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J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104

Tumor blood-flow changes following protoporphyrin IX-based

photodynamic therapy in mice and humans
Mark A. Herman a, David Fromm a,*, David Kessel b
a
Department of Surgery, Wayne State University School of Medicine, 6C-University Health Center, 4201 St. Antoine, Detroit, MI 48201, USA
b
Department of Pharmacology, Wayne State University School of Medicine, 6C-University Health Center, 4201 St. Antoine, Detroit, MI 48201, USA

Received 7 April 1999; accepted 7 September 1999

Abstract

The effects of aminolevulinic acid (ALA)-based photodynamic therapy (PDT) on tumor blood flow are controversial. This study examines
the effects of ALA and Photofrin-based PDT on blood flow of Colon-26 tumors implanted in mice as well as the effects of ALA-based PDT
on blood flow of human colorectal carcinomas and a carcinoid tumor in situ. Tumors are implanted in both flanks of mice. One tumor of each
animal serves as a control. Blood flow is measured using a laser Doppler method. Tumor blood flow in mice not receiving a photosensitizer
but treated with three different light fluences (50, 100 and 150 J/cm2) does not differ significantly from blood flow in the untreated tumor in
the opposite flank. PDT after ALA administration using the three different light fluences does not significantly affect blood flow. In contrast,
PDT after Photofrin administration causes a significant decrease in tumor blood flow with each light fluence, but this change is not as dramatic
as reported in other studies. In contrast to mice, six patients who receive ALA prior to surgery all show a decrease in blood flow (means51.8%,
p-0.001) after PDT using 100 J/cm2. Comparison with other published results suggests that it is likely that flow measurement by the laser
Doppler method underestimates the effects of PDT on tumor blood flow due to the depth of laser penetration. Nevertheless, the present
observations on blood flow suggest that the effects of ALA-based PDT on adenocarcinomas of the colon and rectum as well as an intra-
abdominal carcinoid tumor in humans are more pronounced than would be predicated by some animal studies. q1999 Elsevier Science
S.A. All rights reserved.

Keywords: Photodynamic therapy; Photosensitizers; Tumor blood flow

1. Introduction
Photodynamic therapy (PDT) of malignant disease
involves the administration of a photosensitizing agent and
its subsequent activation by light of the appropriate wave-
length, resulting in death of the cells containing the agent.
The precise mechanism of cell death remains uncertain and
may be different, depending upon the photosensitizer.
Among the mechanisms is a decrease in blood flow. Photo-
frin, the most widely used photosensitizer, has been repeatedly
shown to cause tumor vascular shutdown. These studies
implicate ischemia and resultant tumor hypoxia as a primary,
although not necessarily the only, mechanism of cell death
for Photofrin-based PDT [1–8].
5-Aminolevulinic acid (ALA)-induced protoporphyrin IX
(PpIX) is a very attractive photosensitizer from a clinical
standpoint because of its greater tumor specificity compared
with other drugs [9–11] and its rapid clearance from the
* Corresponding author.; e-mail: dfsurg@aol.com
body [9,11]. However, even less is known about the mech-
anism of cell death following PDT using endogenous pho-
tosensitization with ALA. In contrast to Photofrin, the data
for tumor blood flow after PDT using ALA are controversial
[7,12,13]. Furthermore, it is not known if blood-flow studies
of tumors in experimental animals are comparable to those
for human tumors. The purpose of this study was to examine
the effects of PDT on tumor blood flow of mice after admin-
istration of either ALA or Photofrin as well as in human
patients receiving ALA.
2. Methods
2.1. Approvals
The animal studies were approved by the Wayne State
University School of Medicine Animal Investigation Com-
mittee. The human studies were approved by the Wayne State
University School of Medicine Human Investigation Com-

1011-1344/99/$ - see front matter q1999 Elsevier Science S.A. All rights reserved.
PII S 1 0 1 1 - 1 3 4 4 ( 9 9 ) 0 0 1 0 9 - 8

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100 M.A. Herman et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104
mittees as well as the FDA. Informed consent was obtained
from all participating patients.
2.2. Mice
2.2.1. Tumors
Colon-26 tumor (National Cancer Institute, Frederick
Cancer Research and Development Center, DCT Tumor
Repository, Frederick, MD) was subcutaneously implanted
in both flanks of 38 female CD2F1 mice (Harlan Sprague
Dawley, Indianapolis, IN) weighing 20–40 g. The tumors
were allowed to grow to approximately 0.5 cm=0.5 cm, at
which time the mice were randomly allocated into three
groups based on the photosensitizer used.
2.2.2. Photosensitizers
Twelve mice received ALA, 200 mg/kg (Dusa Pharma-
ceuticals, Tarrytown, NY); 14 mice received Photofrin, 10
mg/kg (QLT Phototherapeutics, Inc., Vancouver, Canada)
and 12 control mice received no drug. All drugs were admin-
istered by tail vein injection. ALA, dissolved in normal saline,
was given 3 h prior to tumor irradiation. Photofrin, dissolved
in 5% dextrose, in water, was given 24 h prior to tumor
irradiation. All mice were kept in darkened cages following
photosensitizer administration.
2.2.3. Irradiation
Following anesthesia using sodium pentobarbital, 55 mg/
kg i.p., the tumor in each flank was exposed by elevating a
flap of overlying skin. One of the two tumors was then
exposed to 633 nm light from an argon dye laser (Spectra-
Physics, Mountain View, CA). The opposite tumor, which
was outside the field of illumination, served as a non-irradi-
ated control. Each of the three groups received a total tumor
light fluence of 50, 100, or 150 J/cm2. The incident fluence
rate ranged from 55 to 68 mW/cm2. Treatment times thus
varied among groups: 15.2 min to deliver 50 J/cm2, 27.8 min
to deliver 100 J/cm2, and 36.6 min to deliver 150 J/cm2.
2.2.4. Tumor photosensitizer level
Concentrations of protoporphyrin IX in colon-26 tumors
3 h after the i.v. administration of the same dose of ALA have
been previously reported as 2.3"0.3 mg/gm [14].
Photofrin concentrations in tumor were estimated using a
previously described method [11,14]. Tumor samples were
protected from light at all times and stored at y708C prior to
measurement. Briefly, 100 mg samples of tumor were
minced, digested over a period of 7–10 days in a 388C incu-
bator using a mixture of water and Scintigest (Fisher Scien-
tific, Pittsburgh, PA). The samples were next diluted with
methanol and centrifuged. The fluorescence emission spec-
trum of the resulting supernatant was measured using broad-
band excitation (400"20 nm) with a monochromator and
CCD system (Instaspec IV, Oriel Corp., Stratford, CT). The
active porphyrins in Photofrin could easily be distinguished
from spurious signals arising from other fluorophores. Pho-
Thursday Nov 25 01:32 PM
tofrin standards were prepared in a manner identical to the
specimens.
2.3. Humans
2.3.1. Tumors
Six patients had either isolated or metastatic adenocarci-
nomas of the colon or rectum and one had a carcinoid tumor.
Each tumor site used for measurement was at least 2 cm in
thickness. Ages ranged from 34 to 68 years (medians54.5
years) and four were women.
2.3.2. Photosensitizer
ALA, 60 mg/kg, (provided by DUSA Pharmaceuticals
Inc., Tarrytown, NY) was mixed in sterile water immediately
before use and given as a single bolus orally to six patients
at various intervals prior to PDT. PDT was done during var-
ious surgical procedures before mobilization of the organ or
tumor to be resected.
2.3.3. Irradiation
Each patient received a total tumor fluence of 100 J/cm2
with an incident fluence rate of 200 mW/cm2 using 633 nm
light from an argon dye laser (Spectra-Physics, Mountain
View, CA) delivered through a microlens delivery system
applied to an easily accessible portion of the tumor.
2.3.4. Tumor photosensitizer detection
Since biopsies to detect tumor concentrations of PpIX in
humans might well alter blood flow, the presence of PpIX
was detected by a previously reported spectrophotofluoro-
metric method [15]. The intended site of treatment showed
unquestionable accumulation of PpIX in each instance and is
in contrast to surrounding normal tissue.
2.4. Blood-flow measurement
A laser Doppler flow probe (Laserflo Blood Perfusion
Monitor, model BPM 403A, TSI Incorporated, St. Paul, MN)
was used to make quantitative determinations of tumor blood
flow. The probe uses a 780 nm diode laser. This wavelength
is well outside the absorption peaks for both Photofrin and
PpIX, eliminating drug activation by the flow probe. Blood-
flow measurements were made by manually holding the tip
of the probe approximately 1 mm from the surface of the
tumor. Since blood flow tended to be heterogeneous, varying
from one section of the tumor to another, all serial measure-
ments were taken from the same point on the tumor surface.
Baseline tumor blood flow was measured immediately prior
to and at the intended site of light treatment.
Blood flow in mice was measured immediately prior to
PDT. Following treatment of one tumor in mice, measure-
ments were made from both tumors at 5 min intervals for 30
min and subsequently at 45 min, and at 60 min. The animals
were then sacrificed and both tumors of the Photofrin animals
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 M.A. Herman et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104
were removed for measurement of the concentration of
photosensitizer.
Blood-flow measurements in humans were made at a time
that vital signs were stable, since induction of anesthesia and
the infusion of i.v. fluids were kept at a constant rate. Only
short intervals of blood flow were measured in humans so as
not to interfere with the operative procedure. In each case,
the tumor was easily exposed so that the blood-flow probe
could be placed and fixed perpendicular to the tumor. Meas-
urements were made immediately before and immediately
after PDT, which took 8 min.
2.5. Statistics
Blood flow prior to treatment with light was compared
with the baseline line value obtained from the tumor not
treated with light using the paired t-test. The means of all
subsequent values for treated and untreated animals were also
compared using the paired t-test.
3. Results
3.1. Mice
3.1.1. Controls
One of the two tumors in control animals, who did not
receive a photosensitizer, was treated with the laser whereas
Fig. 1. Effect of three different light fluences (Ns4 animals for each dose)
on blood flow of Colon-26 tumor subcutaneously implanted into both flanks
of mice who did not receive a photosensitizer. The tumor in one flank was
treated with light and the tumor in the opposite flank was not treated with
light. The differences in blood flow are not statistically significant.
Thursday Nov 25 01:32 PM
101
the tumor lying in the opposite flank received no treatment.
The baseline values for treated and untreated animals are not
significantly different within the three groups of mice, Fig.
1. However, the mean rates of blood flow between the paired
sets of controls for each light fluence (50, 100 and 150 J/
cm2) vary and thus emphasize the importance of measuring
blood flow among tumors in each individual animal in order
to obtain appropriate control values. The reason(s) for this
variability between groups of animals is not clear. Adminis-
tration of a light fluence of 50, 100 or 150 J/cm2 does not
significantly alter blood flow over a 50 min interval, Fig. 1.
3.1.2. ALA
The baseline blood-flow values for treated and untreated
mice are not significantly different within the three groups of
mice. Administration of 50, 100 or 150 J/cm2 to tumors in
animals treated with ALA does not significantly alter blood
flow compared with that in the tumor in the opposite flank
that was not exposed to light, Fig. 2. Since the lack of an
effect on blood flow might be due to limits of detection, the
effect of Photofrin-based PDT was also measured.
3.1.3. Photofrin
The baseline blood-flow values for treated and untreated
mice are not significantly different within the three groups of
Fig. 2. Effect of three different light fluences (Ns4 animals for each dose)
on blood flow of Colon-26 tumor subcutaneously implanted into both flanks
of mice 3 h after receiving ALA, 200 mg, i.v. The tumor in one flank was
treated with light and the tumor in the opposite flank was not treated with
light. The differences in blood flow are not statistically significant.
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102 M.A. Herman et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104
Fig. 3. Effect of three different light fluences (Ns4 animals for 50 J/cm2
and Ns5 animals each for 100 and 150 J/cm2) on blood flow of Colon-26
tumor subcutaneously implanted into both flanks of mice 24 h after receiving
Photofrin, 10 mg/kg, i.v. The tumor in one flank was treated with light and
the tumor in the opposite flank was not treated with light. The differences in
blood flow between tumors treated with light and untreated tumors was
statistically significant for each light dose (see Section 3.1.3).
mice. In contrast to ALA-treated animals, administration of
50, 100 or 150 J/cm2 to tumor in animals treated with Pho-
tofrin results in a significant decrease in blood flow at each
light fluence (p-0.001 for each light fluence), Fig. 3. How-
ever, there does not appear to be a near-complete vascular
shutdown within 1 h of delivery of any of the three light
fluences. Tissue concentrations of Photofrin are shown in
Table 1, which indicates accumulation of the drug.
3.2. Humans
Immediately prior to treatment with light, the relative blood
flow of each tumor was stable, Fig. 4. The blood flow in each
patient decreased after treatment with light, Fig. 4. In one
patient, blood flow was measured for 10 min (rectum –
6.5 h, see Fig. 4) and remained depressed at a steady level
between 30 s and 10 min after treatment. The mean decrease
in blood flow (baseline compared with last measurement)
was 51.8"2.7% (p-0.001).
4. Discussion
Previous studies of blood flow following PDT have
employed differing techniques for measuring vascular
Thursday Nov 25 01:32 PM
Table 1
Tissue concentrations of Photofrin in Colon-26 tumor
Light fluence mg/g tissue
(J/cm2) untreated treated
50 5.6"1.3 5.1"1.1
100 4.6"0.7 3.4"0.7
150 4.0"0.6 3.6"0.4
Light fluences apply only to treated animals. Ns4 in each group.
Valuessmean"SE.
Fig. 4. Effect of 100 J/cm2 light fluence on intra-operative blood flow of
malignant tissue in situ in six different patients who received ALA, 60 mg/
kg, p.o. Blood flow was measured immediately before and 8 min after PDT.
The origin and location of the tumor as well as the time of PDT after receipt
of ALA is indicated for each patient. The mean decrease in blood flow was
51.8"2.7% (p-0.001).
changes. These include radiolabeled microsphere injection
[1,2], direct visualization through transparent sandwich
observation chambers [3], inta-arterial fluorescein dye injec-
tion [4], tumor oxygen tension measurement [5], injection
and extraction of 86RbCl [6,7], magnetic resonance spec-
troscopy using D2O [16,17], microscopic measurement of
changes in blood vessel diameter [12], and ocular angio-
graphy [18]. Laser Doppler flowmetry is another method
that has been used to measure PDT-related blood-flow
changes [4,19–21]. The validity as well as the reliability of
this non-invasive method have been confirmed by a number
of studies, but the latter do not involve tissues exposed to
PDT [22–26]. The primary advantage of laser Doppler flow-
metry is that it is an easily performed, real-time quantitation
of tissue blood flow at depths corresponding to laser light
penetration.
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 M.A. Herman et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104
The 780 nm wavelength of the Doppler will not cause
photoactivation of PpIX or Photofrin, eliminating the possi-
bility of Doppler-induced vascular effects during serial
recordings. However, there are at least two potential draw-
backs of the technique. The first is spatial variation in the
readings. The heterogeneity of tumor tissue combined with
unavoidable hand movements during the manual recordings
can cause changes in blood-flow readings from one area of
the tumor to another. The data of the present study appear to
suggest that serial recordings made from the same site on
each tumor eliminate much of this variability. The second
problem is the depth of tissue penetration by a 780 nm wave-
length. This is greater than that which occurs with the 633
nm laser light source used to activate PpIX and Photofrin.
Thus, Doppler flowmetry carries the possibility of under-
estimating changes in tumor blood flow if the laser Doppler
light penetrates through areas unaffected by PDT and/or into
normal tissues deeper than the tumor.
All studies, including the present, that have examined the
effects of PDT using Photofrin or hematoporphyrin derivative
show a decrease in blood flow measured by fluorescein exclu-
sion, 86RbCl extraction, microsphere, laser Doppler or micro-
scopic methods. This decrease has been observed for
transplanted fibrosarcoma in mice [6,18] and rats [7], trans-
planted urothelial tumors in rats [1,2], SCCVII cells trans-
planted in mice [6], isologous mammary tumors implanted
in rats [3], and spontaneous mouse mammary tumors as well
as transplanted chondrosarcomas in rats [4]. The present
study found a decrease in blood flow in colon tumors
implanted in mice. The decrease in blood flow occurring after
Photofrin-based PDT appears to be independent of the light
fluence. However, another study found that the reduction in
blood flow is directly proportional to the injected dose of
Photofrin and a more extensive vascular effect with increas-
ing light fluences ranging from 2.4 to 90 J/cm2 [4].
While most studies to date using exogenously administered
photosensitizers have reported a decrease in blood flow, there
is controversy about the effects of PDT using endogenous
photosensitization following administration of ALA. Exper-
imental studies suggest that a vascular effect of ALA-based
PDT does not dominate [12]. Fluorescence microscopic
examination also shows low fluorescence in blood vessels of
dimethylhydrazine-induced colonic tumors in rats 1–8 h after
i.v. administration of ALA [13]. These data led to the con-
clusion that the primary effect of PDT using ALA was on the
tumor cells rather than the blood vessels. In contrast, another
fluorescence microscopic study found PpIX in regions imme-
diately adjacent to tumor microvessels of transplanted rat
fibrosarcoma 3 h after administration of ALA, but this study
also used a higher dose of ALA [7]. PDT caused a 93%
reduction in mean total, but relative, vascular perfusion of
the fibrosarcomas. PDT with ALA has also been reported to
decrease blood flow in normal cremaster muscle of rats [12].
Microscopic vasoconstriction has been observed in ALA-
treated rats with transplanted mammary tumor 5 min after
PDT [27]. ‘Partial disruption’ of Colo26 tumor perfusion in
Thursday Nov 25 01:32 PM
103
mice has been reported using fluorescein exclusion tests [28].
In contrast, the present study found no significant effect of
PDT using ALA on the blood flow of colon tumors implanted
in mice. This lack of response to ALA is not due to the absence
of PpIX in the tumor, as we have previously reported signif-
icant concentrations of PpIX in such tumors exposed to the
same dose of ALA and the same time at the start of PDT
[14]. Furthermore, it is unlikely that spontaneous changes in
blood flow (for example, as a result of change in cardiac
output) account for the lack of response, since each animal
treated with PDT also had blood-flow measurement of the
untreated tumor in the opposite flank. While possible, it
appears that the lack of response was not due to inadequate
fluence rate or light fluence. Studies finding an effect of ALA-
based PDT on blood flow used 400 J with 100 mW and 100
J/cm2 with 178 or 300 mW/cm2 [7,12,13], whereas the
present study used 100 J/cm2 with 200 mW /cm2 for humans
and 55–68 mW/cm2 for mice. Tumor resistance to PDT is
also unlikely, as other studies have shown that the Colon-26
tumor is responsive to ALA-based PDT [28–30]. It is also
unlikely that the duration of measurement after PDT was
inadequate in the present study because virtually all studies
of the effect of PDT on blood flow show an early response.
However, the lack of a detectable blood-flow effect of
ALA-based PDT on Colon-26 tumors transplanted in mice
and determined by the laser Doppler method must be inter-
preted with caution. The majority of animal studies indicate
that the vascular effects of hematoporphyrin derivatives are
dramatic, a situation not observed in the present study. Thus,
it is possible that the laser Doppler underestimates flow in
animal tumors. This may be related to the depth of penetration
of the laser and might account for the lack of a detectable
effect of hematoporphyrin derivative on blood flow of human
oral cancer transplanted into mice [19].
In contrast to the lack of a detectable effect of ALA-based
PDT in mice, ALA-based PDT in humans with either ade-
nocarcinomas of the colon and rectum or a carcinoid tumor
is followed by a mean decrease of 52% in blood flow. The
duration of this decrease is not clear because of limitations in
the time of measurement. However, it is at the very least
transient, lasting from 21 s to 10 min. This dramatic decrease,
in comparison to the mouse studies, is most likely not due to
laser Doppler penetration beyond the tumors, because each
measurement was made in tumors that were several centi-
meters thick. However, it should be noted that the laser Dopp-
ler measurements in humans may also be an underestimate
of actual changes due to the depth of penetration of the laser.
The only other study involving changes in blood flow in
humans due to ALA-based PDT (60 J/cm2 with fluence rates
no greater than 100 mW/cm2) that we are aware of involved
topical administration of ALA to basal cell cancers and squa-
mous cell carcinomas in situ [20]. Laser Doppler perfusion
imaging of these lesions showed an increase in perfusion
immediately following PDT. It is not clear if these differing
results in humans are due to differences in tumors, fluence
rates, fluences, penetration depth of the laser or route of ALA
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104 M.A. Herman et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 99–104
administration. Nevertheless, the present observations on
blood flow suggest that effects of ALA-based PDT on ade-
nocarcinomas of the colon and rectum as well as an intra-
abdominal carcinoid tumor in humans is more pronounced
than would be predicted by some animal studies.
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StyleTag -- Journal: JPB (J. Photochem. Photobiol. B: Biol.) Article: 7874