Artigo 02 - cancer stem cells.pdf

8/15/2019 Artigo 02 - cancer stem cells.pdf

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Die Hard: Are Cancer Stem Cells the Bruce Willises ofTumor Biology?

Ákos Fábián,1 Márk Barok,1 György Vereb,1* János Szöllo†si1,2*

AbstractIn recent years, an exponentially growing number of studies have focused on identify-ing cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial incontrolling and curing cancer: through asymmetric division CSCs supposedly drive tu-mor growth and evade therapy with the help of traits shared with normal stem cells

such as quiescence, self-renewal ability, and multidrug resistance pump activity. Here,we give a brief overview of techniques used to confirm the stem cell-like behavior of pu-tative CSCs and discuss markers and methods for identifying, isolating, and culturingthem. We touch on the limitations of each marker and why the combined use of CSCmarkers, in vitro and in vivo assays may still fail to identify all relevant CSC popula-tions. Finally, the various experimental findings supporting and contradicting the CSChypothesis are summarized. The large number of tumor types thus far with a subpopu-lation of uniquely tumorigenic and therapy resistant cells suggests that despite theunanswered questions and inconsistencies, the CSC hypothesis has a legitimate role toplay in tumor biology. At the same time, experimental evidence supporting the estab-lished alternative theory of clonal evolution can be found as well. Therefore, a modelthat describes cancer initiation and progression should combine elements of clonal evo-lution and CSC theory. ' 2008 International Society for Advancement of Cytometry

Key terms

cancer stem cells; progenitor cells; stem cell markers; clonal evolution; therapy resist-ance

THE cancer stem cell hypothesis in its most accepted form states that tumorousgrowth is sustained by only a portion of the tumor cells. These cells uniquely possess

the self-renewal and differentiation capabilities of normal tissue stem cells and are

hence referred to as cancer stem cells (CSC) (1,2). According to the CSC hypothesis

all cancer cells are derived from the multipotent CSCs. These, through asymmetric

division give rise to progenitor cells, which are fast cycling and have limited self-

renewal and differentiation capacity. Mirroring the hierarchical structure of normal

tissues, the daughter cells of progenitors then differentiate to form the heterogeneous

tumor mass. As CSCs would be the only cells attributed with long-term self-renewal

potential, they are the ones supposedly responsible for the occurrence of distant me-

tastases and also for recurrence of malignant disease after initial successful treatment.

A further implication in therapy resistance is that CSCs are thought to possess the

defense mechanisms of normal stem cells, such as multidrug resistance (MDR) pump

activity (3) and by virtue of their stem cell nature are slow cycling, rendering them

immune to traditional chemotherapy targeting fast cycling tumor cells (2).

The cancer stem cell hypothesis witnessed a revival in the last 15 years [for a

detailed timeline, see Ref. (4)], mostly fueled by the recognition that tumor cells are

not equal with respect to their ability to regrow their tumors of origin when trans-

planted into immune compromised mice. It was first shown for acute myeloid leuke-

mia (AML) that significantly fewer malignant cells with a specific cell surface phenotype

1Department of Biophysics and Cell

Biology, Research Center for Molecular

Medicine, Medical and Health Science

Center, University of Debrecen, Hungary

2Cell Biology and Signaling Research

Group of the Hungarian Academy of

Sciences, Research Center for

Molecular Medicine, Medical and Health

Science Center, University of Debrecen,

Hungary

Received 22 October 2008; Accepted 5

November 2008

Grant sponsor: Hungarian National

Research Fund; Grant numbers: OTKA

K68763, K62648, K75752; Grant sponsor:

European Community; Grant numbers: EU

FP6 LSHB-CT-2004-503467, EU FP6 LSHC-

CT-2005-018914, EU FP6 MCRTN-CT-

035946-2, EU FP6 MRTN-CT-2005-019481

*Correspondence to: János Szöl lo†si or

György Vereb, Department of

Biophysics and Cell Biology, Faculty of

Medicine, Medical and Health Science

Center, University of Debrecen, P.O.Box

39, Nagyerdei krt. 98, Debrecen H-4012,

Hungary

Email: [email protected] or [email protected]

Published online 2 December 2008 in

Wiley InterScience (www.interscience.

wiley.com)

DOI: 10.1002/cyto.a.20690

© 2008 International Society forAdvancement of Cytometry

Review Article

Cytometry Part A 75A: 6774, 2009


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are required for transferring leukemia to NOD/SCID (non-

obese diabetic/severe combined immune deficient) mice than

when injecting unsorted cells (5). Interestingly, the only sub-

population of cells capable of initiating the disease in recipi-

ents had a surface marker signature (CD3411CD382) closely

resembling that of normal hematopoietic stem cells (HSC)

(5,6). Since ground breaking work with hematologic malig-

nancies, putative cancer stem cells have been identified in a

plethora of solid tumors, including breast (7), brain (8), lung

(9), prostate (10), colon (11,12), liver (13,14), ovarian (15,16),

head and neck (17), melanoma (18,19), and pancreas (20)

cancers.

Stem cells are found in most adult tissues (21–27) and

might contribute to tissue repair as well as play a role in

malignancies (28). The CSC concept originally stated that

CSCs are mutated versions of these tissue-derived stem cells.

Stem cells seemed a natural source of cancer initiating cells

because they persist for a long time and thus are more likely to

accumulate the genetic changes required for malignant trans-

formation (1,29). The active self-renewal pathway also means

that they require fewer such mutations than differentiated cells

(1), which originally were believed to require at least six muta-

tions to acquire all properties of cancer cells (30). However,

later on it has been shown that retroviral insertion of just three

genes is enough to convert human mammary epithelial cells

(HMEC) to cancer cells (31) and four specific genetic modifi-

cations are sufficient to reprogram diploid human fibroblasts

and convert them to embryonic stem cells (32). Ectopic over-

expression of Wnt-1 in HMECs results in oncogenic transfor-

mation by eliciting a DNA damage response that leads to func-

tional inactivation of p53 and loss of the G1/S checkpoint

(33). Although a further mutation is required to increase

Notch signaling, in this setting essentially one initial mutation

is sufficient for a differentiated human cell to become a tu-

mor-forming cancer cell. These findings further support that

CSCs indeed need not be of stem cell origin. Instead, the CSC

phenotype is most likely an ‘‘All roads lead to Rome’’-type

endpoint of malignant transformation, where irrespective of

the cell of origin, certain common pathways have to be active/

deregulated for self-renewal and tumor growth (Fig. 1).

IDENTIFYING CSCSNo general protocol has been established to confirm pu-

tative CSCs as such, but any study’s aim is to prove the main

Figure 1. The role of CSCs in tumor biology. The CSC phenotype could be a common phenotype acquired by normal tissue stem, progeni-tor or differentiated cells through mutations, which activate/deregulate certain signaling pathways, with the key changes depending onthe cell of origin (1–3). The CSC then fosters progeny that follow an aberrant differentiation pathway. As the cancer progresses any cell

may undergo clonal evolution and thus, influence tumor behavior and potentially mask or disrupt the underlying hierarchical organizationof the cancer cells.

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68 Cancer Stem Cells


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identifying criteria of CSCs—self-renewal and the capacity to

give rise to the heterogeneous lineages that comprise the origi-

nal tumor (2). CSCs should also have a phenotype that is con-

sistently unique to the self-renewing fraction of cells and

allows separation from the rest of the tumor cells.

In vitro assays for confirming self-renewal include serial

colony forming unit (CFU) assays and propagation as tumor-

spheres (34) in stem cell culturing conditions (Table 1) (2). In

the case of culturing tumorspheres, verification is needed that

sphere formation is a result of clonal growth (35) and not sec-ondary aggregation of cells (36). Injection of spheres into

immune compromised mice can confirm whether the cultured

cells also possess the tumor-forming potential required of

CSCs. As stem cell culturing conditions might influence the

biological behavior of cells (37), the cells corresponding to the

surface marker phenotype of sphere-forming cells ideally need

to be isolated from the original tumors and injected without

prior culturing. Differentiation capacity of spheres can be

examined by culturing in differentiating medium and obser-

ving morphological and expression pattern changes, most

notably loss of CSC markers and ‘‘stemness’’-associated gene

expression patterns (23). Asymmetric division can be con-

firmed by culturing separated CSCs and identifying non-CSC

phenotype cells among the progeny. In vitro proliferation and

invasion assays also provide valuable details about the proper-

ties of investigated CSC and non-CSC populations, but on

their own do not predict in vivo behavior (38).

Self-renewal, tumor propagation, and multilineage differ-

entiation can be demonstrated in vivo by xenotransplantation

into immune compromised mice. The putative CSC popula-

tion, the only cell type capable of propagating the tumor

according to CSC theory, gives rise to tumors from fewer cells

than bulk tumor or non-CSC populations. Self-renewal allows

the tumors to be serially transplanted (also known as in vivo

passaging), thus CSCs isolated from secondary tumors are

able to form tumors when injected into mice. As a conse-

quence of multilineage differentiation, the original histological

phenotype of the parent tumor is maintained and recreated in

all tumors grown from CSCs during serial transplantation.

Special care must be taken to assure before inoculation that

differences in tumor-forming capabilities of CSC and non-

CSC populations are not due to differences in cell cycle, viabil-

ity after cell separation or fraction of tumor cells relative to all

injected cells. Verifying viability of cells after injection canprove that non-CSCs engraft and survive in the mouse envir-

onment, but are unable to form tumors on their own (8,12).

WHAT TO LOOK FORThe list of potential markers of CSCs is long (Table 2).

Thus far, CD133 (3,8,10–15,39–43), CD44 (10,16,17,44,45),

CD24 (in combination with CD44) (7,20,35,38), efflux of

Hoechst or Rhodamine dyes [also referred to as Side Popula-

tion (SP)] (3,46–49), CD90 (3,50), CD117 (3,16), CD34 (5,6),

CD20 (18), and aldehyde dehydrogenase (ALDH) (51,52) have

all been used to identify putative CSCs in one or multiple tu-

mor types [a comprehensive overview of normal tissue stem

cell and CSC markers can be found in Ref. (53)]. However,

these markers have certain limitations; most notably they fail

to identify all CSCs (marker negative cells can also have

tumorigenic and clonogenic properties), and merely designate

a subpopulation that is enriched for clonogenic and tumori-

genic activity (54). Also, not all cells with a CSC marker phe-

notype behave as CSCs. Most markers for separating CSCs

were chosen due to their expression on normal stem cells of

certain tissues. A recent study in a mouse model of H. pylori -

induced gastric tumors found that infection of gastric mucosa

induced migration of nonresident, bone marrow derived cells

to the gastric epithelium (55). The neoplastic lesions that

Table 1. Culturing conditions for tumorsphere formation

TUMOR TYPE MEDIUM SUPPLEMENTS CULTURED IN REF.

Breast Serum-free DMEM-12 bFGF, EGF, insulin, BSA 96-well culture dish (35)

Breast RPMI 1640 Glutamine, 10% FCS Low-binding plates (61)

Colon DMEM-F12 EGF, FGF-2, glucose, glutamine, insulin,

progesterone, putrescin, sodiumselenite, apotransferrin, Hepes, heparin,

BSA

NA (42)

Colon DMEM 10% Fetal bovine serum 24 well plate (60)

Colon Serum-free medium EGF, FGF-2 NA (11)

Pancreatic NS-A basal serum-free

medium

EGF, FGF-2, glucose glutamine, insulin,

progesterone, putrescin, sodium

selenite, transferin

Hanging drops (40)

Melanoma Mouse embryonic fibroblast

Conditioned human

embryonic stem cell medium

bFGF Noncoated flasks (18)

Ovarian Serum-free DMEM-F12 bFGF, EGF, insulin, BSA Ultra low attachment plates (16)

bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; BSA, bovine serum albumin; FCS, fetal calf serum; NA, notavailable.

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formed were of the same origin. This indicates that the origi-

nating cell of a malignancy must not necessarily be from the

site of formation and therefore might have a different expres-

sion profile from the resident stem cells.Another question that remains to be answered is how reli-

ably the CSCs are defined by these markers. For instance, in an

experiment in which mammospheres were cultured from

mouse mammary gland cells, cells from fresh tissue dissocia-

tions that could form spheres were CD241, but 6 days of

preculturing led to mammosphere formation from only the

CD242 subpopulation (36). This indicates that the cell popula-

tion exhibiting stem cell properties may well depend on the

conditions under which the tumors grow (56,57) and the envi-

ronmental influences they encounter during separation and

culture. As obtaining single cell suspensions from solid tumors

often involves mechanical and enzymatic disaggregation lasting

hours, the possibility of altering surface marker expressionprofiles has to be taken into consideration. In our own trials,

incubation with digesting enzymes (Collagenase I, Dispase)

substantially downregulated the expression of CD44 even after

1 h (unpublished observation). Therefore, immunohistochem-

istry or fluorescent labeling of both the original tumor tissue

and the isolated cells is necessary to confirm that surface

expression patterns are not an artifact of cancer cell isolation.

The unreliability of the side population to identify CSCs

has been discussed extensively, with the conclusions that nei-

ther do all tumors or cell lines have a consistently identifiable

SP (48) nor are all CSCs necessarily within the SP when there

is one (2,4). Also, the potential cytotoxicity of retained

Hoechst inside the cell is of concern. The marker CD133 has

been used in CSC experiments to identify CSCs in brain, liver,

colon, prostate, and ovarian cancer. Recent papers have shown

that expression of CD133 can be epigenetically regulated

(41,58) and that CD133 expression may be associated with cell

cycle phase (59), although this was previously not seen (15).

Another group found that contrary to previous experiments,

where CD1331 cells were a rare, CSC-enriched population in

colon cancer, basically all colon cancer cells from primary

tumors they studied were CD1331 (60). In the same study,

only a portion of cells from liver metastasis were CD1331, but

both CD1331 and CD1332 cells were capable of tumorsphere

formation in vitro and tumor initiation in vivo. Most interest-

ingly, CD1332 cells had a CD441CD242 phenotype, whereas

CD1331 cells presented a CD44low CD241 phenotype. Upon

investigating primary glioblastomas, Beier et al. demonstratedthat in roughly 25% of tumors the CSC fraction was in the

CD1332 population (43). These cells were capable of asym-

metric division, sphere formation and had similar in vivo

tumorigenicity as the CD1331 CSCs from the other 75%

of examined tumors. In another study of cell lines derived

from BRCA1 deficient breast tumors in mice, CD1331 and

CD441CD242 phenotypes were two non-overlapping popula-

tions that were both enriched for CSCs (61). These experi-

ments suggest that depending on the tumor of origin the

CSCs might be within different phenotypic subpopulations

and that more of these subpopulations can coexist.

CD44 is unique in the list of markers, because it has been

shown to play an active role in tumorigenesis and xenograftformation (62). In experiments conducted with hepatocellular

carcinoma (HCC) cells, putative CD901 HCC CSCs were also

CD441 (50). Treatment of these cells with an anti-CD44 anti-

body induced apoptosis in a dose-dependent manner. In a

strain of intestinal tumor prone mice, CD44 knockouts had a

reduced incidence rate of adenoma, probably by regulating the

amount of DNA damage that the cell attempts to repair before

initiating apoptosis (63). Finally, in a mouse model of chronic

myeloid leukemia (CML), BCR-ABL-1 positive progenitors

required CD44 for efficient bone marrow homing (although

B-cell acute lymphocytic leukemia (B-ALL) initiating cells

with the same mutation did not) (64).

The isolation of CSCs can prove a challenge, even when

working with verified, stably expressed CSC markers. Because

of the relatively small percentage of CSCs in the tumor, large

numbers of bulk cells have to be investigated to acquire

enough CSCs for an experiment. At the same time non-CSC

populations have to be of high purity, since even a small per-

centage of contaminating highly tumorigenic CSCs can dra-

matically influence the results of xenotransplantation experi-

ments. The diversity of investigated malignancies and markers

means that for each sample and source tissue, the appropriate

isolation, labeling and gating strategy has to be optimized

individually and must incorporate the use of proper positive

Table 2. Commonly used markers of CSCs

CSC MARKER ASSOCIATED TUMOR TYPE(S)

CD133 Brain (8,39,43), colon (11,12,42), liver (13,14), lung (3), ovarian (15,41), pancreatic (40), prostate (10)

CD44 Colorectal (44,45), head and neck squamous cell carcinoma (17), liver (50), ovarian (16), prostate (10)

CD24a (with CD44

coexpression)

Breast (7,35,38), pancreatic (20)

Side population Brain (48), breast (48), lung (3), ovarian (46) prostate (48), thyroid (47)

CD90 Breast (3), liver (50), lung (3)

CD34 AML (5,6), lung (9)

CD117 Lung (3), ovarian (16)

CD20 Melanoma (18)

ALDH Breast (52), liver (51)

a In breast tumors CD242, whereas in pancreatic tumors CD241 phenotype is associated with CSCs.

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and negative controls (25,65). For examples of a prudent

approach please refer to figures 1 and 2 of Ref. (25) in this

issue.

INCONSISTENCIES OF THE CSC HYPOTHESISXenotransplantation models, in particular those used for

CSC experiments have received a lot of criticism. Most of

these revolve around the fact that the mouse is a foreign envir-

onment for human cancer cells and therefore, the full tumori-

genetic potential that would be seen with stromal elements

and cytokine signaling more closely resembling human tissues

cannot be revealed. Indeed, in several studies involving con-

genic transplantation into mice, the required number of tu-

mor cells drastically drops as compared to xenografting

(61,66) and transplantability of the malignancy is no longer

confined to a subset of cells (66). The notion that the mouse

environment is somehow selecting which cells can propagate

is further reinforced by findings that Matrigel, co-injected nor-

mal stromal cells or irradiated cancer cells (feeder cells) all

reduce the number of tumor cells needed for reproducible tu-mor growth (31,67). These findings have prompted the use of

a more careful nomenclature, in which the CSCs are referred

to as tumor initiating cells (TIC). This highlights their most

prominent biological feature and at the same time acknowl-

edges tumor initiation may be more characteristic of the host-

graft interaction than the cancer cells themselves.

Another phenomenon the CSC hypothesis has trouble

explaining is why so many bulk cells are needed for tumor for-

mation. More specifically, if a nonselected tumor cell popula-

tion is implanted, the CSC content of the minimum cell dose

for tumor growth is usually 10 times that required when the

same CSCs are used after separation from the bulk. One expla-

nation for this was that somehow the non-CSC population isnegatively regulating the growth of the CSCs (12,68). It is,

however, unclear how injecting more cells overcomes this inhi-

bition, as the relative proportion of CSCs and non-CSCs does

not change with the number of cells injected and an increase

of non-CSCs in the finite volume of a mouse should result in

higher concentrations of hypothetical inhibitory substances.

Along another line of thought, Hill proposed that the small

number of CSCs injected may not provoke an immune

response (56), whereas intermediate numbers from bulk tu-

mor would lead to a tumor rejecting immune response and

high numbers of bulk tumor cells could overwhelm the

immune system and form tumors. This scenario fails to

explain how the growing tumors from CSCs evade rejection

(the extracellular matrix synthesized by the tumor cells may

play a role (69)) and why co-injection of feeder cells (essen-

tially increasing immunogenicity without increasing the num-

ber of cells capable of proliferation) reduces the number of tu-

mor cells required. It is also contradicted by findings of

engrafted, but not proliferating non-CSC tumor cells at the

injection site (8,12).

The enhancing effects of Matrigel and co-injected feeder

cells on tumor growth in the mouse model indicate that can-

cer cells may be lacking some extracellular signals required for

effective proliferation. Hill has proposed that cancer cells capa-

ble of tumor formation after xenotransplantation may possess

an autocrine growth loop (54). A recent study showed that in

colon cancer signaling through IL-4 has a significant role in

tumor growth (42). The ability of CSCs to resist cell death is

mediated by IL-4 and IL-4 is produced by the colon cancer

cells. Breast, thyroid, and lung cancers also produce IL-4 (70).

Another study with melanomas suggested that CSCs might

drive the proliferation of non-CSCs (19). Calculations by Kern

and Shibata imply that some, if not most of the tumorigenic

potential of a tumor must lie in the non-CSC fraction (68).

The discrepancies in the number of cells needed for tumor

growth can be explained if we suppose that non-CSCs have

tumorigenic potential as well, however, they have a higher

threshold for tumor initiating signaling and/or lack effective

autocrine signaling. This higher threshold becomes more

accentuated in mice where normal environmental cues are

sparse and therefore they are dependent on signaling from

other sources. Tumors from bulk cells then can only form

when a critical level of stimulation is provided by the auto/

paracrine signaling of the co-injected CSCs. The non-CSCs atthe same time might essentially act in the way a regular stem

cell niche would and negatively regulate the proliferation of

CSCs.

Serial transplantations have revealed significant genetic

instability in tumors originating from CSCs (39,71,72). One

clear manifestation of this phenomenon is that tumors

become more aggressive with in vivo passaging, with earlier

tumor presentation and faster tumor growth rates

(11,40,71,72). As the number of injected CSCs does not

change, the CSCs re-injected during serial transplantation

must gain additional traits, which the original CSCs from the

primary tumors did not have. The logical conclusion is that

CSCs themselves are not genetically stable over time, whichallows them to evolve and adapt to the mouse environment.

RETHINKING THE CONCEPT OF CSCSDespite the obvious shortcomings of the CSC hypothesis

(Table 3), several lines of experimental evidence suggest that

CSCs might have a crucial role in tumor biology. Several stu-

dies identified cancers that can be clonally initiated and sus-

tained (44,73,74). Disruption of a single signaling pathway has

resulted in malignant transformation (73,75) and was shown

to change the behavior of progenitors and stem cells (73,76).

Asymmetric division of CSCs also explains why cells with cap-

abilities of the putative CSC population do not become the

dominant cell type in tumors, although it can be argued that

CSCs only have a selectional advantage over other tumor cells

under very harsh conditions, such as chemotherapy or xeno-

translpantation.

The methods used to identify putative CSCs are by no

means perfect. However, tumor subpopulations that have rela-

tively low stromal dependence when compared with other tu-

mor cells were readily identified and exhibited more efficient

tumor-forming in mice. They are more therapy resistant in

vitro (16,41,42,61,77) and in vivo (78), effectively increasing

their relative numbers after cytoreductive therapy (40,42,44).

Why CSCs have an intrinsic therapy resistance even in pre-

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viously untreated tumors is hard to explain with clonal evolu-

tion, since no prior selective pressure was applied to produce

this phenotype. Although clonal evolution would allow this

phenotype to occur as a byproduct of constant mutation, it is

unclear why it would be so consistently found as a small frac-

tion of so many tumors.

When designing therapy, it should always be considered

that one aspect of therapy resistance of CSCs is their quiescent

nature. Selective targeting of putative CSCs to reduce tumor

growth and resistance to chemotherapy has proved promising.Antibody targeting of ABCB5 on melanoma cells significantly

reduced tumor growth and tumor formation rate (19). Phar-

macological inhibition of CXCR4 in pancreatic cancer signifi-

cantly reduced tumor metastasis in xenografted mice (40). Yil-

maz et al induced leukemia in mice by deletion of PTEN (73).

Administration of rapamycin—an inhibitor of mTOR in the

PI3K downstream pathway (which is normally negatively

regulated by PTEN)—blocked leukemogenesis, and prolonged

survival of mice with established leukemia. Rapamycin also

restored long-term self-renewal capability of hematopoietic

stem cells with the PTEN deletion. Both in vitro and in vivo

chemotherapy resistance of colon CSCs was reduced by anti-

IL-4 antibody treatment (42). Bauerschmitz et al. identified

tumor specific promoters in CD441CD242 breast tumor cells

(79). By targeting oncolytic viruses to these promoters they

achieved significant in vitro and in vivo killing of the putative

breast CSCs and reduced the size of xenograft tumors. Though

these targeting strategies still require refinement, they also

show that specific knowledge on the particular molecular mar-

kers and/or signaling profile of a given person’s cancer stem

cells may aid in the design of individualized and effective ther-

apy. The specific targeting of CSCs in this setting could help

eradicate a cancer cell subpopulation capable of evading tradi-

tional therapy and so increase disease free survival.

The theories proposed among others by Campbell and

Polyak (80) and Adams and Strasser (81) have tried to unify

the competing models of clonal evolution and CSCs. Accord-

ing to these, evidence can be found for both models and their

prevalence is probably unique to every tumor and may actu-

ally change as the tumor progresses. Although CSCs seem to

be a special subset of tumor cells, newer studies show that

CSCs in themselves are still a heterogeneous population with

different biological properties and that multiple populations

with CSC characteristics can coexist in the same tumor. Maet al. investigated HCC cell lines and were able to separate sub-

populations with different tumorigenic potential based on

CD133 and ALDH expression (51), which however contradicts

the original CSC hypothesis of just one population with tu-

mor-forming capabilities. Herman et al found that

CD1331CXCR42 and CD1331CXCR41 pancreatic cancer

cells do not differ in tumorigenicity, but only the

CD1331CXCR41 population migrates and metastasizes (40).

Work with AML found leukemia-inducing cells to be hetero-

geneous in self-renewal potential (74). In lung cancers, several

subpopulations with different profiles of CSC marker expres-

sion can be identified (3). The CSC hypothesis explains this

heterogeneity with the existence of cancer progenitor cells,

which still possess some residual stem cell traits. It has been

proposed that tumor grade depends on the relative proportion

of progenitors within the tumor. Accordingly, dedifferentia-

tion and aggressiveness may reflect expansion or increased

self-renewal of the fast cycling progenitor population (3).

Whether the heterogeneity of the CSC population is

caused by clonal evolution or partial differentiation of the can-

cer initiating cell is probably more a question of faith at this

time, than an evidence-backed scientific decision. To further

obscure the picture, recent findings indicate that clonal diver-

sity is beneficial for tumor progression (82). It is also becom-

Table 3. Evidence supporting and contradicting the CSC hypothesis

EXPERIMENTAL EVIDENCE

Supporting the CSC hypothesis Contradicting the CSC hypothesis

Large number of cells are needed for xenotransplantation

of tumors

In congenic transplantations substantially fewer cells are needed

The required number of putative CSCs for

xenotransplantation of tumors is relatively small

Xenograft tumors can be serially transplanted, but only

with the CSC subpopulation

Transplantability of malignancies is not restricted to one

subpopulation in congenic transplantations

Non-CSC populations do not initiate tumor growth in

vivo, or require more cells than the CSC population to

do so

A small fraction of tumors cells are capable of sustained

growth under stem cell culturing conditions

CSC markers do not identify a pure CSC population

CSCs have higher clonogenicity in vitro The CSC population is heterogeneous in itself, with differences in

metastatic and tumorigenic potential

Cultured CSCs can give rise to progeny with non-CSC

phenotypesCSCs have intrinsic in vitro and in vivo therapy resistance

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ing clear that several properties, which we thought were

intrinsic to CSCs are modulated by the microenvironment of

the cancer cells (83), and such key traits as metastasizing (84)

and growth (85) may depend on the normal stromal cells that

interact with the cancer cells (86).

CONCLUSIONSThe CSC hypothesis has come a long way since its first

inception. Putative CSCs have been identified in many solid

tumors. Though the methods to identify CSCs have their

uncertainties, the isolated CSCs are a consistently tumorigenic

and therapy-resistant subpopulation of tumor cells. Studies

have proved again and again that putative CSCs are capable of

self-renewal and multilineage differentiation. Selective target-

ing of CSCs seems to be an effective strategy to reduce therapy

resistance and growth of xenograft tumors. However, the CSC

hypothesis faces new challenges. Recent findings indicate that

CSCs themselves may be a heterogeneous population, with

differences in tumor-forming and metastasizing capabilities.

Coexistence of multiple CSC populations with different phe-notypes can also not be ruled out. Data suggest that CSCs may

not be stable over time and could undergo clonal evolution as

well. The stromal environment and CSC niche has a verified

crucial role in the behavior of cancer cells in some cases. The

concerns about the relevance of mouse xenotransplant experi-

ments to human cancers, as well as substantial evidence for

clonal evolution should also not be overlooked. We have to

accept that neither the CSC hypothesis nor clonal evolution

can explain all experimental evidence by itself. By acknowled-

ging the respective strengths and weaknesses of both, a com-

bined model can be constructed in which tumor behavior and

progression can be dictated by CSC biology and modulated or

dominated by clonal evolution and effects of the microenvir-onment. The great challenge of the years to come will be how

we determine the contribution of each model to tumor growth

in a given patient and how we can use that information to

design more effective and hopefully curative therapies.

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