morphometric analysis of tumor serial sections reveals that the vascular space of
solid tumors becomes smaller as the tumor mass grows (Figure l). 4 5 7 In general, as
the tumor increases in size, the vascular surface area decreases.3 8 This decrease, how-
ever, varies extensively from one tumor to another and also within a single tumor. 5
The reduction of the vascular bed is concomitant with a widening of the vessel di-
ameter, 3 4 8 9 an increase in vessel length, 3 4 1 0 and a broadening of the distance between
tumor capillaries.3 4 8 In DS-carcinosarcoma, the mean intercapillary diffusion ranges
are increased threefold during tumor growth from 3 to 12 g, the intraindividual varia-
tions becoming greater, thus indicating greater heterogeneities in vascular patterns
(Figure 2). The general rarefaction of the terminal vascular bed in DS-carcinosarcoma
is accompanied by a 10-fold increase in the vascular flow resistances within the tissue
when the tumor increases in size from 4 to 10 g.11
FIGURE 1. Relationship between tumor wet weight and vascular space (Vcap) in DS-Carcinosarcoma (log
y = -0.163 x + 1.077; r = 0.944). 42023
FIGURE 2. Enlargement of the mean intercapillary distances in DS-Carcinosar-

coma with increasing tumor wet weight. The shaded area represents the range of
the measured values (log y = 0.052 x + 1.519; r = 0.799).
B. Reduced and Heterogeneous Blood Flow in Malignant Tumors

As a consequence of these modifications of the vascular network, the blood supply,
i.e., the blood flow through the vascular bed, is smaller than that in the tissues of
origin.3-4-12-19 Furthermore, as the tumor grows larger, the supply of blood per unit
tumor weight decreases. 3 4 1 4 1 6 1 8 - 2 0 Figure 3 shows the sharp decline of tumor blood
flow in DS-carcinosarcoma with increasing tumor weight, i.e., age of growth.
Tannock and Steel21 have pointed out that there exists a great proportion of blood
which does not exchange with that of the general circulation. Vascular stasis prevents
normal functions of the terminal vascular bed in those areas where circulating blood
is not obvious despite the intactness of vessels. Furthermore, greater heterogeneities
in vascularization cause marked differences between regional blood flow per unit tissue
volume in the central parts and that of the outer tissue layers within the same tumor. 16
Using the 85Kr(0) — clearance technique, it has been shown that there are great
differences not only between tumor center and peripheral areas but also within neigh-
boring parts of the superficial layers.22 The regional blood flow values measured in
different parts of the tumor surface are scattered to a relatively great extent and they
are distributed over the tumor surface rather heterogeneously. Some regions may be
absolutely ischemic.20 Regurgitation and intermittent circulation, i.e., periods of pres-
tasis or stasis, followed by resumption of blood flow, sometimes in a direction opposite
to the previous one, are probably the ' 'normal" features of the intravascular transport
system of neoplastic tissues.5 Stasis occurs since lacuna-like, sinusoidal, and cystiform
blood vessels cannot be drained because "tissue-pressure" due to continuous cell pro-
liferation can prevent efficient circulation. Thrombosis follows and results in the oc-
clusion of the vessels5 20 23
Studies by Bierman et al. 24 showing an increased 0 2 content of neoplasms draining
venous blood indicate a significant degree of arterio-venous shunting instead of circu-
lation through the vascular network. According to many investigators, 2527 the in-
creased incidence of arterio-venous shunts can be veiwed as an essential characteristic
of malignant tumors. It has been estimated that shunt perfusion in DS-carcinosarcoma
represents about 30% of the total perfusion. 28
An additional factor which until now has received little attention is the disturbance
of the microcirculation in malignant tumors as a result of a pronounced tissue acidosis.
147
FIGURE 3. Exponential decrease of total tumor blood flow (TBF) in DS-Carci-

nosarcoma with increasing tumor wet weight, i.e., age of growth (log y = - 0.100
x + 1.685; r = 0.894)4
Because of the high glycolytic rate in tumor cells, low pH values (pH = 6.4 to 7.0),
which cause a definite stiffening of erythrocyte membranes, occur in the tissue. The
decrease of the erythrocyte flexibility or fluidity leads to a drastic reduction of the
microcirculation in malignant tumors. 23
Although the relationship between blood flow and vascularity is very complex, it
can be stated that the metabolically useful blood flow is likely to be rate-limiting for
the delivery of 0 2 and nutrients or drugs, or for the drainage of wastes or cytolytic
products.
After presentation of the basic modulations of vascularization and blood flow which
influence the 0 2 uptake by malignant tumors, to a large degree the critical 0 2 supply
to the tumors will be elucidated in the following sections.
III. O X Y G E N D I F F U S I V I T Y IN N E O P L A S T I C TISSUE
To understand more thoroughly the tumor 0 2 supply conditions, knowledge of the

0 2 diffusivity in neoplastic tissue is required. Exact measurements of the 0 2 diffusion
constants D ( = diffusion coefficient) and K ( = Krogh's diffusion constant) of tumor
tissue had not been carried out up to 1977, and, therefore, previous investigations of
tumor 0 2 supply had to employ estimated values. 2932
The results of those studies are in part contradictory. Using a modified experimental
device for investigating 0 2 diffusion constants in biological media described by
Thews, 3335 the diffusion constants D and K for tumor tissue (DS-Carcinosarcoma)
were determined at different temperatures. The following mean values were obtained
for the conditions of 37°C: 36 37
D = 1.75 X 10~5 cm 2 /s
K = 1.90 X 10"5 mC0 2 /cm/min/atm
The measured 0 2 diffusion constants for tumor tissue correspond to values of normal
tissue with similar water content (82 g H 2 O/100 g tumor wet weight). This close cor-
respondence of the diffusion constants determined in tumor tissue with the appropriate
constants for normal tissues with comparable water content is an important indication
that the 0 2 diffusivity is not altered in neoplasms. Furthermore, it can be stated that
it is possible to predict and to estimate the diffusion coefficient for 0 2 in tumor tissue
if the water content is known, as is also the case with normal tissues.38
IV. O X Y G E N C O N S U M P T I O N O F SOLID T U M O R TISSUE
A. Respiration Rate of Tumors, a Function of 0 2 Delivery

Due to restrictions in tumor vascularization, tumor blood flow, increasing hetero-
geneities of regional microcirculation, and a diminishing 0 2 transport capacity of the
blood as a consequence of progressive tumor anemia, the oxygen uptake per unit
weight decreases exponentially from 1.85 to 0.1 mi O 2 /100 g /min during tumor
growth (Figure 4). 4 - 20 23-39
This must be attributed mainly to a dissociation between 0 2 requirement and 0 2
supply to cancer cells during growth. Similar results have been described by Gullino
et al. 2 2a (fibrosarcoma: 0.33 mi O 2 /100 g/min, Walker carcinoma: 1.1 ml O 2 /100 g/
min, and hepatoma: 1.77 ml O 2 /100 g/min; calculations are based on the assumption
that 1 g fresh tissue is equivalent to 200 mg dry tissue).
The data obtained under in vivo conditions fit well with in vitro data from various
tumors: 0.7 to 5.3 ml O 2 /100 g/min. 29 3 0 3 2 4 0 4 1 The in vivo 0 2 consumption of very
young tumors (1.4 to 1.85 ml O 2 /100 g/min) and the uptake rate of ascites cells of
DS-Carcinosarcoma (3.2 ml O 2 /100 g/min; this value is valid for the 0 2 uptake by
suspended cells in a glucose depleted, but well oxygenated ascitic fluid42) where con-
vective and diffusive transport cannot be limiting factors, are relatively high compared
to the 0 2 uptake of various normal tissues.23 This shows that the low 0 2 consumption
of heavier or older tumors is the result of deficient 0 2 supply conditions but not the
consequence of a respiratory impairment in cancer as postulated by Warburg 43 " about
50 years ago.4-40,43,44 A number of investigations have demonstrated that many tumors
contain a full complement of respiratory enzymes, coenzymes, and mitochondria
which appear normal morphologically and functionally. 4446
Investigations on the respiratory rate of a Flexner carcinoma have shown that there
was no essential difference between tumor 0 2 uptake and that of normal epithelial
tissue.47 Alvarez et al. 40 later found that human endometrium carcinoma had an ap-
proximately 80% higher 0 2 consumption than normal endometrium during the prolif-
erative or secretory stage. Similar findings were made for several other human carci-
nomas (large bowel, stomach, and breast). 48
Today there is clear evidence that the relatively small 0 2 uptake by the tissue of
older tumors is the result of poor 0 2 delivery and not the result of respiratory impair-
ment during sufficient 0 2 supply. The 0 2 consumption of malignant tumors depends
primarily not on the potential capacity, but rather on the inadequate 0 2 supply to the
cell as a result of the disturbed transport conditions. The low 0 2 uptake can be ex-
plained by the discrepancy between normal 0 2 requirements and an inadequate 0 2
supply. The sharp reduction in 0 2 uptake with increasing tumor size can be elucidated
as follows: as a result of the progressive deterioration of the supply conditions (reduc-
tion of the tumor blood flow, exponential increase of the mean intercapillary distances,
149
FIGURE 4. Relationship between tumor wet weight and oxygen consumption

(V02) of DS-Carcinosarcoma (log y = - 0.107 x + 0.531; r = 0.9634 20 23
and reduction of the 0 2 transport capacity), a sufficient 0 2 supply in larger tumor

areas is no longer realized. In addition, the release of 0 2 from the red blood cells
(RBC) to the cancer tissue may be greatly reduced since the erythrocyte flexibility dur-
ing passage through the tumor tissue is decreased. This stiffening of the erythrocyte
membrane leads to an inhibition of the convective transport of H b 0 2 and 0 2 within
the RBC and thereby to deterioration of the release of 0 2 to the tissue.49
B. Heterogeneity of 0 2 Consumption by Malignant Tissues

There seemed to be a discrepancy between the directly measured and the calculated
values for the mean tissue oxygen partial pressure (p0 2 ) in malignant tumors. How-
ever, as Thews and Vaupel39 have pointed out, the differences between the calculated
and the polarographieally measured values became smaller when heterogeneities of the
parameters concerning the 0 2 delivery to the tissue were consistently taken into ac-
count. Mathematical analysis showed that the measured oxygen tensions were quite
plausible when heterogeneities of the supply radii, length of capillaries, capillary per-
fusion, intracapillary hematocrit, and regional tissue perfusion, as well as a heteroge-
neity in 0 2 consumption rates, were considered.
The in vivo studies concerning the in vivo 0 2 uptake by tumor tissues performed by
Gullino et al. 2 and Vaupel4 allowed conclusions regarding only the global or total ox-
ygen consumption of the malignant tissues. Some recently developed techniques, how-
ever, have enabled 0 2 consumption to be measured with a much finer resolution so
that it is possible to measure in microareas and to compare variations in oxygen uptake
with distinct histological structures in the tissue.50 In these experiments, the "oxygen
removal rate", defined as the ratio of the oxygen consumption rate to the oxygen
solubility, is measured polarographieally in samples of recently excised tissue probes
under in vitro conditions. There are considerable heterogeneities in the oxygen removal
rate by tumor tissue, associated with heterogeneities in the histological structures (via-
ble tissue — necrosis). In mouse lung carcinoma a correlation between the 0 2 removal
rate and regions of viable or necrotic tissue was found. Furthermore, the authors could
show that the mean oxygen uptake rate decreased gradually with increasing tumor
mass whereby the spread in values increased. 5152 This behavior is in close agreement
with findings of other groups 4,23 In addition, Constable et al. 51 could show that in
large tumors the 0 2 uptake rate was higher towards the edge than in the center. The
decrease towards the center was due to the existence of areas of low respiration which
were, however, interspersed with areas of the more normal respiration of the tumor
edges.
These results concerning heterogeneous 0 2 consumption are not surprising if one
considers that many blood flow experiments have already indicated marked differences
in tissue perfuson within the same tumor. The experimental findings essentially support
earlier assumptions of Thews and Vaupel39 who had postulated heterogeneous con-
sumption rates in malignant tumors.
C. Oxygen Consumption of Tumors During Reduction of Available Oxygen

If the postulate that the in vivo 0 2 consumption of malignant tumors is limited by
a restricted convective and diffusive transport is valid, any reduction of available oxy-
gen, caused by anemia, hypoxemia within the arterial blood or by lowering of tumor
blood flow, can induce a parallel reduction in the uptake rates.
Acute oxygen shortages were produced by removal of RBCs in tumor-bearing ani-
mals.2 Under these conditions, the malignant tumors improved the 0 2 removal ratios
from the blood; however, the overall utilization of oxygen decreased. During a chronic
shortage of 0 2 due to anemia or low blood flow, the oxygen consumption declined in
proportion to the oxygen available.
The data resulting from a series of experiments on the effect of anemia on the tissue
oxygenation of the uterine cervix cancer suggested that anemia unfavorably alters 0 2
delivery and the effectiveness of radiotherapy. The impairment of radiosensitivity in
these experiments could be explained by the deficiency of tissue oxygenation as a result
of reduced oxygen transport within the blood. 52
The influence of a progressive lowering of tumor blood flow on respiratory gas
exchange was examined by Guenther et al. 54 and Vaupel. 4 The results of these investi-
gations on the 0 2 uptake of DS-carcinosarcoma after progressive lowering of tumor
blood flow are shown in Figure 5. As the blood flow decreases, the arterio-tumorven-
ous 0 2 concentration difference (AVD 02 ) of the lighter tumors (dots, mean wet weight
= 4.9 g) rises under steady state conditions and reaches that of the heavy tumors
(circles, mean wet weight = 8.9 g), when the blood flow values reach 20% of the initial
values. The AVD 02 of the heavy tumors remains practically unchanged during the re-
duction of blood flow. When looking at the 0 2 uptake (V 02 ), one sees that the smaller
tumors have a distinctly higher 0 2 consumption than the heavy tumors. During the
progressive fall of the tumor blood flow, the 0 2 consumption of both groups contin-
ually decreases and, at 20% of the initial blood flow values, reaches 0 2 uptake rates
of 0.3 and 0.05 mi O 2 /100 g/min, respectively. These results clearly show that already
in young tumors an increase in the 0 2 extraction of the blood cannot fully compensate
the reduction of tumor blood flow. In older tumors which already show an initially
high 0 2 extraction of the blood, the AVD 02 cannot be effectively increased under these
conditions.
During steady state hypoxemia induced by respiratory hypoxia, the depleted oxygen
supply to the cancer cells cannot be compensated by a more extensive 0 2 extraction.
Consequently, the oxygen uptake by the tumor tissue decreases significantly. With a
reduction of the arterial oxygen tension from 89 mmHg (1 mmHg = 0.133 kPa) under
normoxic conditions to 64 mmHg, the 0 2 consumption of tumors significantly de-
creased by 18%. An opposite response pattern was found for the glycolytic rate ( +
47%). 55
151
FIGURE 5. Effect of progressive lowering of total tumor blood flow

(TBF) on the arterio-tumorvenous 0 2 concentration difference
(AVD02) and on the 0 2 uptake by small (dots; mean wet weight = 4.9
g) and by heavy tumors (circles; mean wet weight = 8.9 g). Values
are means ± SD.
D. Oxygen Consumption of Tumor Tissue During Abundance of Oxygen

The results during abundance of oxygen caused by either respiratory hyperoxia or
polycythemia showed that under in vivo conditions malignant tumors are able to utilize
more oxygen if they can receive more of it.2 This ability cannot be exhausted in vivo.
From these experiments it was concluded that it is impossible to "saturate" the oxygen
consumption of tumors in vivo. This is another hint that the small respiration rate is
probably due to a lack of 0 2 supply rather than to the inability of utilization, i.e.,
"damaged respiration".
Under steady state conditions of respiratory hyperoxia (pure oxygen) a significant
increase of the 0 2 uptake ( + 14%) by the tumor tissue can be observed.55 During a
rise of the arterial p 0 2 to 480 mmHg, a significant drop of the glycolytic rate occurs
( - 17%).
E. In Vivo Oxygen Consumption of Tumors During Hyperthermia

A number of studies have unequivocally demonstrated that hyperthermia retards the
growth rate of certain types of malignant tumors and completely eradicates some tu-
mors with minimal damage to normal tissues. 5658 This differential response of normal
tissues and in vivo tumors to hyperthermia has been attributed to the difference in the
tissue 0 2 concentration, among other things. 59 However, basic experiments of temper-
ature modifications on tumor blood supply, on tumor oxygen consumption, and on

respiratory gas exchange under in vivo conditions are very few.
A recent objective on this topic was to measure directly the influence of moderate
and tolerable levels of hyperthermia when applied to "tissue-isolated" tumors in ex-
perimental animals.
During isolated perfusion of DS-Carcinosarcoma in rat kidneys at 37.0, 39.5, and
42.0°C, Vaupel et al. 60 could show that after a rise in tumor temperature from 37.0 to
39.5°C, an increase in tumor blood flow of about 30° occurred (Figure 6). This was
due to a decrease of total tumor vascular resistance. Under these conditions, the oxy-
gen uptake by the tumor tissue rose for about 33 °/o. As cause for this increase in 0 2
consumption at 39.5°C, the increase in blood flow within the tissue must be primarily
implicated. Mueller-Klieser et al. 42 have pointed out that the 0 2 uptake by ascites cells
of DS-carcinosarcoma suspended in a glucose-depleted but well oxygenated ascitic
fluid increased about 14% when the temperature was raised from 37.0 to 39.5°C. From
these results it can be concluded that under in vivo conditions variations of temperature
may influence the 0 2 uptake by tumor tissue primarily by changes of tumor blood
flow. This can be readily demonstrated by raising the temperature from 39.5 to 42.0°C.
Although the 0 2 uptake of ascites cells under these conditions showed a further in-
crease of about 14%, the 0 2 consumption of solid tumors falls back to values which
lie in the range of the initial values for 37.0°C (Figure 6). This decline of the 0 2 con-
sumption to initial values under in vivo conditions could be observed despite a further
increase of the respiratory activity by the single cell as demonstrated by ascites cells.42
As cause for this reduction of 0 2 uptake by the tissue, a corresponding reduction of
tumor blood flow to starting levels at 37.0°C must be discussed. The cause of the
reduced blood flow at 42.0°C is probably complex (disturbance of the microcirculation
during 42°C-hyperthermia and pronounced tissue acidosis).
As von Ardenne and Reitnauer 61 have pointed out, a hyperthermia-induced ampli-
fication of tumor acidification and its feedback effect on microcirculation causes a
steep decrease of tumor blood flow if the local hyperthermia exceeds 41 °C. As a result,
the in vivo 0 2 uptake by the cancer cells is directly dependent on the blood flow, i.e.,
upon the convective transport of 0 2 during normothermia as well as during hyper-
thermic conditions.
Observations on the effect of heating at 43 °C on vascular volume and vascular
permeability of subcutaneous Walker 256 tumors revealed that there was no change
in these vascular functions during extreme hyperthermia. In contrast, normal tissues
of rats showed an increase in the functional vascular volume. 58 The results of Vaupel
et al. 60 on the relationship between perfusion rate or oxygen consumption and temper-
ature differ somewhat from the recently published findings of Gullino et al. 62 in that
tumor blood flow and 0 2 uptake tended to be uniform when the temperature was
raised from 37°C to hyperthermic conditions. Using rat mammary carcinomas, Gul-
lino et al. 62 could show that warming of tumors that were initially at a temperature
lower than physiologic temperature consistently increased the blood flow and the 0 2
consumption. Warming of tumors that already were at physiologic temperature only
occasionally produced changes in the blood supply and in the 0 2 utilization.
In both in vivo investigations, however, it was found that temperature changes led
to changes in the 0 2 uptake by tumor tissue only when the tumor blood flow was
influenced by the temperature changes. The 0 2 uptake by cancer tissues under these
conditions occurs parallel with the changes in tumor blood flow.
V. T H E O R E T I C A L A N A L Y S I S O F T H E 0 2 S U P P L Y IN T H E
I N T E R C A P I L L A R Y R E G I O N O F T U M O R TISSUE
Before the development of micromethods for measuring the 0 2 supply conditions
153
FIGURE 6. Total tumor blood flow (TBF) and 0 2 consumption

(V02) of tumors during normothermia and hyperthermia at 39.5 and
42.0°C. Values are means ± SEM, n = number of animals, signifi-
cance is based on paired t-test.
in microareas of tissue, 0 2 supply models were developed in order to gain an insight

into the basic "laws" of oxygen supply. 63 Recently, models were also used to interpret
the results of measurements using micromethods. However, all models known to date
are too schematic for a subtle analysis. Since capillary networks in malignant tumors
are quite irregular, all mathematical models must be used with some reservations. 64
Nevertheless, diffusion models can allow general conclusions concerning the 0 2 supply
to tissue as well as provide an interpretation of results for individual organs or for
tumor tissues.4 63 In the following, an analysis of the 0 2 supply in cancer tissue is
considered, using a diffusion model with concurrent capillary blood flow direction
(Krogh's cylinder model).

In order to calculate the intercapillary p 0 2 distribution on the basis of a large num-
ber of measured data, the 0 2 supply to tumor tissue has been simulated by a digital
model, the tumor capillaries running rectilinear and parallel to one another. 28 In addi-
tion, the arterial inflows, and thus the blood flow direction within tumor capillaries,
were varied as desired. The following microcirculatory units were chosen to represent
the different variation possibilities:63
1. Concurrent capillary flow (Krogh's cylinder structure)
2. Countercurrent capillary blood flow
3. Partial concurrent or countercurrent capillary flow
4. Spirally arranged arterial inflows and venous outflows, shifted against one another
by half the length of tumor capillaries ("helix structure")
The computation of the intercapillary p 0 2 distribution using in vivo data has shown
that the p 0 2 distribution of the different microcirculatory units differ only very
slightly. For this reason, and in order to gain some simplification, concurrent capillary
flow is assumed throughout the following discussion.
A. Computation of the Critical Supply Radii for 0 2 in Tumor Tissue

By means of the 0 2 partial pressures at the capillary wall, it is possible to estimate
the radius of the so-called boundary cylinder, i.e., the supply radius which guarantees
an adequate 0 2 supply. A partial differential equation of 0 2 diffusion, first presented
by Krogh65 and his mathematician Erlang, is employed for this analysis. The formula-
tion of the equation according to Thews66 is as follows:
(1)
where p crif = critical cellular p 0 2 , Pcap = p 0 2 value at the capillary wall, A = 0 2

consumption of the tumor tissue, K - Krogh*s diffusion constant, Rcap••= capillary
radius, and Rcrir = critical supply radius. The following assumptions are used:
1. Each capillary supplies its respective surrounding circular tissue cylinder.

2. Longitudinal-diffusion, i.e., diffusion parallel to the capillary, is neglected.
3. The diffusion coefficient and the 0 2 consumption of the tumor tissue are distrib-
uted homogeneously throughout the tissue cylinder and are constant.
4. Steady state conditions are present.
The following data form the basis for calculating the critical supply radii:
Al = 1.8 mC O 2 /100 g/min ( 0 2 uptake of small tumors)
A2 = 3.2 me O 2 /100 g/min ( 0 2 uptake of isolated tumor cells
of DS-Carcinosarcoma suspended in well-oxygenated but
glucose-depleted ascitic fluid42 )
P
crit = 2mmHg 6 7
K = 1.9 mfi 0 2 / c m / m i n / a t m 3 6 ' 3 7
R = 3
cap ^m-
155
Using measured p 0 2 values for the arterial blood of tumor-bearing animals, 4 the
critical supply radius for 0 2 at the arterial end of perfused capillaries amounts to about
75 to 80 /¿m if an 0 2 consumption of 1.8 mi/100 g/min is assumed (Figure 7). Since
intercapillary distances of about 150 to 160 pm can already be found in tumors with
wet weights over 8 g (Figure 2), it can be stated that in larger tumors insufficiently
supplied areas can already be observed at the arterial end of some capillaries. This
means that in those tumors hypoxia and anoxia in peripheral areas at the arterial end
of a capillary can occur. Assuming that there is a p 0 2 at the venous end of a tumor
capillary of about 40 mmHg, the critical supply radius for 0 2 is calculated to be 60
¡Am. However, this p 0 2 at the capillary end is an unrealistic value because arteriove-
nous shunting causes this value to be substantially higher than the effective p 0 2 at the
venous end of metabolically useful capillaries. If a p 0 2 value of 20 mmHg is considered
for computation, the critical supply radius amounts to 45 \xm. Under these circumstan-
ces critical 0 2 supply conditions in the tissue of DS-carcinosarcoma are to be expected
when the distance between neighboring tumor capillaries exceeds 90 \xm at the venous
end. Therefore, in malignant tumors having fresh weights greater than 5 g, oxygen
deficiency areas at the venous end of the capillaries already involve large regions. This
result corresponds closely with the fact that necrosis can be detected in those tumors.
It must be stressed at this point that all of these considerations reflect only the optimal
case for 0 2 supply in tumor tissue. It is very probable, however, that the supply situa-
tion is even worse than has been previously described since heterogeneities of microcir-
culation have not been considered and since the effective, i.e., functionally important,
intercapillary distances of metabolically useful capillaries can be considerably larger.
This assumption is valid since many tumor capillaries are seen as vessels during the
morphometric analysis but, at least temporarily, do not take part in the perfusion.
When one assumes a shunt perfusion of about 30%, the 0 2 partial pressure at the
venous end of a large number of metabolically useful capillaries is about 5 to 10
mmHg. 28 This would mean, however, that even in smaller tumors hypoxic and anoxic
areas occur at the venous end. Morphological findings support these speculations. The
results of intercapillary p 0 2 and intracapillary oxyhemoglobin saturation measure-
ments likewise confirm this assumption (see Sections VI and VII).
Figure 7 refers to the significance of 0 2 consumption for the calculation of the sup-
ply radii. With an increase of the 0 2 uptake by the cells from 1.8 (curve Ax) to 3.2
mi/100 g/min (curve A 2 ), the critical supply radius is distinctly reduced. A difficulty
in all calculations, in addition to determination of the effective p 0 2 values at the ve-
nous end of a metabolically useful capillary, lies in determining the 0 2 consumption
of cancer cells which are adjacent to the supplying capillary. The 0 2 uptake of these
cells strongly influences the diffusion range for 0 2 in the surrounding tissue. Unfortu-
nately, it is not yet possible to measure the 0 2 consumption of these cells which are
adequately supplied in solid tumors.
Using published values of parameters of the respiratory gas exchange, a range of
estimates of oxygen diffusion radius for mouse tumor has been calculated by Tan-
nock29 30 to lie between 50 \¿m ( 0 2 consumption of the tumor tissue = 5.3 mi/100 g/
min) and 90 \xm ( 0 2 consumption = 1.4 mi/100 g/min). The agreement between esti-
mated diffusion radius and mean radius of the tumor cell cords in these studies was
consistent with hypoxia at the periphery of the tumor cords. The results suggested that
hypoxia or anoxia may be one factor involved in tumor cell death and necrosis.68
None of the parameters has been measured for the tissue constituting the lung tu-
mors which have been examined by Thomlinson and Gray. 32 Therefore, their results
together with Tannock's calculations can give only an approximate estimate of the 0 2
gradients to be expected within the tumors which they investigated. Further estimates
were made by Rajewsky69 as well as by Goldacre and Sylvén70 but in these evaluations
156 Turn or Blood Circula tíon
FIGURE 7. Relationship between critical supply radius (Rcr/t) and

0 2 partial pressure at the capillary wall (p0 2 cap.) in malignant tumors
using Krogh's cylinder model. For comparison two different 0 2 con-
sumption rates are given (Ai or A 2 ).
either physiological values were lacking or data were used that had been obtained under
in vitro conditions.
B. p 0 2 Distribution Within a Tissue Cylinder and Its Respective Tumor Capillary

The p 0 2 distribution within a tissue cylinder surrounding a tumor capillary can be
calculated from the following equation:
(2)
where r = distance from the capillary center, p = p 0 2 in the circular tissue cylinder
depending on r, and R = radius of the Krogh cylinder.
A relief-representation of computed p 0 2 values within malignant tumor tissues is
shown in Figure 8. As can be seen from this figure, the limiting radius decreases con-
tinuously from the arterial to the venous end of a metabolically useful capillary. A
slightly waist-form truncated cone results as supply area. The shaded areas portray the
regions of hypoxia or anoxia in a tumor weighing about 10 g, whereby an intracapillary
distance of 160 /urn is given. 0 2 deficiency areas are a common feature in the entire
periphery. Additionally, the arterial and tumorvenous truncated areas are given in the
figure.
By means of a special analog computer, it is possible to simulate the conditions in
the range of very low p 0 2 values, the 0 2 uptake being dependent on p 0 2 values. 7173
Taking into account Michaelis-Menten kinetics and utilizing in situ parameters for
simulation of the critical supply conditions in malignant tumors, it has been possible
to show that under these conditions a slightly improved 0 2 supply situation in capillary
networks is evident. Employing the reaction kinetics of the respiratory enzymes at very
157
FIGURE 8. Relief representation of computed 0 2 partial pressures

(p0 2 ) in tumor tissue assuming constant 0 2 consumption until critical
0 2 tensions are reached. The shaded areas portray the regions of hy-
poxia and anoxia in a tumor weighing about 10 g (intercapillary dis-
tance = 160 ¿mi). The arterial and tumorvenous truncated areas of
the waist-form cone are given additionally.
low 0 2 partial pressures, it can be demonstrated that the diffusion range can be im-
proved by about 20%. In a study concerning the "laws" of substrate supply in the
intercapillary region of cancer tissue, von Ardenne 74 75 has also pointed out that the
critical supply radii increase if the 0 2 consumption decreases in the unsaturated region
of respiration. However, even if the kinetics of 0 2 uptake at very low levels of 0 2
partial pressures are considered, it becomes obvious that regional hypoxia and anoxia
obligatorily occur during tumor cell proliferation. 71
VI. M E A S U R E M E N T O F T H E P 0 2 D I S T R I B U T I O N W I T H I N
N E O P L A S T I C TISSUE
From the theoretical analysis of the 0 2 supply in the intercapillary region of malig-
nant tissues, it must be concluded that during tumor growth hypoxia or even anoxia
imminently occur. In order to prove this statement, direct measurements of the local
tissue p 0 2 values were performed with membrane-covered gold microelectrodes.
A. p 0 2 Distribution in Tumor Tissue During Normoxia

In addition to the possibility of determining the p 0 2 distribution in tumor tissue by
means of mathematical analysis using known diffusion equations, the microdimen-
sional p 0 2 distribution can be experimentally investigated utilizing 0 2 microelectrodes
(tip diameter = 2 to 5 /¿m).
Polarographic measurements of local oxygen tensions in DS-carcinosarcoma using
needle electrodes reveal that in superficial layers (0 to 180 ^m) the p 0 2 profiles have a
characteristic course which quantitatively can be attributed to the diffusion of atmos-
pheric oxygen into the tissue.4 76 In deeper parts of the tumor tissue which are supplied
by blood vessels only, there exist marked maxima and minima only in regions with
sufficient vascularization. Only in these areas are variations of the single values ob-
vious as is characteristic for normal tissues. In most areas the p 0 2 profiles are quite
uniform with relatively slight regional differences (Figure 9). Similar results were ob-
tained by other investigators in the tissue of various solid tumors.77"92 However, in the
latter experiments electrodes with greater tip diameters were used. Therefore with this
method only integrated values for the 0 2 partial pressure in larger tissue areas could
be measured. It can also be expected that larger destructions of the tissue and capillary
compression within the area where the electrode is inserted will occur. Furthermore,
large electrodes have a relatively high 0 2 consumption. From studies with gold microe-
lectrodes it becomes evident that the p 0 2 gradients in the tumor tissue are almost com-
pletely flat. A monotonous pattern of very low p 0 2 values predominates in the tissue
of solid tumors (Figure 9); only relatively slight regional differences can be found.
Therefore, p 0 2 histograms, i.e., tissue p 0 2 frequency distributions, show typical fea-
tures. Figure 10 illustrates this pattern of p 0 2 distribution in four tumors of different
ages, whereby wet weight increases with duration of implantation. The histograms are
shifted to lower p 0 2 values that means tilted to the left and more limited in variability.
FIGURE 9. p 0 2 profiles in central areas of the tumor tissue. The profiles are quite uniform
without greater regional differences. A monotonous pattern of very low p 0 2 values predominates
(s = insertion depth of the microelectrodes).4
159
FIGURE 10. p 0 2 frequency distribution in tumors of different wet weights. The

arrows mark the mean arterial and tumorvenous 0 2 partial pressures for the tumors
presented in this figure.4 20 23
This regularity is particularly evident if the mean p 0 2 values in the neoplastic tissue
is described as a function of tumor weight. Figure 11 shows that there exists a signifi-
cant logarithmic correlation between these two parameters. During a gain of weight,
the p 0 2 values ranging from 0 to 5 mmHg increase from 50 to 80%. Furthermore, the
investigations have shown that in major parts of the tumor tissue the actual p 0 2 is
below a critical value, i.e., in a hypoxic range, so that the activity of the respiratory
enzymes is reduced. Thus, the 0 2 consumption of these areas depends not only on the
complement of the respiratory enzymes and coenzymes in cancer cells but also on the
0 2 transport conditions in the tissue, especially on the capillarization, i.e., primarily
on the convective transport of oxygen by the blood stream.
There is an indication that Warburg's theory suggesting that neoplasms may be
caused by anoxia must be regarded as a gross oversimplification of carcinogenesis. A
large number of investigations have shown that partial hypoxia or anoxia is not the
cause but the result of malignant growth.
B. Dynamic Behavior of p 0 2 Values in Tumor Tissues During Respiratory Hyperoxia

During respiratory hyperoxia the p 0 2 distribution pattern is changed only insignifi-
cantly. The in vivo investigations of the dynamic tissue p 0 2 behavior during hyperoxia
indicate that in most cases increases of the tissue p 0 2 values within the tumor tissue
are very small. Very often there is no response at all. 0 2 pulses evoke significant p 0 2
changes only in those areas where the initial tissue p 0 2 values are relatively high.4 55
The reason for the frequent absence of 0 2 responses during respiratory hyperoxia
can easily be found when the intracapillary p 0 2 course is analyzed theoretically under
these conditions. During isobaric oxygenation, arterial hyperoxemia improves the sup-
ply conditions only at the arterial end, i.e., in the first quarter of a tumor capillary,
since physically dissolved oxygen diffuses out while passing through the first quarter
FIGURE 11. Relationship between tumor wet weight and mean 0 2

partial pressure (p0 2 ) in the tissue (log y = - 0.070 x + 1.292; r =
0.928)4 20 "
of a vessel. Thus, insufficient supply conditions or hypoxia can be improved only at

the arterial end of the tumor capillaries if the hosts are permitted to breathe pure
oxygen. The peripheral areas at the venous end are not influenced at all.
A summary of the results of polarographic p 0 2 measurements in tumor tissue shows
that the hypoxic and anoxic areas occurring during tumor growth as predicted by math-
ematical analysis can, in fact, be demonstrated, whereby the supply situation progres-
sively deteriorates as the tumor mass grows larger.
VIL INTRACAPILLARY H b 0 2 SATURATION IN MALIGNANT

TUMORS
According to the results of the polarographic measurements in malignant tumors,

one would expect that in solid tumor tissue very low intracapillary hemoglobin oxygen
saturation (Hb0 2 ) values predominate. In order to verify this assumption, Hb0 2 satu-
ration values within tumor capillaries are determined utilizing a cryophotometric mi-
cromethod whereby the intracapillary hemoglobin absorption spectra are measured in
frozen tissue slices at —100°C in a vacuum-isolated microscope cryostat and transferred
into intracapillary Hb0 2 saturation values using a multicomponent analysis.93 The re-
sults of measurements on tissue specimens from tissue-isolated preparations reveal that
during normoxia 53% of the data obtained lie in the range of 0 to 10% Hb0 2 satura-
tion. 24% of the measured values show zero levels. Only 8% of the values exceed 50%
saturation (Figure 12). These measurements demonstrate that very low Hb0 2 satura-
tions predominate in the tissue of malignant tumors.28 93
These results support the conclusions from p 0 2 measurements in tumors as well as
those from theoretical analysis of 0 2 supply in malignant tumors as were described in
Section V.
Regional differences can be found only in some tissue areas of malignant tumors
where a sufficient vascularization still exists. At this point, another finding of the mea-
surements of intracapillary Hb0 2 saturation must be emphasized: since approximately
80% of the measured intracapillary Hb0 2 values lie below the measured Hb0 2 values
for tumor venous blood, it may be concluded that both a considerable arterio-venous
161
FIGURE 12. Frequency distribution of measured Hb0 2 saturation values in tumor

capillaries ( + 3 - 8 pirn) on tissue-isolated tumors of DS-Carcinosarcoma during
normoxia (solid line) and respiratory hyperoxia (shaded area), n = number of mea-
surements (1 mmHg = 0.133 kPa)
shunting and a marked heterogeneity of perfusion in the microarea occur. This is sub-
stantiated by the results of many investigations on tumor blood flow and vasculariza-
tion.
During respiratory hyperoxia only slight increases in the Hb0 2 saturation values
within tumor capillaries are obvious (Figure 12). 40% of the data obtained under these
conditions are in the range of 0 to 10% Hb0 2 saturation. Only 11% of the measured
values exceed 50% saturation when the tumor-bearing animals are breathing pure ox-
ygen spontaneously. 18% of all measured Hb0 2 saturation values during respiratory
hyperoxia are at zero levels.
Comparing the data of measurements of the intracapillary HbOz saturation during
normoxia with the results obtained during respiratory hyperoxia, it becomes obvious
that the mean Hb0 2 saturation value during hyperoxia has increased only about 5%.
In general, it can be stated that during respiratory hyperoxia the intracapillary Hb0 2
saturations have increased only slightly and, therefore, respiratory hyperoxia does not
yield any practical improvement of the oxygen supply to solid tumors in situ.
Considerable interest has been shown in the oxygenation, in the growth pattern, as
well as in the radiation responses of tumors where the blood supply and the drainage
are either central or peripheral.94 In spite of the similarity between some malignant
tumors, the response to radiation was found to be rather different in each, especially
in relation to oxygenation (for a general review concerning the relation between radi-
osensitivity and oxygenation, see Reference 30).
As the vascular pattern of malignant tumors is determined not only by the temporal
sequence but also by the location of the tumor growth,27 95 different locations were
chosen for implantation of DS-carcinosarcoma. Subcutaneously growing tumors of
different types show mostly peripheral blood supply and drainage with a dense periph-
eral tumor vascularization around a more or less avascular center,27 95 96 whereas
tissue-isolated preparations of the same tumor type in the rat kidney have a blood
supply pattern of exclusively central arrangement.
Measurements of intracapillary Hb0 2 saturation in malignant tumor tissue at differ-
ent implantation sites have shown that low Hb0 2 saturations predominate in the tissue
of all tumors. However, comparison of tumors at the same stage of growth shows that
there are slightly higher intracapillary H b 0 2 saturation values within subcutaneously

growing tumors than within tissue-isolated tumors (Figure 13). These higher H b 0 2
saturations reflect a better oxygenation in the tissue probes of subcutaneous tumors
during normoxia. 95 96
VIII. INTERRELATIONSHIPS BETWEEN 0 2 CONSUMPTION A N D

GLUCOSE UPTAKE BY MALIGNANT TUMORS
Most living cells depend on oxygen and glucose utilization to obtain the chemical
energy necessary to maintain their functions and growth. Tumor cells are no exception
to this rule although they may differ from other cells in their mechanisms of control
of 0 2 and glucose utilization. 97
Respiration and glycolysis are closely interrelated in both normal and neoplastic
cells. The availability of oxygen usually depresses the glycolytic rate. This effect of
oxygen is termed the Pasteur effect. Alternatively, when tumor cells respire in the
presence of sufficient glucose so as to maintain maximal glycolytic rates, the 0 2 con-
sumption decreases. This effect has been termed the Crabtree effect. Although the
Pasteur and the Crabtree effects are still not well understood, they represent focal
points for deciphering the relationships between glycolysis and respiration. 97 98
Quite novel findings on the critical role played by the pyruvate kinase in the regula-
tion of glycolysis by respiration (Pasteur effect) and vice versa (Crabtree effect) were
reported by Gosalvez and Weinhouse. 97
A. In Vivo Investigations on the Pasteur Effect in Solid Tumor Tissues or Ascites Cells
The Pasteur effect is usually defined as the inhibition of glycolysis by oxygen in
tissues showing both the aerobic and anaerobic pathway of glucose breakdown. In the
presence of 0 2 , glycolysis is restricted in favor of the oxidative pathway. Since the
aerobic pathway is the most economic and effective way to utilize glucose, the glucose
consumption by the cell can generally be limited if there has been no previous absolute
or relative glucose deficiency. In vivo investigations on the inoculated DS-Carcinosar-
FIGURE 13. Frequency distribution of intracapillary Hb0 2 saturation values in

tissue-isolated preparations of neoplasms (t.i.t.; solid line) and within subcutane-
ously growing tumors of DS-carcinosarcoma (s.c.t.; shaded area; 1 mmHg = 0.133
kPa).
163
coma in the rat kidney also show a typical Pasteur effect when the conditions are
changed from arterial normoxemia to respiratory hyperoxia. Following an increase in
the arterial 0 2 partial pressure from 89 to 480 mmHg, a drop in glycolytic rate by
about 10% is observed although the improved 0 2 supply benefits only the first quarter
of the area adjacent to a capillary i.e., the arterial end of the capillary. The release of
lactate from the tumor tissue into the blood is reduced as a consequence of the dimin-
ished glycolytic rate; the lactate-pyruvate ratio drops correspondingly. 98 However, no
significant reduction of the glucose uptake was observed. A decrease in the glucose
uptake could not be expected under these conditions because of a deficient glucose
supply to the tumor cell in vivo.
Thus, a drop of the glucose consumption together with an improvement of the 0 2
supply conditions will remain the privilege of well-supplied cells in vitro where suffi-
cient glucose supply is guaranteed.
Studies on the ability of the tumor respiration in vivo to fulfill its physiological
functions have shown that the in vivo respiration of cancer cells which were suspended
in ascitic fluid is not able to affect glycolysis and to spare glucose. Only extra 0 2 supply
induced the Pasteur effect."
B. In Vivo Observations on the Crabtree Effect in Solid Tumors

Investigations on tumor metabolism under in vitro conditions have shown that the
0 2 uptake by tumors decreased after the addition of glucose to the incubation me-
dium.100 After a rise of the arterial glucose concentration in tumor-bearing animals, a
temporary decrease of the in vivo 0 2 consumption by the tumor tissue occurred.98 101
This result corresponds closely to phenomena observed under in vitro condi-
tions.98 101 In accordance with in vitro data, the extent of the decrease of the 0 2 con-
sumption and the duration of this effect depend on the intracellular glucose concentra-
tion, i.e., on the amount of glucose added. The phenomenon was reversible (Figure
14). An inhibition of the Crabtree effect during high C 0 2 partial pressures or acidosis
can be observed.
FIGURE 14. Temporary decrease of the 0 2 uptake (V02) by solid tumors after
addition of glucose to the tumor-bearing animal (Crabtree effect). The time of glu-
cose addition is marked by the arrow. Curves A, B, and C represent experiments
on tumor bearing animals with increasing acidosis in the arterial blood. 4 98
IX. 0 2 D E F I C I E N C Y , A L I M I T I N G F A C T O R FOR C E L L
P R O L I F E R A T I O N A N D R A D I O S E N S I T I V I T Y IN M A L I G N A N T
TUMORS
From a series of in vitro studies, it is well known that low oxygen concentrations,
low 0 2 partial pressures as well as low glucose concentrations, and low pH values may
act as limiting agents for tumor cell proliferation. 102
A critical review of all known in vivo results leads to the conclusion that hypoxia is
indeed also a growth limiting factor for cancer cell growth under in vivo conditions. 102
From the compilation of results coming from either experimental or theoretical inves-
tigations and presented here, there is clear evidence that during tumor growth hypoxia
and anoxia are common features forming a hostile environment for cancer cells and
conferring partial radioresistance. This hostile environment (hypoxia and anoxia, glu-
cose depletion and severe acidosis) must be regarded as a major factor in the causation
of cell death and necrosis or declining growth rate, the very phenomena which accom-
pany increasing tumor weight.
REFERENCES
1. Gullino, P. M. and Grantham, F. H., Studies on the exchange of fluids between host and tumor. I.
A method for growing "tissue-isolated" tumors in laboratory animals, / . Natl. Cancer Inst., 27,
679, 1961.
2. Gullino, P. M., Grantham, F. H., and Courtney, A. H., Utilization of oxygen by transplanted tumors
in vivo, Cancer Res.,27, 1020, 1967.
2a. Gullino, P. M., In vivo utilization of oxygen and glucose by neoplastic tissue, in Oxygen Transport
to Tissue-II, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press, New York, 1976, 521.
3. Vaupel, P., Guenther, H., Grote, J., and Aumueller, G., Atemgaswechsel und Glucosestoffwechsel
von Tumoren (DS-Carcinosarkom) in vivo. I. Experimentelle Untersuchungen der versorgungsbes-
timmenden Parameter, Z. Ges. Exp. Med., 156, 283, 1971.
4. Vaupel, P., Atemgaswechsel und Glucosestoffwechsel von Implantationstumoren (DS-Carcinosar-
kom) in vivo, in Funktionsanalyse biologischer Système, Vol. 1, Thews, G., Ed., Steiner, Wiesbaden,
1974.
5. Gullino, P. M., Extracellular compartments of solid tumors, in Cancer, Vol. 3, Becker, F. F., Ed.,
6. Gullino, P. M. and Grantham, F. H., Studies on the exchange of fluids between host and tumor.
III. Regulation of blood flow in hepatomas and other rat tumors, / . Natl. Cancer. Inst., 28, 211,
1962.
8. Vogel, A. W., Intratumoral vascular changes with increased size of a mammary adenocarcinoma:
new method and results, J. Natl. Cancer Inst., 34, 571, 1965.
9. Hilmas, D. E. and Gillette, E. L., Morphometric analyses of the microvasculature of tumors during
growth and after X-irradiation, Cancer, 33, 103, 1974.
10. Jirtle, R., Clifton, K. H., and Rankin, J. H. G., Measurements of mammary tumor blood flow in
unanaesthetized rats, J. Natl. Cancer Inst., 60, 881, 1978.
11. Vaupel, P., Interrelationship between mean arterial blood pressure, blood flow, and vascular resist-
ance in solid tumor tissue of DS-Carcinosarcoma, Experientia, 31, 587, 1975.
12. Schwarz, W., Schulz, V., Kersten, M., Woerz, R., and Vaupel, P., Durchblutung und Sauerstoffver-
brauch gewebsisolierter Impftumoren (DS-Carcinosarkom) in vivo.Z. Krebsforsch., 75, 161, 1971.
13. Gullino, P. M., and Grantham, F. H., Studies on the exchange of fluids between host and tumor.
II. The blood flow of hepatomas and other tumors in rats and mice, J. Natl. Cancer Inst., 27, 1465,
1961.
transplanted tumors, / . Natl. Cancer Inst., 29, 389, 1962.
165
15. Rogers, W., Edlich, R. F., Lewis, D. V., and Aust, J. B., Tumor blood flow. I. Blood flow in
transplantable tumors during growth, Surg. Clin. North Am.,47, 1473, 1967.
16. Vogel, A. W. and Haynes, J., Mammary adenocarcinoma (72j) blood flow in mice treated with
ThioTEPA, / . Natl. Cancer Inst., 37, 293, 1966.
17. Gullino, P. M., Blood flow and vascular stroma of tumors, Acta Union Int. Contra Cancrum, 20,
1645, 1964.
18. Peterson, H.-L, Appelgren, L., and Kjartansson, L, Tumor blood flow and tumor vessel permeabil-
ity, Bibl. Anat., 15, 277, 1977.
18a. Appelgren, L., Kjartansson, L, and Peterson, H.-L, Quantification of tumor blood flow and blood
flow distribution, Bibl. Anat., 15, 255, 1977.
19. Gump, F. E. and White, R. L., Determination of regional tumor blood flow by krypton-85, Cancer
Philadelphia), 21, 871, 1968.
21. Tannock, I. F. and Steel, G. G., Quantitative techniques for study of the anatomy and function of
small blood vessels in tumors, J. Natl. Cancer Inst., 42, 771, 1969.
22. Schulz, V., Vaupel, P., Guenther, H., and Thews, G., Gesamtdurchblutung (tTBF) und régionale
Gewebsdurchblutung (rTBF) isolierter Impftumoren (DS-Carcinosarkom) in vivo, Verh. Dtsch. Ges.
Inn. Med., IS, 140, 1972.
23. Vaupel, P., Thews, G., and Wendling, P., Kritische Sauerstoff-und Glucoseversorgung maligner
Tumoren, Dtsch. Med. Wochensch. , 101, 1810, 1976.
24. Bierman, H. R., Kelly, K. H., and Singer, G., Studies on the blood supply of tumors in man. VI.
The increased oxygen content of venous blood draining neoplasms, / . Natl. Cancer Inst., 12, 701,
1952.
25. Aumueller, G., Vaupel, P., and Guenther, H., Etudes morphologiques sur la vascularisation de tu-
meurs implantées chez le rat, Bull. Cancer, 59, 225, 1972.
26. Delarue, J., Abelanet, R., and Chomette, G., La vascularisation des tumeurs malignes, Presse Med.,
73, 1517, 1965.
27. Habighorst, L. V., Tierexperimentelle Untersuchungen zur Tumorvaskularisation, Radiologe, 17,
111, 1977.
28. Vaupel, P., Grunewald, W. A, Manz, R., and Sowa, W., Intracapillary H b 0 2 saturation in tumor
tissue of DS-Carcinosarcoma during normoxia, in Oxygen Transport to Tissue-Ill, Silver, L A . ,
Ereciñska, M., and Bicher, H. L, Eds., Plenum Press, NewYork, 1978, 367.
29. Tannock, I. F., The relation between cell proliferation and the vascular system in a transplanted
mouse mammary tumor, Br. J. Cancer, 22, 258, 1968.
30. Tannock, I. F., Oxygen diffusion and the distribution of cellular radiosensitivity in tumors, Br. J.
Radiol., 45, 515, 1972.
31. Rieger, F., Diffusionskoeffizienten und Kapillarversorgungsradien des 0 2 — und des Glucosestoff-
wechsels beim DS-Karzinosarkom. Zur mathematischen in-vivo-Theorie der Begrundung des Krebs-
Mehrschritt-Therapie-Konzeptes 1973, Arch. Geschwulstforsch.,43, 52, 1974.
32. Thomlinson, R. H., and Gray, L. H., The histological structure of some human lung cancers and
the possible implications for radiotherapy, Br. J. Cancer, 9, 539, 1955.
33. Thews, G., Ein Verfahren zur Bestimmung des 0 2 — Diffusionskoeffizienten, der 0 2 — Leitfàhigkeit
und des 0 2 — Loeslichkeitskoeffizienten im Gehirngewebe, Pfluegers Arch., 271, 227, 1960.
34. Grote, J. and Thews, G., Die Bedingungen flir die Sauerstoffversorgung des Herzmuskelgewebes,
Pfluegers Arch., 276, 142, 1962.
35. Grote, J., Die Sauerstoffdiffusionskonstanten im Lungengewebe und Wasser und ihre Temperatur-
abhaengigkeit, Pfluegers Arch., 295, 245, 1967.
36. Grote, J., Suesskind, R., and Vaupel, P., Oxygen diffusion constants D and K of tumor tissue (DS-
Carcinosarcoma) and their temperature dependence, in Oxygen Transport to Tissue-Ill, Silver, I. A.,
Ereciñska, M., and Bicher, H. L, Eds., Plenum Press, New York, 1978, 361.
37. Grote, J., Suesskind, R., and Vaupel, P., Oxygen diffusivity in tumor tissue (DS-Carcinosarcoma)
under temperature conditions within the range of 20 to 40°C, Pfluegers Arch., 372, 37, 1977.
38. Vaupel, P., Effect of percentual water content in tissues and liquids on the diffusion coefficients of
0 2 , C 0 2 , N 2 , and H 2 , Pfluegers Arch., 361, 201, 1976.
39. Thews, G. and Vaupel, P., 0 2 supply conditions in tumor tissue in vivo, in Oxygen Transport to
Tissue-II, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press, New York, 1976, 537.
40. Alvarez, E., Okagaki, T., and Richart, R. M., Oxygen consumption of endometrial adenocarcinoma,
Am. J. Obstet. Gynecol., 109, 874, 1971.
41. Weinhouse, S., The Warburg hypothesis fifty years later, Z. Krebsforsch., 87, 115, 1976.
42. Mueller-Klieser, W., Zander, R., and Vaupel, P., Oxygen consumption of tumor cells (DS-Carcino-
sarcoma) suspended in glucose-depleted ascitic fluid at 1 to 42°C, Pfluegers Arch., in press.
43. Shapot, V. S., Some biochemical aspects of the relationship between the tumor and the host, Adv.
Cancer Res., 15, 253, 1972.
43a. Warburg, O., Ueber den Stoffwechsel der Carcinomzelle, Klin. Wochenschr.,4, 534, 1925.
44. Weinhouse, S., Glycolysis, respiration, and anomalous gene expression in experimental hepatomas:
G.H.A. Clowes Memorial lecture, Cancer Res., 32, 2007, 1972.
45. Chance, B., Dynamics of respiratory pigments of ascites tumor cells, Trans. N.Y. Acad. Sri., 16,
74,1953.
46. Chance, B. and Hess, B., On the control of metabolism in ascites tumor cell suspensions, Ann. N. Y.
Acad. Sri.,63, 1008, 1956.
47. Minami, S., Versuche an ueberlebendem Carcinomgewebe, Biochem. Z., 142, 334, 1923.
48. Macbeth, R. A. L. and Bekesi, J. G., Oxygen consumption and anaerobic glycolysis of human malig-
nant and normal tissue, Cancer Res., 22, 244, 1962.
49. Zander, R. and Schmid-Schoenbein, H., Influence of intracellular convection on the oxygen release
by human erythrocytes, PfluegersArch., 335, 58, 1972.
50. Constable, T. B., Rogers, M. A., and Evans, N. T. S., Comparison between the oxygen removal
rate and the histological structure of normal and tumour tissues, Pfluegers Arch., 373, 145, 1978.
51. Constable, T. B., The effect of irradiation on the oxygen removal rate of the SSBIa rat fibrosarcoma,
Eur. J. Cancer, 12, 963, 1976.
52. Constable, T. B. and Evans, N. T. S., The distribution of oxygen consumption rates in some tissues
before and after X-irradiation, in Oxygen Transport to Tissue-II, Grote, J., Reneau, D., and Thews,
G., Eds., Plenum Press, New York, 1976, 611.
53. Schreiner, P., Siracká, E., Siracky, J., and Mânka, I., The effect of anemia on the radiotherapy
results of the uterine cervix cancer, Neoplasma, 22, 655, 1975.
54. Guenther, H., Vaupel, P., and Thews, G., Einfluss der Durchblutung auf den Atemgas- und Gluco-
sestoffwechsel von Implantationstumoren (DS-Carcinosarkom), Verh. Dtsch. Ges. Inn. Med., 78,
136,1972.
55. Vaupel, P., Effect of hyperoxia, hypoxia and hypercapnia on 0 2 supply of malignant tumors in
situ,Bibl. Anat., 15, 288, 1977.
56. Overgaard, J., Effect of hyperthermia on malignant cells in vivo, Cancer, (Philadelphia), 39, 2637,
1977.
57. Suit, H. D., Hyperthermic effects on animal tissues, Radiology, 123, 483, 1977.
58. Song, C. W., Effect of hyperthermia on vascular functions of normal tissues and experimental tu-
mors, J. Natl. Cancer Inst., 60, 711, 1978.
59. Dewey, W. C , Thrall, D. E., and Gillette, E. L., Hyperthermia and radiation — a selective thermal
effect on chronically hypoxic tumor cells in vivo, Int. J. Radiât. Oncol. Biol. Phys., 2, 99, 1977.
60. Vaupel, P., Ostheimer, K., and Thome, H., Blood flow, vascular resistance, and oxygen consumption
of malignant tumors during normothermia and hyperthermia, Microvasc. Res., 13, 272, 1977.
61. von Ardenne, M. and Reitnauer, P. G., Amplification of the selective tumor acidification by local
hyperthermia, Naturwissenschaften, 65, 159, 1978.
62. GuUino, P. M., Yi, P.-N., and Grantham, F. H., Relationship between temperature and blood supply
or consumption of oxygen and glucose by rat mammary carcinomas, J. Natl. Cancer Inst., 60, 835,
1978.
63. Grunewald, W. A. and Sowa, W., Capillary structures and 0 2 supply to tissue, Rev. Physiol.
Biochem. Pharmacol., 77, 149, 1977.
64. Boag, J. W., Oxygen diffusion in tumor capillary networks, Bibl. Anat., 15, 266, 1977.
65. Krogh, A., The rate of diffusion of gases through animal tissues with some remarks of the coefficient
of invasion, / . Physiol. (London), 52, 391, 1918/1919.
66. Thews, G., Die Sauerstoffdiffusion im Gehirn. Ein Beitrag zur Frage der Sauerstoffversorgung der
Organe, PfluegersArch. Ges. Physiol,21 \, 197, 1960.
67. Hegner, D. and Glossmann, H., Polarographische Messungen der Atmung bei niedrigen Sauerstoff-
drucken, Z. Naturforsch., 20 b, 234, 1965.
68. Tannock, I. F., Oxygen distribution in tumours: influence on cell proliferation and implications for
tumor therapy, in Oxygen Transport to Tissue-II, Grote, J., Reneau, D., and Thews, G., Eds.,
69. Rajewsky, M. F., In vitro studies of cell proliferation in tumours. II. Characteristics of a standardised
in vitro system for the measurement of 3 H-thymidine incorporation into tissue expiants, Eur. J. Can-
cer, 1, 281,1965.
70. Goldacre, R. J. and Sylvên, B., On the access of blood-borne dyes to various tumor regions, Br. J.
Cancer, 16, 306, 1962.
71. Vaupel, P., Hutten, H., and Thews, G., Critical diffusion ranges for oxygen and glucose in tumor
tissue considering Michaelis-Menten kinetics, Bibl. Anat., 13,313, 1975.
72. Vaupel, P., p 0 2 distribution in tumor tissue of DS-Carcinosarcoma, Oncology, 30, 475, 1974.
167
73. Huttcn, H., Thews, G., and Vaupel, P., Some special problems concerning the oxygen supply to
tissue, as studied by an analog computer, in Oxygen Supply, Kessler, M., Bruley, D. F., Clark, L.
C , Luebbers, D. W., Silver, I. A., and Strauss, J., Eds., Urban & Schwarzenberg, Munich, 1973,
25.
74. von Ardenne, A. and von Ardenne, M., Solution of the diffusion field equation of substrate concen-
tration in the intercapillary region considering the decrease in substrate consumption in the unsatu-
rated region of cancer cell glycolysis and respiration, in Oxygen Transport to Tissue-II, Grote, J.,
Reneau, D., and Thews, G., Eds., Plenum Press, New York, 1976, 555.
75. von Ardenne, M. and von Ardenne, A., Gesetzmaessigkeiten der Substratversorgung, der Zellkinetik
und der Therapiemechanismen im Interkapillarraum der Krebsgewebe, Z. Naturforsch., 30 c, 91,
1974.
76. Guenther, H., Vaupel, P., Metzger, H., and Thews, G., Stationaere Verteilung der 0 2 — Drucke im
Tumorgewebe (DS-Carcinosarkom).I. Messungen in vivo unter Verwendung von Goldmikroelektro-
den, Z. Krebsforsch.,11, 26, 1972.
77. Bergsj0, P. and Evans, J. C., Tissue oxygen tension of cervix cancer. Comparison of effects of
breathing a carbon dioxide mixture and pure oxygen, Acta Radiol. Ther. Phys. Biol., 7, 1, 1968.
78. Bergsj0, P. and Evans, J. C , Oxygen tension of cervical carcinoma during the early phase of external
irradiation. I. Measurements with a Clark microelectrode, Scand. J. Clin. Lab. Invest., 22, (Suppl.
106), 159, 1968.
79. Cater, D. B., Oxygen tension in neoplastic tissues, Tumori, 50, 435, 1964.
80. Cater, D. B. and Silver, I. A., Quantitative measurements of oxygen tension in normal tissue and in
the tumours of patients before and after radiotherapy, Acta Radiol., 53, 233, 1960.
81. Cater, D. B., Schoeniger, E. L., and Watkinson, D. A., Effect of oxygen tension of tumours of
breathing oxygen at high pressures, Lancet, 2, 381, 1962.
82. Evans, N. T. S. and Naylor, P. F. D., The effect of oxygen breathing and radiotherapy upon the
tissue oxygen tension of some human tumours, Br. J. Radiol., 36, 418, 1963.
83. Gray, L. H. and Scott, O. C. A., Oxygen tension and the radiosensitivity of tumours, in Oxygen in
the Animal Organism, Dickens, F., and Neil, E., Eds., Pergamon Press, London, 1964, 537.
84. Jamieson, D. and van den Brenk, H. A. S., Comparison of oxygen tensions in normal tissues and
Yoshida sarcoma of the rat breathing air or oxygen at 4 atmospheres, Br. J. Cancer, 17, 70, 1963.
85. Jamieson, D. and van den Brenk, H. A. S., Oxygen tension in human malignant disease under hy-
perbaric conditions, Br. J. Cancer, 19, 139, 1965.
86. KanaFyanov, G. S. and Kauashev, S. K., Partial oxygen pressure and tissue respiration in PC-1
tumors during growth, Bull. Exp. Biol. Med. (U.S.S.R.), 77, 782, 1974.
87. Kolstad, P., Vascularization, oxygen tension, and radiocurability in cancer of the cervix, Norwegian
Monographs on Medical Science, Universitetsforlaget, Oslo, 1964.
88. Kolstad, P., Intercapillary distance, oxygen tension, and local recurrence in cervix cancer, Scand. J.
Clin. Lab. Invest., 22, (Suppl. 106), 145, 1968.
89. Kruuv, J., Inch, W. R., and McCredie, J. A., Effects of breathing gases containing oxygen and
carbon dioxide at 1 and 3 atmospheres pressure on blood flow and oxygenation of tumors, Can. J.
Physiol. Pharmacol., 45, 49, 1967.
90. Urbach, F., Pathophysiology of malignancy. I. Tissue oxygen tension of benign and malignant tu-
mors of the skin, Proc. Soc. Exp. Biol. Med., 92, 644, 1956.
91. Urbach, F., The oxygen tension of cutaneous tumor tissue, measured in vivo, Proc. Am. Assoc.
Cancer Res., 2, 154, 1956.
92. Kruuv, J. A., Inch, W. R., and McCredie, J. A., Blood flow and oxygenation of tumors in mice,
Cancer, (Philadelphia), 20, 51, 1967.
93. Vaupel, P., Manz, R., Mueller-Klieser, W., and Grunewald, W. A., Intracapillary HbO z saturation
in malignant tumors during normoxia and hyperoxia, Microvasc. Res., in press.
to tumour responses to single large doses of radiation, Eur. J. Cancer, 14, 237, 1978.
95. Manz, R., Vaupel, P., and Mueller-Klieser, W., Measurements of H b 0 2 saturations within tumor
capillaries of subcutaneously growing malignant tumors, Pfluegers Arch., 373, R39, 1978.
96. Mueller-Klieser, W., Manz, R., Grunewald, W. A., and Vaupel, P., Intracapillary H b 0 2 saturations
in malignant tumours with central or peripheral blood supply and drainage, / . Physiol. (London), in
press.
97. Gosalvez, M. and Weinhouse, S., Control mechanisms of oxygen and glucose utilization in tumours,
in Oxygen Transport to Tissue-II, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press, New
York, 1976, 587.
98. Vaupel, P. and Thews, G., Pathophysiological aspects of glucose uptake by the tumor tissue under
various conditions of oxygen and glucose supply, in Oxygen Transport to Tissue-II, Grote, J., Re-
neau, D., and Thews, G., Eds., Plenum Press, New York, 1976, 547.
99. Shapot, V. S., Studies on the ability of the tumor respiration in vivo to fulfill its physiological func-
tions, in Oxygen Transport to Tissue-II, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press,
New York, 1976,581.
100. Crabtree, H. G., Observation on the carbohydrate metabolism of tumors, Biochem. J., 23, 536,
1929.
101. Vaupel, P., Guenther, H., and Grote, J., Einfluss einer Hyperglykaemie auf die Sauerstoff-und Glu-
coseaufnahme von Implantationstumoren (DS-Carcinosarkom) in vivo, Z. Krebsforsch., 11, 17,
1972.
102. Vaupel, P., Low oxygen and glucose concentrations, and tissue acidosis as limiting factors for cell
proliferation in malignant tumors, Klin. Wochensch., in press.
169
Chapter 10
M A T H E M A T I C A L M O D E L S O F T R A N S P O R T P H E N O M E N A IN
N O R M A L A N D N E O P L A S T I C TISSUE
Joseph F. Gross and Aleksander S. Popel
TABLE OF CONTENTS
I. Introduction 169
II. Modeling of Oxygen Transport in the Microcirculation 172
III. Modeling of Transport of Indicators in Tissue 175
IV. Modeling of Convective Transport in Tissue 176
V. Modeling of Drug Distribution 179
VI. Summary 180
References 180
The mathematical modeling of transport phenomena in the microcirculation has re-

ceived a great deal of attention in recent years (see reviews by Gross and Aroesty, 1
Middleman, 2 Lightfoot, 3 Leonard and Jdrgensen, 4 and Fletcher5). A series of remark-
able advances in making velocity, pressure, and concentration measurements at the
microscopic level has made it possible to obtain a body of data which can be incorpo-
rated into detailed models for fluid and material transport, both intra- and extralumin-
ally.6 7 It is well known that a large body of whole-organ or macroscopic transport
data exists, but the very complicated and presently unknown scaling relationships be-
tween the microscopic phenomenological behavior of the system and its macroscopic
manifestation make it very difficult to interpret this data. In the case of tumor micro-
circulation, little quantitative data exists at the microscopic level at the present time.
The goal of the present work is to provide a brief review of mathematical modeling
of transport phenomena in normal and neoplastic microcirculation. This theme inter-
faces with several areas that have received extensive attention, not explicitly focusing
on neoplastic tissue. Consequently, no effort is made to include a complete bibliog-
raphy in each of the subtopics discussed here.
It should be noted at the outset that the structure and form of the phenomenological
equations used to describe the transport processes in the microcirculation are the same
for both normal and neoplastic tissue. The differences — and they may be important
— lie in the morphology and physical properties of the two tissues, e.g., the microves-
sel morphology appears much more regular and ordered in normal tissue than in tumor
tissues. There will also be significant differences in the transport properties of the
tissues in the normal and tumor microcirculations It will be important to establish if

the major variations in transport behavior between normal and neoplastic tissue are
caused by fundamental differences in internal tissue structure or result from morpho-
logic variations.
The dimensional scale of the microcirculation has very important implications for
the structure of the equations used to describe the transport phenomena occurring in
the intraluminal flow, transluminal^ across the endothelial wall, and in the extravas-
cular space. A detailed description of capillary blood flow is a formidable task due to
the presence of red blood cells passing single file through the lumen of the capillary.
The character of the flow in the capillary can be defined by a dimensionless ratio of
inertial to viscous forces called the Reynolds number. For flows at the microcirculatory
level, the Reynolds number is about 10"2 or 10"3 and this implies that viscous and
pressure forces are most important in determining flow behavior in the microvessels.
It is beyond the scope of this chapter to discuss in detail the large body of work relating
the mechanical aspects of capillary blood flow. The reader is referred to reviews by
Fung and Zweifach,8 Skalak, 9 Gross and Aroesty, 1 and Goldsmith and Skalak.10
Temporal changes in microcirculatory blood flow resulting from systemic pulsatility
have been quantitatively reported by Intaglietta et al. 6 However, these time-dependent
components are small compared with the absolute values of pressure and flow. In a
series of papers by Aroesty and Gross 1112 and Gross et al., 13 it was demonstrated that
the time-dependent behavior of flow and pressure in the microcirculation could be
analytically represented and that a quasi-steady solution provided an adequate descrip-
tion for the flow. The work on pulsatile microhemodynamics is summarized in a review
by Gross.14
We now consider the problem of material transport in microcirculatory systems.
There are basically two methods by which material is transported. Convection de-
scribes material transport which occurs as a result of macroscopic movement of the
volume element in which the material is found. Molecular diffusion results from the
random motion of the molecules of the material and depends on the molecular weight
of the material, concentration gradient, and other factors. Depending on the behavior
and state of the surrounding medium, either or both of these processes may play an
important role in transporting fluid and material from the lumen to the target cells.
Figure 1, taken from Swabb et al., 15 is given as an example of the regions in which
different transport processes occur as a function of the molecular weight of the mate-
rial and the glycosaminoglycan content of the extravascular space. As the molecular
weight of the material increases, the diffusion coefficient decreases and diffusive trans-
port also decreases. The tissue glycosaminoglycan content provides a measure of the
density of the polysaccharide network in the tissue and hence of the resistance of the
material to species transport. Different tumors are indicated in the figure by their gly-
cosaminoglycan content and some typical drugs, nutrients, and materials that can be
transported through tissue are shown on the right ordinate as a function of their mo-
lecular weight. The ratio of diffusion to convective transport is given by a parameter
À (1/À is generally known as the Peclet number), which appears on the figure as lines
of constant À. When À < 1, convective transport dominates and when À > 1, diffusive
transport is the important mechanism. The region at the left is defined by that combi-
nation of tissue and species for which convection plays an important role in the trans-
port process and the lower right region defines where diffusion is dominant. The figure
shows that oxygen and glucose transport in tumor tissue result primarily from diffu-
sional processes. It can be seen that there is some macroscopic data for both the dif-
fusion and convective regions. It is clear that because of the broad range of glycosa-
minoglycan content of tumors and molecular weight of cytotoxic drugs the
171
FIGURE 1. Diffusive and convective mass transport in the extravascular space of

normal and neoplastic tissue at 37 to 38°C.
extravascular transport of anticancer drugs in neoplastic tissue may be convective, dif-

fusive, or a combination of the two.
Most mathematical models of capillary tissue transport are based on a concept pro-
posed by August Krogh16 in 1919. His model considers an elementary microvascular
unit composed of a cylindrical blood vessel surrounded by a co-axial cylindrical tissue
volume (Figure 2).
Krogh assumed that each capillary was surrounded by parallel capillaries with iden-
tical characteristics (geometry, blood flow, inlet concentration, physical properties of
the tissue). This symmetry led him to formulate a condition that no material flux ex-
isted at the external surface of the tissue cylinder. This assumption is a great oversim-
plification since it neglects tissue heterogeneity entirely. Considering the complexity
FIGURE 2. Geometry of Krogh tissue cylinder model.
and inhomogeneity of neoplastic tissue, it can be anticipated that the Krogh cylinder
model will not be able to provide an adequate description of transport in tumor tissue.
However, because it can often provide analytical solutions, the Krogh model is used
to obtain qualitative results even when the necessary assumptions are not satisfied.
Under certain conditions, the radial gradients of concentration in the tissue may be
negligible which makes it possible to consider "one-dimensional" transport problems.
If axial gradients can also be neglected, then the tissue volume can be regarded as a
well-mixed reservoir. Such models using the concept of a well-mixed tissue volume in
either radial or both radial and axial directions are called compartmental microcircu-
latory models.
A mathematical model of material transport in tissue should generally include con-
vection, diffusion, and consumption by the tissue cells. In the case of certain species,
some of these effects may be insignificant and this makes it possible to construct rela-
tively simple models of the transport process. Mathematical models of transport phe-
nomena in the microcirculation have been very useful in the interpretation of experi-
mental data as well as predicting the physical characteristics of living systems. These
models have been employed to map the spatial and temporal distribution in the tissue
of such materials as oxygen, carbon dioxide, glucose, various tracers, and drugs. In
the present chapter, we review several aspects of transport phenomena in the microcir-
culation with special reference to tumor tissue when it is appropriate.
The mathematical models of transport in the microcirculation that we discuss here
will be divided into four sections:
1. Modeling of oxygen transport in the microcirculation

2. Modeling of transport of indicators in tissue
3. Modeling of convective transport in tissue
4. Modeling of drug distribution
II. MODELING OF OXYGEN TRANSPORT IN THE

MICROCIRCULATION
Although one of the chapters in this book is devoted to oxygen transport in tumors,
it is necessary, in the interest of completeness of the present review of mathematical
173
models, to discuss the analytical formulation which describes oxygen transport. Fur-
thermore, the equations and methods of solution developed for oxygen transport are
applicable to the study of other lipid-soluble species. The model illustrated in Figure 2
consists of a group of capillaries each having radius Rc and length L and surrounded
by a concentric tissue cylinder of radius Rf. Each cylinder is bounded at the arterial
and venous ends by plane surfaces normal to the capillary. Introducing a cylindrical
coordinate system with the coordinate r normal to the capillary axis (0 < r < Rf) and z
parallel to the axis (0 < z < L), we can write the governing equations for the capillary
and tissue oxygen concentration, respectively, in the following form (e.g., Middle-
man) 2 :
(1)
(2)
where c(z,t) is the concentration of oxygen dissolved in the blood, 4* is percent oxygen
saturation of hemoglobin, N is the hemoglobin binding capacity at 100% saturation,
u is the average velocity of blood in the capillary, j is the oxygen flux density at the
capillary-tissue interface, c,(r,z,t) is the oxygen concentration in the tissue, Df is the
diffusivity coefficient in the tissue and g is the oxygen consumption rate. Therefore,
oxygen convection along the capillary axis, oxygen diffusion through the capillary
wall, radial and axial diffusion of oxygen, and oxygen consumption in the tissue region
are all included in the model. Such mechanisms as axial diffusion of oxygen in the
capillary and convective transport in the tissue have been shown to be negligible.
It should be noted that the aforementioned quantities are considered as averaged in
a certain statistical sense, so that irregularities due to the presence of red blood cells
in the capillary, and due to cell, interstitial fluid, and other structures in the extralu-
minal space, are smoothed out.
Since there is no resistance to the passage of oxygen molecules through the capillary
wall, the capillary and tissue oxygen concentration should be in equilibrium at the
interface.
(3)
where a = S 6 /S, is the partition coefficient equal to the ratio of the solubility coeffi-
cients of oxygen in blood and tissue, respectively. Continuity of the oxygen flux
through the capillary wall implies
(4)
At the cylindrical surface r = R„ 0 < z < L, and at the plane surfaces z = 0, 1, Rc <
r < Rf, the flux of oxygen must vanish
(5)
where n is a vector normal to the surface. Further, the oxygen concentration at the
arterial end of the capillary is usually given:
(6)
This latter boundary condition deserves a brief comment. According to studies by

Duling and his co-workers, Duling and Berne17 and Duling and Pittman, 18 the oxygen
concentration at the capillary inlet can be significantly lower than the concentration
in the systemic macrocirculation due to oxygen loss through the walls of small arteries
and arterioles. Recent theoretical investigations by Popel et al. 19 confirmed that tran-
sluminal diffusion may be responsible for large precapillary longitudinal oxygen gra-
dients. These findings indicate that caution should be exercised in assigning the inlet
capillary oxygen concentration.
In certain situations, time-dependent solutions of the equations may be of interest;
for example, the oxygen distribution following blood flow occlusion or restoration.
For such temporal cases, appropriate initial conditions such as the initial concentration
distribution in the capillary and in the tissue must be given.
The governing Equations 1 and 2, contain a number of constant and variable coef-
ficients that can be determined from appropriate experimental data. The quantities N
and H'(c) generally characterize the oxygen-binding characteristics of the blood, (e.g.,
Altman and Dittmer 20 ), the function ^(c) is usually approximated by the two-parame-
ter Hill equation 2
(7)
where k and n are empirical constants.

Oxygen diffusivity in the tissue and the oxygen consumption rate are characteristics
of the tissue; the oxygen consumption rate is usually described by Michaelis-Menten
kinetics2
(8)
where A and B are experimentally determined constants. The data on the diffusivity
and consumption rate for neoplastic tissues were reported, e.g., by Grote et al., 21 Gul-
lino,22 and Boag. 23
Solutions to the governing Equations 1 and 2 can be obtained in an analytical form
only under special conditions. The first such solution for the tissue region in steady
state was derived by Krogh's collaborator Erlang24 under the assumptions that the axial
diffusion of oxygen was negligible, and the oxygen consumption rate constant. This
solution has been the most widely used in physiological studies:
(9)
The analytical studies following the pioneering work of Ki ogh and Erlang 24 have
been done by Thews, 25 Blum,26 Hudson and Cater, 27 Hyman,2* Salathe et al., 29 as well
as others. These solutions have provided some important insights into the problem of
oxygen transport. However, if nonlinear oxygen consumption kinetics are taken into
account, then it is necessary to use numerical methods. In order to solve the relevant
nonlinear partial differential equations, Reneau et al., 3 0 3 1 Gonzalez-Fernandez and
Atta, 32 and Hyman with co-workers, 33 34 carried out comprehensive numerical studies
using the method of finite differences. Halberg et al. 35 utilized Monte Carlo methods,
and Hutten et al. 36 obtained solutions using an analog computer.
175
The solutions for the Krogh cylinder model (or slight modifications) were applied
by Vaupel,37 Hutten et al., 36 von Ardenne and von Ardenne, 38 39 and Boag,40 to deter-
mine oxygen availability in neoplastic tissues.
It was pointed out above that morphological studies of capillary structures in tumors
indicate a high degree of heterogeneity. Therefore, the results based on the Krogh
model generally should not be expected to be in good agreement with the experimental
data which has been confirmed by Thews and Vaupel. 41 In order to eliminate the dis-
crepency, these authors proposed a heuristic approach to oxygen transport considering
a "conglomerate" of Krogh cylinders with different dimensions (without allowance
for interaction between adjacent cylinders) in an attempt to simulate tissue heterogene-
ities. The results of such an approach agreed with the experimental data more closely
than those based on the model of a single Krogh cylinder. More general models taking
into account tissue heterogeneity, and, in particular, allowing for interaction between
the capillaries, have been developed by Griinewald,42 Metzger,43-44 and Popel. 45 A re-
cent review by Griinewald and Sowa46 contains many examples of applications of Grii-
newald's model to the simulation of oxygen transport in skeletal muscle. This model
was also used to study oxygen distribution in tumors. 47 The results of these calculations
are very encouraging.
The models described above can be applied to study the transport of different spe-
cies. To the present time, the Krogh model has been used to simulate transport of
glucose,38 3 9 4 8 4 9 and carbon dioxide49 in normal and neoplastic tissues.
III. M O D E L I N G O F T R A N S P O R T O F I N D I C A T O R S IN TISSUE
The method of injecting certain indicators (dyes, radioactive tracers, etc.) into the
arterial inflow and obtaining their concentrations in the venous outflow is used to
obtain information on such parameters as the blood flow rate, volumes of intravascu-
lar and extravascular spaces, in particular, of the interstitial and intracellular spaces,
capillary and cell permeability, and surface area.
It should be emphasized that the indicator-dilution method is a whole-organ mac-
roscopic method which can only yield average characteristics of the capillary bed, e.g.,
'average' ' capillary permeability. Considering that some tissues exhibit a high degree
of heterogeneity, one can understand that interpretation of whole organ data is a very
complex problem, and the possibility of translating these data to the microscopic level
appears to be rather limited.
Different mathematical models of indicator transport in the tissue have been devel-
oped to facilitate the analysis of such experimental data. The models aimed at provid-
ing the information on the regional blood flow from indicator-dilution method are
based on the integral relationships of conservation of mass and do not consider details
of the microscopic transport in the tissue. Such models are described in reviews by
Zierler50 and Bassingthwaighte and Holloway. 51
It is usually assumed that molecular exchange of indicators occurs at the capillary
tissue level. The degree of sophistication of the models describing such exchange is
suggested by the operational transport mechanisms such as radial and axial diffusion
in the capillary and tissue, and resistance of the capillary tissue barrier. Most of the
models dealing with transport of species in the microcirculation utilize the concept of
the Krogh cylinder described above. In essence, the governing equations describing
transport of indicators are similar to Equations 1 and 2 of the preceding section with
the difference that the terms describing radial and axial diffusion in the capillary may
also be included, and that the consumption term is usually omitted. The boundary
condition, Equation 3, in the case of finite diffusion permeability of the capillary mem-
brane assumes the form
j = P(c-act) (10)
where P is the permeability coefficient which reduces to Equation 3 as P -* °°. Table

1 demonstrates the major accomplishments in the mathematical modeling relevant to
the indicator-dilution method. For additional references concerned with radial diffu-
sion in the capillary, the reader is advised to consult the review by Leonard and Jçír-
gensen.4 Levitt62 carried out the analysis further by allowing mass transfer between the
neighboring Krogh cylinders.
The aforementioned models have been repeatedly applied in studies of capillary tis-
sue exchange of various species in the myocardium, lungs, liver, skeletal muscle, and
other organs and tissues. Similar studies of neoplastic tissues using the indicator-dilu-
tion method could be carried out with parallel development of relevant mathematical
models.
IV. M O D E L I N G O F C O N V E C T I V E T R A N S P O R T IN TISSUE
Convective transport of species becomes important when high convective fluid ve-
locities occur, or when the molecular weight is sufficiently high that diffusional proc-
esses are very slow. Because of data which indicate that the endothelial wall in neo-
plastic tissue is very permeable to fluid movement, a high extravascular fluid velocity
is to be expected. The problem is to link the flows in the different elements of the
transport process from the blood flow in the capillary to the cells in the extravascular
tissue space.
The equations governing convective flows through the endothelial wall separating
the intra- and extraluminal spaces and through the extravascular tissue itself are a
balance between the pressure gradients acting as a driving force for fluid motion and
the resistance due to friction between the fluid and tissue. The major pressure forces
result from variations in the hydrostatic and osmotic pressure in the microvascular
system. The variation in osmotic pressure is due to the differences in protein concen-
tration that exist in the intra and extraluminal compartments.
Fluid movement across the endothelial wall is governed by an equation first enunci-
TABLE1
Summary of Mathematical Models of Indicators Exchange in

Tissue
Capillary Tissue Tissue Capillary

axial radial axial tissue
diffusion diffusion diffusion barrier Ref
N" I* N Fc 52
N I N N 53
N I I F 54
F I F N 55
N I I F 56
N I N F 57
F F F F 58
F I F F 59
N F N F 60
N F N F 61
• This mechanism of diffusion is not taken into account.

6
Infinite diffusion rate.
Finite diffusion rate.
177
ated in 1896 and known as Starling's hypothesis13 which states that the transluminal
hydrostatic and osmotic pressure gradients are the key factors in fluid transport:
(ID
where K(x) = Starling's coefficient, p, = intraluminal hydrostatic pressure, pe = ex-
traluminal hydrostatic pressure, TT, = intraluminal osmotic pressure, ne — extraluminal
osmotic pressure.
The Starling's coefficient is a factor that includes the conductivity properties of the
endothelial wall and other phenomenological transport properties. It is obtained ex-
perimentally by occluding a single capillary and observing the change in position of
erythrocytes or the concentration of an impermeable marker near the point of occlu-
sion. Either of these methods leads to a determination of K for a single capillary. In
normal capillaries, it has been shown that the Starling's coefficient increases from the
arteriolar to venular side64 and that this increase can be as great as a factor of ten. 65
The work of Peterson et al. 66 and Gullino67 has shown that the endothelial wall in
tumors has significantly greater permeability coefficients than normal vessels. This
latter result can be very important because it may represent one of the major differ-
ences between normal and neoplastic microcirculation.
The highly complex nature of the extravascular tissue makes analytical description
difficult but a porous medium model has often been used as a good approximation.
The interstitial fluid velocity is assumed to be proportional to the interstitial pressure
gradient. This relationship is known as Darcy's Law and can be written:
(12)
Kf(PeX.) is the Darcy coefficient and is a function of the interstitial pressure and prop-
erties of the medium. In principle, this is a very complicated function, but it is usually
assumed to be constant or a function of a single property of the medium such as
porosity. An and Salathe 68 have obtained an estimate of both K, and the functional
relationship between porosity of tissue and the interstitial pressure. The governing
equation for interstitial pressure is given by:
(13)
The solution to this equation maps the pressure field in the extravascular tissue space
when Kf is known. The experimental determination of local values of the interstitial
fluid pressure has not yet been achieved, but volume average values can be measured
using a capsule technique. 69 70 These data indicate that fluid pressure can change de-
pending on the flow rate and composition of the intraluminal fluid. The spatial varia-
tion of fluid pressure is that large pressure gradients may occur in the region near the
capillary wall but it becomes constant at distances beyond a few capillary diameters. 71
It has been shown experimentally as indicated above that a change in the intralumi-
nal flow can influence extraluminal fluid pressure and flow distribution. Blake and
Gross72 have studied this problem by simultaneously solving the equations for intra-
luminal flow and Darcy's Law for the fluid movement in the extravascular space with
the Starling's hypothesis as a capillary wall condition to obtain pressure and flow map-
ping in the extravascular space between different geometric arrays of capillaries for
various intraluminal pressure gradients. Their analytical results showed that the fluid
transport through the endothelial wall was independent of neighborhood capillaries,

but the distribution of fluid pressures and flow streamlines in the tissue space depended
strongly on the geometry of the capillary array. They assumed that K, was constant
and had a value of about 10"7 (cm/sec) (cm/cm H 2 0), based on work by Intaglietta
and DePlomb, 73 Winters and Kruger,74 and Guyton et al. 75 Examples of their results
are shown in Figures 3 and 4. In this case, the geometry is a circular array of capillaries
with a single capillary at the center similar to Figure 2. The figures show results for a
plane through the center capillary and two peripheral capillaries. The circular lines
radiating outward are the isobars and the lines with arrows are the flow streamlines.
Figure 3 shows the situation when the intraluminal pressure gradient is small, and it
can be seen that most of the fluid transferred through the endothelial wall moves away
from the capillary array to the outer tissue space. When the intraluminal pressure gra-
dient is increased, the flow map changes completely as shown in Figure 4. Now the
fluid which is filtered through the capillary walls at the arteriolar end is reabsorbed at
the venous end and very little fluid escapes into the outer tissue region. This example
demonstrates that convective transport patterns in the tissue can be quite sensitive to
even a single intraluminal flow property.
It is well known that tumor tissue exhibits a higher degree of inhomogeneity than
does the normal tissue and this will certainly have an important effect on the manner
in which materials are transported from the capillary to the target cells. No detailed
microscopic time-dependent concentration profiles have yet been obtained, but such
experiments are presently being carried out by Endrich and Intaglietta.76 The molecular
weights of materials to be convected range from about 102 to 104 and in this range it
FIGURE 3. Extraluminal flow for Krogh geometric array, small intraluminal pressure difference.
179
has been shown by Swabb et al. 15 that the tissue diffusion coefficients are about 10"5
to 10"7 cmVsec. For intercapillary lengths of the order of 100 ^m, this would yield a
diffusional velocity of 10"3 to 10"5 cm/sec. In vitro filtration data on tumor tissue have
yielded fluid flux velocities of about 10~6 cm/sec. These are all order-of-magnitude
estimates but it appears that in the range of lower molecular weights, the major con-
tribution to transport is by diffusion just as in normal tissue.
V. M O D E L I N G O F D R U G D I S T R I B U T I O N
As was pointed out in the introduction, the transport of drugs in neoplastic tissues
may be carried out by different forms of diffusion and convection, depending upon
the properties of drug and tissue. Therefore, it is apparent that the mathematical
models described in the preceding paragraphs are relevant as well to the transport of
drugs in the microcirculation. The majority of studies of drug transport in the cardio-
vascular system are concerned with the distribution of drugs among the blood and
different organs of the body described as "well-mixed'' compartments. Such an ap-
proach called "macroscopic pharmacokinetics' ' is well described in the literature. 7779
On the contrary, only few examples of more detailed analysis of drug distribution in
the capillary tissue region have been developed, among which we should mention the
pioneering work by Bellman et al., 80 who formulated the equations of mass balance
in the capillary, interstitial space and cells taking into account the capillary tissue and
FIGURE 4. Extraluminal flow for Krogh geometric array, large intraluminal pressure difference with
balanced absorption and filtration.
interstitial fluid cell barriers. Very often, the lack of detailed information on physical
parameters required by such a model somewhat restricts its application to realistic
situations. It is hoped that the future cooperative theoretical and experimental efforts
will provide a complete picture of drug distribution in neoplastic tissue on the micro-
circulatory level.
VI. S U M M A R Y
We have attempted to summarize briefly the basic elements of mathematical mod-

eling of species transport in the normal microcirculation and how these methods may
be applied to neoplastic tissue. A body of whole organ and regional data now exists
for tracer and drug studies in neoplastic tissue and detailed measurements at the micro-
scopic level are now being carried out. The latter, together with the appropriate
models, provide the framework for the scaling laws to link the whole organ results
with the microscopic data. This should yield a quantitative and predictive method for
describing species transport in tumors.
REFERENCES
1. Gross, J. F. and Aroesty, J., Mathematical models of capillary flow: a critical review, Biorheology,
9,225, 1972.
2. Middleman, S., Transport Phenomena in the Cardiovascular System, Interscience, New York, 1972,
chap. 3 and 4.
3. Lightfoot, E. N., Jr., Transport Phenomena and Living Systems, Interscience, New York, 1974.
4. Leonard, E. F. and J0rgensen, S. B., The analysis of convection and diffusion in capillary beds,
Annu. Rev. Biophys. Bioeng., 293, 1974.
5. Fletcher, J. E., Mathematical modeling of the microcirculation, Math. Biosci., 38, 159, 1978.
6. Intaglietta, M., Richardson, D. R., and Tompkins, W. R., Blood pressure, flow, and elastic proper-
ties of microvessels of cat omentum, Am. J. Physiol., 221, 922, 1971.
7. Nakamura, Y. and Wayland, H., Micromolecular transport in the cat mesentery, Microvasc. Res.,
9,21, 1975.
8. Fung, Y. C. and Zweifach, B. W., Microcirculation: mechanics of blood flow in capillaries, Annu.
Rev. FluidMech., Van Dyke, M. and Vincenti, W. G., Eds., 189, 1971.
9. Skalak, R., Mechanics of the microcirculation, in Biomechanics: Its Foundations and Objectives,
Fung, Y. C , Perrone, N., and Anliker, M. N., Eds., Prentice-Hall, Englewood Cliffs, N. J., 1972,
457.
10. Goldsmith, H. L. and Skalak, R., Hemodynamics, Annu. Rev. Fluid Mech., 7, 213, 1975.
11. Aroesty, J. and Gross, J. F., The mathematics of pulsatile flow in small vessels. I. Casson theory,
Microvasc. Res., 1, 1972.
12. Aroesty, J. and Gross, J. F., Pulsatile flow in small blood vessels. I. Casson theory, Biorheology, 9,
33, 1962.
13. Gross, J. F., Intaglietta, M., and Zweifach, B. W., Network models of pulsatile hemodynamics in
the microcirculation of the rabbit omentum, Am. J. of Physiol., 226, 1117, 1974.
14. Gross, J. F., The significance of pulsatile microhemodynamics, in Microcirculation, Vol. 1, Kaley,
G. and Altura, B., Eds., University Park Press, Baltimore, Md., 1977, 365.
15. Swabb, E. A., Wei, J., and Guillino, P. M., Diffusion and convection in normal and neoplastic
tissues, Cancer Res., 34, 2814, 1974.
16. Krogh, A., The Anatomy and Physiology of Capillaries, Hafner, New York, 1959.
17. Duling, B. R. and Berne, R., Longitudinal gradients in periarteriolar oxygen tension: a possible
mechanism for the participation of oxygen in local regulation of blood flow, Circ. Res., 27, 669,
1970.
18. Duling, B. R. and Pittman, R. N., Oxygen tension: dependent or independent variable in local control
of blood flow?Fed. Proc. Fed. Am. Soc. Exp. Biol., 37, 2012, 1975.
181
19. Popel, A. S., Gross, J. F., and LaLone, B. J., Simulation of precapillary oxygen transport in skeletal
muscle, submitted for publication, 1978.
20. Altman, P. L. and Dittmer, D. S., Eds., in Respiration and Circulation, Federation of American
Societies for Experimental Biology, Bethesda, Md., 1971, 19.
21. Grote, J., Süsskind, R., and Vaupel, P., Oxygen diffusivity in tumor tissue (DS-carcinosarcoma)
under temperature conditions within the range of 20—40°C, PfluegersArch., 372, 37, 1977.
22. GuUino, P. M., In vivo utilization of oxygen and glucose by neoplastic tissue, in Oxygen Transport
to Tissue — //, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press, New York, 1976, 521.
23. Boag, J. W., Cell respiration as a function of oxygen tension, Int. J. Radiât. Biol., 18, 475, 1970.
24. Krogh, A., The number and the distribution of capillaries in muscles with the calculation of the
oxygen pressure head necessary for supplying the tissue, / . Physiol. (London), 52, 409, 1919.
25. Thews, G., Uber die mathematische Behandlung physiologischer diffusion Prozesse in zylinderfttr-
migen Objekten, Acta Biotheor., 10, 105, 1953.
26. Blum, J., Concentration profiles in and around capillaries, Am. J. Physiol., 198, 991, 1960.
27. Hudson, J.A. and Cater, D. B., An analysis of factors affecting tissue oxygen tension, Proc. R. Soc.
London, B161, 247, 1964.
28. Hyman, W. A., A simplified model of the oxygen supply function of capillary blood flow, in Oxygen
Transport to Tissue, Pharmacology, Mathematical Studies and Neonatology, Bruley, D. and Bicher,
H., Eds., Plenum Press, New York, 1973, 835.
29. Salathe, E. P., Wang, T . - C , and Gross, J. F., A mathematical analysis of oxygen transport to tissue,
submitted for publication.
30. Reneau, D., Bruley, D., and Knisely, M., A mathematical simulation of oxygen release, diffusion,
and consumption in the capillaries and tissue of the human brain, in Chemical Engineering in Medi-
cine and Biology, Hershey, D., Ed., Plenum Press, New York, 1967, 135.
31. Reneau, D., Bruley, D., and Knisley, M., A digital simulation of transient oxygen transport in cap-
illary-tissue systems (cerebral gray matter), AIChE J., 15, 916, 1969.
32. Gonzales-Fernandez, J. M. and Atta, S. E., Transport and consumption of oxygen in capillary-tissue
structures, Math. Biosci.,2, 225, 1968.
33. Hyman, W. A., Grounds, D. J., and Newell, P. H., Oxygen tension in a capillary-tissue system
subject to periodic occlusion, Microvasc. Res., 9, 49, 1975.
34. Hyman, W. A. and Artigue, R. S., Oxygen and lactic acid transport in skeletal muscle: effect of
reactive hyperemia, Ann. Biomed. Eng., 5, 260, 1977.
35. Halberg, M., Bruley, D., and Knisley, M., Simulating oxygen in the microcirculation by Monte-Carlo
methods, Simulation, 15, 206, 1970.
36. Hutten, G., Thews, G., and Vaupel, P., Some special problems concerning the oxygen supply to
tissue, as studied by an analogue computer, in Oxygen Supply, Kessler, M., Bruley, D. F., Clark, L.
C , Lubbers, D. W., Silver, I. A., Strauss, Eds., University Park Press, Baltimore, Md., 1973, 25.
37. Vaupel, P., Atemgaswechsel und Glucosestoffwechsel von Implantationstumoren (DS-Carcinosar-
com) in vivo, in Funktionsanalyse biologischer Système, Vol. 1, Thews, G., Ed., Steiner, Wiesbaden,
1974,78.
38. von Ardenne, A. and von Ardenne, M., Solution of the diffusion field equation of substrate concen-
tration in the intercapillary region considering the decrease in substrate consumption in the unsatu-
rated region of cancer cell glycolysis and respiration, in Oxygen Transport to Tissue — //, Grote, J.,
39. von Ardenne, A., and von Ardenne, M., Solution of the diffusion field equation of substrate concen-
tration in the intercapillary region considering the increase in substrate consumption in the unsatu-
rated region of cancer cell glycolysis and respiration, in Oxygen Transport to Tissue—//, Grote, J.,
40. Boag, J. W., Oxygen diffusion in tumor capillary networks, Bibl. Anat., 15, 266, 1977.
41. Thews, G. and Vaupel, P., 0 2 supply in tumor tissue, in Oxygen Transport to Tissue — //, Grote,
J., Reneau, D., G., Eds., Plenum Press, New York, 1976, 547.
42. Griinewald, W., Computer calculation for tissue oxygenation and the meaningful presentation of the
results, in Oxygen Transport to Tissue — //, Grote, J., Reneau, D., and Thews, G., Eds., Plenum
Press, New York, 1976, 783.
43. Metzger, H., Geometric considerations in modeling oxygen transport processes in tissue, in Oxygen
Transport to Tissue — //, Grote, J., Reneau, D., and Thews, G., Eds., Plenum Press, New York,
1976,761.
44. Metzger, H., Distribution of oxygen partial pressure in a two-dimensional tissue supplied by capillary
meshes and concurrent and countercurrent systems, Math. Biosci., 5, 143, 1969.
45. Popel, A. S., Analysis of capillary tissue diffusion in multi-capillary systems, Math. Biosci., 1978.
46. Griinewald, W. A. and Sowa, W., Capillary structures and 0 2 supply to tissue, Rev. Physiol.
Biochem. Pharmacol., 11, 149, 1977.
47. Vaupel, P., Grunewald, W. A., Manz, R., and Sowa, W., Intracapillary H b 0 2 saturation in tumor
tissue of DS-carcinosarcoma during normoxia, in Oxygen Transport to Tissue — ///, Silver, I. A.,
Erecinska, M., and Bicher, H. I., Eds., Plenum Press, New York, 1978, 367.
49. Davis, E. J., Cooney, D. O., and Chang, R., Mass transfer between capillary blood and tissues,
Chem. Eng. J. (Lausanne), 7, 213, 1974.
50. Zierler, K. L., Circulation times and the theory of indicator-dilution methods for determining blood
flow and volume, in Handbook of Physiology, Section 2, Vol. 1, Hamilton, W. F., and Dow, P.,
Eds., American Physiology Society, Washington, D. C , 1962, 585.
51. Bassingthwaighte, J. B. and Holloway, G. A., Jr., Estimation of blood flow with radioactive tracers,
Semin. Nucl. Med .,6, 141, 1976.
52. Sangren, W. C. and Sheppard, C. W., Mathematical derivation of the exchange of a labeled sub-
stance between a liquid flowing in a vessel and an external compartment, Bull. Math. Biophys., 15,
387,1953.
53. Goresky, C. A., A linear method for determining liver sinusoidal and extravascular volumes, Am.
J. Physiol, 204, 626, 1963.
54. Johnson, J. A. and Wilson, T. A., Model for capillary exchange, Am. J. Physiol, 210, 1299, 1966.
55. Perl, W. and Chinard, F. P., A convective diffusion model of indicator transport through an organ,
Circ. Res., 22, 273, 1968.
56. Levitt, D. G., Evaluation of the early extraction method of determining capillary permeability by
theoretical capillary and organ models, Circ. Res., 21, SI, 1970.
57. Goresky, C. A., Ziegler, W. H., and Bach, G. G., Capillary exchange modeling: barrier-limited and
flow limited distribution, Circ. Res., 27, 739, 1970.
58. Bassingthwaighte, J. B., Knopp, T. J., and Hazelrig, J. B., A concurrent flow model for capillary
tissue exchange, in Capillary Permeability, Crone, C. and Lassen, N. A., Eds., Academic Press, New
York, 1970, 60.
59. Bassingthwaighte, J. B., A concurrent flow model for extraction during transcapillary passage, Circ.
Res., 35, 483, 1974.
60. Lee, J. S. and Fronek, A., An analysis of the exchange of indicators in single capillaries, Microvasc.
Res., 2, 302, 1970.
61. Kuo, Y. M., Gustafson, W. A., and Friedman, J. J., Radial diffusion effects in a finite extravascular
space surrounding a single capillary, Microvasc. Res., 5, 148, 1973.
62. Levitt, D. G., Theoretical model of capillary exchange incorporating interactions between capillaries,
Am. J. Physiol.,220, 250, 1971.
63. Starling, E. N., On the absorption of fluids from the convective tissue spaces, / . Physiol (London),
19,312, 1896.
64. Intaglietta, M., Evidence for a gradient of permeability in frog mesenteric capillaries, in Bibl Anat.,
9,465, 1967.
65. Gore, R. W., Schoknect, W., and Bohlen, H. G., Filtration coefficients of single capillaries in rat
intestinal muscle, in Microcirculation I, Grayson, J. and Zingg, W., Eds., Plenum Press, New York,
1976,331.
66. Peterson, H.-L, Appelgren, L., Lundborg, G., and Rosengren, B., Capillary permeability of two
transplantable rat tumors as compared with various normal organs of the rat, Bibl. Anat., 12, 511,
1973.
67. Gullino, P. M., Extravascular compartments of solid tumors, in Cancer, Vol. 3, Becker, F. F., Ed.,
68. An, K. N. and Salathe, E. P., A theory of interstitial fluid motion and its implication for capillary
exchange, Microvasc. Res., 12, 103, 1976.
69. Guyton, A. C , Concept of negative interstitial pressure based on pressures in implanted perforated
capsules, Am. J. Physiol, 216, 460, 1963.
70. Stromberg, D. D. and Wiederhielm, C. A., Effects of oncotic gradients and enzymes on negative
pressures in implanted capsules, Am. J. Physiol, 219, 928, 1970.
71. Salathe, E. P., An analysis of interstitial fluid pressure in the web of the bat wing, Am. J. Physiol,
1,297, 1977.
72. Blake, T. R. and Gross, J. F., A theoretical study of the relationship between single and multiple
capillary fluid exchange, Bibl. Anat., 15, 148, 1977.
73. Intaglietta, M. and DePlomb, T., Fluid exchange in tube and tunnel capillaries, Microvasc. Res., 6,
153,1973.
74. Winters, A. D. and Kruger, S., Drug effects on bulk flow through mesenteric membranes, Arch.
Int. Pharmacodyn. Ther., 173, 213, 1968.
183
75. Guyton, A. C , Scheel, K., and Murphree, D., Interstitial fluid pressure. III. Its effect of resistance
to tissue fluid mobility, Circ. Res., 19, 412, 1966.
76. Endrich, B. and Intaglietta, M., Microcirculation and molecular transport in mammary carcinoma,
Progress Report, Gross, J. F., Ed., Breast Cancer Task Force, National Cancer Institute, National
Institutes of Health, Bethesda, Md., 1978.
77. Bischoff, K. B. and Brown, R. G., Drug distribution in mammals, Chem. Eng. Prog. Symp. Ser.,
62,33,1966.
78. Bischoff, K. B., Some fundamental considerations of the application of pharmacokinetics to cancer
chemotherapy, Cancer Chemother. Rep. Part 1,59, 777, 1975.
79. Gross, J. F. and Dedrick, R. L., Macroscopic pharmacokinetics and cancer chemotherapy, in Proc.
AIChE-GVC Conf., Vol. 4, American Institute of Chemical Engineers, New York, 1974, F4.
80. Bellman, R., Jacquez, J. A., and Kalaba, R., Some mathematical aspects of chemotherapy. I. One-
organ models, Bull. Math. Biophys.,22, 181, 1960.
Taylor & Francis
185
Chapter 11
A N G I O G R A P H Y A N D P H A R M A C O A N G I O G R A P H Y IN H U M A N A N D
EXPERIMENTAL TUMORS
Leif Ekelund
TABLE OF CONTENTS
I. Angiography 185
II. Pharmacoangiography 189
III. Conclusions 196
References 201
I. A N G I O G R A P H Y
The role of angiography in the diagnosis of malignant tumors is established. The

angiographic diagnosis of malignancy is usually based upon the demonstration of
either coarse or tiny tortuous, irregular "tumor vessels", often in combination with
an abnormal early arteriovenous shunting of contrast medium within the tumor. Ac-
cumulation of contrast medium in vascular lakes — pooling — and arterial encasement
provide additional evidence of neoplastic origin as well as the accumulation of contrast
medium within the lesion, so-called tumor stain. Some of these findings are typically
illustrated in the classical angiographic picture of a renal carcinoma (Figure 1). How-
ever, confusion still exists among angiographers concerning the concept of *'tumor
vessels". Are those vessels that can be demonstrated angiographically in a malignant
tumor pathologically altered normal vessels, or are they really newly-formed vessels?
In order to investigate the morphologic background of tumor angiography, Billing and
Lindgren, 1 as early as 1944, injected contrast medium into arteries in 14 operative
specimens of renal carcinoma and 8 of gastric carcinoma, and studied the angioarchi-
tecture of these tumors radiographically and histologically. In renal carcinomas vessels
without elastine in the walls, measuring 1 to 3.75 mm in diameter, were found, whereas
tiny, pathological vessels were seen in the gastric carcinomas. Ljunqvist and Lager-
gren,2 in a thorough, combined angiographic, microangiography, and histological
study of 24 renal adenocarcinomas, were able to demonstrate four microangiographic
tumor patterns. Irrespective of the caliber of the tumor vessels, the wall was of capil-
lary type with no evidence of either a muscle coat or elastic tissue. In order to elucidate
this subject further, Ekelund et al., 3 correlated the angiographic and histopathologic
findings in a number of renal and pancreatic carcinomas, as well as in a group of
malignant gliomas. An overall good correlation was found between the angiographic
and microscopic pictures of tumor vascularization. As to renal carcinomas, no corre-
lation was seen between tumor differentiation, cell type, and angiographic findings.
FIGURE 1. Angiography of a huge renal carcinoma. Abundant tu-

mor vessels in middle and lower portion of the kidney with arteriove-
nous shunting caudally (arrows).
The degree of vascularization, as demonstrated by angiography, was found to be pri-

marily correlated to the extent of necrosis within the tumor. Histologically, the vascu-
lar pattern in the vital parts of renal carcinomas was dominated by capillary and lacu-
nary vessels. The vascular endothelium covered the tumor cells directly without any
intervening smooth muscle cells in the wall (Figure 2). These structures obviously rep-
resented newly formed tumor vessels and were seen more often in angiographically
hypervascularized tumors.
In malignant gliomas the formation of new vessels is a well known phenomenon.
As in renal carcinomas, these vessels were lined only by endothelial cells, but in gliomas
this often occurred with a pronounced proliferation, several layers of swollen cells
being found in the wall, which was therefore thickened, with a consequent narrowing
of the vascular lumina. This corresponded well with the angiographic picture charac-
terized by a varying number of tiny tumor vessels.
If newly formed tumor vessels thus certainly exist in renal carcinomas and malignant
gliomas, whether they may also be found in pancreatic carcinoma is far more question-
able. Angiographic demonstration of such vessels has been reported by several authors,
including Lunderquist, 4 and Bookstein et al., 5 and Tylen.6 Histological evidence for
this concept was provided by Molas et al., 7 who found thin-walled capillary vessels in
187
FIGURE 2. Highly differentiated renal carcinoma of clear cell type. Numerous capillar-
ies and a venous lake, coated by endothelium, directly surrounded by tumor cells. Hema-
toxylin-erythrosin x 100. (From Ekelund, L., Jonsson, N., and Lunderquist, A., Radiol-
oge, Springer-Verlag, Heidelberg, 17, 1977, 95.)
operative specimens of pancreatic carcinoma. These vessels, however, were too small
to be demonstrated at in vivo angiography. They also found arterial occlusions, and
concluded that from an angiographic point of view, pancreatic carcinoma must be
regarded as avascular. This opinion was shared by Marions and Wiechel,8 who per-
formed angiography of surgically removed pancreatic carcinomas without being able
to demonstrate newly formed vessels. Microscopically, however, they found a small
amount of wide capillary vessels within the tumors. Similar vessels were also found in
the histological examinations of Ekelund et al., 3 while, as judged from angiography,
9 out of 10 pancreatic carcinomas were avascular. These authors also pointed to the
remarkable paucity of veins within the neoplasms, and if a vein was found at micros-
copy it was usually occluded by a thrombus. This corresponds very well with the find-
ings in selective venography of pancreatic carcinomas, where venous obliterations are
important features. 9
The different vascularization of various tumors may perhaps be explained by the
recently isolated tumor angiogenesis factor that is mitogenic to endothelial cells and
stimulates rapid formation of new capillaries in animals. 10 It might be that this factor
is produced in, e.g., renal carcinomas and malignant gliomas, but not by the scanty
cells in a pancreatic carcinoma.
Experimental tumors may also be investigated by in vivo angiography. The rat is
widely used in experimental cancer research, but until recently no technique has been
available for the angiographic study of these tumors. In 1970 Ekelund and Olin11 de-
veloped a technique for selective arterial catheterization in the rat. The catheterization
is performed under general ether anesthesia, the radiopaque catheter (OPP 10, Portex,
England, O.D./I.D. 0.65/0.25 mm), being introduced from the femoral artery by a
cutdown technique. With the aid of magnification fluoroscopy, the preformed tip of
FIGURE 3. Selective renal angiogram in a rat with a dimethylnitro-

samine-induced well-differentiated kidney tumor. Abundant neovas-
cularity and arteriovenous shunting of contrast medium cranially. Up-
per pole of kidney (white arrows). Radiopaque catheter (black arrow).
the catheter can be directed into most branches of the aorta, e.g., the renal, celiac, or
superior mesenteric artery, as well as the carotid or vertebral artery. A small film chan-
ger for industrial film is used to obtain serial angiograms. With this technique it is
possible to obtain films of high quality; at a selective renal angiography, it is thus
possible to visualize the glomeruli. Using this technique, various experimental tumors
have been investigated, including renal, urinary bladder, and hepatic neoplasm. 121314
Renal tumors were induced by dimethylnitrosamine. The best time to examine these
tumors by angiography appeared to be 6 to 8 months after the introduction of carcin-
ogen. These neoplasms were often anaplastic and, angiographically, appeared hypo-
vascular, but well-differentiated tumors presenting as hypervascular lesions at angiog-
raphy were also seen (Figure 3).
Liver tumors were induced either chemically with dibuthylnitrosamine or by direct
inoculation with living tumor cells intr apor tally. One drawback of chemical carcino-
genesis is the slow tumor growth, while tumors induced by direct inoculation could
already be demonstrated at angiography after 2 to 3 weeks (Figure 4).
With the present technique it has become possible to study the growth of experimen-
tal tumors by repeat angiography, which should be of great help in the future experi-
mental development of various therapeutic procedures; surgical, cytostatic, as well as
immunologic. By transcatheter arterial embolization of experimental hepatic tumors,
it has thus been found possible to arrest the arterial supply of neoplasms temporarily
189
FIGURE 4. Celiac angiography in a rat three weeks after intraportal in-

oculation with 106 living colon carcinoma cells. Lateral projection. Large
tumor, supplied from wide and tortuous arteries (arrows).
with ensuing extensive tumor necrosis15 (Figure 5). This might provide an alternative
to surgical hepatic dearterialization in certain patients. Transcatheter embolic occlu-
sion of abdominal tumors is receiving increasing interest, and may be applied either
preoperatively in order to facilitate surgery (decrease bleeding) or to control hemor-
rhage, or palliate local tumor symptoms in patients who are not surgical candidates. 16
Gross hematuria may thus be eliminated in patients with inoperable renal carcinoma
(Figure 6).
II. P H A R M A C O A N G I O G R A P H Y
The angiographic diagnosis of richly vascularized tumors usually offers no diffi-

culty, while small cystic or sparsely vascularized neoplasms may escape detection alto-
gether. Since the walls of newly formed tumor vessels are usually made up of endoth-
elial cells alone and lack contractile elements, they are not as capable as normal vessels
of responding to a vasoactive stimulus. This structure of neoplastic vessels is seen in
human as well as in experimental tumors (Figures 2, 7), and it provides the morpho-
logic background for the positive results that may be obtained from pharmacoangiog-
FIGURE 5. Same case as in Figure 4. After transcatheter arterial emboli-

zation with small gelatin, the arterial supply of the neoplasm is arrested.
raphy. In order to direct the largest possible amount of contrast medium into these
tumor vessels, it seems reasonable to use a vasoconstrictor to reduce the blood flow
to surrounding normal vessels. Epinephrine is the drug that has been used most often
for this purpose, and mainly in the kidney. 1718 However, pitfalls have been reported
in which epinephrine caused constriction of larger renal arteries supplying a peripher-
ally located tumor with subsequent nonvisualization of the neoplasm. 19 20
Acting upon the results of Elkin and Meng, 21 who found that in dogs angiotensin
acted further peripherally in the vascular bed than epinephrine, at least in the kidney,
the former drug was introduced in clinical renal pharmacoangiography by Ekelund et
al.22 Their hypothesis was that the risk of constricting arteries feeding the tumor should
be smaller with angiotensin. The results were so promising that we now always include
an angiotensin study in equivocal cases (Figure 8). Ekelund and Gõthlin 23 studied the
effect of angiotensin on normal human renal circulation by means of angiography and
blood flow measurements, using a dye dilution technique. They concluded that angiog-
raphy should be performed 10 to 60 sec after the adminitration of angiotensin into the
renal artery, and found the optimum dose to be 0.5 /¿g angiotensin for renal tumors.
A survey of pharmacoangiography of the kidney was given by Ekelund in 1973.24
Angiotensin has also been used with good results in areas other than kidneys, includ-
191
FIGURE 6. Same case as in Figure 1. This patient had pulmonary metastases:

surgery was therefore not considered. Suffered from intractable hematuria. The
renal artery has been occluded by percutaneous transcatheter embolization and the
hematuria stopped immediately.
ing liver, pancreas, bone, and soft tissues. Thus, Ekelund and Lunderquist25 investi-
gated the diagnostic effect of angiotensin in 80 patients, employing selective catheteri-
zation of various arteries. The diagnostic value of angiography was improved in about
70% of cases. The improvement usually consisted of enhanced filling of smaller arter-
ies with a higher concentration of contrast medium. In three patients (one with liver
metastasis, one with a fibrosarcoma in the soft tissue of the right upper extremity, and
one with an adenocarcinoma of the cecum), the diagnosis was possible, thanks only
to the findings obtained with pharmacoangiography when no abnormal changes were
seen in control angiograms (Figure 9). The authors concluded that pharmacoangiog-
raphy with angiotensin is not the definitive answer to the problem of angiographic
FIGURE 7. Chemically induced rat renal tumor with endothelium-

coated vessel without muscle cells in the wall. Hematocylin-eosin x
100. (From Ekelund, L., Gothlin, J., Jonsson, N., and Sjogren, H.
O., Acta Radiol. Diagn., 17, 329, 176.
differentiation between benign and malignant lesions. However, this drug can enhance
visualization of tumors and will sometimes help to demonstrate tumors which are not
apparent on routine angiography.
Among vasoconstrictors, vasopressin has also been used in pharmacoangiography.
Carlsson and Eriksson26 employed this drug in nephroangiography and found that
vasopressin contracted the arteries, did not change the circulation time, and increased
the retrograde flow of contrast medium, thus indicating some decrease in renal blood
flow. Tumor vessels were not affected by the drug. Using a dye dilution technique,
Gothlin27 found that vasopressin induced a biphasic response in normal human kid-
neys, with an initial decrease followed by a compensatory increase above normal. A
reverse response to vasopressin was found in three hypervascularized renal carcinomas,
and it was suggested that tumor vessels might react in some degree to vasoconstricting
drugs. A similar suggestion was recently made by Mattsson et al., 28 who studied intra-
tumoral blood flow distribution in transplantable rat tumors by isotope technique, and
found that noradrenaline infusion reduced the intratumoral blood flow distribution
significantly, as well as that in normal muscle. These observations suggested that tumor
vessels might be influenced by vasoactive drugs acting on alpha receptors. However,
193
FIGURE 8. (A) Control selective renal angiogram in a patient with hematuria. Avascular mass in middle
portion of the left kidney. (B) Angiography after the injection of 0.5 /¿g angiotensin in the renal artery.
Normal arteries constricted, but numerous tumor vessels are now demonstrated within the lesion. Histolog-
ical diagnosis: necrotic renal carcinoma.
the similar response in tumor and normal muscle to noradrenaline infusion might also
indicate that changes of blood flow distribution in tumors are secondary to induced
changes in normal tissue blood flow distribution in the transplantation area. Ob-
viously, this matter needs further evaluation before any certain conclusions can be
drawn about the possible ability of tumor vessels to react to vasoactive stimuli.
Vasodilators have also been tried in connection with angiography in order to en-
hance demonstration of tumor vessels. Thus, Kahn and Callow29 tried tolazoline in
FIGURE 8B.
order to improve the diagnostic information in bronchial, celiac, and femoral angiog-
raphy, and recently Hawkins 30 and Hawkins and Hudson 31 have advocated the use of
this drug to demonstrate poorly vascularized visceral, bone, and soft tissue tumors.
With very few exceptions, no clinical comparison of the effect of vasoactive drugs on
tumor vascularity in the individual case has been undertaken. As experimental tumors
should provide an excellent model for such comparative pharmacoangiographic inves-
tigation, Ekelund et al., 32 tested three vasoconstrictors and one vasodilator in rat renal
and hepatic tumors. Vasoconstrictors (angiotensin, norepinephrine, and vasopressin)
were found to give more diagnostic information than the vasodilator tolazoline. An-
giotensin turned out to be the superior drug (Figure 10). This comparative pharma-
coangiographic study was later extended into the clinic, using a number of bone and
195
FIGURE 9. Subclavian angiograms of a 58-year-old woman with a fibrosarcoma in the upper portion of
the right arm, employing photographic subtraction. (A) No tumor demonstrated. (B) After 10 \jig of angi-
otensin, a faint accumulation of contrast material in the tumor (arrowheads) and arteriovenous shunting
are seen. Feeding artery (open arrow) and draining vein (black arrow). (From Ekelund, L. and Lunderquist,
A., Radiology, 110, 533, 1974.
soft tissue tumors. 33 One vasoconstrictor (angiotensin) and one vasodilator (tolazoline)
were tested. The results clearly indicated that angiotensin was superior in improving
diagnosis. Following pharmacoangiography with tolazoline, tumor vessels were often
"drowned" within normal vasculature and significant arteriovenous shunting of con-
trast medium was usually masked (Figure 11). Jonsson et al. 34 recently tried another
vasodilator, prostaglandin Elf in pharmacoangiography of bone and soft tissue tu-
mors. Except in one case involving a hemangioma, the diagnostic information about
tumor vascularity and delineation of the extent of disease decreased as compared to
control. On the basis of this combined experimental and clinical experience, it may be
concluded that in order to demonstrate poorly vascularized tumors at pharmacoan-
giography, vasoconstrictors should be preferred to vasodilators. This also seems logi-
cal; as tumor vessels react less (or not at all) to vasoactive stimuli, to direct the greatest
amount of contrast medium into these vessels, normal vessels surrounding the lesion
should be constricted.
One interesting feature of angiotensin is the different optimal dose level in various
vascular fields. While 0.5 /¿g was found to be optimal for pharmacoangiography of
the kidneys, higher doses were required in the extremities. The dose-response relation-
ship also seemed to vary between individuals, among other things, probably owing to
the status of the electrolyte balance. Thus, it has been shown in rabbits that sodium
FIGURE 10. Celiac angiograms in a rat 14 days after intraportal inocula-

tion with 106 living cells from a colon carcinoma. Lateral projection. (A)
Control series. Several tumors of varying size (arrows). (B) After angiotensin
more abnormal vessels are filled in the largest tumor. Another lesion, not
visible at control series, is now obvious posteriorly (arrow). (C) Post-norep-
inephrine series. Less information obtained from this series compared to con-
trol. Neither vasopressin nor tolazoline increased the diagnostic information
as compared to control angiography. (From Ekelund, L., Gõthlin, J., Jons-
son, N., and Sjogren, H., Acta Radiol. Diagn., 17, 329, 1976.)
restriction results in a marked reduction in the vascular response to angiotensin. 35 Fur-

thermore, in normal man sodium restriction reduces the sensitivity to angiotensin ten
times.36 In our own experience, 10 to 15 /ng of angiotensin is a convenient and accept-
able dose in the axillary, iliac, and femoral arteries.
III. C O N C L U S I O N S
Even if it is evident that pharmacoangiography may provide increased diagnostic

information about tumor vessels, it must be emphasized that pitfalls do exist. The
angiographer has to be aware of the optimum dose levels to work with, as too high a
dose of a vasoconstrictor may prevent contrast medium from reaching the lesion. It
must also be realized that inflammatory granulation tissue may contain vessels which
lack contractile elements in the wall; hence, the same diagnostic effect may be achieved
as in malignant tumors. As always in diagnostic radiology, clinical correlation is nec-
essary.
197
FIGURE ÍOB.
FIGURE 10 C.
FIGURE 11. Femoral angiograms of a 54-year-old woman with a poorly differentiated

fibrosarcoma in the left thigh. (A) Control series. (B) Increased accumulation of contrast
medium within the tumor after 15 /¿g of angiotensin. Arteriovenous shunting is also visible
although not demonstrated in the control series. Normal arteries constricted. (C) After 25
mg of tolazoline, diagnostic information is impaired, compared to the control. (From Ek-
elund, L., Laurin, S., and Lunderquist, A., Radiology, 122, 95, 1977.)
199
FIGUREI IB.
FIGURE ÎIC.
201
REFERENCES
1. Billing, L. and Lindgren, A. G. H., Die pathologisch-anatomische Unterlage der Geschwulstarterio-
graphie. Eine Untersuchung der arteriellen Gefasse des Hypernephroms und des Magenkarzinoms,
Acta Radiol.,25, 625, 1944.
2. Ljunqvist, A. and Lagergren, C., The arterial vasculature of renal adenocarcinomas, Acta Pathol.
Microbiol. Scand.,61, 55, 1966.
3. Ekelund, L., Jonsson, N., and Lunderquist, A., Tumor vessels. Angiographic-histopathologic cor-
relation, Radiologe, 17, 95, 1977.
4. Lunderquist, A., Angiography in carcinoma of the pancreas, Acta Radiol., Suppl., (235), 1965.
5. Bookstein, J. J., Reuter, S. R., and Martel, N., Angiographic evaluation of pancreatic carcinoma,
Radiology, 93,757, 1969.
6. Tylén, U., The accuracy of angiography in the diagnosis of carcinoma of the pancreas, Acta Radiol.
Diagn., 14,449, 1973.
7. Molas, G., Potet, F., Mignot, J., and Barge, J., L'angiographie du pancreas normal et pathologique.
Etude anatomique sur pièces opératoires et nécropsiques, Ann. Anat. Pathol., 14, 275, 1969.
8. Marions, O. and Wiechel, K.-L., Rontgenologisk ikterusutredning, Opuse. Med., Suppl., (32), 1974.
9. Lunderquist, A. and Tylén, U., Phlebography of the pancreatic veins, Radiologe, 15, 198, 1975.
10. Folkman, J., Tumor angiogenesis: therapeutic implications, N. Engl. J. Med.,285, 1182, 1971.
11. Ekelund, L. and Olin, T., Catheterization of arteries in rats, Invest. Radiol., 5, 69, 1970.
12. Ekelund, L. and Jonsson, N., Angiography in dimethyl-nitrosamine-induced rat renal tumors, Acta
Radiol. Diagn., 11, 489, 1971.
13. Ekelund, L., Gothlin, J., and Henriksson, H., Angiography in dibuthylnitrosamine-induced rat blad-
der tumors, Acta Pathol. Microbiol. Scand., Sect. A, 80, 691, 1972.
14. Ekelund, L., Henrikson, H., Olin, T., and Sjogren, H. O., Angiography in hepatic cysts and tumors
in the rat, Invest. Radiol., 9, 396, 1974.
15. Ekelund, L., Stigsson, L., Jonsson, N., and Sjogren, H. O.., Transcatheter arterial embolization of
normal livers and experimental hepatic tumors in the rat, Acta Radiol. Diagn., 18, 641, 1977.
16. Goldstein, H. M., Wallace, S., Anderson, J. H., Bree, R L., and Gianturco, L., Transcatheter occlu-
sion of abdominal tumors, Radiology, 120, 539, 1976.
17. Abrams, H. L., Effect of epinephrine on the renal circulation, in Angiography, Little, Brown, Bos-
ton, 1971.
18. Bosniak, M. A., Ambos, M. A., Madayag, M. A., Lefleur, R. S., and Casarella, W. J., Epinephrine-
enhanced renal angiography in renal mass lesions: is it worth performing? Am. J. Roentgenol., 129,
647, 1977.
19. Kahn, P. C , The epinephrine effect in selective renal angiography, Radiology, 85, 301, 1965.
20. Ross, L. S. and Baltaxe, H. A., The value of epinephrine in the diagnosis of epidermoid carcinoma
of the kidney, Am. J. Roentgenol., 112, 600, 1971.
21. Elkin, M. and Meng, C.-H., The effect of angiotensin on renal vascularity in dogs, Am. J. Roent-
genol., 98, 927,1966.
22. Ekelund, L., Gothlin, J., and Lunderquist, A., Diagnostic improvement with angiotensin in renal
angiography, Radiology, 105, 33, 1972.
23. Ekelund, L. and Gothlin, J., Effect of angiotensin on normal renal circulation determined by angiog-
raphy and a dye dilution technique, Acta Radiol. Diagn., 18, 39, 1977.
24. Ekelund, L., Pharmako-Angiographie der Niere, Radiologe, 13, 279, 1973.
25. Ekelund, L. and Lunderquist, A., Pharmacoangiography wih angiotensin, Radiology, 110, 533, 1974.
26. Carlsson, B. and Eriksson, U., Renal angiography under the influence of vasopressin and bradykinin,
Am. J. Roentgenol., 109, 161, 1970.
27. Gothlin, J., Effect of vasopressin on human renal circulation investigated by angiography and a dye
dilution technique, Acta Radiol. Diagn., 17, 763, 1976.
28. Mattsson, J., Appelgren, L., Karlsson, L., and Peterson, H.-L, Influence of vasoactive drugs and
ischemia on intra-tumour blood flow distribution, Eur. J. Cancer, 14, 761, 1978.
29. Kahn, P. C. and Callow, A. D., Selective vasodilation as an aid to angiography, Am. J. Roentgenol.,
94,213, 1965.
30. Hawkins, Jr., J. F., Tolazoline for arterial enhancement in angiography, Excerpta Med., 301, 222,
1973.
31. Hawkins, Jr., J. F. and Hudson, T., Priscoline in bone and soft-tissue angiography, Radiology, 110,
341, 1974.
32. Ekelund, L., Gothlin, J., Jonsson, N., and Sjogren, H. O., Pharmacoangiography in experimental
tumors. Evaluation of vasoactive drugs, Acta Radiol. Diagn., 17, 329, 1976.
33. Ekelund, L., Laurin, S., and Lunderquist, A., Comparison of a vasoconstrictor and a vasodilator in
pharmacoangiography of bone and soft-tissue tumors, Radiology, 122, 95, 1977.
34. Jonsson, K., De Santos, L. A., Wallace, S., and Anderson, J. H., Prostaglandin E, (PGE,) in an-
giography of tumors of the extremities, Am. J. Roentgol., 130, 7, 1978.
35. Streuler, G. J., Heinricks, K. J., Guiod, L. R., and HoUenberg, N. K., Sodium intake and vascular
smooth muscle responsiveness to norepinephrine and angiotensin in the rabbit, Ore. Res., 31, 758,
1972.
36. HoUenberg, N. K., Solomon, H. S., Adams, D. F., Abrams, H. L., and Merrill, J. P., Renal vascular
responses to angiotensin and norepinephrine in normal mass. Effect of sodium intake, Circ. Res.,
31,750, 1972.
203
Chapter 12
HEPATIC DEARTERIALIZATION A N D INFUSION TREATMENT OF

LIVER TUMORS
Stig Bengmark, Eva Peterson-Dahl, and Per E. Fredlund
TABLE OF CONTENTS
I. Introduction 203
II. Predominant Arterial Blood Supply to Liver Tumors 203
III. Hepatic Artery Ligation in Experimental Tumors 205
IV. Hepatic Artery Ligation in the Treatment of Human Liver Tumors 205
V. Hepatic Dearterialization Does Not Prolong Survival 206
VI. Regional Infusion Chemotherapy For Hepatic Tumors 206
VII. Combined Chemotherapy And Hepatic Dearterialization 207
VIII. Alternative Methods for Infusion And Hepatic Artery Occlusion 211
IX. Future Aspects 212
References 214
I. INTRODUCTION
Only a small proportion of all hepatic tumors in man are localized to a restricted
part of the liver in such a way that they can be radically removed by surgery. Radio-
therapy has offered little help to the numerous patients with primary or secondary
liver tumors. Much effort has therefore been exerted to develop new methods for the
treatment of liver malignancies, utilizing the possibilities offered by the unique dual
blood supply to the liver. Interruption of the arterial blood supply and regional infu-
sion of drugs into the hepatic artery are the methods which have most often been used
with various techniques and results.
II. P R E D O M I N A N T ARTERIAL BLOOD SUPPLY TO LIVER TUMORS
More than 50 years ago, Segall1 demonstrated that hepatic tumors were supplied
chiefly by blood from the hepatic artery. At this time, both experimental and clinical
evidence seemed to indicate that interruption of the arterial flow to the liver caused
severe liver damage and death. Unintentional division of the hepatic artery was consid-
ered to be a fatal complication to surgery. In 1949 Markowitz2 showed that after he-
patic artery ligation in dogs, penicillin therapy could prevent liver necrosis and death.
The same author 3 later suggested that hepatic artery ligation could be used for the
treatment of liver tumors.
The vascularity of malignant neoplasms in the liver has been investigated by many
authors during the last decades, confirming the predominantly arterial supply to the
tumors. Bierman4 et al. investigated the vascular pattern of liver malignancies in man
by means of an angiographic technique. Breedis and Young5 studied the tumor circu-
lation of experimental liver neoplasms and autopsy specimens of patients using various
dyes, gelatin, and vinylacetate for injection into hepatic arteries and veins. Dye was
demonstrated within the tumors after hepatic artery injections, but only in their pe-
riphery after portal injections. These authors estimated that 80 to 100% of the human
liver tumor circulation was derived from the hepatic artery. Similar results were re-
ported by Honjo and Matsumura 6 in rat tumors with colored gelatin solutions injected
into the portal vein and hepatic artery. Healey7 studied human livers with metastatic
cancers, injecting vinylacetate into the vessels. He found some cases with a practically
normal vascular pattern of the liver, while in others there was more or less complete
exclusion of the portal vein vessels. Blanchard et al. 8 used isotope-labeled microspheres
for injection into the hepatic artery and portal vein systems in rabbits with implanted
liver tumors. They found that tumor/liver ratios averaged 4.2 after hepatic artery in-
jections, but only 0.3 after injections into the portal vein. Nilsson and Zettergren 9
investigated induced primary hepatic carcinoma in rats by injecting barium sulfate and
formalin into the hepatic vessels and used an X-ray technique to visualize the vascular
penetration of the tumors. In some neoplasms the contrast material from the portal
vein filled vessels resembling sinusoids, although the main filling occurred from the
hepatic artery. Around the periphery of the tumor a zone of vessels was filled with
contrast material from the portal vein.
In a series of studies Ackerman 10ad and associates examined the blood supply of
experimental liver metastases. Using isotope-labeled microspheres, they found that
smaller tumors, less than 30 mg in weight, were fed by both the hepatic artery and the
portal vein systems. Larger tumors had a predominantly arterial blood supply. Using
a perfusion technique with colored silicone rubber, they confirmed that implanted liver
tumors in rats received their blood supply from the hepatic artery, and that tumor
vessels formed a plexus or ring at the periphery of the malignant growth with connec-
tions from the hepatic artery. After hepatic artery ligation, the tumor plexus in many
animals could be filled by perfusion via the portal vein. Small tumors, less than 2 mm
in diameter, seemed to induce the development of new vessels, encircling the tumors.
Mixing freely, these vessels generated from either the arterial or the portal circulation
or from both. Larger tumors had a well developed arterial circulation and a wide va-
riety of vascular patterns was discerned with vascularity varying from sparse to well
developed. Shunting of blood through the tumor vessels to the surrounding liver tissue
seemed to occur.
Interruption of the portal flow gave an immediate reaction of decreased relative
perfusion to the tumors via the arterial system. Investigating 117 patients with primary
or secondary carcinoma of the liver, Suzuki et al. 11 found a highly varying vascularity
in the tumor tissue. They divided the liver tumors into four grades according to their
vascularity and compared the arteriography with microangiography of resected speci-
mens and with histological findings. Marked hypervascúlarity was present in 13% of
the tumors and the authors suggest that the treatment should be diversified according
to vascularity. Extremely hypo vascular tumors were likely to respond better to portal
vein branch ligation while a suitable treatment for hypervascular tumors would be
hepatic artery ligation.
205
Measuring the distribution of isotope-tagged microspheres, Lindell12 and associates

were able to show that small experimental tumors had normal or increased portal cir-
culation, while medium sized and large metastases had a much reduced portal blood
flow as compared to that of the normal liver. The reduction of portal blood flow in
larger tumors was compensated by arterial hyper fusion. After occlusion of the artery
the hyperfusion changed into ischemia.
III. HEPATIC ARTERY LIGATION IN EXPERIMENTAL TUMORS
Breedis and Young5 were the first to test a theory put forward by Markowitz3 in
1952 that hepatic tumors might be treated by hepatic artery ligation. In a small series
of rabbits with experimental liver tumors, they found no tumor regression after liga-
tion of the hepatic artery. The explanation offered for this finding was that a rich
collateral arterial supply led to incomplete deprivation of arterial blood to the tumors.
Also Fischer and Fischer13 were unable to find any effect of hepatic artery ligation on
tumor-take or on growth of newly implanted tumors in rat livers. In fact, ligation of
the hepatic artery three days prior to tumor cell injection into the portal vein, or ten
minutes after intraportal injection, facilitated the development of metastases. Studying
the effect of hepatic artery ligation in experimental liver carcinomas in rats, Nilsson
and Zettergren14 found that the ligation resulted in subtotal or total necrosis of the
liver tumors. The survival time of the rats was significantly prolonged. The results
seemed to be better in rats with secondary tumors obtained by the intraportal injection
of sarcoma cell suspensions than in animals with primary liver cancer induced by 3-
methyl-4-dimethylaminoazobenzene.
IV. HEPATIC ARTERY LIGATION IN THE TREATMENT OF H U M A N

LIVER TUMORS
In 1953 Reenhoff and Woods15 reported on ligation of the hepatic and splenic arter-
ies in treatment of cirrhosis with ascites. One of the patients had extensive metastatic
tumor growth in the liver and after hepatic artery ligation he left the hospital improved
as far as ascites was concerned. This report seems to have passed unnoticed. In 1966,
Mori et al.16 reported a case of stomach carcinoma with liver metastasis in which the
hepatic artery was accidentally obstructed at operation. The patient died and at au-
topsy severe necrosis of the liver tumors was revealed with only slight damage of sur-
rounding liver tissue. The authors suggested that temporary occlusion of the hepatic
artery could be used for the treatment of liver cancer.
The first report of intentional ligation of the hepatic artery for the treatment of
human liver tumors was published by Nilsson17 in 1966. In one patient with leiomy-
osarcoma, a new operation was performed 2 months later and it was then possible to
resect the remaining metastases which had decreased considerably in size. Some tumors
seemed to have completely disappeared. Microscopic examination revealed only a few
living tumor cells on the borders of the neoplasms.18
Initially, the results of hepatic artery ligation seemed to be very promising with re-
markable regression of tumor size and clinical symptoms. Various enzyme elevations
in the circulating blood signaled the ischemic damage induced to the liver and to the
tumor tissue.19
It soon became evident that the enzyme response was variable and sometimes
failed.20 Postoperative angiography revealed that collaterals or aberrant vessels were
often overlooked at operation, resulting in a most dubious effect of the treatment.
Furthermore, it was found that rapid revascularization of the tumors occurred within
weeks after hepatic artery ligation. 2122
Numerous arterial collaterals reaching the liver by up to 26 potential routes have

been reported.23 The liver arteries are not end arteries. By arteriography, Mays and
Wheeler24 have demonstrated that intralobal vessels will supply the whole liver with
arterial blood if the artery to one part of the liver is left undivided. It can also be
shown that occlusion of the main hepatic artery alone is often quite ineffective in
depriving the liver tumors of their arterial supply.
Hepatic artery ligation is a relatively simple operation to perform and has been ex-
tensively used by many authors. 2535 In order to disrupt the collateral flow and improve
the effectivity of the arterial occlusion, the original operative procedure was extended
to include not only the distal ligation of the hepatic artery, but also a meticulous dis-
section of all other structures carrying arterial collaterals to the liver. In the hepatod-
uodenal ligament the common duct and the portal vein were denuded and other struc-
tures ligated and divided. The falciform and triangular ligaments were divided and all
connections between the diaphragm and the liver were cut. The lesser omentum was
opened and branches from the phrenic arteries were sought and divided. The complete-
ness of the procedure could be controlled by means of wash out studies after injection
of radioactive Xenon. The surgical method described above has been called total or
complete hepatic dearterialization, and is a far more extensive and difficult operation
than simple hepatic artery ligation. For clinical purposes complete dearterialization is
considerably more effective 36 and several reports of this method have appeared during
recent years. 3741 Many patients, though, present enormously enlarged livers and it may
be practically impossible to break all collaterals. It may also be suspected that the more
extensive procedure will cause a higher complication rate and the preoperative mortal-
ity has been around 20% in larger materials.37»42
V. HEPATIC DEARTERIALIZATION DOES NOT PROLONG

SURVIVAL
In 1972 Almersjò* et al.37 reviewed the results of their first 44 patients treated with
hepatic dearterialization. Some patients had been further treated with oncolytic drug
infusion or liver resection. The overall impression was that hepatic liver dearterializa-
tion as the sole procedure could not prolong survival, although considerable tumor
regression could be noticed in many patients. Necrosis of most of the tumor cells was
demonstrated in patients later operated on with liver resection or biopsy. Combined
treatment with cytotoxic drugs was recommended. Most of the more recent reports
about hepatic artery ligation or dearterialization reveal that combination treatment has
been used, although the techniques for infusion have varied from systemic administra-
tion to regional intraarterial and intraportal infusion.26 2 8 2 9 3 1 3 6 4 1 4 3
VI. REGIONAL INFUSION CHEMOTHERAPY FOR HEPATIC

TUMORS
As long as chemotherapeutic agents for tumor treatment have been available, it has
been postulated that such drugs are more effective if locally administered into the
tumor circulation. The vascular arrangements of the liver and the predominantly arte-
rial supply to liver tumors will make it possible to reach the tumors with high concen-
trations of cytotoxic drugs by infusion into the hepatic artery. The development of
infusion methods has run parallel to the development of angiography. In 1961, Byron
et al.44 reported a technique for catheterization of the left brachial artery for chemo-
therapy of hepatic and other intraabdominal malignancies. Two main routes are now
used for intraarterial liver chemotherapy. Besides the percutaneous placement of cath-
207
eters via the brachial artery, a catheter can also be placed at operation in the hepatic
artery, introduced from the gastroduodenal artery as described by Watkins and co-
workers. 45
Numerous reports on the techniques and results of infusion of various drugs into
the hepatic artery circulation from the percutaneously placed brachial artery catheter
are now available and a few of the more recent reports will be referred to here. Ac-
cording to Virtanen, 46 a thin catheter for long-term infusion can be placed in the he-
patic artery and left there for up to three or four months. The prevailing drug for
infusion treatment is 5-fluorouracil (5-FU),47"50 but methotrexate, actinomycin-D, and
adriamycin have also been used.29-51 Although percutaneous introduction of a catheter
into the arterial system is by far the most convenient method for the patient, requiring
no surgery, this procedure has some disadvantages. The extent of tumor growth within
and outside the liver cannot be evaluated as thoroughly as after laparotomy. Aberrant
liver arteries are frequent, especially from the superior mesenteric artery and the left
gastric artery, and these vessels are sometimes difficult to reach with a catheter. At
operation such arteries can be located and divided. Dislocation of the catheter will
sometimes occur and replacement can be a difficult procedure.
Many operations are carried out in order to perform liver resection, but it is often
found that removal of all tumor tissue is technically impossible. One alternative for
treatment may be to introduce a thin catheter into the hepatic artery with the tip in
proximal direction as described by Watkins. 45 The route through the gastroepiploic
artery is recommended as this will diminish the risk for bleeding, catheter displace-
ment, and leakage of the cytostatic agent into the abdominal cavity, which may have
disastrous consequences. The catheter will be fixed into position, brought out through
the abdominal wall via a separate stab wound, and connected to infusion equipment,
e.g., a pressure infusion bag or a portable pump. The original device developed by
Watkins 45 used 5-fluorodeoxyuridine (FUDR) as cytotoxic agent. In our experience
another piece of equipment, the Sigma® motor pump (Middleport, N. Y.) is more easy
to handle and can be used also for 5-FU, which is universally available. A small
amount of heparin is added to the infusion solution to avoid clotting. The portable
infusion pump can be used both for the intrabrachial catheter and for the abdominal
catheter with the same dosage and the patients can be fully ambulatory with continuous
infusion of cytotoxic substances for up to three months, or as long as the catheter
functions properly. The pump container has to be refilled with cytostatic solution and
the pump batteries recharged at regular intervals, usually every 3 to 5 days.
The effects of the treatment with regional infusion of oncolytic drugs are difficult
to evaluate. The criteria for the selection of patients for the treatment may vary from
group to group and no properly controlled or randomized series is available. In the
report of Ansfield,48 419 patients, most of them with metastatic tumors in the liver
from gastrointestinal primaries, were treated with regional 5-FU. Three quarters of
the patients had been previously treated with 5-FU systemically and were switched over
to regional treatment because of progression of the disease. The complication rate was
minimal and tumor regression was registered in 55%, which is considerably higher
than after systemic treatment with the same drug. A prolongation of life was claimed
in this series, although survival rates are impossible to evaluate as matched controls
are lacking. Improvement of about 60% of the patients is also reported in other series
with similar treatment. 45 4 7 4 9 5 2
VII. COMBINED CHEMOTHERAPY A N D HEPATIC

DEARTERIALIZATION
Most liver tumors grow as rounded nodules, expanding, but not infiltrating the liver
parenchyma. They often have a necrotic center and an outer zone with rapidly multi-
plying cells, surrounded by a well developed vascular plexus. The expansive growth
seems to occur at the periphery. The dearterialization procedure may cause major ne-
crosis of the central part of the tumor, but available experience34 37 indicates that dear-
terialization will have little or no effect on the outer zone with rapidly multiplying
cells. As these cells may be more susceptible to cytotoxic drugs, a combination of
chemotherapy and dearterialization should be rational. Dearterialization will greatly
diminish the cell population and the drug may abolish the remaining, actively multiply-
ing cells.
As systemic treatment seems to be less effective, various techniques for regional
infusion have been tried. Immediately after dearterialization, when the surviving cells
are nourished by the portal flow, an intraportal infusion may be more effective. Such
treatment was chosen by Almersjo et al., 42 who reported on 40 patients with primary
or metastatic liver malignancies treated with hepatic dearterialization, 19 of whom
were given intraportal 5-FU infusion. The combined treatment resulted in a somewhat
prolonged survival. Development of new arterial collaterals to the tumors, will proba-
bly limit the effect of the treatment.
In 1972, Gulesserian et al. 28 described another interesting method of regional infu-
sion after hepatic artery ligation. They used the distal end of the divided artery for
insertion of a catheter for infusion. Modifications of this method have been used by
several other authors, 29 3 1 3 3 5 3 but the results are still to be evaluated. One major com-
plication seen after hepatic artery ligation followed by regional chemotherapy is liver
abscesses, which occurred in 6 of 64 patients as reported by Jochimsen. 53
After unintentional occlusion of the hepatic artery with reconstruction in a case with
hepatic metastases from a gastric carcinoma, Mori et al. 16 in 1966 found massive ne-
crosis of the hepatic tumors. They suggested that temporary occlusion of the hepatic
artery might be used for the treatment of hepatic tumors. A method for transient
occlusion of the hepatic artery was developed by us and described in 1974. 3643 The
method includes a dissection of the liver similar to that performed in total dearteriali-
zation. All collaterals in the ligaments and from the diaphragm are divided, and in the
hepatoduodenal ligament the common duct and portal vein are dissected free. The
gallbladder is removed to avoid necrosis. Instead of dividing the hepatic artery, stran-
gulating slings of fine polyethylene catheters (Pe 50) are placed around the hepatic
artery, one sling distal and another proximal to the junction between the proper hepatic
artery and the gastroduodenal artery (Fig 1). The pair of strangulating slings is drawn
through a thicker catheter (Pe 320) and together they are pulled through a separate
stab incision in the abdominal wall and fixed with sutures in their proper position. No
occlusion of the main hepatic artery is performed during the operation, but by pulling
the thin catheters an occlusion can be achieved at any time in the postoperative period.
During the operation a thin infusion catheter will also be introduced into the gastroe-
piploic artery, passing through the gastroduodenal artery and positioned with its tip
in the proximal direction in the hepatic artery in a similar way to that described by
Watkins. 45
In order to minimize liver abscess development, a broad spectrum antibiotic is ad-
ministered for a few days postoperatively, partly by infusion into the intraarterial cath-
eter. When the patient has recovered from the immediate effects of the operative
trauma, that is, after 2 to 3 days, the cathether is flushed with heparin and the thin
polyethylene slings are pulled and fixed with a hemostatic clamp against the thick cath-
eter to ensure complete occlusion of the artery. For optimal tumor necrosis it will
probably be sufficient to occlude the artery for 8 to 12 hr, but we have found it possible
to maintain the occlusion for up to 16 hr without causing permanent clotting of the
209
FIGURE 1. In preparation for temporary hepatic artery occlusion, thin

catheter slings are placed around the artery, one on each side of the gas-
tro-duodenal artery. Each pair of slings is drawn through a thicker tube.
An infusion catheter for regional chemotherapy is introduced into the
hepatic artery from the gastro-epiploic and gastro-duodenal arteries.
artery. The procedure can be performed with little or moderate pain and discomfort
for the patient.
Angiographic control of the patency of the artery and the catheter position can be
carried out when the strangulating slings are opened. The slings can be left in place
for several months, but can also be easily removed without operation. It is possible to
commence regional infusion of oncolytic drugs a few days after the occlusion. Accord-
ing to recent experimental evidence in pigs with the same model for hepatic artery
occlusion,54 it may also be possible to infuse cytotoxic drugs to the liver during the
occlusion or immediately after the release of the slings. In the experimental model, it
was found that much less damage was caused to the liver parenchyma if the hepatic
artery was occluded three days after the operation instead of immediately after the
laparotomy. This is probably an effect of a physiological adjustment of the organism
after the operative trauma and not due to collateral development. Clinical experience
has shown that the patients are much less affected by the operation if the surgery and
the occlusion of the hepatic artery do not coincide. In our first series of 23 patients
treated with temporary hepatic artery occlusion for liver metastases, no peroperative
deaths occurred and the tendency for liver insufficiency and bleeding disorders was
much less pronounced than with permanent liver dearterialization. The main advan-
tages of the described procedure will be the safety, the facility of angiographic inves-
tigations after the treatment, and the possibility of combining the dearterialization
with long term intraarterial oncolytic infusion. The cytotoxic drugs may be given with
a similar technique and dosage as previously described for regional infusion.
When we first started our present series, we thought that patients with metastatic
lesions outside the liver could also benefit from this treatment when the major tumor
burden seemed to be located in the liver. Although tumor regression was recorded in
most patients, it soon became evident that the progress of the extrahepatic tumors
continued and survival was not at all or very slightly prolonged. By selecting cases
with no known extrahepatic tumor growth, the results of the treatment will probably
be better. On the other hand, such selection will leave only a few patients for the
randomized study we have started for the comparison of this combined treatment with
regional chemotherapy as the sole procedure. Figure 2 shows the survival time in 15
patients with hepatic metastases from colon-rectal cancer treated with temporary liver
dearterialization followed by intraarterial 5-FU infusion.
Temporary hepatic artery occlusion seems to be especially well suited for the treat-
ment of slow-growing hepatic metastases from tumors with endocrine activity, such
as carcinoids. A dramatic decrease in urinary excretion of 5-HIAA can be found after
this type of dearterialization, and the patients will often be rid of flush and other
manifestations of the carcinoid syndrome for years afterwards. In Figure 3 the effect
FIGURE 2. Fifteen patients with liver metastases from colon-rectal cancer have been treated with
temporary hepatic artery occlusion, followed by regional 5-FU infusion. The left part of the figure shows
the time from diagnosis to operation, the right part demonstrates postoperative survival. Longest survival
time from operation is 42 months.
211
FIGURE 3. A 52-year-old woman with hepatic metastases from small intestinal car-
cinoid. (A) Liver angiography before dearterialization: multiple, well-circumscribed hy-
pervascular tumors. (B) One week after temporary occlusion of the hepatic artery: cen-
ter of lesions avascular indicating necrosis, liver markedly enlarged. (C) One year after
treatment: size of liver and metastases much decreased.
of temporary occlusion of the hepatic artery on hypervascular carcinoid metastases is

shown.
VIII. ALTERNATIVE METHODS FOR INFUSION A N D HEPATIC

ARTERY OCCLUSION
Several other methods have been presented for the treatment of hepatic tumors by
arterial infusions of various substances. Ariel and Pack in 196755 reported a method
of treatment with microspheres loaded with the radioactive isotope 90Yttrium. These
microspheres made of ceramic or plastic material and with a diameter of 10 to 20 /¿m
were intended to be trapped and lodged within the small tumor vessels giving an inter-
nal irradiation to the tumor tissue. This ingenious method has not been widely accepted
in tumor treatment. Another interesting method of treatment with microspheres, has
recently been presented. Lindell et al.56 have achieved a temporary arrest of arterial
blood flow by injection of degradable microspheres consisting of crosslinked polysac-
charide derivatives. This material is degraded in human blood by amylase activity after
15 to 20 min. The period of ischemia is probably too brief for any substantial tumor
necrosis to develop, but the infusions can be repeated. The microspheres can be sus-
pended in a solution containing a cytotoxic drug, such as 5-FU and can then act as
carriers of the drug to the tumor area during the temporary arrest of the circulation,
while the systemic toxicity is diminished.
Occlusion of the hepatic artery can also be performed with various catheterization
212 Tumor Blood Circula tion
FIGURE 3B.
procedures. A balloon catheter can be selectively placed into the hepatic artery,57 but
remaining collateral circulation will probably make this method less useful for tumor
treatment.
More or less permanent occlusion of the hepatic artery can be achieved by a percu-
taneous angiographic technique with injection of thrombotic material into the artery.
In 1974 Doyon and associates,58 reported hyperselective hepatic arterial embolization
by injection of fragmented gelatin sponge with thrombin. This method is claimed to
cause coagulation and obliteration of all the peripheral arterial branches, thus prevent-
ing revascularization of the tumor area.59 A similar technique has been used by Alison
et al.60 in the treatment of carcinoid liver metastases. Intraarterial injection of a poly-
merizing acrylate has also been used for similar purposes in treatment of liver tumors.61
IX. FUTURE ASPECTS
This review has given a fragmentary survey of how the vascular anatomy of the liver
and liver tumors can be utilized today. So far no reports of permanent cure with these
methods have appeared. Tumor regression can be demonstrated with several treatment
modalities, but prolongation of life has been very difficult to prove. Most series are
small and difficult to evaluate. No properly randomized investigation of the effective-
213
FIGURE 3C.
ness of treatment has been presented. The efforts are still on an experimental level and
will probably be so for several years to come.
This article has shown several methods for the manipulation of the tumor circulation
which may be further developed in the future. We can control the blood flow to a
tumor area, halt it for a certain period and change the composition of the flowing
medium, thereby altering the external milieu for the cancer cell. We have created con-
ditions in which numerous factors of importance for the life and growth of the cell
can be varied and studied. The tumor area may be exposed to variations in nutritive
flow, pH, enzyme activity, or other metabolic factors. Cytotoxic preparations can be
given locally in enormously high concentrations. The proliferating cancer cells may
theoretically be synchronized into the same cell cycle phase. A small dose of a potent
mitotic toxin administered over a short period would thus be highly effective in killing
cells in a mitotic phase. Tumor reduction achieved by the described methods may be a
prerequisite for effective infusion of immunologically active preparations of cellular
or humoral antitumor factors. Such factors, if developed, might require local admin-
istration to the restricted tumor area under controled conditions. More potent cyto-
toxic drugs with high systemic toxicity but with short time activity may be used if
tumor circulation is under proper control.
214 Tumor Biood Circulation
REFERENCES
1. Segall, H. N., An experimental anatomical investigation of the blood and bile channels of the liver,
Surg. Gynecol. Obstet., 37, 152, 1923.
2. Markowitz, J., Rappaport, A., and Scott, A. C , Prevention of liver necrosis following ligation of
hepatic artery, Proc. Soc. Exp. Biol. Med.,70, 305, 1949.
3. Markowitz, J., The hepatic artery, Surg. Gynecol. Obstet.,95, 644, 1952.
4. Bierman, H. R., Byron, R. L., Jr., Kelley, K. H., and Grady, A., Studies on the blood supply of
tumors in man. III. Vascular patterns of the liver by hepatic arteriography in vivo, / . Natl. Cancer
Inst.,\2, 107, 1951.
5. Breedis, C. and Young, G., The blood supply of neoplasms in the liver, Am. J. Pathol., 30, 969,
1954.
6. Honjo, I. and Matsumura, H., Vascular distribution of hepatic tumors: experimental study, Rev.
Int. Hepatol., 15, 681, 1965.
7. Healey, J. E., Jr., Vascular patterns in human metastatic liver tumors, Surg. Gynecol. Obstet., 120,
1187, 1965.
8. Blanchard, R. J. W., Grotenhuis, I., La Fave, J. W., and Perry, J. F., Jr., Blood supply to hepatic
V2 carcinoma implants as measured by radioactive microspheres, Proc. Soc. Exp. Biol. Med., 118,
465, 1965.
9. Nilsson, L. A. V. and Zettergren, L., Blood supply and vascular pattern of induced primary hepatic
carcinoma in rats, Acta Pathol. Microbiol. Scand.,71, 179, 1967.
10a. Ackerman, N. B., Lien, W. M., Kondi, E. S., and Silverman, N. A., The blood supply of experimen-
tal liver metastases. I. The distribution of hepatic artery and portal vein blood to "small" and
"large" tumors, Surgery, 66, 1067, 1969.
10b. Lien, W. M. and Ackerman, N. B., The blood supply of experimental liver metastases. II. A micro-
circulatory study of the normal and tumor vessels of the liver with the use of perfused silicone rubber,
Surgery, 68, 334, 1970.
10c. Ackerman, N. B., Lien, W. M., and Silverman, N. A., The blood supply of experimental liver me-
tastases. III. The effects of acute ligation of the hepatic artery or portal vein, Surgery, 71, 636, 1972.
lOd. Ackerman, N. B., The blood supply of experimental liver metastases. IV. Changes in vascularity
with increasing tumor growth, Surgery, 75, 589, 1974.
11. Suzuki, T., Sarumaru, S., Kawabe, K. and Honjo, I., Study of vascularity of tumors of the liver,
Surg. Gynecol. Obstet., 134, 27, 1972.
12. Lindell, B., Aronsen, K. F., Rothman, U., and Sjogren, H. O., The circulation in liver tissue and
experimental liver metastases before and after embolization of the liver artery, in Transient Liver
Ischemia by Intra-arterial Injection of Degradable Microspheres, Lindell, B., Thesis, Malmo, 71,
1977.
13. Fisher, B., Fisher, E. R., and Lee, S. L., The effect of alteration of liver blood flow upon experimen-
tal hepatic metastases, Surg. Gynecol. Obstet., 112, 11, 1961.
14. Nilsson, L. A. V. and Zettergren, L., Effect of hepatic artery ligation of induced primary liver car-
cinoma in rats, Acta Pathol. Microbiol. Scand. 71, 187, 1967.
15. Rienhoff, W. F., Jr. and Woods, A. C , Jr., Ligation of hepatic and splenic arteries in the treatment
of cirrhosis with ascites, JAMA, 152, 687, 1953.
16. Mori, W., Masuda, M., and Miyanaga, T., Hepatic artery ligation and tumor necrosis in the liver,
Surgery, 59, 359, 1966.
17. Nilsson, L. A. V., Therapeutic hepatic artery ligation in patients with secondary liver tumors,Rev.
Surg., 23, 374, 1966.
18. Almersj'd, O., Bengmark, S., Engevik, L., Hafstrom, L. O., and Nilsson, L. A. V., Hepatic artery
ligation as pretreatment for liver resection of metastatic cancer, Rev. Surg., 23, 377, 1966.
19. Almersjo, O., Bengmark, S., Engevik, L., Halfstrom, L. IO., Loughridge, B. P., and Nilsson, L.
A. V., Serum enzyme changes after hepatic dearterialization in man, Ann. Surg., 167, 9, 1968.
20. Bengmark, S., Btfrjesson, B., Fredlund, P. E., and Vang, J. O., Plasma activities of lysosomal en-
zymes after hepatic dearterialization in man, Am. J. Surg., 132, 363, 1976.
21. Bengmark, S. and Rosengren, K., Angiographic study of the collateral circulation to the liver after
ligation of the hepatic artery in man, Am. J. Surg., 119, 620, 1970.
22. Plengvanit, U., Chearanai, O., Sindhvananda, K., Damrongsak, D., Tuchinda, S., and Viranuvatti,
V., Collateral arterial blood supply of the liver after hepatic artery ligation: angiographic study of
twenty patients, Ann. Surg., 175, 105, 1972.
23. Michels, N. A., Collateral arterial pathways to the liver after ligation of the hepatic artery and re-
moval of the coeliac axis, Cancer (Phila. in 1953), 6, 708, 1953.
215
24. Mays, E. T. and Wheeler, C. S., Demonstration of collateral arterial flow after interruption of he-
patic arteries in man, N. Engl. J. Med., 290, 993, 1974.
25. Plengvanit, U., Viranuvatti, V., and Chearanai, O., Treatment of primary liver carcinoma, Med.
Chir.Dig., 3, 301, 1974.
26. Murray-Lyon, I. M., Dawson, J. L., Parsons, V. A., Rake, M. O., Blendis, L. M., Laws, J. W.,
and Williams, R., Treatment of secondary hepatic tumors by ligation of hepatic artery and infusion
of cytotoxic drugs, Lancet, 2, 172, 1970.
27. Koudahl, G. and Funding J., Hepatic artery ligation in primary and secondary hepatic cancer, Acta
Chir. Scand., 138, 289, 1972.
28. Gulesserian, H. P., Lawton, R. L., and Condon, R. E., Hepatic artery ligation and cytotoxic infusion
in treatment of liver metastases, Arch. Surg. (Chicago), 105, 280, 1972.
29. Fortner, J. G., Mulcare, R. J., Solis, A., Watson, R. C , and Golbey, R. B., Treatment of primary
and secondary liver cancer by hepatic artery ligation and infusion chemotherapy, Ann. Surg., 178,
162, 1973.
30. Jugdutt, B. I., Watanabe, M., and Turner, F. W., Hepatic artery ligation in treatment of carcinoid
syndrome, Can. Med. Assoc. J., 112, 325, 1975.
31. Zike, W. L., Safaie-Shirazi, S., and Gulesserian, H. P., Hepatic artery ligation and cytotoxic infusion
in treatment of liver neoplasms, Arch. Surg. (Chicago), 110, 641, 1975.
32. Mokka, R. E. M., Larmi, T. K. I., Huttunen, R., and Kairaluoma, M. I., Evaluation of the ligation
of the hepatic artery and regional arterial chemotherapy in the treatment of primary and secondary
cancer of the liver, Ann. Chir. Gynaecol. Fenn.,64, 347, 1975.
33. Sparks, F. C , Mosher, M. B., Hallauer, W. C , Silverstein, M. J., Rangel, D., Pássaro, E., Jr.,
and Morton, D. L., Hepatic artery ligation and postoperative chemotherapy for hepatic metastases:
clinical and pathophysiological results, Cancer (Philadelphia), 35, 1074, 1975.
34. Ramming, K. P., Sparks, F. C , Eilber, F. R., Holmes, E. C , and Morton, D. L., Hepatic artery
ligation and 5-fluorouracil infusion for metastatic colon carcinoma and primary hepatoma, Am. J.
Surg., 132, 236, 1976.
35. Farndon, J. R., The carcinoid syndrome: Methods of treatment and recent experience with hepatic
artery ligation and infusion, Clin. Oncol.,3, 365, 1977.
36. Bengmark, S., Fredlund, P., Hafstrom, L. O., and Vang, J., Present experiences with hepatic dear-
terialization in liver neoplasm, Prog. Surg., 13, 141, 1974.
37. Almersjb*, O., Bengmark, S., Rudenstam, C. M., Hafstrom, L. O., and Nilsson, L. A. V., Evaluation
of hepatic dearterialization in primary and secondary cancer of the liver, Am J. Surg., 124, 5, 1972.
38. McDermott, W. V., Jr., and Hensle, T. W., Metastatic carcinoid to the liver treated by hepatic
dearterialization, Ann. Surg., 180, 305, 1974.
39. McDermott, W. V., Jr., Paris, A. L., Clouse, M. E., and Meissner, W. A., Dearterialization of the
liver for metastatic cancer, Ann. Surg., 187, 38, 1978.
40. Nagasue, N., Inokuchi, K., Kobayashi, M., Ogawa, Y., Iwaki, A., and Yukaya, H., Hepatic dear-
terialization for nonresectable primary and secondary tumors of the liver, Cancer (Philadelphia), 38,
2593, 1976.
41. Balasegaram, M., Complete hepatic dearterialization for primary carcinoma of the liver, Am. J.
Surg., 124, 340, 1972.
42. Aimers jo, O., Bengmark, S., Hafstrom, L. O., and Leissner, K.-H., Results of liver dearterialization
combined with regional infusion of 5-fluorouracil for liver cancer, Acta Chir. Scand., 142, 131, 1976.
43. Fredlund, P. and Bengmark, S., Dearterialization procedures in the treatment of liver tumors, Fe-
gato, 21, 475, 1975.
44. Byron, R. L., Jr., Perez, F. M., Yonemoto, R. H., Bierman, H. R., Gildenborn, H. L., and Kelly,
K. H., Left brachial arterial catheterization for chemotherapy in advanced intra-abdominal malignant
neoplasms, Surg. Gynecol. Obstet., 112, 689, 1961.
45. Watkins, E., Jr., Khazei, A. M., and Nahra, K. S., Surgical basis for arterial infusion chemotherapy
of disseminated carcinoma of the liver, Surg. Gynecol. Obstet., 130, 581, 1970.
46. Virtanen, G. W., Percutaneous transbrachial artery infusion catheter techniques, Am. J. Roent-
genol., 117, 696, 1973.
47. Tandon, R. N., Bunnell, I. L., and Copper, R. G., The treatment of metastatic carcinoma of the
liver by the percutaneous selective hepatic artery infusion of 5-fluorouracil, Surgery, 73, 118, 1973.
48. Ansfield, F. J., Ramirez, G., Davis, H. L., Wirtanen, G. W., Johnson, R. O., Bryan, G. T., Manalo,
F. B., Borden, E. C , Davis, T. E., and Esmaili, M., Further clinical studies with intrahepatic arterial
infusion with 5-fluorouracil, Cancer (Philadelphia), 36, 2413, 1975.
49. Burrows, J. H., Talley, R. W., Drake, E. H., San Diego, E. L., and Tucker, W. G., Infusion of
fluorinated pyrimidines into hepatic artery for treatment of metastatic carcinoma of liver, Cancer,
Philadelphia, 20, 1886, 1967.
50. Massey, W. H., Fletcher, W. S., Judkins, M. P., and Dennis, D. L., Hepatic artery infusion for
metastatic malignancy using percutaneously placed catheters, Am. J. Surg., 121, 160, 1971.
51. Shah, P., Baker, L. H., and Vaitkevicius, V. K., Preliminary experiences with intra-arterial Adria-
mycin, Cancer Treat. Rep.,61, 1565, 1977.
52. Labelle, J. J., Lucas, R. J., Eistenstein, B., Reed, M. D., Vaitkevicius, V. K., and Wilson, G. S.,
Hepatic artery catheterization for chemotherapy, Arch. Surg. (Chicago), 96, 683, 1968.
53. Jochimsen, P. R., Zike, W. L., Shirazi, S. S., and Pearlman, N. W., Iatrogenic liver abscesses: a
complication of hepatic artery ligation for tumor, Arch. Surg. (Chicago), 113, 141, 1978.
54. Jeppsson, B., Dahl, E. P., Fredlund, P. E., Stenram, U., and Bengmark, S., Hepatic necrosis in the
pig produced by transient arterial occlusion, submitted for publication, 1978.
55. Ariel, M. I. and Pack, G. T., Treatment of inoperable cancer of the liver by intra-arterial radioactive
iostopes and chemotherapy, Cancer (Philadelphia), 20, 793, 1967.
56. Lindell, B., Aronsen, K.-F., Nosslin, B., and Rothman, U., Studies in pharmacokinetics and toler-
ance of substances temporarily retained in the liver by microsphere embolization, Ann. Surg., 187,
95,1978.
57. Dunnick, N. R., Doppman, J. L., and Brereton, H. D., Balloon occlusion of segmental hepatic
arteries: control of biopsy-induced hemobilia, JAMA, 238, 2524, 1977.
58. Doyon, D., Mouzon, A., Jourde, A.-M., Regensberg, C , and Frileux, C , L'embolisation artérielle
hépatique dans les tumeurs malignes du foie, Ann. Radiol., 17, 593, 1974.
59. Harry, G., Roche, A., Weingarten, A., and Doyon, D., L'embolisation artérielle hépatique, Med.
Chir.Dig.,6, 193, 1977.
60. Allison, D. J., Modlin, I. M., and Jenkins, W. J., Treatment of carcinoid liver metastases by hepatic-
artery embolisation, Lancet,!, 1323, 1977.
61. Shermeta, D. W., Golladay, E. S., and White, R., Jr., Preoperative occlusion of the hepatic artery
with isobutyl 2-cyanoacrylate for resection of the "unresectable" hepatic tumor, Surgery, 83, 319,
1978.
217
Chapter 13
REGIONAL PERFUSION WITH ANTICANCER DRUGS FOR

TREATMENT OF MALIGNANT TUMORS
Larsolof Hafstrõm and Per-Ebbe Jõnsson
TABLE OF CONTENTS
I. Introduction 217
II. History 217
III. Cytostatic Agents and Dosage for Perfusion of Malignant Melanoma 218
IV. Leakage from the Extremity During Perfusion 218
V. Present Technique for Regional Perfusion 218
VI. Results 220

A. Malignant melanoma 220
B. Soft tissue sarcoma 221
C. Own material 221
VII. Comments 221
References 222
I. INTRODUCTION
In a regional perfusion system, the tumor-bearing region or organ is subjected to

the influence of a high concentration of anticancer drug without other parts of the
body being contaminated by the drug to any important degree. This technique for
perfusion is accomplished by isolating the region or organ and maintaining the blood
circulation there by means of a pump-oxygenator.
II. HISTORY
In 1950, Klopp1 described how he had administered nitrogen mustard directly into
the arteries supplying a tumor-bearing area, the aim being to increase locally the dosage
and the concentration of the drug and avoid system toxic effects. The experience with
this technique was stimulating.2 3 Since these preliminary reports, many other reports
about the infusion technique have been published. Among the pioneers should be men-
tioned Sullivan and Watkins.4
In 1958, Creech et al.5 developed the regional infusion technique into the regional
isolation-perfusion technique, in which the tumor-bearing area was excluded from the
general circulation by means of an extracorporeal circuit consisting of pump and oxy-
genator. With this technique also, the goal was to deliver a high amount of the drug
into the tumor with a minimal effect on the rest of the body. With the oxygenation,
they aimed at a high oxygen tension in the tumor-bearing area which might potentiate
the effect of the cytostatic drug. 6 7 They obtained good preliminary results. 5 6
After Creech's work there were some reports without any further innovations until
Cavalière et al. 8 reported their results of hyperthermic perfusion without any cytostatic
agent. They experienced remarkable effects on certain tumors, especially melanomas.
Stehlin9 in 1969 reported good results when combining perfusion with heat. He has
further developed this technique, and so far has reported only consistently performed
series of hyperthermic perfusions for adjuvant as well as palliative purposes for malig-
nant melanoma. 10 His work can be criticized because he uses historical controls, but it
has to serve as a landmark in this field up to the present.
III. C Y T O S T A T I C A G E N T S A N D D O S A G E U S E D FOR P E R F U S I O N O F
MALIGNANT MELANOMA
Phenyl-alanine-mustard (PAM) has been the drug most consistently used for perfu-
sion, particularly for malignant melanoma. Other alkylating agents such as nitrogen
mustard and thioTEPA have also been used.11 Actinomycin D has been tried in a
restricted number of patients. 12
The basis for using PAM is experimental studies in rats with transplanted mela-
noma. 13 PAM would carry cytotoxic radicals into mélanine metabolism of the tumor
cells. The duration of the effect of PAM is about 6 hr. The recommended dose varies
between 0.6 to 1.0 mg/kg bodyweight for the upper extremity and 1.0 and 1.5 mg/kg
bodyweight for the lower in normothermic perfusion. In a hyperthermic perfusion,
the dose of PAM has to be reduced by 25%.
The dose of PAM added to the perfusion system is equivalent to a single systemic
dose corresponding to a concentration of about eight times the level that can be ob-
tained by systemic administration.
As PAM is more rapidly inactivated by increasing temperature above 40.5 to 41 °C,
the hyperthermia has to be controlled below that level.
IV. L E A K A G E F R O M T H E E X T R E M I T Y D U R I N G P E R F U S I O N
The ultimate aim of isolation-perfusion is to maintain the drug in the perfused region
without leakage to the rest of the body. It is evident that it is not enough just to
measure the volume of the perfusate before and after the procedure, as there might
be an exchange in both directions. Various methods for estimation of the leakage from
the system have been worked out. 14 The most used are 51Cr-tagged red cells or 131I-
albumin.
Measuring the activity of 131I-albumin with scintillation probe, rate meter, and rec-
tilinear recorder, Stehlin15 found almost no leakage when he perfused the lower extrem-
ity distal to the inguinal ligament. After 1 hr perfusion through the iliac vessels, the
leakage was measured at 10% to 30%. It is more difficult to avoid leakage when the
upper extremity is perfused.
To avoid leakage, it is important to keep the blood pressure in the perfusion circuit
slightly lower than the systemic blood pressure.
V. P R E S E N T T E C H N I Q U E F O R R E G I O N A L P E R F U S I O N
The lower extremities can be perfused through the iliac, femoral, or popliteal vessels,
219
and the upper extremities through the axillary or the proximal brachial vessels. The
procedure is carried out during general anesthesia. Before dissection of the vessels, the
extremity is externally warmed and isolated by a heat mattress containing circulating
water at 38°C and isolated by a plastic weave. Thermistor® probes are inserted into
the skin, and into the subcutaneous tissues and muscles, for monitoring the tempera-
ture continuously during the perfusion. The artery to and the vein from the extremity
are surgically exposed and isolated. The cannulation is carried out after systemic he-
parinisation. The cannules have as large a caliber as possible, and are connected to an
extracorporeal circuit consisting of pump-oxygenator and heat exchanger. To prevent
leakage through collaterals a tourniquet is applied above the cannules (Figure 1). The
perfusion is started with a temperature of 32 °C in the extremity, and when the muscle
temperature reaches 38°C the cytostatic agent is added. After 15 to 20 min muscle
temperature has reached 40 to 40.5°C, and then the temperature of the blood is ad-
justed to maintain this level during the whole perfusion (Figure 2). The cytostatic drug
PAM is administered in a dose of 0.9 mg/kg/bodyweight for the lower extremity and
0.45 for the upper. To get reduced peaks and a more even concentration of PAM in
the perfusate, this dose is divided into two or more, according to the length of perfu-
sion. The duration of perfusion varies according to the clinical stage of the disease.
After perfusion is finished, the extremity is washed out with Ringer solution. Postop-
eratively, cephalotin is administered as a prophylactic. As thrombosis prophylaxis and
blood flow stimulus, low molecular weight dextran is given.
FIGURE 1. Schematic view of technique for regional perfusion of

lower extremity with cytostatics and controlled hyperthermia. The ex-
tremity is isolated with a heating mattress. Thermistor® probes are
inserted for continuous registration of the temperature in the extrem-
ity.
FIGURE 2. Temperature registered before and during perfusion.

Dotted area indicates period for hyperthermia.
VI. RESULTS
A. Malignant Melanoma
When reviewing other literature for regional perfusion for malignant melanomas,
one is impressed to notice how inaccurate reports are about the amount of drug, the
perfusion time with drug, the temperature and how the temperature is controlled, and
also how the leakage is controlled. Furthermore, there is great inaccuracy in tumor
staging, in describing which surgery was primarily performed, and in the thoroughness
of the followup. So far, no studies have been reported where standard treatment is
tested against perfusion in a randomized way, nor have any well controlled studies
come to light.
Since the report from Creech, there have been approximately 15 papers presenting
the results of regional perfusion. More than half of them have been presented during
the three years since 1975 to 1977.1016_25 Four of these papers describe controlled hy-
perthermic perfusion, and the others perfusions with body temperature of the perfus-
ate.
When perfusion has been performed for manifest melanoma a good objective tumor
regression is reported. More than 50% tumor regression is reported in most patients
by several authors. 1 0 1 7 2 1 2 3 2 5 2 6 The results concerning 5-year survival in different ma-
terials are impossible to analyze properly. Figures vary between 22 to 48.5%. 1 0 1 2 1 7
Stehlin made a comprehensive study of patients with in-transit metastases of malignant
melanoma subjected to normothermic perfusion in 1951 to 1964, and to hyperthermic
perfusion in 1967 to 1974. He found improved figures in the later series.10
When regional perfusion has been performed as adjuvant therapy to standard surg-
ical therapy, 5-year survival is reported as approximately 80%, 1 0 - 1 2 1 6 , 2 ° 2 3 and the fre-
quency of local recurrences within 5 years as approximately 5%. 1619 ' 20 ' 23 As a compar-
ison, it can be mentioned that many series report a 5-year survival of 67 to 78% for
patients with malignant melanoma, clinical stage one, subjected to standard surgical
therapy. 16 2728
221
Complications of regional perfusion must be considered to be of low frequency. In

more than 2000 perfusions, 17 postoperative deaths have been reported. Further, in
13 cases an amputation of the perfused leg has been performed mainly as a result of
tissue necrosis. Other complications are thromboembolism and neuritis. Myelosup-
pressive effects of the cytostatic agent are limited and seen in approximately 4%.
B. Soft Tissue Sarcoma

A major problem in soft tissue sarcoma of the extremity is to achieve local control
without mutilating surgery such as amputation. With this aim in view, regional perfu-
sion with or without irradiation has been tried as an adjunct to local excision. Standard
technique for perfusion has been used, mostly without hyperthermia. The drugs which
have been used are PAM and actinomycin D in combination.
Because of the heterogeneity of tumor type and tumor stage in limited materials, it
is very difficult to draw any general conclusions from experiences presented in four
publications. 2932 The recurrence rate is reported as 9 to 24% 29 30 32 and 5-year survival
as 62 to 66%. 3 0 3 2
C. Own Material
Since January 1976, 22 perfusions on an adjuvant basis have been performed at our
department. In these cases there was no evidence of tumor remaining after primary
surgery. The indication for these perfusions was clinical stage I and III B, Clark's level
IV and V. Therapeutic perfusion was performed on 16 patients, of whom two were
perfused twice. In the adjuvant group, no recurrence has been detected so far and all
patients are alive, 2 to 30 months after perfusion. In 11 patients in the therapeutic
group, it was possible to register the effect of perfusion and in all of them an objective
tumor regression of more than 50% was observed. In one patient a total tumor necrosis
was instituted, verified by histopathologic examination. Ten of the patients perfused
for therapeutic purposes are still alive, and have had no recurrence during the obser-
vation time of 2 to 24 months. In the remaining patients in this group, there was local
or general recurrence after 5 to 12 months. Three of them are now dead.
VII. C O M M E N T S
Opinions as to the value of regional cytostatic perfusion with or without hyperther-

mia range from enthusiasm to maximal negativism. All authors who have employed
the technique are pleased with the results. The negative criticism of the technique is
reported among others by Lawrence and Terz, 33 who express the opinion that regional
artery chemotherapy will never have a major impact on clinical cancer therapy except
in those unusual circumstances, where the agent is rapidly absorbed and fixed by the
tissue being treated. They also say that it is quite conceivable that the approach has
some merit in selected circumstances, but clinical trials have effectively compared this
approach with less inconvenient alternatives. There may be a place for this type of
therapy in the future, but this approach should be limited to those individuals and
institutions equipped to answer these yet unanswered questions in a scientific manner.
Little is so far known about the theoretical background for the effect of the perfu-
sion technique and the synergistic effect of hyperthermia with radiation and with al-
kylating agents. These questions have to be solved in experiments. Further, little is
known about the optimal temperature, the optimal duration of the perfusion as well
as blood flow, oxygenation, and pH.
It could be of interest if vasoactive drugs could be used to deviate the blood flow
during perfusion to the tumor to get as massive a dose as possible of the cytostatic
drug, or to restrict the blood flow in the tumor to get an increased temperature in the
tumor, using the blood as a cooling agent.
There is no doubt that the perfusion technique in melanoma of the extremity is

directed towards local control of disease in the same way as adjuvant radiation therapy
for breast cancer is given for local control. Regional perfusion on an adjuvant basis
could be added to the treatment of patients with a high risk of local recurrences. As
the technique entails a very low rate of complication and low morbidity (5 to 7 days
hospital care), there is justification for trying it as a complement to a more restricted
surgical attitude. This trial should be performed in a controlled randomized manner
against standard surgical treatment for patients with melanoma infiltrating to Clark's
level IV and V.
If the findings reported concerning an increased immunological effect are correct,
perhaps even a systemic effect for control of widespread occult tumor growth can be
established.10
As to manifest tumor regional perfusion of either local recurrences or intransit me-
tastasis of melanoma should be tried in a randomized controlled manner against stand-
ard treatment, which is local surgery plus cytostatic drug (mainly at present DTIC)
and some kind of immunological treatment (mainly BCG). If these trials show that
perfusion has some advantages, the further developments have to establish optimal
temperature, duration, drug, etc. In conclusion, there is an enormous amount of clin-
ical research work still to be performed in this field. Certainly, controlled randomized
studies would be most welcome.
REFERENCES
1. Klopp, C. T., Fractionated intra-arterial cancer chemotherapy with methyl-bis-amine-hydrochloride:

a preliminary report, Ann. Surg., 132, 811, 1950.
2. Klopp, C. T., Ba teman, J. C , Bery, N., Alford, C , and Winship, O., Fractionated regional cancer
chemotherapy, Cancer Res., 10, 229, 1950.
3. Bateman, J. C , Klopp, C. T., and Cromer, J. K., Hematologic effects of regional mustard therapy,
Blood;6, 26, 1951.
4. Sullivan, R. D. and Watkins, E., Jr., Arterial infusion cancer chemotherapy, in Progress in Clinical
Cancer, Vol. 1, Ariel, J. M., Ed., Gruñe & Stratton, New York, 1965, 321
5. Creech, O., Jr., Krementz, E. T., Ryan, R. F., and Winblad, J. N., Regional perfusion utilizing an
extracorporeal circuit, Ann. Surg., 148, 616, 1958.
6. Creech, O., Jr., Krementz, E. T., Ryan, R. F., Reemtsma, K., and Winblad, J. N., Experiences with
isolation-perfusion technics in the treatment of cancer, Ann. Surg., 149, 627, 1959.
7. Krementz, E. T. and Knudson, L., The effect of increased oxygen tension on the tumoricidal effect
of nitrogen mustard, Surgery, 50, 266, 1961.
8. Cavalière, R., Ciogatto, E. G., Covanella, B. ., Heidelberger, C , Johnsson, R. O., Margottino, M.,
Mondovi, B., Moricca, G., and Rossi-Fanell, A., Selective heat sensitivity of cancer cells, Cancer
(Philadelphia), 20, 1351, 1967.
9. Stehlin, J. S., Jr., Hyperthermic perfusion with chemotherapy for cancer of the extremities, Surg.
Gynecol. Obstet., 129, 305, 1969.
10. Stehlin, J. S., Jr., Giovanella, B. C , de Ipolyi, P. P., Muenez, L. R., and Andersson, R. F., Results
of hyperthermic perfusion for melanoma of the extremities, Surg. Gynecol. Obstet., 140, 338, 1975.
11. Krementz, E. T., and Ryan, R. F., Chemotherapy of melanoma of the extremities by perfusion;
Fourteen years clinical experience, Ann. Surg., 175, 900, 1972.
12. Golomb, F. M., Perfusion of melanoma, Oncology, 26, 197, 1972.
13. Luck, J. M., Action of p-Di (2-chloro-ethyl)-amino-L-phenylalanine on Harding-Passey Mouse Mel-
anoma, Science, 123, 984, 1956.
14. Clarkson, B., and Lawrence, W., Jr., Perfusion and infusion techniques in cancer chemotherapy,
Med. Clin. North Am., AS, 689, 1961.
15. Stehlin, J. S., Jr., Clark, R. L., Jr., and Dewery, W. C , Continuous monitoring of leakage during
perfusion, Arch. Surg. (Chicago), 83, 943, 1961.
223
16. McBride, C. M., Sugarbaker, E. V., and Hickey, R. C , Prophylactic iosolation-perfusion as the
primary therapy for massive malignant melanoma of the limbs, Ann. Surg., 182, 316, 1975.
17. Weaver, P. C , Wright, J., Brander, W. L., and Westbury, G., Salvage procedures for locally ad-
vanced malignant melanoma of the lower limb (with special reference to the role of isolated limb
perfusion and radical lymphadenectomy), Clin. Oncol., 1, 45, 1975.
18. Brown, A. S., Wallack, M. K., Horstmann, J. T., Hamilton, R. W., Johnson, J. L., and Roseto,
F. E., Perfusion therapy for extremity melanoma, Arch. Surg. (Chicago), 111, 961, 1976.
19. Wagner, D. E., A retrospective study of regional perfusion for melanoma, Arch. Surg. (Chicago),
11,410, 1976.
20. Sugarbaker, E. V. and McBride, C. M., Survival and regional disease control after isolation-perfu-
sion for massive stage I melanoma of the extremities, Cancer (Philadelphia), 37, 188, 1976.
21. Hansson, J. A., Simert, G., and Vang, J., The effect of regional perfusion treatment on recurrent
melanoma of the extremities, Acta Chir. Scand., 143, 33, 1977.
22. Lejeune, F. J., Mathieu, M., and Ken is, Y., Hyperthermic isolation-perfusion with melphalan, a
preliminary appraisal of local and general effects in malignant melanoma, Tumori, 63, 289, 1977.
23. Schraffordt-Koops, H., Oldhoff, J., van der Ploeg, E., Vermey, A., and Eibergen, R., Regional
perfusion for recurrent malignant melanoma of the extremities, Am. J. Surg. 133, 221, 1977.
24. Schraffordt-Koops, H., Oldhoff, J., van der Ploeg, E., Vermey, A., Eibergen, R., and Beekhvis,
H., Some aspects of the treatment of primary malignant melanoma of the extremities by isolated
regional perfusion, Cancer (Philadelphia), 39,27', 1977.
25. Jõnsson, P.-E., Hafstrom, Lo., Hansson, J. A., Holmin, T., Sundqvist, K., and Sjogren, H.-O.,
Regional hyperthermic perfusion therapy for melanoma of the lower extremities — technique and
immediate results, Eur. Soc. Surg. Res.,9, (Suppl.l) 37, 1977.
26. Englund, N.-E., Lindstedt, E., and Vang, O. J., Regional perfusion in treatment of sarcomas of the
extremities, Acta Chir. Scand. 137, 243, 1971.
27. Olsen, G., The malignant melanoma of the skin, Acta Chir. Scand. Suppl. 365, 1966.
28. Mattsson, W., Gynning, I., Hogeman, K.-E., Jacobsson, S., and Linell F., A retrospective study of
304 cases of malignant melanoma in Malmo* 1952-71, Scand. J. Plast. Reconstr. Surg., 10, 189, 1976.
29. McBride, C. M., Sarcomas of the limbs: results of adjuvant chemotherapy using isolation perfusion,
Arch. Surg., (Chicago), 109, 304, 1974.
30. Stehlin, J. S., Jr., de Ipolyi, P. D., Giovanella, B. C , Gutierrez, A. E., and Anderson, R. F., Soft
tissue sarcomas of the extremity: multidisciplinary therapy employing hyperthermic perfusion, Am.
J.Surg., 130, 643, 1975.
31. Schraffordt-Koops, H., Eibergen, R., Oldhoff, J., van der Ploeg, E., and Vermey, A., Isolated
regional perfusion in the treatment of soft tissue sarcomas of the extremities, Clin. Oncol., 2, 245,
1976.
32. Krementz, E. T., Carter, R. D., Sutherland, C. M., and Hutton, J., Chemotherapy of sarcomas of
the limbs by regional perfusion, Ann. Surg., 185, 555, 1977.
33. Lawrence, W., Jr. and Terz, J. J., Principles and techniques of cancer chemotherapy, in Cancer
Management, Lawrence, W., Jr., and Terz, J. J., Eds., Grune & Stratton, New York, 1977,
chap.3.
Taylor & Francis
225
INDEX
A decrease with tumor age and volume, 106

influence of vasoactive drugs, 132-133
Actinomycin D, 207, 218 intratumor distribution, 108-113
effect on transplants, 18 in vivo observation, 115-116
Adenocarcinoma, 58 flow rate, 121-126
Adrenergic innervation, 130 methodology, 116-118
Adriamycin, 207 vascular growth, 118-121
Age, correlation with oxygen consumption, 148 irradiation, see Irradiation, effect on tumor
Algire access-type chamber, 118 blood flow
Alimentary canal, neoplasm, vascularity, 11 local, 107-108
Alpha receptor, 131 measuring principles, 88-89
Anastomoses, arterio-venous, 38 restriction in malignant tumors, 146
Anemia, 150 Blood vessel
Anesthesia, influence on tumor blood flow endothelium, 34, 36, 53
Angiogenesis, 49-51 inervation, 129-131
chemical mediator, 56-58 irradiation induced damage, 138
fetal, 51-56 morphological classification, 33-38
fibroblast growth factor, see fibroblast growth permeability, 125
factor Boundary cylinder, 154
induction, 62-64 Brady Kinin, 132
brain tumor, 60 Bromosulphonphtalein, 104
breast lesions, 61-62 Bronchogenic carcinoma
skin tumor, 60-61 arterial pattern, 8
morphology, 64-72 vascularity, 11
nerve growth factor, see Nerve growth factor
Angiography, 39, 121-122, 185-189
Angiotensin, 190-191
Anoxia, 155
,4
c
C-Antipyrene, tumor tissue uptake, 95-96, 105
,25
I-Antipyrene Capillary
intratumordistribution, 108-109, 111 angioarchitecture, 9-10
tumor-tissue uptake, 94-95 basement membrane, 34
13I
I-Antipyrene, 105 bed, 175
Antivasculogenic agent, 62 blood flow, 170
Arterial inflow, direct measurement, 89 fenestrated endothelium, 34-35
Arteriography, 40 giant, see Giant capillary
Arteriole, 33 permeability, 77, 83-84, 175
Arteriovenous shunting, 134, 146 isotopic measurement, 82-84
Artery, 33 sprout, 34, 69, 71-72
Autoradiography, 96 formation, 50
tissue transport, 171
volume, 30
widening in the vicinity of tumor transplants,
B 120
Capillary sprout, see Capillary, sprout
Basement membrane, 50, 54 Carbon dioxide, 175
Blood Carcinogenesis, 13
born dye, acess to tumor region, 17 Cardiac output, fractional distribution, 94
circulation, see Circulation Cell proliferation, oxygen deficiency as a limiting
plasma, see Plasma factor, 164
vessel, see Blood vessel Central necrosis, 119-120
Blood flow Chemotherapy, see Infusion chemotherapy
anesthesia, see Anesthesia, influence on tumor Circulation, see also Microcirculation
blood flow radioactive tracer study
autoregulation, 104 application, 92-99
capillary, see Capillary, blood flow tissue as a black box, 91-92
concepts, 87-88 tracer substances, 90-91
correlation with 02 consumption, Colon, carcinoma angiography, 189, 196-197
Convective transport, 170-171 G

modeling in tumor tissue, 176-179
Crabtree effect, 162 Gastric carcinoma, vasculature, 10
in vivo observation, 163-164 Giant capillary, 34, 37
Critical supply radius, 154-156 Glioma, 8
Cryophotometric micromethod, 160 induction of neuro-vascularization, 60
Glucagon,132
Glucose, 175
D uptake in malignat tumors, 162-163
Glycolysis, 162
Glycosaminoglycan, tumor tissue content, 170
Darcy'slaw, 177
Granulosa tumor, 50-51
Diffusion, 170-171
vascular system, 16
coefficient, 147 Guerin carcinoma, uptake of 86Rb, 105
ò/s-Diketopiperazine, antitumor activity, 38
1, 2, bis(3, 5-Dioxopiperazine-l-yl) propane, see
ICRF159
Distribution volume, 94 H
Dorsal air sac, 56
Drugs Haemangioma, 57
distribution in tumor tissue, 179-180 Hamster cheek-pouch chamber, 21, 116
vasoactive, see Vasoactive drug Heat clearance method, determination of tumor
Dye dilution technique, 192 blood flow, 96-97
Height over area method, blood flow
determination, 96
Hemangiopericytoma, 30-31
E Hemoglobin, intra-capillary oxygen saturation,
160-162
ECV, see Extracellular volume Hepatic artery
EGF, see Epidermal growth factor ligation
Endothelial growth promoting factor, 60 experimental tumor, 205
Endothelial sprouting, 54 treatment of human liver tumors, 205-206
Endothelial tube, 56 occlusion, 208-210, 211-213
Hepatic dearterialization, 207-211
Endothelium, conductive properties, 177
Hepatic tumor
Epidermal growth factor (EGF), 58-59
blood supply, 11,203-205
Epinephrine, 132, 190
hepatic artery ligation, 107
Extracellular volume (ECV), 80
infusion chemotherapy, see Infusion
Extraction fraction, 94
chemotherapy
Extravascular fluid, distribution, 125
Hepatoma
Extravascular space, 77, 79
collagen content, 16
composition, 125
microcirculation, 16
isotope measuring technique, 79-81
vascular morphology, 29-30
Eye, neovascularization, 62
Hilleguation, 174
5-Hydroxytryptamine, 132
Hyperemia, 14
Hyperoxia, 151, 161
F dynamic behavior of po2 values in tumor tissue,
159-160
Fetal angiogenesis, see Angiogenesis, fetal Hyperthermia, oxygen consumption, 151-153
Fibrinogen, localization in tumor tissue, 16, 82 Hypoxemia, steady state, see Steady state
Fibroblast growth factor, 59-60
hypoxemia
Fibrosarcoma
Hypoxia, 150, 155
collagen content, 16
femoral angiogram, 198-200
subclavian angiogram, 195
vascular pattern, 23 I
5-Fluorodeoxyuridine (FUDR), 207
5-Fluovouvacil (5-FU), 207 ICRF 159, antitumor activity, 38
FUDR, see 5-Fluorodeoxy-Uridine Imperial Cancer Research Fund agent number
Functional vascular volume, 78 159, see ICRF 159
227
Indicator, transport in tumor tissue, 175-176 Michaelis-Menten Kinetics, 156, 174

Infusion chemotherapy, 206-211 Microangiography, 40
Intercapillary region, oxygen supply, 152-157 serial, see Serial microangiography
Interstital fluid, velocity, 177 Microarteriography, 40
Interstitial pressure, equation, 177 Microcirculation, see also Circulation
Interstital space, see Extravascular space compartmental model, 172
Intraocular transplantation, 13 correlation with tumor necrosis, 12
Intraportal infusion disturbance of, 146-147
Irradiation organization, 31
fractional, 139-140 Microcirculatory module, 1
single dose, 137-139 Microelectrode, 158
Ischemia, 133 Microsphere, see Radioactive microsphere
Isoproterenol, 132 Mouse-back chamber, 116
Isotope-clearance technique, see Tissue clearance MPS, see Methyl prednisone
technique Multicomponent analysis, 160
K
N
Ketamine hydrochloride, 134
Knisely quartz-rod technique, 21 Necrosis, 149, 155, 186
Krogh tissue cylinder, 171-172 central, see Central necrosis
extraluminal flow, 178-179 Neoplasm
p0 2 distribution, 156 fetal characteristics, 56
Krough diffusion constant, 147, 154 po2 distribution, 157-160
"Krypton, tissue clearance, 97-98, 146 reaction of host vasculature, 18
Nephroangiography, 192
Nerve growth factor (NGF), 58-59
L Neuroblastoma, 57
NGF, see Nerve growth factor
Lax tumor, 24, 120 Nitrogen mustard, 218
LIA, see Lymphocyte induced angiogenesis Noradrenaline, 193
Liquid scintillation counter, 96 Norepinephrine, 132-133
Lissamine green, 105, 108 Normothermia, oxygen consumption, 153
Liver Normoxia, 160
tumor, see Hepatic tumor pQ2 distribution in neoplastic tissue, 158-159
Lymph-node, metastasis, 40
Lymphocyte, induction of angiogenesis, 63
O
M
Optical transparency, 116
Macrophage, induced angiogenesis, 63 Oxygen
Mammary gland consumption
carcinoma, 57 abundance, 151
microcirculation, 121 hetergeneity, 149-150
vascular morphology, 25-28 hyperthermia, 151-152
Mass balance, equation, 179 interrelationship with glucose uptake, 162-
Material flow, 88 163
Material transport, 170 low available oxygen, 150-151
Mean flow, 88 respiration rate, 148
Melanoma diffusion, 147-148
malignat growth, 65-68 distribution, 157-158
regional perfusion, 220-221 normoxia, 158-159
vascular morphology, 22-31 hyperoxia, 159-160
Mesenchymal cell, 54 intercapillary region
Metastic tumor hemoglobin saturation, 160-162
animal model, 64 supply, 152-157
vascular pattern, 19 removal rate, 149
Methotrexate, 207 transport, 172-175
Methyl prednisone (MPS), 58 restricted, 144-147
P Sarcoma, 58
fibrinogen uptake, 39
PAM, see Phenyl-alanine-mustard vascular occlusion, 25
Pancreatic carcinoma, angiography, 187 Serial microangiography, 122-123
Partition coefficient, 107 quantification, 124
Pasteur effect, in vivo investigation, 162-163 Sheetlike growth pattern, 116
Peclet number, 170 Sinusoidal vessel, 34-35
Perfusion rate, 124-126 Skin, neoplasm, 11
Pericyte, 54 Sodium pentobarbital, 134
Permeability coefficient, 176 effect on tumor blood flow, 107-108
Permeability-surface-area product (PS), 83-84 Soft tissue sarcoma, regional perfusion, 221
Pharmacoangiography, 189-196 Spindle-cell sarcoma, vascular pattern, 23
Pharmacokinetics, macroscopic, 179 Starling's coefficient, 177
Phenyl-alanine-mustard (PAM), 218 Steady state hypoxemia, 150
Plasma Stereologic technique, 41
velocity, 122-123 Stromal response, 15
volume (PV), 80 Subcutaneous tissue, tumor vascular morphology,
Plethysmography, 89-90, 105, 132 23-25
Polarography, measurement of 0 2 tension, 158 Systemic blood pressure, 132
Polycythemia, 151
Propranolol, 132
Prostaglandin E,, 195 T
Prostaglandin E2, 132
Provisional capillary network, 54 TAF, see Tumor angiogenesisis factor
Pseudostable state, 94 Temperature
PS, Permeability-surface-area product effect on oxygen consumption, 152
Pulmonary neoplasm, blood supply, 40 effect on tumor blood flow, 152
PV, see Plasma, Volume effect on tumor growth, 118
Pyranin, 122 Tense tumor, 24, 119-120
Thickness-restricted chamber, 116-117
Thoma's laws, 54
R Thymidine labelling index, 60
Tissue acidosis, 146
Radioactive isotope, see also specific isotopes, 91 Tissue-blood partition coefficient (A), 97
Radioactive microsphere, measuring technique, Tissue clearance technique, 97-99, 107
93-94, 105,204,211 simultaneous clearance, 98-99, 108
Radiosensitivity, 164 Tissue-isolated implantation tumor, 144
RCV, see Red cell volume Tissue stroma, 79
Red cell volume (RCV), 80 Tolazoline, 193
Redundancy index, 42 Total tumor blood flow, see also Blood flow, 88,
Regional perfusion 104-107, 147
cytostatic agents, 218 Transcatheter embolic occlusion, 189-191
history, 217-218 Transparent-chamber technique, 21
leakage, 218 Transplantation, 104
results, 220-221 Transport
technique, 218-220 convective, see Convective transport
Renal carcinoma, angiography, 186-188, 193-194 diffusive, see Diffusion
Respiration, 162 material, see Material transport
Reynolds number, 170 mathematical model, 169-180
Round-cell sarcoma, vascular pattern, 23 transvascular, see Transvascular transport
Rous sarcoma, 58 Trans vascular transport, 81
86
Rubidium Tumor angiogenesis factor (TAF), 57
intratumor distribution, 108-109, 133 Tumor cell volume, 79
tumor-tissue uptake, 94-95 Tumor diffusible factor, 56
S V
Sanders-Shubik chamber, 21 Vascular bed, relative resistance, 26

Sandwich chamber system, 21, 120-121 Vascular density, 41
Sapirstein method, 94-95 Vascular-density index, 7
229
Vascular irritation, 7 Volume flow, 87

Vascular morphology, 1-5, 30-31 direct measurement, 88-90
architectural organization, 31-39
historical review, 5-6
animal tumors, 12-20 w
human tumors, 6-12
experimental tumors, 20-22 Walker adenofibroma, 25
literature, 3-5 Walker 256 carcinoma, 57
quantitation, 41-42 vascular morphology, 28-29
radiographic observation, 39-41 Weight
Vascular permeability, 81-84 correlation with 0 2 consumption, 149
Vascular space, 77-79 p0 2 distribution as a function of, 159
correlation with tumor weight, 145 Wilms' tumor, 57
isotope measuring technique, 79-81 Window system, 120-121
Vascular statis, 146
Vasculogenesis, 53
Vasoactive drug, influence on tumor blood flow,
X
132-133
Vasopressin, 192
'Xenon,96
Vasoproliferation stimulation of, 56
tissue clearance, 97-98, 107, 112
Vein, 38
washout, 108
Venous concentration-time curve, 92
Venous outflow, 104
direct measurement, 88-89
Venule, 38 Y
Vessel, see Blood vessel
Videoimage analysis, 124 'Yttrium, 211

PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

PDF

Uploaded by

Copyright:

Available Formats

TUMOR

Boca Raton London New York

CRC Press is an imprint of the

Reissued 2019 by CRC Press

© 1979 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

A Library of Congress record exists under LC control number:

ISBN 13: 978-0-367-24532-0 (hbk)

Early morphological studies of tumor vascularization have given a fairly detailed

Hans-Inge Peterson, M.D., Ph.D., is Chief Surgeon, Department of Surgery,

K. Lennart Appelgren, M.D., Ph.D. Jan Mattsson, M.D.

Stig Bengmark, M.D., Ph.D. Eva Peterson-Dahl, M.D.

Per E. Fredlund, M.D., Ph.D. Aleksander S. Popel, Ph.D.

THE VASCULAR MORPHOLOGY OF TUMORS

I. Introduction and Basic Concepts 1

II. Historical Review of the Literature to 1966 5

III. The Vascular Morphology of Certain Experimental Tumors 20

IV. The Organization of the Vascular Architecture in Tumors 31

V. Radiographic Observations of Vascular Morphology in Man and Animals . . .39

VI. Methods of Quantitation of Vascular Morphology 41

The Main Themes of Papers Relating to the Vascular Morphology of

1. Diagnostic use of the vascular patterns in tumors

Principal topic Authors Date Ref.

Capillary network in Virchow 1863 5

Blood vessels in tumors Goldmann 1907 7

Principal topic Authors Date Ref.

Observations on the Warner 1964 53

A. The Themes Involved

1. The use of the vascular morphology of tumors in diagnosis

1. A general description of the vascularity of human tumors at autopsy and on surg-

Lindgren. Only undifferentiated tumor types showed so uncharacteristic a capillary

1. The injection into blood vessels of barium sulphate followed by roentgenographic

as common in cerebral neoplasms is not a specific characteristic of brain neoplasms,

Cowdry and Sheldon, in 1946,29 reported on the significance of hyperemia around

1. In the center, there was a zone of avascular necrosis.

III. T H E V A S C U L A R MORPHOLOGY OF EXPERIMENTAL TUMORS

A. Tumors of Melanin-Forming Tissue

al 56.72 e x a m i n e c i two melanomas of the hamster. Originally described by Fortner 73 as

B. Sarcomas and Tumors of Subcutaneous Tissues

C. Tumors of the Mammary Gland

D. The Walker Carcinosarcoma 256

F. Tumors of the Blood-Vascular System

IV. ORGANIZATION OF THE VASCULAR ARCHITECTURE IN

1. A simple route where a single capillary bed is perfused.

The Varieties and Classification of Blood Vessels in the Sub-

Class 1 Arteries and arterioles

within, sometimes massively, infiltrating growths. He considered it "exceedingly rare"

B. Vascular Density Changes and Redundancy Index of Tumor Vessels

It is a measure of the meandering of a tortuous vessel, and indicates the increase in

Cancer, University of Nebraska Medical Center, Omaha, during a period of sabbatical

II. General Aspects of Angiogenesis 50

III. Fetal Angiogenesis 51

IV. Factors from Tumor Tissue 56

V. Angiogenesis Induced by Specific Tumors and Cells 60

VI. The Morphology of Tumor Angiogenesis 64

Tumor angiogenesis is the formation of capillary sprouts induced by a group of

Capillary growth, constituting as it does one of the main elements of granulation

Ill. FETAL ANGIOGENESIS

1. Dissociation of cells of the angioblastic cord as a result of release of the adhesion

Fetal Angiogenesis: Table Showing Different Types of Vascular Development6

Type of development that

A. Chemical Mediators and Angiogenesis: Tumor Angiogenesis Factor or Factors

B. Nerve Growth Factor (NGF) of Levi-Montalcini