Download Tumor Heterogeneity - Barbara Ann Karmanos Cancer Institute

[CANCER RESEARCH 44, 2259-2265,
June 1984]
Perspectives in Cancer Research
Tumor Heterogeneity
GlorÃ-aH. Heppner1
Michigan Cancer Foundation, Detroit, Michigan 48201
In 1977, Dan Dexter and I, with colleagues at Roger Williams
General Hospital, submitted a manuscript to Cancer Research in
which we reported the isolation of four distinct tumor subpopu
lations from a single, spontaneously arising mouse mammary
cancer. We speculated that these subpopulations were evidence
for intraneoplastic heterogeneity and that heterogeneity was a
general phenomenon, knowledge of which was important in the
treatment of cancer. Editorial review of this manuscript, if not
swift, was unambiguous: it was unworthy of publication in Can
cer Research. Two reasons were given: our results were not
novel; and anyway "everyone knew" that cancers are mono
clonal. The first was certainly true; previous investigators, most
notably Levan and Hauschka (48), Klein and Klein (47), Makino
(50), Henderson and Rous (34), Gray and Pierce (28), Prehn (76),
Mitelman (59), and Foulds (23, 24), had reported evidence of
multiple tumor subpopulations within single cancers. With regard
to the second objection, it seemed that the reviewers were
implying that single-cell origin somehow ruled out subsequent
variation as the population grew. I could only reflect that I too
was originally monoclonal but became undeniably heteroge
neous. Ultimately, the skeptical reviewers gave in to the force of
this simple logic (15).
At about the same time, Fidler and Kripke (22) published an
analysis of tumor heterogeneity as exemplified by metastasis.
These reports fell on fertile ground; witness that in the first 9
months of 1983, Cancer Research contained more than 20
papers dealing directly with some aspect of tumor heterogeneity.
Symposia have been held on the topic (62, 68), and a new
periodical has appeared (Invasion and Metastasis) subtitled A
Journal of Cancer Dissemination and Tumor Cell Heterogeneity.
It might seem then that our early speculations were well founded.
However, now finding myself more in demand as a reviewer than
as an author, I see a new merit in skepticism. Furthermore, I am
becoming increasingly distressed by what I see as a focus on
the difference between tumor cell subpopulations, without the
necessary parallel understanding that tumor cells share a fun
damental unity. The populations that we observe and therapeuticaiiy attack are not only contiguous in space and origin but are
also complex ecosystems with characteristics that transcend
those of their individual members. To understand how these
systems develop, it is necessary to fuse the concepts and
methods of developmental and population biology with those of
cell biology. In short, this is my perspective on research in tumor
heterogeneity.
What I Mean by "Tumor Heterogeneity"
An inevitable result of the increased use of any term that
1Supported by NIH Grants CA-27437 and CA-27419, the Concern Foundation,
the E. Walter Albachten bequest, and the United Foundation of Metropolitan Detroit.
To whom requests for reprints should be addressed, at Michigan Cancer Founda
tion, 110 East Warren Avenue, Detroit, Ml 48201.
Received December 19,1983; accepted March 2,1984.
encompasses a popular concept is a gradual blurring of its
meaning until it becomes an umbrella that covers, and perhaps
shields from close examination, phenomena that are only periph
erally related. This has been the fate of the "stem cell," and it is
happening to the term "tumor heterogeneity." Tumors are vari
able in several ways. Their characteristics change with organ
site and cell origin. Numerous host variables, such as age and
hormonal status, also introduce differences. The same cancer
can even vary in putatively similar hosts. However, this intertumor variation is not what I, and I think most investigators, mean
by tumor heterogeneity.
The types of variability usually meant by the term tumor
heterogeneity are cellular differences within a single neoplasm.
Even here, however, there is room for confusion. Tumors are
architecturally complex, differing regionally in vasculature, host
infiltrates, connective tissue components, and other character
istics which can alter the phenotype of otherwise identical cells.
Marked differences in the proliferation behavior of tumor cells
within a single cancer are commonplace. Some cells, perhaps
most, are reproductively dead; others are out of cycle; and still
others are cycling but are, at a given time, at different stages in
the process. Furthermore, many cellular phenotypes, such as
antigen expression (12, 17), membrane composition (7, 69),
response to chemotherapy (87), metastatic proclivity (84, 89), to
name a few, are themselves functions of the cell cycle. These
differences, which we might call "secular" (as distinguished from
those transmittable genetic and epigenetic differences which
determine cell lineages), may be of biological and clinical signifi
cance, but their analysis is only confused by lumping them with
lineage differences under the single term, tumor heterogeneity.
In this essay, "tumor heterogeneity" will be reserved for those
cases in which tumor cell differences are believed to be due to
differences in cell lineage, i.e., due to the presence of distinctly
different subpopulations capable of breeding true. (The extent of
temporal stability of subpopulations will be discussed below.)
This definition is not intended to discriminate between variability
that arises from genetic, as distinguished from nongenetic or
epigenetic, processes. Cells within clones are certainly not iden
tical; they too are subject to secular variation. However, this
usage allows a focused experimental analysis of at least one
type of neoplastia variability.
Evidence for Tumor Heterogeneity
Numerous reviews have appeared recently citing evidence of
subpopulations within tumors (14, 21, 36, 54, 74). Subpopulations have been isolated from cancers of every major histological
type and organ site and from both experimental and human
cancers. They have been isolated from tumors induced by chem
ical, physical, or viral agents; from long-term cell lines; and from
tumors of recent origin.
The list of characteristics
by which subpopulations
differ is
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also extensive: cellular morphology; tumor histology; karyotype
and other cytogenetic markers; growth rate; Å“il products; re
ceptors; enzymes; immunological characteristics; metastatic abil
ity; and sensitivity to therapeutic agents. In addition, since sister
cells usually remain contiguous in solid tumors, sublines tend to
be localized regionally or zoned (20, 31, 76).
There might seem to be overwhelming evidence for tumor
heterogeneity. It is important to recognize, however, that much
of the evidence is similar in kind. In the first place, cultured,
tumor-derived subpopulations are by definition obtained through
isolation; consequently, the finding that they differ is subject to
the interpretation that the differences are isolation induced. Dur
ing isolation, cells are released from variability-inhibiting in vivo
controls, and the special circumstances of cloning, i.e., separa
tion of cells and their subsequent growth, can be stimuli for
production of new phenotypes (32, 73). Secondly, the finding of
heterogeneity in situ, as by immunohistochemical methods, is
compatible with clonal heterogeneity but does not prove it, since
"secular" explanations are also possible. A third argument, that
tumors are heterogeneous because metastatic and primary tu
mors differ in cellular composition, is also not definitive but,
rather, circular, since this interpretation depends on the prior
conviction that métastasesarise from a nonrepresentative sub
set of parental cells to begin with.
It seems to me that convincing evidence for tumor heteroge
neity in nature consists of the demonstration of lineage variation
in situ, coupled with the experimental dissection of the same
variation in cloned, true-breeding subpopulations. This type of
evidence is not abundant. The usual evidence is more ambigu
ous; results from my laboratory are typical. While we have shown
that different mammary tumor karyotypes (and marker chromo
somes) can be found in subpopulations of clonal origin, we have
never demonstrated the direct descent of these phenotypically
distinct isolates from their putative parental cells. One would like
to see studies in which lineage heterogeneity is demonstrated in
fresh uncultured tumor specimens and the lineage markers are
then used as a basis for isolating and characterizing the different
cell populations. This ideal has been approached in the Shapiros'
laboratory (82) where karyotype heterogeneity was first used as
a marker for subpopulations in fresh primary human gliomas and
then was used as a reference to identify these subpopulations
in clones isolated from the same tumors. The subpopulations
differed in several parameters, including sensitivity to drugs (91)
and genetic stability (81). Karyotype differences have been de
scribed in many other kinds of tumors by conventional techniques
(59) and by flow cytometry (2), but to my knowledge the Shapi
ros' work is unique in being a prospective study, one in which
subpopulations are first identified in fresh tumors and their
existence is subsequently confirmed in culture. Changes in kar
yotype have been used to monitor tumor progression in situ (40).
Insofar as progression reflects differential reproduction of subpopulations (63), karyotype frequency changes are proof of
tumor heterogeneity. On the other hand, if differences in subpopulation generation are only assumed, this argument is also
circular.
These comments should not be taken as reflecting serious
doubt on my part that tumor heterogeneity exists (Reviewers:
take note), but I do argue that every claim for tumor heteroge
neity, or homogeneity for that matter, should be subjected to
skeptical examination and that the quality of evidence be evalu
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ated. Weakness in an experimental proof remains a weakness
whether the experiment is done once or 100 times.
Origin of Tumor Heterogeneity
As mentioned, tumor heterogeneity can be viewed as suspect
because superficially it appears at variance with the idea that
cancers arise by the transformation of a single cell (19). Setting
aside the evidence that not all tumors arise as single cells (77),
the suspicion seems unjustified when one considers that most
eukaryotic organisms begin as single cells but soon become
heterogeneous. Tumors also appear to undergo developmental
and differentiative changes, at least some of which result from
altered gene expression. Heterogeneity is not a property unique
to tumors, but one they share with other organs. Indeed, Griffen
ef al. (29) found that nonneoplastic skin fibroblasts from normal
carriers of 5<*-reductase deficiency can be cloned into subpopulations with a wide range of enzyme activity. Heterogeneity in
lineages derived from a common stem cell has been documented
in many normal cells, most thoroughly in those of the hemopoietic
system (85).
Heterogeneity is a feature of neoplastia development that can
precede the tumor itself. Normal cells differ in susceptibility to
carcinogens (6, 43), and the heterogeneity in characteristics of
different SV40-transformed clones has been shown to reflect the
heterogeneity of the normal cells from which they are derived
(67). Evidence of cellular heterogeneity has been reported in
preneoplastic hepatocytes (65) and mammary epithelial cells (1).
In addition, even cells that are not transformed by a carcinogen
are often altered physiologically by the exposure. Such carcino
gen-altered, nontransformed cells constitute an abnormal envi
ronment for the initiated cells and, as Rubin (79) has discussed,
can be shown to influence the expression of transformation.
Cellular heterogeneity must be viewed then as a feature of
both normal and precancerous tissues. It seems not unlikely that
the mechanisms that are responsible for variability under these
circumstances could also be responsible for generating tumor
heterogeneity. Studies on teratocarcinoma (71), on small cell
carcinoma of the lung (3), and on mammary carcinoma (4, 30,
80) all attest that differentiation of neoplastic "stem cells," quite
analogous to that in normal tissues, results in tumor heteroge
neity. Pierce and Cox (71) described the development of heter
ogeneity within teratocarcinoma as a "caricature of embryogenesis."
Besides "normal" mechanisms, heterogeneity may arise by
tumor specific mechanisms. Increased genetic instability is a
case in point (63). This instability leads to more errors in tumor
cell DNA (point mutations, genomic rearrangements, chromo
some losses, gene amplification, etc.) and is reflected in in
creased phenotypic variability. Evidence comes from observa
tions of karyotypic abnormalities that accompany neoplastic
progression (40) and from the work of Cifone and Fidler (11) who
found in fibrosarcoma cells that rates of spontaneous mutation
are greater in metastatic than in nonmetastatic subpopulations.
However, considerable caution is necessary when one compares
mutation rates in different cell populations. Observed mutation
frequencies depend on several factors that are difficult to control;
these include gene copy number, parental and mutant cloning
efficiencies, cell size, and cell-cell interactions. Elmore et al. (16)
used stringent criteria and could not measure a difference in
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mutation frequency between normal and transformed diploid
human skin fibroblasts. Furthermore, although one might antici
pate that aneuploidy itself would result in variant formation, and
indeed some aneuploid tumor lines are unstable (81), in our
experience aneuploid mammary lines in vivo can be highly vari
able or relatively stable (30, 57).
Recent determinations of cell variant production rates have
tended to direct attention away from mutation to extra- or
epigenetic mechanisms as the chief source of variability in tu
mors. For example, Bosslet and Schirrmacher (8), Harris et al.
(32), and Peterson et al. (70) have reported rates of 10"2 to10~5
variations/generation, which exceed by several orders of mag
nitude the rates of 10"6 to 10~8 mutations/generation usually
considered to be consistent with genetic mutation. Of course,
the high rates could reflect a specific carcinogen-induced hypermutability (25). Nor are the differences in the rates of phenotypic
variant appearance and mutation in normal versus neoplastia
cells as large, on close examination, as the above frequencies
imply. The data of Peterson ef al. (70) indicate mutation rates in
cultures of normal breast epithelial cells that are nearly as high
as those of their malignant counterparts. This suggests that
some as yet unrecognized variable (genetic or epigenetic), found
in normal and neoplastic cells, causes the high rates of pheno
typic variation. In any case, the cognoscenti have, for better or
for worse, shifted their attention in recent times to epigenetic
mechanisms as an important factor in the generation of tumor
heterogeneity.
A hypothesis that narrows the apparent differences between
genetic and epigenetic mechanisms was provided by Frost and
Kerbel (26), who suggested that DNA hypomethylation, and the
activation of otherwise repressed genes, may be the cause of
tumor variant production. In support of this view are the follow
ing: (a) hypomethylation accounts for the parallel between neo
plastic and normal tissue development since alteration in DNA
methylation plays a role in the latter (10,42); (b) hypomethylation
is consistent with the cases of inappropriate gene expression in
neoplastic progression (39); (c) methylation patterns are herita
ble, passed on from parent to daughter cells (5); (d) methylation
patterns change with high frequencies and rates reminiscent of
variant formation (26). The evidence provided by these obser
vations is circumstantial; direct evidence for the hypomethylation
hypothesis is more limited. However, Feinberg and Vogelstein
(18) have reported differential methylation of specific genes in
different cells of a colon carcinoma and an instance of progres
sive hypomethylation in a primary lung carcinoma and its (liver)
métastases. In addition, the hypomethylating agent, 5-azacytidine, has been shown to induce cell variants in several tumor
lines (26). Although suggestive, each of these studies is still
incomplete proof. Missing is convincing evidence, in the differ
ential methylation studies, that the changes observed are actually
intraclonal and, in the 5-azacytidine studies, adequate measure
ment of concentration and kinetic parameters to establish that
the action of the agent is actually attributable to hypomethylation
(specifically, hypomethylation of those genes for which putative
expression is altered).
Somatic cell fusion, another epigenetic process, has also been
suggested as a mechanism of variant production (13,44). What
ever the mechanism(s), it is subject to influences from outside
the tumor cell. For example, in some tumor systems, the rates
at which variants appear in a cell lineage are greater in vivo than
in vitro (9, 30). Furthermore, variant production is not always a
random event. Kiang ef al. (46) reported the periodic appearance
and disappearance of several characteristics during the serial
transplantation of GR mouse mammary tumors. Similarly, Vaage
(86) found that, on repeated testing, serially passaged pieces of
the same C3H mammary tumor underwent characteristic
changes at about the same time. The histories of the several
tumors passaged by Vaage were different, but among the lin
eages derived from a given tumor, the temporal relations were
identical. These examples of tumor progression, which is thought
to result from the successive production and selection of new
variants (63), suggest a degree of orderliness unexpected from
a stochastic process.
Some Complications
Reports of tumor heterogeneity have in general been well
received because they seem to explain troubling observations
and experiences. The weakness of experimental cancer research
has always been variability. Generalizations are hard to pin down.
Experiments are often difficult to reproduce. By definition, vari
ability is difficult to duplicate. In a way, the study of tumor
heterogeneity answers a prayer; ¡(reproducible results are no
longer a problem. Indeed, they become evidence, another ex
ample of tumor heterogeneity!
To the clinician, heterogeneity can be a serviceable explanation
for the failure of a therapy: "What is lacking is a drug to which
the few invasive and metastatic cells are sensitive! The remainder
of the cancer is irrelevant." I have described this approach as a
horse opera scenerio: the good tumor cells are in white hats, the
bad ones in black. We "target" our magic bullets and await the
hero's (heroine's, for a very few) reward (35).
These simple notions ignore important complications. One is
that the data which distinguish particular subpopulations as, for
example, highly metastatic or nonmetastatic, fast or slow grow
ing, drug resistant or sensitive are always obtained by studying
isolated subpopulations or clones. Cloning is a way of multiplying
almost identical components and is useful for magnifying the
individual properties of tumor cells. However, it is now clear that
tumors have a "societal" aspect which is changed or lost when
the numbers of one component are varied at the expense of
another. Study of isolated tumor subpopulations shows the
behavior of cells when cloistered like monks, not when in their
relevant tumor society.
My associates and I have studied the ability of mammary
tumor subpopulations to influence each other's growth and
behavior. We found that subpopulations behave very differently
when isolated and when growing and interacting together. Cell
interactions alter properties as diverse as growth rate (37, 52),
immunogenicity (56), sensitivity to drugs (51), and ability to
metastasize (55). Parallel findings have been reported by
Hauschka (33), Klein and Klein (47), Nowotny and Grobsman
(64), Janssen and Revesz (41), and recently by Keyner eÃ-al.
(45), Newcomb ef al. (61 ), Olsson and Ebbesen (66), Wang ef al.
(88), and Woodruff (90).
Especially interesting is the effect of subpopulation interaction
on metastatic behavior described by Poste ef al. (73) and Miner
ef al. (58). Cloned tumor cells may be unstable, changing from
highly to poorly metastatic (and vice versa) from generation to
generation. By contrast, the behavior of tumor lines formed
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G. H. Heppner
without cloning (and consequently possessing some of the di
versity of the parental tumor) is far more stable. These mixed
lines faithfully transmit a characteristic metastatic behavior
through successive generations. This stability implies either (a)
the presence in mixed populations of several lines, each mutable,
but with mutability masked by averaging or because the meta
static conversion in one line signals the reverse conversion of
another or (b) the interaction of the unlike lineages in the mixed
population in ways that suppress their intrinsic instabilities. Poste
ef al. (73) and Miner ef al. (58) provided convincing evidence for
the latter explanation by showing that donai lines, each of which
is unstable when grown in isolation, are stabilized when grown
together (either in vitro or in vivo). Not all clonal lines can be
stabilized or can stabilize, and these abilities exist only among
clones from the same tumor type, not among types of different
histological origin.
A general mechanism to explain tumor subpopulation interac
tion has not been described (38). Our group has uncovered
numerous examples of the ways that subpopulations can inter
act. Some interactions require host participation (51, 52); others
occur in vitro (37, 51). Some interactions involve a diffusable
factor (37); others require cell contact (53). What is certain,
however, is that tumor cells are similar to members of other
societies. The ways in which they interact depend upon their
potential and the circumstances in which they find themselves.
We have recently extended this picture of a tumor ecosystem to
include host cell components, lymphocytes, and macrophages.
These emigres, like the autochthonic components, influence and
are influenced by their tumor neighbors (49, 78). Interactions
complicate the study of tumor heterogeneity because, while
heterogeneity is detected by isolating cell subpopulations, the
properties of the parental tumor cannot be deduced by the simple
addition of its component parts; a purely reductionist analysis is
bound to fail. A second, related complication is cell line instability.
Several recent studies have raised doubts about the signifi
cance of tumor heterogeneity by showing that daughter lines
subcultured from variant populations behave differently than
does the parental line; that is, that tumor subpopulations are
reproductively unstable. At the outset, it should be recognized
that this is not a generalization that holds for all tumor subpopulations and clones. Phenotype stability is yet another charac
teristic by which different subpopulations vary, that is, are het
erogeneous (57, 60). The systems used to demonstrate highfrequency instability are, in general, long-term cell lines. If genetic
instability increases as a function of tumor progression (63), one
would expect such lines to represent the furthest end of the
spectrum. Nevertheless, as Stackpole (83) thoroughly docu
mented, B16 melanoma clones have unstable characteristics
with regard to metastasis, so that new lines derived from estab
lished metastatic lines are themselves not necessarily metastatic,
and nonmetastatic lines can give rise to metastatic clones. Poste
ef al. (75) have shown that growing metastatic foci, of proven
clonal origin, become heterogeneous with time. In other words,
events in the primary tumor are recapitulated in the metastasis.
Similar results using the KHT sarcoma line have already been
cited (32). The group responsible for the latter work, under the
leadership of Victor Ling, has proposed a model to explain their
observations. Their "dynamic heterogeneity" model contrasts
with models in which tumor subpopulations are thought of as
stable units, subject only to selection by the host environment.
In the dynamic heterogeneity model, although tumors are also
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considered to consist of multiple clones, the genotype of these
clones is changeable. At a given time, a particular clone may
give rise to metastatic cells, but, having done so, its progeny
may back-mutate, producing a nonmetastatic phenotype. In this
view, a tumor is a collection of unstable cells, the instability of
which is masked in the whole by mutually canceling "forward"
and "backward" mutations in the individual clones.
To my mind, the "dynamic heterogeneity" model, white ingen
ious, fails to explain critical experimental data. The model is in
fact one of the alternative hypotheses tested by Poste et al. (73)
and Miner ef al. (58) (see above). The dynamic heterogeneity
model predicts that, if several unstable lines are grown in polyclonal mixtures, each will maintain a high rate of mutation. Poste
ef al. (73) and Miner ef al. (58) found that clonal instability is
suppressed, not just masked, in polyclonal cultures. While Ling's
group (32) worked with tumor systems different from those of
Poste ef al. (73) and Miner ef al. (58), the stabilization phenom
enon has been demonstrated in several different tumor types,
so there is no reason to believe that it is restricted to a particular
system.
The observation that clonal instability is suppressed by clonal
interactions does not explain the origin of the instability. At a
certain level, instability can be seen as a continuation of the
processes discussed under "The Origin of Tumor Heterogeneity"
as well as evidence of their dynamic nature. At issue in both
cases is not the fact of change, however, but the frequency with
which it occurs. Most investigations of clonal instability have
been focused on the ability to metastasize. We may assume that
metastasis, like virtually every complex phenotypic property that
has been thoroughly studied, is not determined uniquely by a
single gene but is an "emergent" property, resulting from both
the direct and pleiotropic actions of many genes. Furthermore,
there is more to the cell than genes and chromosomes. In
somatic cell duplication, much information is presumably carried
by cytoplasmic and membrane components, and because of the
parallel binary nature of replication and cell duplication, extranuclear transfer can resemble the segregational information transfer
of the genotype. Depending upon the nature of the informationbearing material (e.g., mRNA, organelles), extranuclear informa
tion may remain an influence for varying periods of time and
through varying numbers of cell divisions. Insofar as such infor
mation influences gene penetrance, the variation in its transfer
properties may suggest an instability in the genome itself. The
problem that confronts us is, "What fills the enormous gulf that
exists between the genotype and the phenotype of a cell?"
Rather than implying that tumor heterogeneity, as defined
here, does not exist, the apparent instability of the metastatic
phenotype may bear witness to its extent and significance.
Metastasis is considered to be a process in which only some of
the cells in the primary tumor can engage and, as a complex
process, it depends on a constellation of characteristics each of
which can be transmitted to daughter cells and is required for
maintenance of the metastatic phenotype. Change in any of the
determinants of the process will lead to a change in metastatic
potential. If the determinants are genes (by no means a necessity,
as emphasized above), mutation at any of the several loci (per
haps dozens) will appear phenotypically as a change in meta
static potential. Obviously, under these circumstances, mutation
rate deduced from phenotypic change would appear far higher
than the true rate experienced by each gene. When pleiotropy is
present, one cannot expect to obtain meaningful mutation rates
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from observation of phenotypic change. Without detailed knowl
edge of both the genetic and extragenetic mechanisms that
underlie a particular change in phenoty pe, no rational basis exists
for choosing a class of mechanism with a frequency of change
that can be taken as a norm. Certainly, when the complexity of
change in cellular systems is considered, it is logically inappro
priate to adopt point mutation frequency as a standard of com
parison, as is conventional, especially since point mutation may
yet prove to be among the rarest sources of cell variability.
A multigenic basis for the metastatic phenotype has other
experimental consequences. Consider a single cell populating a
new metastatic locus and duplicating (the argument does not
change in cases when a few cells are involved). Early in the
colonization process, the population will be homogeneous with
respect to the metastatic phenotype. However, because several
"determining" genes are at risk of mutation, there will be, in a
short time, an accrual of nonmetastatic cells which cloning will
divulge. Thus, it should be no wonder that clones of cells from
metastatic foci may be no more capable of metastasis than cells
from their parent tumor. Both are mixed cell populations. The
development of a tumor is a complex process, characterized by
the early appearance of cell line heterogeneity, constant produc
tion of new cell lines, and evolving cell line interdependence.
Although these processes might be modified by the experimen
talist, none of them stops when he enters the picture.
In addition to the problem of pleiotropy, there are other inherent
difficulties to a meaningful assessment of cellular variability.
Definition of the extent of variability depends upon the number
of measurable characteristics and the precision with which the
measurements can be made. It is likely that the number of
different subpopulations in a tumor exceeds the number of
measurable phenotypes. Furthermore, some characteristics,
such as metastatic ability, do not lend themselves to stringent
quantitative analysis. Under these circumstances, the distinction
between experimental variability and phenotypic instability is
clouded. Other characteristics, such as sensitivity to drugs or
expression of a particular marker protein, may be capable of
precise measurement in any particular experiment, but investi
gators have come to accept a degree of variability between
experiments as the norm. Tumor heterogeneity, then, is always
going to be a "working definition," and a minimal one at that.
The reality may be that the mosaic of characteristics
isolated cell is different from that of every other.
of every
tem in which evolution through natural (and artificial) selection
might take place and, at least superficially, tumor progression
resembles evolution. However, change is not always evolution.
Paradoxically, even though variability is necessary to evolution,
the great heterogeneity within tumors can be interpreted as
suggesting that selection is not a major determinant in the
population biology of cancer. Presence of strong selective forces
leads to homogeneous, not heterogeneous, populations, and on
this basis one may be led to discount the role of selection in
tumor progression. Before doing so, however, it may be valuable
to look to organismal evolution for arguments that, by analogy,
may be applicable to cancer biology.
Instrumental in my own thinking on these problems is the work
of Wright (92), especially his shifting balance theory of the
evolution of species. A feature of this theory is its emphasis on
the multifaceted processes that stand between genotype and
phenotype, such that each phenotype is determined by many
genes and each gene has numerous pleiotropic effects. The
result is that evolution is determined by "natural selection
among interactive systems." Wright argues that, in complex
organisms which have thousands of genetic loci, among which
there are numerous opportunities for interactions that favor
survival, the observed phenotypes will be variable and will tend
"to wander continually" because of accidents of sampling, selec
tive migration, changes in the environment, etc. This "microevolution" allows for relatively rapid population adjustment as com
pared to the rates to be expected were selection operating on a
simple one gene-one phenotype organism. More rapid "macroevolution" occurs when a population encounters the opportunity
for expansion offered by new niches in previously uninhabited
territory, after surviving a catastrophe that has eliminated other
species or when reaching, through microevolution, a new adap
tive level that opens up previously nonexistent niches.
The pleiotropy of the "malignant" genotype, the inherent vari
ability of neoplastic populations, the interactions among tumor
subpopulations, and the opportunity for niches in the cancer cell
environment all suggest that a Wright-like "shifting balance"
approach may be useful in understanding the temporal devel
opment of tumors. As examples, one might begin by considering
the dynamics of cell cloning or of tumor repopulation after
chemotherapy as finding their analogues in the macroevolutionary thrust provided by expansion into new territory or survival of
a catastrophe.
Tumor Heterogeneity and Population Biology
Conclusions
The importance of multigenic determinants and pleiotropy,
discussed above, is in the implication that tumor heterogeneity
is not only extensive but also the "natural" condition of these cell
Understanding of the nature of tumor heterogeneity has under
gone change in the last 5 years. From early attempts to dem
onstrate tumor subpopulations, we have progressed to studying
their biological significance. It is clear that our first ideas about
tumor heterogeneity were too simple. As isolation of tumor
subpopulations became more routine, the phenotypic differences
between them have come to seem less important. We began as
tumor taxonomists seeking to classify the characteristics and
origin of different neoplastic populations and now are develop
mental biologists studying their ontogeny, structure, interactions,
and collective behavior. In this maturing view, a particular isolated
tumor subpopulation is unimportant except as a reminder of the
diversity of the cell society from which it came. Recognition of
tumor heterogeneity is essential to any theory of neoplastic
development, as well as to experimental design and clinical
populations. The variable cancer cell population appears to be a
likely object upon which selective forces might act. The environ
ment of a cancer is one in which selective forces are present.
There are, for example, regional differences in oxygen supply,
acidity, nutrient supply, and the presence or absence of immunocompetent infiltrates which give growth advantage to some,
but not other, cells within their spheres of influence. Likewise,
different organs also offer different microenvironments, and
these will be advantageous to certain metastatic tumor variants.
There are also powerful negative selection forces provided by
the therapeutic efforts of the clinician. The combination of transmittable variability and environmental diversity suggests a sys
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G. H. Heppner
treatment. Tumor societies are highly adapted for survival. They
survive natural and artificial (therapeutic) selection through het
erogeneity by producing new variants to "outflank" it and by
utilizing subpopulation interactions to counteract its destructive
influence (52). Goldie and Goldman (27) have analyzed the ther
apeutic implications of variant production, showing that early
and combined therapy is required to defeat its protective effects.
Cellular interaction also offers opportunity for intervention (36).
Having recognized their complexity, we must now learn to anni
hilate tumor societies.
Acknowledgments
I gratefully acknowledge the influence of all my colleagues, particularly Drs. Dan
Dexter, Fred Miller, and Bonnie Miller, on the development of the ideas presented
here. I also am grateful to Dr. Sam Horowitz for his generous and critical help in
writing this perspective and to Pat Piiion for her cheerful and speedy typing of all
its numerous versions.
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