Neoplasms, Differentiations and Mutations

G. Barry Pierce, MD

Evidence has been presented to support the concept that malignant tumors are
postembryonic differentiations superimposed upon the process of tissue main-
tenance and renewal. Mfalignant stem cells are derived from normal stem cells.
They have a capacity for proliferation and differentiation that operates at a different
level of control than the normal. Even so, malignant stem cells are responsive
to environmental control, suggesting that it may be possible to direct their differ-
entiation or at least to control their ability to replicate. A tumor is a caricature
of normal tissue and appears undifferentiated because of the preponderance of
undifferentiated proliferating stem cells in relationship to the number of cells
that have differentiated and become benign (Am J Pathol 77:103-118, 1974).

IT IS THE PURPOSE OF THIS PAPER to present a point of view

about tumors that has been developing in our laboratorv over the
past decade or more. Mv initial interest was in the histogenesis of
testicular tumors, studies that led to the demonstration that differentia-
tion occurs in malignant tumors,' and that some of the progenv of
malignant stem cells differentiate to benign postmitotic cells. Thus,
the idea "once a cancer cell, alwavs a cancer cell" did not hold for
the three types of tumor studied.24 Recently,, we have been interested
in the position of tumors in the flow of biology, including the cell of
origin, and reviewed data supporting the speculation that cancer is
a pathology of cells involved in tissue renewal.5 In this respect it is
probably less a "pathology" or accident of cell biology than a
differentiation in it own right.
Specifically, two points will be reviewed and new evidence will
be presented to support a third: a) A tumor is a dynamic system com-
posed of malignant stem cells and their well-differentiated and benign
progeny, admixed to form a caricature of the tissue of origin. b) Malig-
nant stem cells are responsive to environmental controls, an observa-
tion that should make us rethink the concepts of progression and
autonomyr. c) Malignant stem cells are derived from normal stem
cells by a process equivalent to a postembrvonic differentiation. Neo-
Presented at the Fiftv-eighth Annual Meeting of the Federation of American Societies
for Experimental Biology-, Atlantic City, NJ, April 7-12, 1974.
Supported in part by Grant DT-14 from the American Cancer Society and Grant AM-
15663 from the National Institutes of Health.
Address reprint requests to Dr. G. Barry Pierce, Department of Pathologv, University
of Colorado Medical Center, 4200 East Ninth Aye, Denver CO 80220.
103
104 PIERCE American Joumal
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plasms have their origin in the process of tissue maintenance and

renewal.
In approaching these points, it is now common knowledge that tissues
such as those lining the gut are constantly undergoing renewal. Senes-
cent cells are replaced by an equivalent number of new cells which
are the progeny of stem cells. The latter are located in the crypts.
Half of the progeny of the stem cells migrate up the sides of the
villi where they differentiate and perform the specialized functions
of gastrointestinal epithelium. After a life-span of about 4 days, senes-
cent cells are sloughed from the tips of the villi as necrotic debris.
This system is under precise control, and loss of cells is matched by
formation of cells; even so, the system is responsive to environmental
changes that require rapid production of new cells. Many of the stem
cells in the crypts are in G-0, that is, they are not part of the active
mitotic cycle, yet they can commence synthesizing DNA and become
part of the proliferative pool when the occasion demands.07 The sta-
bility and heritability of this differentiation is dependent upon stable,
heritable controls of gene expression. How these controls operate is
not known, although in recent years it has become apparent that
certain steroids administered to the embryo can cause sex reversal
with major changes in differentiation of gonads and accessory sex
organs.8'9 These steroids are believed to have specific cytoplasmic and
nuclear receptors and somehow influence control of gene expression.10
Isn't it interesting that steroids can be responsible for a differentiation
in the embryo through control of genome, yet in adults steroid-induced
tumors are believed by most oncologists to arise by mutation?
The idea that a tumor is composed of a heterogeneous collection
of stem cells and well-differentiated cells is not readily apparent from
light microscopic examination of many tumors. If a section from a
benign tumor of smooth muscle is compared to one from a highly
malignant leiomyosarcoma, the phenotypic characteristics of normal
smooth muscle cells are readily apparent in the cytoplasm of the benign
cells and lacking in that of the malignant cells. This has led most
oncologists to the erroneous conclusion that malignant cells have lost
features of differentiation. Loss implies a mechanism. It implies that
tumor cells had their origin from a well-differentiated cell, and then
through some mechanism lost features of the differentiated phenotype.
An alternative explanation for the morphologic appearance of malig-
nant tumors has been proposed: possibly tumors develop from normal
but undifferentiated cells, and rather than losing features of differen-
tiation, the cells do not acquire them.5 Obviously, some of the cells
Vol. 77, No. 1 DIFFERENTIATION AND MUTATION IN NEOPLASMS 105
October 1974

of a leiomyosarcoma have better formed cvtoplasm than others, and

when this type of malignancy is examined with the electron micro-
scope, it becomes apparent that it is composed of cells heterogeneous
in degrees of differentiation. This phenomenon is better illustrated
in studies of teratocarcinoma.
Transplantable testicular teratocarcinoma of strain 129 mice were
first described by Stevens and Little.11 The tumor is ustuallx composed
of twelve or more somatic tissues, including brain, bone, teeth, glands,
etc. Illustrated in Figure 1 are a gland, skin, brain, primitive connec-
tive tissue and embr-vonal carcinoma cells; the latter are extremely
malignant. It has been shown by cloning experiments that a single
embryonal carcinoma cell, when transplanted into the peritoneal cav-
itv of a mouse, can give rise to a tumor that contains all of the tissues
of the teratocarcinoma.12 When these tissues were separated from the
cancer cells and transplanted in compatible hosts, thev survived and
functioned. At the conclusion of the experiment, they had caused their
host no apparent discomfort and were obviouslv benign.2 Thus, the
dogma that once a cancer cell, always a cancer cell did not hold for
this type of tumor. Often, the tissues formed in these tumors were
arranged in patterns reminiscent of mouse embryos, and these structures
have been called embrvoid bodies. Thev are a caricature of embrvo-
genesis.
The idea that a tumor is a caricature of a normal tissue is illustrated
better in studies of squamous cell carcinoma. The tumor used was first
isolated by Dr. Katherine Snell from the lip of an Irish rat and was
composed of masses of undifferentiated cells in which were found
structures known to pathologists as squamous pearls (FiguLre 2). Pearls
are excellent replicas of squamous epithelium.
When animals with these tumors were injected with tritium-labeled
thvmidine, it turned out that the cells in the pearls were not labeled
at 2 hours, but manv of the undifferentiated cells were labeled. By
96 hours, labeled cells began to appear in the pearls, supporting the
conclusion that previously undifferentiated cells had migrated into
the pearls.3 WNhen these labeled cells in the pearls were examined
by autoradiographv with the electron microscope, it turned out that
they had in fact differentiated into squamous epithelial cells. When
the pearls were dissected from the tuLmor and transplanted into the
subeutaneous space of other animals. they never produced tumors.
Under the conditions of the experiment, equivalent amounts of undiffer-
entiated tissue did produce tumors in a third of the cases.:
Just as the teratoma was a caricatuire of embrvogenesis, the squamous
106 PIERCE American Joumal
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cell carcinoma was a caricature of tissuegenesis. Each of these tumors

was a dynamic system composed of proliferating undifferentiated
stem cells which gave rise to undifferentiated cells, as was expected,
and also to ones which had the capacity to undergo differentiation
to a benign state.
How is the development of benign cells and tissues from malignant
stem cells explained? If the mechanism of malignancy is mutation,
is this apparent reversal of the process explained bv reverse mutation?
On the basis of the number of mutations required, this would be
impossible. We preferred to explain the mechanism of this phenomenon
as analogous to that in nuclear transplantation.13 In the nuclear trans-
plantation experiments of Briggs and King 14 and Gurdon,"5 it is evi-
dent that cytoplasm controls nuclear expression. At least, when the
nucleus of a differentiated endodermal cell was placed in the fertilized
enucleated cytoplasm of an amphibian egg, development was in
accord with the state of differentiation of the cytoplasm rather than
with the state of differentiation of the nucleus. Since the overt features
of differentiation occur in the cvtoplasm, and since the nucleus of the
malignant cells was already programmed for a particular differentiation,
it might be concluded that as the differentiated products gathered
in the cytoplasm, they in turn established a new control of nuclear
function which led to cessation of synthesis of DNA, loss of fetal
antigens and the acquisition of the antigens characteristic of the adult
state, etc.13 Again, rather than invoke mutation, it would appear much
more reasonable to suppose that what is going on is really further
differentiation.
But at this point you mav ask, but what of viruses and cancer?
When King and McKinnel `3 and King and DiBerardino 17 transplanted
the nuclei from renal cell carcinoma of the frog (incidentally these
tumors were caused by a DNA tumor virus), and placed these nuclei
into the enucleated cytoplasm of a fertilized egg, thev did not observe
the development of a mass of cancer cells which sturvived until the
yolk of the egg was exhausted. That is the natural historv of cancer.
Rather, blastulae developed from these cells, and although their growth
was arrested and they died, it would appear that under the circum-
stances, cytoplasm controlled not onlv nuclear expression but also the
expression of an integrated oncogenic viral genome.
There is no question that viral particles can be injected into an
appropriate animal and cause the development of a tumor. The ques-
tion is what happens in real life? It is conceivable that the genetic
information contained in a DNA oncogenic vinis is part of the genome
Vol. 77, No. 1 DIFFERENTIATION AND MUTATION IN NEOPLASMS 107
October 1974

of a normal cell of some species. If this information is activated,

transformation may result (oncogene).1819 The mechanism of activa-
tion would be equivalent to the mechanism of differentiation. It is also
conceivable that the information contained in an oncogenic virus may
be responsible for a phenotypic trait in its normal cell of origin, and
if removed from that cell (infectious virus) and then integrated in
the genome of another suitable cell, would not express the original
phenotypic trait, but would, through abortive expression, transform
the cell. This situation would be analogous to what happens in the
laboratory models of viral transformation. In this situation, it is worth
noting that although almost 100l of cells in a plate of fibroblasts may
be infected by a DNA oncogenic virus, only a few cells are trans-
formed. Apparently, the viral genome is integrated and partly expressed
because all of the cells are T positive.2° Is the milieu appropriate for
the transformational (differentiative) event in only a few cells? What
are the factors involved? Thus, the real question is where do viruses
come from and how are they activated under normal circumstances
to produce tumors. Since thev are genomic, I would presume that
the process by which they are activated is one of differentiation.
Now I should like to focus your attention on the second point, that
malignant cells are responsive to some types of control. Specifically,
I would like to refer to an experiment performed more than a decade
ago by Mendelson.21 Mendelson perfused mice bearing spontaneous
mammary adenocarcinomas for 5 days with tritium-labeled thymidine
and observed that about half of the tumor cells were not labeled
and proposed that these cells were stem cells arrested in G-0. We
wondered if the unlabeled cells had not, in fact, differentiated and
become postmitotic. The experiments of Mendelson were repeated
using autoradiography with the electron microscope to determine
whether the unlabeled cells were senescent as a result of differentiation
or were stem cells arrested in G-O.
In confirmation of the observation of Mendelson, about half of the
cells of spontaneous adenocarcinomas synthesized DNA during the
5-day interval of observation.4 Degrees of differentiation of neoplastic
cells were graded from one to three, three was the most differentiated,
as illustrated in Figure 3. The cells with evidence of secretion were
graded 3+, those with endoplasmic reticulum and Golgi complexes
were graded 2+, those lacking these differentiations were graded 1+.
When autoradiograms of three tumors were examined, 11, 14 and
35% of nonlabeled cells were grade 3. In a pulse experiment, it was
shown that tumor cells with grade 3 differentiation did not synthesize
108 PIERCE American Journal
of Pathology

DNA and were senescent, therefore, as a result of terminal differ-

entiation.4
Mendelson was proved correct in his assumption that there were
a significant number of cells arrested in G-0. The cells arrested in
G-O must be responsive to some kind of environmental stimulation.
It is known that tumor cells can be stimulated to more rapid division by
hormones and other agents, but I would suggest that the control with
which we are concerned here is more intimate and has to do with
cell-to-cell types of interaction.22 We have been remiss in our relative
lack of appreciation paid to the social relationships of cells. These
are important for the understanding of dormancy, a situation in which
a few cells are trapped in an unsuitable environment for optimal pro-
liferation, and are unable to express their phenotype and metastasize
for an interval often measured in years. When enough of these cells
aggregate and create an optimal environment, they behave as cancer.
We are beginning to understand certain things about contact inhibi-
tion and effects of certain molecules in stimulating growth and cell
aggregation, but when one realizes that as many as a third of the cells
in any malignant tumor may not be proliferating because of some
unknown kind of intimate growth controL it seems a problem of major
proportions is being ignored.
These observations have implications for the concepts of progression
and autonomy. It would appear that a major element in progression
is a sorting out of populations of cells best able to survive under the
conditions of -growth.13 This would explain the gain and losses of
unit characteristics as described by Foulds.23 It is probably not the
complete explanation,24 but it is the most important component.
At this point, I should like to focus on the final of the three subjects:
the origin of malignant stem cells. This point is of importance because
if tumors arise by the process of differentiation, then their origin,
rather than being in differentiated cells, should be in undifferentiated
ones. Consider the mammary adenocarcinoma just described. The rest-
ing mammary gland of the C3H mouse has the appearance illustrated
in Figure 4. Notice there are just a few scattered ducts surrounded
by adipose tissue. This appearance changes during pregnancy when
the fat is largely replaced by glandular acini which develop through
proliferation and differentiation of the duct cells. These cells become
functional during lactation (Figure 5).
Although tumors may develop during resting phases, pregnancy
or lactation, it is clear that the ductal cells of the breast are the stem
cells capable of forming acinar cells and milk when properly stimu-
Vol. 77, No. 1 DIFFERENTIATION AND MUTATION IN NEOPLASMS 109
October 1974

lated. We examined these ducts with the electron microscope to

determine their state of differentiation and found those from a non-
involved breast of an animal bearing a tumor in another breast to
be composed of undifferentiated cells (Figure 6). The host was in-
jected with tritium-labeled thymidine, and autoradiography with the
electron microscope was performed to determine the state of differen-
tiation of the stem cells of these ducts. Ihe undifferentiated appearance
of the basilar cytoplasm and of the apical cytoplasm are illustrated
in Figures 7 and 8, respectively. They lack endoplasmic reticulum,
and Golgi complexes do not contain secretion droplets. There is little
resemblance to lactating epithelium. Compare this appearance to that
of the mammary tumor (Figure 3). The cells in the resting ducts are
comparable in degree of differentiation to the stem cells of the tumor.
The normal duct cell and the neoplastic stem cell derived from it are
each capable of differentiating into lactating epithelium.
Thus, undifferentiated cells are available in the breast to serve as
a target in carcinogenesis, and there is no need to postulate dediffer-
entiation from well-differentiated cells to account for the mechanism
of this malignancy.
But in addition to histiotypic differentiation there is another form
of differentiation going on in these cells. Illustrated in Figure 3 is
the histiotypic differentiation of mammary tissue with its various
kinds of secretion droplets. Cells that contain many viral particles
usually do not display this phenotype; rather, the viral particles asso-
ciated with a multitude of small vesicles which are illustrated in
Figure 9. Since the virus is RNA in type and made on a DNA copy
incorporated in the genome of the cell, is it not conceivable that this
differentiation is associated with a specialized product for which the
viral RNA is the messenger? The multitude of small vesicles present
could be the ultrastructural machinery attempting to support the pro-
duction of this gene product. Again, it is conceivable that a species
exists in which the genetic information that codes for the production
of ths RNA is responsible for the production of a hormone or an
enzyme. This is why I believe that it is important for us to identify
the cell of origin of viral genomes. I believe they will be normal con-
stituents of some cells, but when placed in other cells, they may
lack the essentials for normal function, upset controls and cause cancer.
It is also conceivable that as components of the normal genome they
may be repressed oncogenes, and when activated by the processes
of differentiation (carcinogenesis), cause the elaboration of gene prod-
ucts that, when fed back into the nucleus, change the control of the
110 PIERCE American Journal
of Pathology
genetic apparatus of the entire cell, leading to the malignant phenotype.
In previous seminars, I have pointed out that the genome of the
normal cell contains all of the information necessary for expression
of the malignant phenotype, and there is no need to reiterate that
here.5
The cell of origin of teratocarcinomas has been identified in elegant
studies performed by Dr. Leroy Stevens 25 and will be briefly reviewed.
Stevens has shown that teratocarcinomas take origin from primordial
germ cells in strain 129 mice. These may be considered to be the stem
cells of the species, so it is little wonder that the tumor is a caricature
of embryogenesis. The experiments were performed in the following
way: genital ridges from 12-day-old mouse embryos were transplanted
beneath the renal capsule, where they then differentiated. Half differ-
entiated into fetal testes, and 80% of these contained teratocarcinomas
by the seventh day after transplantation. If Dr. Stevens added a gene
for the congenital absence of germ cells, the incidence of terato-
carcinomas approached 0. The smallest teratocarcinoma observed 7
days after transplantation of a genital ridge was composed of 23
embryonal carcinoma cells which had close ultrastructural resemblance
to the primordial germ cells transplanted into the testes 7 days pre-
viously. Each was exceedingly undifferentiated with few membranous
proffles.-6 Subsequently, Stevens obtained teratocarcinomas by trans-
planting fertilized eggs beneath the testicular capsule.-"
Thus, it has been established that one kind of malignant cancer
takes origin in an undifferentiated stem cell, and that in the breast
there are myriads of undifferentiated cells available for carcinogenesis.
These cells are the ones charged with either maintaining the species,
in the case of teratocarcinoma, or of maintaining and replenishing
the mammary epithelium in the case of the breast. This leads to the
conclusion that the process of carcinogenesis is superimposed upon
that of cell renewal.
The process of cell renewal is conceptualized in the lower portion
of Figure 1.28 The stem cell is capable of division only, and one of its
cells undergoes degrees of maturation and divides again. At some
point the next division results in a cell that has the phenotypic char-
acterisfics of the mature tissue and after at least one more division,
gives rise to a cell that is postmitotic and senescent.
If carcinogenesis is superimposed upon this process of cell renewal,
then it should be possible to explain the occurrence of benign as well
as malignant tumors. This has been a particularly perplexing area of
oncology. If polyoma virus is injected into an animal, benign and
Vol. 77, No. 1 DIFFERENTIATION AND MUTATION IN NEOPLASMS 111
October 1974

malignant tumors develop.`9 If chemical carcinogen is painted on the

skin of an animal, benign tumors develop before malignant ones.30
This has led some to believe that benign tumors are a stage in the
development of malignant ones. I believe there are better explanations
for the phenomenon. Since benign tumors closely resemble the adult
counterpart and since the same chemical can give rise to benign or
malignant cells, it would suggest that the state of differentiation of
the targeted stem cell would determine whether or not a tumor was
benign or malignant. If undifferentiated cells capable of synthesizing
DNA were the target, malignant tumors would result; if well-differ-
entiated cells still capable of one more division were the target, then
a benign tumor would develop. This concept, that benign or malignant
tumors develop, depending upon the state of differentiation of the
target cell, is illustrated in Text-figure 1.28 The benign tumor appears
first because its social requirements are little different from those of
the normal tissue, and they are able to maximally express their growth

- MITOTICALLY ACTIVE -----POSTMITOTIC -

-~-MAIGNAT CELLS
MALIGNANT CELS ~t DIFFERENTIATED__
BENIGN CELLS

GRADE 3 OR 4
\i< I\ MALIGNANCY

I~ ~

CD' SD c / W-0
MALIGNANCY
c, C' C

El C C BENIGN
Nor mITUMOR
0;____
° u;
s_ 2 ___ TISSUE___

oE
0 ~~~TISSUE
RENEWAL

Normal Senescent
Stem Cell Differentiated Cell

TErr-Fic 1- Concept of carcinogenesis, predicting that the state of differentiation of

the cell targeted in carcinogenesis will determine whether the tumor will be benign or
malignant. (Reprinted by permission from Pierce et aP88)
112 PIERCE American Joumal
of Pathology

potential. The malignant cells are scattered among normal cells and
have different social requirements from the normal cells and are
unable to proliferate optimally. A prolonged latent period ensues.
When enough of these cells are accumulated to provide an optimal
environment, then the phenotypic expression of cancer occurs.
The concept that neoplasms develop from stem cells involved in
tissue renewal should not be confused with Conheim's proposal that
neoplasms take origin from embryonic cells that somehow escape
normal developmental influences. Normal stem cells do not escape
developmental influences, they develop and function normally until
carcinogenesis alters intercellular controls.
References
1. Pierce GB Jr, Dixon FJ Jr: Testicular teratomas. I. Demonstration of terato-
genesis by metamorphosis of multipotential cells. Cancer 12:573-583, 1959.
2. Pierce GB, Dixon FJ Jr., Verney EL: Teratocarcinogenic and tissue forming
potentials of the cell types comprising neoplastic embryoid bodies. Lab Invest
9:583-602, 1960
3. Pierce GB, Wallace C: Differentiation of malignant to benign cells. Cancer
Res 31:127-134, 1971
4. Wylie CV, Nakane PK, Pierce GB: Degrees of differentiation in nonprolif-
erating cells of mammary carcinoma. Differentiation 1: 11-20, 1973
5. Pierce GB, Johnson LD: Differentiation and cancer. In Vitro 7:140-145,
1971
6. Lajtha LG: Problems of bone marrow cell kinetics. Postgrad Med J 38:41-47,
1962
7. Becker H, Stanners CP, Kudlow JE: Control of macromolecular synthesis in
proliferating and resting Syrian hamster cels in monolayer culture. II.
Ribosome complement in resting and early G1 cells. J Cell Physiol 77:43-50,
1972
8. Riddle 0, Dunham HH: Transformation of males to intersexes by estrogen
passed from blood of ring doves to their ovarian eggs. Endocrinology 30:
959--968, 1942
9. Whittier BH, Rawles ME, Kock RC: Biological differences in the action
of synthetic male hormones on the differentiation of sex in the chick embryo.
Proc Natl Acad Sci USA 24:176-182, 1958
10. Tonkins GM, Martin DW, Stellwagen RH, Baxter JD, Mamont P, Levin-
son BB: Regulation of specific protein synthesis in encaryotic cells. Cold
Spring Harbor Symp Quant Biol 35:635-640, 1970
11. Stevens LC, Little CC: Spontaneous testicular teratomas in an inbred strain
of mice. Proc Natl Acad Sci USA 40:1080-1087, 1954
12. Kleinsmith LJ, and Pierce GB: Multipotentialitv of single embryonal
carcinoma cells. Cancer Res 24:1544-1551, 1964
13. Pierce GB: Teratocarcinoma: model for a developmental concept of cancer,
Current Topics in Developmental Biology, Vol. 2. Edited by A. A. Moscona,
A. Monroy. New York, Academic Press, Inc, 1967, pp 223-246
 Vol. 17, No. 1 DIFFERENTIATION AND MUTATION IN NEOPLASMS 113
October 1974

14. Briggs R, King TJ: Transplantation of living nuclei from blastula cells
into enucleated frogs' eggs. Proc Natl Acad Sci USA 38:455-403, 1952
15. Gurdon JB: Nuclear transplantation in amphibia and the importance of
stable nuclear changes in promoting cellular differentiation. Q Rev Biol
38:54-78, 1963
16. King TJ, McKinnell RG: An attempt to determine the developmental poten-
tialities of the cancer cell nucleus by means of tansplantation, Cell Physiology
of Neoplasia. Austin, Texas, University of Texas Press, 1960, pp 591-617
17. King TJ, DiBerardino MA: Transplantation of nuclei from the frog renal
adenocarcinoma. I. Development of tumor nuclear-transplant embryos. Ann
NY Acad Sci 126:115-126, 1965
18. Todaro GJ, Huebner RJ: The viral oncogene hypothesis: New evidence.
Proc Natl Acad Sci USA 69:1009-1015, 1972
19. Huebner RJ, Kefoff GJ, Sarma PS, Lam WT, Turner HC, Gilden RV,
Orozlan S, Meier H, Myers DD, Peters RL: Group-specific antigen expression
during embryogenesis of the genome of the C-type RNA tumor virus: impli-
cations for ontogenesis and oncogenesis. Proc Nat] Acad Sci USA 67:366-376,
1970
20. Lehman JM, Defendi V: Changes in deoxyribonucleic acid synthesis regu-
lation in Chinese hamster cells infected with Simian virus 40. J Virol 6:738-
749, 1970
21. Mendelson ML: Autoradiographic analysis of cell proliferation in spon-
taneous breast cancer of C3H mouse. HI. The growth fraction. J Natl Cancer
Inst 28:1015-1028, 1962
22. Leighton J: Spread of Cancer. New York, Academic Press, Inc, 1967
23. Foulds L: The experimental study of tumor progression: a review. Cancer
Res 14:327-39, 1954
24. Gray JM, Pierce GB: Relationship between growth rate and differentiation
of melanoma in vivo. J Natl Cancer Inst 32:1201-1210, 1964
25. Stevens LC: Origin of testicular teratomas from primordial germ cells in
mice. J Natl Cancer Inst 38:549-552, 1967
26. Pierce GB, Stevens LC, Nakane PK: Ultrastructural analysis of the early
development of teratocarcinomas. J Natl Cancer Inst 39:755-773, 1967
27. Stevens LC: The devekloment of teratomas from intratesticular grafts of
tubal mouse eggs. J Embryol Exp Morphol 20:329-341, 1968
28. Pierce GB, Nakane PK, Mazurkiewicz JE: The natural history of the
malignant stem cell Differentiation and Control of Malignancy of Tumor
Cells. The Fourth Intemational Symposium of the Princess Takamatsu Cancer
Reearch Fund, Tokyo, Japan, 1973 (In press)
29. Defendi V, Lehman J M: Biobogical characteristics of primary tumors
induced by polyoma viu in hamsters. Int J Cancer 1:525-540, 1966
30. Yamagiwa, K, Ichikawa K: Experimental study of the pathogenesis of carci-
noma. J Cancer Res 3:1-21, 1918

Acknowledgments
The author wishes to acknowledge the technical assistance of Mr. Alan Jones. Drs.
P. K. Nakane and R. H. Fennell offered valuable suggestions and criticism of the manu-
script.
114 PIERCE American Journal
of Pathology

[I& ws folow]
 2~~~~~~~~~

ida

Fig 1-Teratocarcinoma strain 129 mice. Illustrated are glands, skin, brain, primitiw con-
nectiw tissue and in the upper right, embryonal carcinoma cells (x 180). (Reprinted by
permission from Pierce and Verney, Cancer 14: 1017-1029, 1961). Fig 2-Squamous
cell carcinoma, Irish rat Note the well-differentiated squamous cell forming a pearl with a
keratotic center. Undifferentiated cancer cells surround the pearl (x 480). Fig 3-
Spontaneous mammary adenocarcinoma CsH mouse. A well-differentiated cell (grade 3) is
with secretion droplets lines the acinus. A grade 2 cell with much endoplasmic reticulum
centrally placed and two undifferentiated stem cells (grade 1) are in the upper left (X 700).
(Reprinted by permission from Wylie et at.)
... ....

4k

Fig 4-Normal mammary ductule from resting breast of CnH female (x 320). Fig 5-
Lactating mammary gland. These cells are derived from the type of ductular cells illus-
trated in Figure 4. Fig 6-Ductule from noninvolved breast of an old CMH female bearing
a spontaneous mammary adenocarcinoma. The cells appear undifferentiated, some viral
particles are present (x 4500).
 7~~~~~~~~~~~~~~~~~~

AVO,~~
~~~~~~~~~~~~~~~~~~~~~~~~~~P;. . ew

Fig 7-Autoradiogram illustrating undifferentiated appearance of basilar cytoplasm of cells

capable of synthesizing DNA in resting mammary ductules (X 26,000). Fig 8-Auto-
radiogram illustrating undifferentiated appearance of apical cytoplasm of cells capable of
snhsizing DNA in resting mammiary ductules (X 26,000).
Fig 9-Autoradiogram illustrating the smooth walled vesicles associated with mouse mam-
mary tumor virus production. Note the absence of normnal mammary phenotypes
(X 23,000).