The Hitchhikers Guide to Cancer Stem Cell Theory:

Markers, Pathways and Therapy

A

kos Fabian,
1
Gyorgy Vereb,
1,2*
Janos Szollo

si
1,2
*

Abstract
Cancer stem cell (CSC) biology is a rapidly developing field within cancer research.
CSCs are postulated to be a unique cell population exclusively capable of infinite self
renewal, multilineage differentiation and with ability to evade conventional cytotoxic
cancer therapy. These traits distinguish CSCs from their more differentiated counter-
parts, which possess only limited or no potential for self renewal and tumor initiation.
Therefore, CSCs would be the driving motor of malignant growth and therapy resist-
ance. Accordingly, successful cancer treatment would need to eliminate this highly
potent group of cells, since even small residual numbers would suffice to recapitulate
the disease after therapy. Putative CSCs has been identified in a broad range of human
malignancies and several cell surface markers have been associated with their stem cell
phenotype. Despite all efforts, a pure CSC population has not been isolated and often
in vitro clonogenic and in vivo tumorigenic potential is found in several cell popula-
tions with occasionally contradictory surface marker signatures. Here, we give a brief
overview of recent advances in CSC theory, including the signaling pathways in CSCs
that also appear crucial for stem cells homeostasis in normal tissues. We discuss evi-
dence for the interaction of CSCs with the stromal tumor environment. Finally, we
review the emerging potentially effective CSC-targeted treatment strategies and their
future role in therapy. ' 2012 International Society for Advancement of Cytometry

Key terms
cancer stem cells; cancer stem cell markers; cancer stem cell niche; therapy resistance
CANCER stem cell (CSC) theory hypothesizes that heterogeneity within tumors is
not a mere consequence of random mutation and clonal evolution, but results from
an intrinsic hierarchy of cells, with the putative CSC at the apex of the hierarchy
(1,2). The CSC is thought to share several key features with normal stem cells: unlim-
ited capacity for self renewal, including maintenance of the CSC population through
asymmetric division, the ability to differentiate into several cell lineages and intrinsic
resistance against cytotoxic therapies through drug-efflux mechanisms and slow cell
cycling (2). CSC theory postulates that not all tumor cells are equal with regard to
self-renewal, tumor initiation, and maintenance potential, these traits being reserved
for the CSC population. Additionally, cellular heterogeneity and hierarchy within the
tumor originates from CSCs, which give rise to daughter cells that proliferate and dif-
ferentiate into the cell mass that compromises a significant portion of the bulk tumor
(1). Further, CSCs are thought to be responsible for therapy resistance, minimal resid-
ual disease and relapse after initial successful therapy, their stem-like features making
them able to evade conventional treatment modalities (3). It has also been implied
that these stem cell traits allow CSCs to play a leading role in metastasis (46).
Since the first experiments verifying that a specific phenotype within AML cells
enriches for cells that recapitulate the original disease upon transplantation into
NOD/SCID mice (7,8), putative CSCs have been identified in several human malig-
nancies such as CML (9,10), brain (1115), breast (14,1621), colon (2226), endo-
metrial (27), head and neck (28,29), gastric (30,31), liver (3236), lung (19,3739),
1
Department of Biophysics and Cell
Biology, Medical and Health Science
Center, University of Debrecen, Hungary
2
MTA-DE Cell Biology and Signaling
Research Group, Medical and Health
Science Center, University of Debrecen,
Hungary
Received 25 March 2012; Revision
Received 22 August 2012; Accepted 23
August 2012
Grant sponsor: Hungarian National
Research Fund; Grant numbers: OTKA NK
101337, K75752; Grant sponsors; New
Hungary Development Plan, European
Social Fund, European Regional Devel-
opment Fund; Grant number: TAMOP-
4.2.1/B-09/1/KONV-2010-0007; Grant
sponsor: Baross Gabor Program Grant
number: REG_EA_09-1-2009-0010
*Correspondence to: Gyorgy Vereb and
Janos Szollosi, Department of
Biophysics and Cell Biology, Medical
and Health Science Center, University of
Debrecen, P.O. Box 39, Nagyerdei krt.
98, H-4012 Debrecenm Hungary
Email: vereb@med.unideb.hu and
szollo@med.unideb.hu
Published online 20 September 2012 in
Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.22206
2012 International Society for
Advancement of Cytometry
ReviewArticle
Cytometry Part A 83A: 6271, 2013
melanoma (4042), ovarian (4347), pancreatic (4851),
prostate (14,52), renal cell (53,54) and thyroid carcinomas
(55). Despite the large body of work in CSC research, there is
still a considerable amount of confusion and controversy sur-
rounding the CSC hypothesis. Part of this stems from the fact,
that to date, the CSC phenotype is a functional one, based on
the cells behavior in an array of in vitro and in vivo experi-
ments designed to test for renewal and differentiation capacity,
as well as tumorigenic potential. Recent addition of testing for
stemness-related gene expression patterns (31,36,38,39,53,
54,56,57) has helped to further verify stem-like properties at
the molecular level, but there is still not a set of surface mar-
kers in any malignancy that not only enriches, but is exclusive
for cells displaying CSC behavior. Often, cells lacking the sur-
face marker phenotype of putative CSCs still retain various
degrees of tumor initiation capacity (14,20,27,31,41,49,58). In
extremis, mutually exclusive surface marker phenotypes have
been shown to harbor cells with CSC traits (12,5961). To fur-
ther obscure the picture, a significant amount of evidence is
mounting, that CSC behavior is substantially influenced by
the tumor microenvironment (62) and CSCs can alter their
expression profiles, resulting in a moving target for CSC
research and raising questions about the adequacy of the
assays used in vitro and in vivo. Whether CSCs have exclusive
tumorigenic potential within the tumor population and are
responsible for all the heterogeneity and the establishment of
all cell lineages, remains to be seen.
Nevertheless, there is clearly a functionally distinct popu-
lation within most malignancies with an intrinsic resistance
against conventional cytotoxic therapy (63,64). This in vitro
and in vivo resistance is in line with recent findings that CSC-
associated expression patterns in bulk tumors have clinical sig-
nificance and are independent prognostic markers for patient
survival (6570). Further, treatment modalities developed
along the lines of CSC theory and targeting putative CSCs
show promising results and are effective in preliminary studies
(71,72). Taken together, the CSC population isolated with the
admittedly limited functional assays and surface markers has
biological relevance and broadens our appreciation for the
complexity of tumor biology.
EVIDENCE SUPPORTING CANCER STEM CELL THEORY
The CSC theory, although widely accepted, has had to
face justified criticism. Several caveats are known, starting
with the identification of putative CSCs (discussed in detail
later). CSCs were originally thought to arise from normal
stem cells of tissues through malignant transformation, but lit-
tle irrevocable evidence exists linking CSCs to their normal
counterparts. One major reason for CSCs having remained
elusive is that CSC isolation techniques only select an
enriched, nonhomogeneous population. Therefore, a direct
investigation of CSCs and their possible evolution from non-
malignant tissue has largely not been possible. At the same
time, the dysregulation of several key pathways that are impor-
tant in the maintenance and differentiation of normal stem
cells were shown to play crucial roles in carcinogenesis.
Amongst others, pathways regulated by Wnt (73), Hedgehog
(74,75), Notch (76), Nestin (77), Nanog (78,79), and trans-
forming growth factor beta (TGF-b) (80,81) were shown to be
functionally active and relevant in cancer. More and more
active stemness-associated pathways are also identified in pu-
tative CSCs (8284). Not only are these pathways active in
CSCs, but they also seem to be essential as evidenced by
experiments in which selective inhibition led to loss of CSC
function. Invasion and migration of prostate CSCs was not
possible after knockdown of sex determining region of Y box
2 (SOX-2) and octamer-binding transcription factor 3/4
(OCT3/4) (85), while small interfering RNA (siRNA) knock-
down of SOX-2 eliminates tumorigenicity of side population
(SP) cells in lung adenocarcinoma (86). Knockdown of b-cate-
nin led to tumor regression and loss of CSCs in chemically
induced murine skin tumors and in human squamous cell car-
cinoma, and reduced the growth of xenografted human tumor
cell lines (87). Combined inhibition of sonic hedgehog and
mammalian target of rapamycin (mTOR) together with gem-
citabine depleted CSCs in human pancreatic cancer cell lines
(88), while antagonism of hedgehog signaling induced apo-
ptosis in CSCs (89). Downregulation of SOX-2 or twist-related
protein 1 (TWIST-1) lead to differentiation of tumorsphere
forming CD133
1
glioblastoma cells (90). Blocking hedgehog
signaling at the level of Smoothened by cyclopamine inhibited
chronic myeloid leukemia (91) and prostate CSCs (92).
Although active stemness-associated pathways do not neces-
sarily prove stem cell origin of CSCs, they do show a common
functional heritage.
Biological tumor behavior does not always conform to
the linear-hierarchical organization dictated by the strictly
taken CSC theory. For instance, Quintana et al. showed that
the cells in various types of melanoma tumors are not hier-
archically organized and have a reversible phenotype with
tumorigenic potential equal among different phenotypes (93).
Similarly, in BCR-ABL1 lymphoblastic leukemia, functionally
defined CSC populations proved to be genetically diverse, of-
ten with multiple genetically distinct CSC subclones in the
same patient, indicating a branching multi-clonal evolution
(94). While these cases demonstrate that not all tumors adhere
to the CSC model, there is mounting evidence that clonal
expansion and multi-lineage differentiation is sufficient to
drive tumor growth and heterogeneity (95,96). Lathia et al.
separated CSCs and non-CSCs from glioblastomas and then
lentivirally labeled the cells with either GFP or YFP (97). After
inoculating mice with a cell suspension containing CSCs and
non-CSCs in a 1:10 ratio, they monitored tumor growth with
intravital microscopy. The CSCs initially localized in the peri-
vascular region and then outgrew the non-CSC population,
with cells of dominantly CSC origin constituting the bulk tu-
mor. Since tumors evolving in this manner eventually
resembled the parent tumors at the histological level, the case
can be considered exemplary of the potential of CSCs for
intratumor evolution and diversification. The same group also
found CSCs capable of symmetric or asymmetric cell divi-
sions, depending on the environmental conditions (98). The
coexistence of these division types is a long standing corner-
stone of CSC theory and was thought to be a prerequisite for
REVIEW ARTICLE
Cytometry Part A 83A: 6271, 2013 63
CSC derived tumor heterogeneity and CSC renewal. Interest-
ingly, both differentiated progeny and CSCs could be gener-
ated by either division type, questioning the dogma, that one
type is responsible for self-maintenance and the other for cel-
lular expansion. In a truly groundbreaking work, Zhu et al.
first identified Prominin-1 (Prom1 or CD133) cells as normal
tissue stem cells in the mouse small intestine (99). Then en-
dogenous Wnt signaling was activated in the mice using a mu-
tant beta-catenin allele, leading to gross dysplasia and intrae-
pithelial neoplasia. Through lineage tracking with a YFP
encoding reporter allele, they showed a disproportionate
expansion of the Prom1
1
cells and that cells within the neo-
plastic lesion were progeny of Prom1
1
cells. In line with CSC
theory, only a fraction of cells retained the Prom1
1
phenotype
and only a fraction of these cells were proliferating. This, to
our knowledge, was the first direct proof that a solid tumor
can arise from malignant transformation of a tissue stem cell
and that all tumor cells are descendents of CSCs. Previously,
leukemia CSCs were found to be heterogeneous in self renewal
potential and to follow a hierarchy reminiscent of the normal
hematopoietic stem cell compartment (100). While this can be
interpreted as indirect evidence of stem cell origin, a recent
study suggests that CSCs need not originate from the pinnacle
of cellular hierarchy. Goardon et al. showed that in human
acute myeloid leukemia, two molecularly distinct CSC harbor-
ing populations are present (101). These populations are hier-
archically ordered, in so far as one can give rise to the other,
but not vice versa. Global gene expression profiling showed
that the CSC populations resembled normal progenitors and
not hemopoetic stem cells. Therefore, stem cell origin is not
necessary for CSC functionality. Based on these data it is
highly probable, that depending on the type and origin of
malignancy, a spectrum of cells can be responsible for giving
birth to the common CSC phenotype. Newly developed in
vivo imaging modalities could aid in tracking and identifying
putative CSCs (102104) and thereby bring us closer to the
origins of CSCs. Recent advances in detecting circulating tu-
mor cells (105108) and monitoring minimal residual disease
(109,110) should also help establish the role of CSCs in metas-
tasis formation and disease propagation.
IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
The in vitro and in vivo assays for identifying cell popula-
tions harboring CSCs have been extensively discussed else-
where (2,111113). Briefly, colony forming capacity and
tumorsphere formation in specific nonattached culturing con-
ditions, maintained through serial passaging, has been asso-
ciated with a stem-like phenotype. Activity of pathways asso-
ciated with stemness is increasingly being used to verify stem-
like behavior. Additionally, loss of stemness-associated molec-
ular and surface markers parallel to induced differentiation is
also used. In this respect, growth of daughter cell lineages with
differentiated marker signatures distinct from that of parent
cells and/or other progeny are hallmarks of multi-lineage dif-
ferentiation capacity.
Xenotransplantation has become the gold standard of in
vivo testing. Putative CSCs should be tumorigenic in vivo,
reproducibly giving rise to tumors when transplanted into
immune-compromised mice. The cell numbers required for
tumor initiation are preferentially lower than when inoculat-
ing unsorted or non-CSC phenotype cells. The tumors should
ideally recapitulate the morphology and histological character-
istics of parent tumors, verifying multi-lineage differentiation
and hierarchical heterogeneity. The tumors should also be ca-
pable of serial passaging, that is, CSCs derived from primary
xenograft tumors should initiate tumor growth and give rise
to secondary tumors. While exemplifying the special capabil-
ities of CSCs, xenotransplantation has also come under the
most scrutiny as an adequate method for assessing tumori-
genic potential (114116). The question that frequently arises
is whether we are indentifying all cells capable of tumor initia-
tion or only a subset that has the unique capacity to propagate
in mice. Experiments have shown that even non-CSCs can
engraft and remain viable in the mouse environment, but fail
to proliferate and form tumors (11,23,97). As more and more
information surfaces about the active role of the tumor micro-
environment in tumor growth, concerns amount that other-
wise tumorigenic cells may lack stromal and interstitial factors
in the xeno-environment and therefore fail to thrive (115).
While one can argue that CSCs identified under these circum-
stances still have survival advantage and growth autonomy
over the non-CSC population, whether this automatically
translates into the same biological behavior and tumorigenic
potential in the human body is an open question. The efficacy
of the xenotransplant system was also stressed in an elegant
work by Quintana et al. (117), who showed that the addition
of Matrigel lowers the number of melanoma cells needed for
tumor initiation in NOD/SCID mice, indirectly lending sup-
port to the significance of the inoculation environment. More
importantly, by using NOD/SCID IL2Rc
null
mice, which lack
the natural killer activity of wild type NOD/SCID mice, the
number of melanoma cells required for tumor initiation was
drastically reduced to almost the single cell level and tumor
initiation capability did not show an association with any par-
ticular cell surface phenotype. While this might be a unique
property of melanoma, which is a highly aggressive malig-
nancy known to metastasize early and at small tumor size; it
clearly signifies that even in the immune-compromised NOD/
SCID mice tumorigenic cells are lost due to immune-surveil-
lance. Again, the biological significance and specific human
relevance of cells with tumorigenic potential that cannot
escape the immune-surveillance of NOD/SCID mice can be
debated. One way to circumvent the potential negative influ-
ence of the xeno-environment is by using syngeneic murine
model systems of human disease (118,119). Of course, in these
cases care needs to be taken to verify that the relevant cellular
markers and signaling pathways are also present and impli-
cated in the human counterpart.
Using the methods mentioned above, CSC populations
with a characteristic cell surface expression profile were dis-
covered in a plethora of human malignancies (for a summary
and references, please see Table 1). Several of the most com-
monly used CSC markers are CD133, CD44, CD24, and
ALDH1 in solid tumors and CD34 in hematological malignan-
REVIEW ARTICLE
64 Cancer Stem Cells
cies. Recently, several new markers have been identified,
including CD105 (53), granulin-epithelin precursor (GEP)
(36), c-Met (50), and integrin a6 (120). It is important to real-
ize that all the markers used only enrich for the putative CSC
population and the signature is not unique to CSCs. Often,
the population negative for one or more CSC markers also has
the potential for tumor initiation, although usually a very lim-
ited capability. One of the most controversial markers is
CD133 (Prominin-1). Initial studies proved CD133 as a
marker for brain and colon CSCs and later on also liver, lung,
ovarium, pancreatic and prostate cancer. Several authors also
found CSC activity in CD133

populations of glioblastoma
and colon tumors (12,61,121), in some instances CSC func-
tionality being exclusive to the CD133

population (122,123).
Gunther et al. showed that under stem cell culturing condi-
tions, cell lines derived from patient glioblastomas can form
tumorspheres which, depending on the cell line, are either
formed by CD133
1
or CD133

cells (60). Although the cell

lines with CD133
1
tumorspheres proved to be more tumori-
genic, the CD133

tumorspheres were also capable of initiating

tumor growth. This controversy of CD133 expression in glio-
blastoma CSCs can be reconciled through the work of Chen
et al., whom from PTEN-deficient glioblastomas isolated three
subtypes of neurosphere forming cells, all capable of multi-lin-
eage differentiation (59). Type I cells were CD133

, produced
CD133
1
and CD133

daughter cells, showed the most aggres-

sive tumor growth in xenotransplants and gave rise to histolo-
gically less differentiated tumors; Type II cells were CD133
1
,
produced CD133
1
and CD133

daughter cells, showed inter-

mediate tumor growth and differentiation; Type III cells were
also CD133

, but produced only CD133

daughter cells,
showed the slowest tumor growth and produced the most dif-
ferentiated tumors. The authors concluded that in PTEN-defi-
cient glioblastomas, several CSC lineages may be present,
which are hierarchically organized and have different cell
surface markers. An example of CSC marker plasticity was
supplied by the work of Bae et al. (85). They showed that E-
cadherin, which is expressed by prostate CSCs, was transiently
downregulated during the migration of CSCs in a Matrigel
assay and only returned to premigration levels several hours
afterwards. It should also be mentioned that CSC markers are
not necessarily universal in a given tumor type. For instance,
the CD44
1
CD24

phenotype thought specific for CSCs in

breast tumors has been shown to be only present in basal-type
breast cancers and ALDH1
1
is only applicable as a CSC
marker in basal-type and HER21breast tumors (124).
It also remains to be seen whether all CSC markers have
an essential part in maintaining the functional CSC phenotype
or their expression is just a byproduct of stemness. The role of
CD44 is crucial for cell viability of hepatocellular carcinoma
CSCs (33), intestinal carcinogenesis of mice (125), and bone
marrow homing of chronic myeloid leukemia (CML) cells
(126). Du et al. found that in colorectal cancer, CD44
1
cells
were enriched for CSCs with clonogenic and in vivo tumori-
genic potential and both properties were impaired after siRNA
knockdown of CD44 (58). Similarly, Pham et al. showed in
primary cultures of malignant breast tumors that small hair-
pin RNA (shRNA) knockdown of CD44 in CD44
1
/CD24

breast CSCs reduced expression levels of Wnt, Hedgehog and

Akt pathway associated markers to that seen in non-CSCs
(57). The in vivo tumorigenicity of CSCs was also impaired
with CD44 knockdown. In gastric CSCs, lentiviral knockdown
of CD44 was shown to impair tumorsphere formation and tu-
mor growth (30). Recently, Li et al. reported that they identi-
fied c-Met, a receptor tyrosine kinase, as a marker for CSCs in
human pancreatic adenocarcinomas (50). In their study, lenti-
viral knockdown of c-Met inhibited in vivo tumor formation
and pharmacological blockade of c-Met inhibited tumor-
sphere formation, induced CSC apoptosis and decreased tu-
mor growth while concurrently reducing the CSC population
Table 1. Cancer stem cell markers and associated tumor types
TUMOR TYPE
CSC MARKER
CD133 CD44 CD24 ALDH CD90 CD34 OTHER REF.
Brain 1/ 1 SP, integrin-a6 (1115,120)
Breast 1 1 1 1 SP (14,1621)
Colon/colorectal 1 1 1 (2226)
Endometrium 1 (27)
Gastric 1 1 (30,31)
Head and Neck 1 1 (28,29)
Leukemia (AML, CML) 1 CD38
2
(710)
Liver 1 1 1 1 GEP (3236)
Lung 1 1 1 1 SP, CD117 (19,3739,86)
Melanoma 1 CD20, ABCB5 (4042,117)
Ovarian 1 1 SP, CD117 (4347)
Pancreatic 1 1 1 1 c-Met, EpCam (4851)
Prostate 1 1 SP (14,52)
Renal cell CD105 (5354)
Thyroid SP (55)
GEP, granulin-epithelin precursor; SP, side population.
REVIEW ARTICLE
Cytometry Part A 83A: 6271, 2013 65
of the tumor. In glioblastoma multiforme, integrin a6 was
shown to enrich for CSCs (120). The shRNA knockdown of
integrin a6 inhibited in vitro CSC growth and tumorsphere
formation as well as in vivo tumor formation and tumor
growth. The same effects were achieved using a blocking anti-
body against integrin a6. It has also been suggested that the
ABCG2 multidrug-transporter not only causes the SP pheno-
type of putative CSCs through dye-efflux mechanisms, but
also regulates Hedgehog signaling in these cells (127).
In general, whenever feasible, knockdown or blockade of
a new CSC marker should be attempted to determine whether
it influences CSC behavior and function. Ideally, gene expres-
sion should be monitored simultaneously, preferentially focus-
ing on known stemness-associated pathways.
CSC INTERACTIONS WITH THE TUMOR
MICROENVIRONMENT
While initial research focused on the autonomous tumor
initiation and growth properties of CSCs, recent studies have
highlighted the important influence of the tumor environment
not only on tumor behavior, but on CSCs themselves. Early
on it was hypothesized that similar to their normal stem cell
counterparts, a niche may exist that provides CSCs with envir-
onmental cues that help maintain stemness and may partially
regulate CSC fate (128). Support for this theory was gained
from histological and immunohistochemical slides showing
cells with a CSC-associated marker phenotype preferentially
enriched in perivascular regions of tumors (15,120,129,130).
As previously mentioned, initial perivascular localization of
xenografted CSCs was recently visualized with intravital mi-
croscopy (97). There is mounting evidence that this is not a
mere spatial coincidence, but also harbors a functional rela-
tionship. Beck et al. found that vascular endothelial growth
factor receptor 2 (VEGFR-2) blockade decreased tumor mi-
crovascular density and also proportionally reduced CSC pool
size in papillomas (130). Most remarkably, this was not solely
owed to the reduced availability of nutrients, but also to a
direct inhibition of CSC proliferation. They successfully veri-
fied a neurolipin-dependent autocrine VEGF loop, in which
VEGF regulates CSC stemness-associated gene expression and
proliferation. Similarly, underscoring the crucial interaction of
tumor cells with surrounding stroma, paracrine hedgehog sig-
naling with stromal elements was found to be essential for car-
cinogenesis and tumor angiogenesis (131134). Even the nor-
mal stem cell compartment is implicated, as mesenchymal
stem cells were shown to affect the phenotype of tumor cells,
as reviewed by Grisendi et al. (135). In a mouse breast cancer
model, Malanchi et al. verified that putative CSCs are the only
cell population capable of forming distant metastasis (136).
This function of CSCs, however, was dependent on inducing
periostin (POSTN) expression in the host stroma via trans-
forming growth factor b3 (TGF-b3). POSTN was shown to
augment Wnt signaling in CSCs, and loss of POSTN or block-
ade of TGF-b3 action inhibited metastasis formation. Further
evidence that CSCs are susceptible to environmental factors
was provided by a study showing that stemness of glioma
CSCs can be inhibited by human umbilical cord blood stem
cells (HUCBSC), resulting in reduced in vitro migration and
in vivo reduction of tumor volumes (90). The inhibitory effect
of HUCBSCs was found to arise from reducing SOX-2 and
TWIST1 expression in CSCs, which induced differentiation of
the cells. The tumor environment can also significantly contri-
bute to therapy resistance of tumors. Lonardo et al. found that
despite successful targeting of Nodal signaling in CSCs in a
pancreatic cancer cell line leading to diminished in vivo tumor
growth in combination with gemcitabine, initial results with
primary tumor xenografts were discouraging (137). They dis-
covered that efficient accumulation of the therapeutic agent
was inhibited; however, blocking the hedgehog pathway in
stromal cells increased drug delivery by a factor of 10 and
restored therapeutic efficacy, in line with a previous study
showing stromal depletion as an effective measure for enhan-
cing target site delivery of chemotherapeutics (138).
Hypoxia within tumors has been suggested to be a deter-
minant of CSC behavior (139). It was shown that in glioblas-
tomas, CSCs differentially express hypoxia inducible factors
(86), with the HIF2a subtype preferentially expressed in CSCs
(140). While knockdown of HIF1a inhibited both CSCs and
non-CSCs, knockdown of HIF2a selectively decreased in vitro
CSC cell survival and impaired in vivo tumor formation.
Another group identified a set of signature genes differentially
expressed in SP cells from glioblastoma cell lines and primary
glioblastomas (141). The expression of signature genes as well
as of CD133 was upregulated by culturing under hypoxic con-
ditions. Overexpression of HIF2a had a similar effect, while
knockdown of HIF2a inhibited hypoxic upregulation of genes.
In human pancreatic cancer cell lines, hypoxia also elevated
the expression of CD133 and increased invasiveness of cancer
cells (142). In this study, overexpression of HIF1a increased
metastatic and tumorigenic ability. Finally, in another study
with prostate cancer cell lines, culturing under hypoxic condi-
tions induced expression of HIF1a and HIF2a with an upregu-
lation of Oct 3/4 and Nanog (143). Hypoxia also induced
expression of CD44 and ABCG2 and led to higher clonogeni-
city and increased sphere formation. The hypoxia induced
changes were already evident at a moderate oxygen level of
7%. This oxygen level, thought to be representative of physio-
logical concentrations in human tissues, was also shown to
increase HIF2a expression in glioblastoma CSCs (144). Upreg-
ulation of Oct 4and Sox2, and an increase in proliferation rate
and self-renewal potential of CD133
1
cells were also wit-
nessed. An interesting signaling pathway was identified by
Feldman et al., who found leptin receptor to be expressed via
an OCT4/SOX2 mechanism on hepatocellular carcinoma cell
line CSCs (145). In CSCs, leptin induced phosphorylation of
STAT3 and induction of Oct4 and Sox2. Additionally, intraper-
itoneal injection of leptin into xenografted mice led to faster
tumor growth and CSCs formed tumors faster in obese mice
than in lean littermates. Immunostaining of hepatocellular
carcinoma specimens demonstrated leptin receptor expression
in putative CD133
1
CSCs. Leptin driven Notch-1 signaling
has also been identified in breast cancer cell lines (146). The
clinical relevance of leptin augmented tumor development
could be especially high in developed countries with obesity-
REVIEW ARTICLE
66 Cancer Stem Cells
prone populations. The diversity of identified environmental
factors shows that CSC-niche interactions are at least as com-
plex as the CSCs themselves. It has also been hypothesized,
that several different CSC niches may coexist, with different
pathways active in perivascular, hypoxic, and metastatic envir-
onments (62).
In summary, the stromal compartment may play as much
a pivotal role in determining tumor growth and metastasis, as
the intrinsic properties of CSCs. However, the CSC niche need
not necessarily evolve from nontumorous cell populations. In
two studies focusing on angiogenesis in glioblastomas, vascu-
lature of tumors was shown to be of partially neoplastic origin.
Ricci-Vitiani et al. showed a portion of endothelial cells in
glioblastomas carry the same genetic alterations as tumor cells
(147). They also found that in xenograft tumors grown from
glioblastoma CSCs, the endothelial lining of vessels is primar-
ily composed of cells of human origin. Wang et al. also showed
somatic mutations to be shared by tumor cells and a fraction
of endothelial cells within the tumor (148). Through lineage
analyses they were able to trace the endothelial cells to a spe-
cial subset of CD133
1
glioblastoma CSCs, which can differ-
entiate into endothelial progenitors which in turn give rise to
the endothelial population. They found the transition from
CSC to endothelial progenitor to be VEGF dependent,
whereas further maturation to endothelial cells relied on effec-
tive Notch-1 signaling. A new layer of complexity is added if
we consider that hedgehog signaling was shown to induce
VEGF production in fibroblasts (131), which in this context
may not only promote angiogenesis per se, but also the cross-
differentiation of CSCs. Results similar to those with glioblas-
toma were seen in xenografts of human renal cell carcinoma,
where tumor vasculature was also of human origin (53). These
studies shed light on a new facet of CSC function and hint
that the role of CSCs in tumor biology may go beyond main-
taining the strictly taken tumor cell population. Interestingly,
the capacity of CSCs to differentiate into endothelial cells
seems to be a trait shared with normal pluripotent- and stem
cells, which were shown to be capable of cross-lineage differ-
entiation (149152). Therefore, cross-differentiation might be
a further normal stem cell property conserved in CSCs.
PROGNOSTIC VALUE OF CSCS AND THEIR IMPLICATIONS
FOR THERAPY
The clinical significance of CSCs has been a subject of
much debate. It was speculated that stemness features of CSCs
would allow them to evade conventional anticancer therapy
and maintain minimal residual disease, eventually leading to
tumor relapse (153,154). While the therapy resistance in vitro,
and in xenotransplanted tumors has been well documented
(64,155,156), there was little proof that poor therapy out-
comes in patients can be directly linked to CSCs. Recently, it
has been shown that immunohistochemically verifiable CSC
marker expression in histological slides from patient tumors
has prognostic value and is associated with a poorer clinical
outcome (6570,157,158). Similarly to circulating cancer cells
(159162), the presence of circulating CSCs was proven to be
a prognostic factor for faster disease progression (163165).
Further, a relationship was established between myeloma CSC
numbers in patients and progression free survival after treat-
ment with rituximab (153). In breast cancer, the number of
breast CSCs in patients core needle biopsies increased after
chemotherapy, demonstrating a survival advantage of CSCs
over the bulk tumor population (166). While CSCs were
thought to resist therapy through drug efflux mechanisms,
antiapoptotic ability, efficient DNA repair and slow cycling
(167), two recent studies have suggested other coping mechan-
isms that may be active in the CSC population. Chaterjee et al.
found that breast CSCs can enter dormancy upon treatment
with farnesyl transferase inhibitors, a class of drugs designed
to inhibit proper maturation of Ras (168). This induced dor-
mancy was maintained by CSCs through autophagy and the
cells were able to reenter the cell cycle upon withdrawal of the
drugs. Viale et al. showed p21 dependent cell cycle restriction
to be functional in CSCs in a mouse leukemia model (169).
The cell cycle restriction not only prevented DNA damage
accumulation, but also functional exhaustion of CSCs and
therefore contributed to maintenance of stemness. The role of
CSCs in minimal residual disease and relapse was supported
by the work of Hamilton et al., who showed that a subpopula-
tion of Bcr-Abl positive CML CSCs are able to survive the
withdrawal of growth factors in addition to combined Bcr-Abl
knockdown and pharmacological inhibition by dasatinib, a ty-
rosine kinase inhibitor (170). These cells were quiescent under
the harsh conditions and although they did not proliferate,
remained viable. Upon withdrawal of dasatinib and addition
of growth factors, the cells reentered the cell cycle and began
to proliferate.
It has become clear that traditional treatment regiments
are inefficient at eliminating the CSC population and new
therapeutic approaches are needed that take into account the
special properties of CSCs. Basically, a reduction in the CSC
population can be achieved by inducing differentiation/apo-
ptosis in CSCs by targeting receptors or signaling pathways
crucial for CSC self-maintenance and viability. This approach
has started to show promising results. The paracrine signaling
of colon CSCs through interleukin 4 (IL-4) was successfully
blocked by anti-IL-4 antibody treatment, reducing in vitro
and in vivo chemotherapy resistance (24). In human renal cell
carcinomas, IL-15 treatment of CSCs abolished tumorigenic
potential and blocked CSC self-renewal (54). A heterodimer of
bone morphogenetic protein efficiently inhibited TGF-b de-
pendent breast CSC function, decimating CSCs in vitro and
inhibiting metastases formation in vivo (171). Reduction in
Nanog-1 expression in breast CSCs by 3-O-methylfunicone
inhibited sphere formation and colony formation (172). An
off-target shRNA against neostemin, lentivirally transfected
into glioblastoma cells, led to preferential apoptosis of
CD133
1
putative CSCs and significantly slowed xenotrans-
planted tumor growth (173). Another study found shRNA
knockdown of ubiquitine ligases to induce apoptosis and dif-
ferentiation in cell line derived glioblastoma CSCs (174). As
previously discussed, blockade of TGF-b3 action with a
secreted decoy receptor inhibited metastasis of mouse breast
CSCs (136) and antibody targeting of integrin a6 in glioblas-
REVIEW ARTICLE
Cytometry Part A 83A: 6271, 2013 67
toma inhibited CSC driven in vivo tumor initiation and
growth (120). Recent development of subtype-specific Notch
antibodies demonstrated efficacy by inhibiting Notch-depend-
ent xenograft tumor growth without the side effects of pan-
Notch blockade (175). A selective antibody targeting Notch-1
induced apoptosis in a breast cancer cell line, reduced the CSC
population and irreversibly impaired CSC mammosphere for-
mation (176). Oxymatrine was also shown to diminish the
side population in a breast cancer cell line by decreasing
b-catenin levels and activation (177).
The successful targeting of CSCs may, however, not be
sufficient to achieve adequate disease control. A computa-
tional modeling of CSC and non-CSC population dynamics
concluded that differentiation therapy of slow cycling, self-
renewing populations or cytotoxic therapy of a larger, faster
proliferating population alone cannot eliminate tumorous
growth (178). These calculations did not utilize any specific
assumptions about tumor growth regulation, they only
hypothesized that relative population densities regulate prolif-
eration and asymmetric cycling of CSCs. A recent study
demonstrated the viability of this assumption, with CSC den-
sities reaching the same level in in vitro mixed cultures of
CSCs and non-CSCs, irrespective of the initial ratio of the
populations after plating (179). Therefore, efficient tumor
therapy is suggested to be dependent on parallel administra-
tion of cytotoxic agents in combination with targeted CSC
elimination. Several studies have found a systematic synergis-
tic effect between traditional chemotherapeutics and newer,
CSC-targeted agents. Checkpoint kinase 1 inhibition in com-
bination with chemotherapy greatly enhanced in vitro cyto-
toxity and in vivo efficacy of chemotherapy against non-small
cell lung CSCs (180). Acting through ataxia telangiectasia
mutated, an oncolytic virus and the alkylating agent temozo-
lomide synergistically killed glioblastoma CSCs while sparing
neurons (181). In pancreatic cancer, inhibition of c-Met
reduced the CSC fraction in xenograft tumors; however, full
growth inhibition was only achieved after adding gemcitabine
to the treatment regimen (50). Similarly, stable disease and
growth inhibition of primary pancreatic cancer xenografts was
only accomplished with triple therapy consisting of gemcita-
bine, knockdown of Nodal in CSCs and blockade of smooth-
ened in stromal elements (137). Also in pancreatic cancer,
combined gemcitabine, sonic hedgehog- and mTOR inhibi-
tion abolished metastatic activity and reduced xenograft tu-
mor growth (88). Finally, retinoid compounds blocked the
increase in ALDH1
1
cells after carboplatin therapy of ovarian
cancer cell lines and in combination with carboplatin signifi-
cantly reduced in vivo tumor growth (182). While the novel
combination therapies show great potential, tolerability in the
clinical setting and especially the long term effects on normal
stem cells still need to be determined. In a special setting, the
evolving approach of tumor therapy with the patients repro-
grammed and expanded own T cells (183) can be ideally
exploited to target CSC specific surface antigens (72). This
approach has first been confirmed for targeting CD20 on
CSCs in a xenograft model of human melanoma cells (184)
and then successfully applied in a clinical case (185).
CONCLUSION
Our understanding of CSC biology is rapidly expanding.
Attention is gradually shifting from merely identifying mar-
kers that enrich for CSCs to untangling the complex web of
signaling pathways that determine the stem-like behavior.
These pathways are often dysregulated counterparts of net-
works functionally important in the maintenance of normal
tissue stem cells. We are also beginning to recognize that CSCs
are not lonely fighters, they often interact with and modulate
signaling of stromal elements to elucidate a paracrine environ-
ment favoring tumor growth and invasion. To what extent
stromal factors influence CSC behavior and how essential
these factors are for tumor propagation still remains to be
seen. An intriguing new aspect of CSC biology is their poten-
tial to not only give rise to the various malignant cell lines
within a tumor, but also to produce progeny that can differ-
entiate into various supporting tissues and structures. The
clinical significance of CSCs is growing, with the CSC pheno-
type representing an independent prognostic factor for poorer
patient outcome. Indirect and direct evidence for therapeutic
consequences of CSC behavior, such as therapy resistance,
minimal residual disease and relapse are mounting. Luckily,
every identified marker and pathway represents a new poten-
tial target for effective therapy. Initial experience with targeted
CSC therapy is promising and hopefully the preliminary in
vitro and in vivo results, whether alone or in combination
with traditional cytotoxic agents, can be carried over into clin-
ical trials and eventually lead to successful cancer therapy.
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