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The cancer stem cell: premises, promises
and challenges
© 2011 Nature America, Inc. All rights reserved.
Hans Clevers
Over the last decade, the notion that tumors are maintained by their own stem cells, the so-called cancer stem cells,
has created great excitement in the research community. This review attempts to summarize the underlying concepts
of this notion, to distinguish hard facts from beliefs and to define the future challenges of the field.
Central to the cancer stem cell (CSC) concept is the observation that
not all cells in tumors are equal. The CSC concept postulates that,
similar to the growth of normal proliferative tissues such as bone
marrow, skin or intestinal epithelium, the growth of tumors is fueled
by limited numbers of dedicated stem cells that are capable of selfrenewal. The bulk of a tumor consists of rapidly proliferating cells
as well as postmitotic, differentiated cells. As neither of these latter
two classes of cells has the capacity to self-renew, the contribution of
these non-CSC tumor cells to the long-term sustenance of the tumor
is negligible.
A major attraction of the CSC concept rests in the explanations it
provides for several poorly understood clinical phenomena. CSCs are
tacitly believed to have acquired the molecular armaments of normal
stem cells: CSCs can renew themselves, and they are built to last a lifetime, to be resilient to electromagnetic and chemical insults, to be able
to slumber for prolonged periods of time and to colonize other parts
of the body. Thus, the CSC hypothesis could explain what is commonly known: a person with cancer can generally not be considered
cured, even when his or her initial response to radiation or chemotherapy is encouragingly robust. Rare CSCs may be able to survive
these therapeutic regimens, thus explaining why local recurrence is
the almost-inevitable outcome of effective treatment of solid tumors
by radiation or chemotherapy. Quiescent CSCs that have drifted to
distant sites may be responsible for metastases that can appear many
years after curative surgical treatment of a primary tumor. Metastatic
relapse in breast cancer, for instance, can occur more than a decade
after initial treatment1. Lastly, the CSC hypothesis promises the development of more effective treatments, aimed not at reducing tumor
bulk, but rather at targeting the ‘beating heart’ of the tumor, the CSC.
An avalanche of primary papers, reviews, workshops and symposia
has been devoted to this concept. As often occurs in rapidly developing fields of science, the opinions and conclusions of authors, reviewers and editors alike may have been colored by scientific enthusiasm
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and
University Medical Center Utrecht, Utrecht, The Netherlands. Correspondence
should be addressed to H.C. ([email protected]).
Published online 7 March 2011; doi:10.1038/nm.2304
nature medicine VOLUME 17 | NUMBER 3 | MARCH 2011
and clinical promise. Indeed, an attitude of healthy caution seems to
be developing in the maturing cancer stem cell community.
History
Stem cell concepts and their application to cancer are many decades
old2 (Fig. 1). Since the nineteenth century, tumors have been known
to show explicit histological heterogeneity. In 1937, Furth and Kahn
established that a single cell from a mouse tumor could initiate a new
tumor in a recipient mouse3. Subsequently, the frequency of tumorinitiating cells in solid tumors and leukemias was found to be variable
but low, requiring 103 to 107 cells4–6. The resulting tumors typically
showed the morphologic heterogeneity of the original tumor.
In a series of landmark experiments, Pierce showed that malignant
teratocarcinomas contain highly tumorigenic cells that, as single cells,
can differentiate into multiple differentiated, nontumorigenic cell
types7. Thus, teratocarcinomas loosely follow normal development.
Incidentally, these insights laid the foundation for the embryonic
stem cell field. In the mid-1900s, techniques became available to
identify proliferating cells by radiolabeling. Combined with autoradiography8, this allowed measurements of cellular proliferation,
lifespan and hierarchical organizations within normal tissues9. By
using this approach on a mouse squamous cell carcinoma in 1971,
Pierce showed that early labeling occurred almost exclusively in the
undifferentiated areas. At later time points, the DNA label appeared
in the well-differentiated areas, which were thus shown to derive from
undifferentiated cells. These well-differentiated cells did not form
tumors when transplanted into compatible hosts10. These and other
experiments led Pierce to the following early definition of the CSC
concept11: “a concept of neoplasms, based upon developmental and
oncological principles, states that carcinomas are caricatures of tissue
renewal, in that they are composed of a mixture of malignant stem
cells, which have a marked capacity for proliferation and a limited
capacity for differentiation under normal homeostatic conditions, and of the differentiated, possibly benign, progeny of these
malignant cells.”
Hematological tumors were also found to show proliferative heter­o­
geneity12,13 and a hierarchical organization14,15, leading to the prediction that slow-cycling leukemic stem cells caused tumor relapse14,16,17.
Investigators then observed that leukemic stem cells entered into the cell
313
© 2011 Nature America, Inc. All rights reserved.
review
cycle after chemotherapy, much like normal
Clonal evolution
Cancer stem cells
stem cells. These findings provided the rationRous discovers his sarcoma virus
1911
ale for combining chemotherapeutic agents18.
The focus of cancer research shifted in
1937
Furth and Kahn graft single mouse tumor cell
the 1970s, when mutations in oncogenes
1946
Belanger and Leblond develop autoradiography
and tumor suppressor genes were found to
and document stem cell hierarchies
cause most human cancers. This let Nowell
to formulate the clonal evolution concept 19,
1960
Pierce: teratomas contain pluripotent stem cells
stating “it is proposed that most neoplasms
1963
Till and McCulloch discover
arise from a single cell of origin, and tumor
hematopoietic stem cells
1965
progression results from acquired genetic
Nobel Prize for Peyton Rous
1966
variability within the original clone allowMultiple studies report stem-like
1971
Knudson defines tumor suppressor
cells in hematological malignancies
ing sequential selection of more aggressive
‘two-hit’ hypothesis
sublines. Tumor cell populations are appar1975
Varmus and Bishop discover the
ently more genetically unstable than normal
Src protooncogene
1976
cells. The acquired genetic instability and
Nowell formulates the clonal evolution
associated selection process, most readmodel of multihit carcinogenesis
ily recognized cytogenetically, results in
1986
advanced human malignancies being highly
Weinberg and colleagues clone the
first tumor suppressor Rb
individual karyotypically and biologically.
Hence, each person’s cancer may require
Nobel for Varmus and Bishop
1989
individual specific ­ therapy, and even this
1995
may be thwarted by emergence of a genetiJohn Dick revives CSC theory by
xenografting human AML
cally variant subline resistant to the treatment.” Fearon and Vogelstein formulated a
2003
Clarke demonstrates CSCs in
breast cancer by xenografting
clonal evolution model for colon cancer, in
which the progression from early adenoma
to invasive carcinoma reflects the stepwise Figure 1 A timeline of the important discoveries in the fields of clonal evolution and cancer stem
cells. Clonal evolution is shown on the left, and CSCs are shown on the right.
acquisition of mutations in specific cancer
20
genes . The concept of clonal evolution
provided a ready explanation for the relentless advance toward ever cancer cells, and the two fractions showed equal cell cycle kinetics24,
more malignant behavior within established tumors.
yet the tumors seemed hierarchically organized when tested functionally. This paper was rapidly followed by similar studies on other solid
Revival of the CSC concept. In the early nineties, research on hemato­ tumors such as brain cancers25 and colon cancers26–28. For a comprepoietic stem cells was flourishing, and bone marrow transplantation hensive recent overview of these efforts, the reader is referred to ref. 29.
had successfully been introduced in the clinic. Crucial technologies In each case, small numbers of cells defined by specific markers were
had been developed, such as FACS combined with large sets of well- able to transfer disease into immunodeficient mice. As in earlier
validated cell surface markers and mouse xenograft assays for human studies, the frequencies of tumorigenic cells were variable and often
hematopoietic stem cells. Dick and his colleagues revived the study low. Typically, the transplanted tumors tended to recapitulate at least
of functional heterogeneity within leukemias21 and found that most some of the heterogeneity of the original tumors.
Most studies of CSCs follow a common scenario: a marker or marker
subtypes of acute myeloid leukemia (AML) could be engrafted reliably in immunodeficient mice. Leukemic engraftment could only be combination is found to be expressed in a heterogeneous fashion in
initiated from CD34+CD38− fractions. Moreover, the xenograft assay a certain tumor type. On the basis of this marker heterogeneity, suballowed measurement of the frequency of the initiating cell; it was populations of cells are sorted from primary tumors and transplanted
found to be on the order of one per million tumor cells. Thus, a CSC into immunodeficient mice by limiting dilution, after which tumor
growth is scored some weeks or months later. Different capacities
was identified in AML22,23.
Clarke and his colleagues applied these concepts and experimen- for tumor initiation between tumor cell subsets can be interpreted
tal approaches for the first time to a solid breast cancer tumor. In a as evidence for the presence of CSCs in the primary tumor, and it is
landmark paper that appeared in 2003 (ref. 24), it was shown that then often said that the tumor adheres to the CSC model. To date,
breast tumors comprise heterogeneous populations of breast cancer many theoretical and experimental caveats to the CSC model have
cells. In the xenograft assay, as few as 100 CD44+CD24−/low cells were remained unexplored, largely because of technological challenges.
tumorigenic, whereas tens of thousands of cells with alternate pheno- Below I discuss a number of these issues.
types were not. Of note, normal human leukocytes, endothelial cells,
mesothelial cells, and fibroblasts were eliminated from the transplant Caveats to theory and to experimental strategy
by the use of a set of defined lineage markers (‘Lineage−’). The tumori- Is the CSC phenotype a stable trait? The xenograft assay provides
genic subpopulation could be serially passaged: each time, cells within a snapshot of the state of cancer cells at the time of removal of a
this population generated new tumors containing CD44+CD24−/low tumor. Does a positive outcome of the xenograft assay identify a CSC
tumorigenic cells as well as the phenotypically diverse nontumorigenic population that would have been stable in time in the original tumor,
cells present in the initial tumor. No clear morphological distinction or would cancer cells fluctuate between CSC and non-CSC states?
was obvious between the tumorigenic and nontumorigenic breast Dick has termed these two situations the hierarchical and stochastic
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© 2011 Nature America, Inc. All rights reserved.
review
models, respectively2. The stability of the CSC phenotype has not
yet been experimentally probed. Nevertheless, two recent studies
shed some light on the plasticity of CSC tumor cells. In their study
on melanoma, Morrison and his colleagues30,31 showed that tumors
arising both from CD133− cells and from CD133+ cells sorted from
an original melanoma re-establish the original ratios of CD133− and
CD133+ cells. This experiment indicates that individual cancer cells
can recapitulate the marker heterogeneity of the tumors from which
they derive. In a follow-up study32, the same authors showed that
all of 15 commonly used CSC markers behaved similarly to CD133.
Roesch et al.33 report differential expression of the H3K4 demethylase JARID1B in subpopulations of human melanoma cells in culture.
JARID1B+ cells cycled more slowly than JARID1B− cells, but they
also generated more progeny and were more tumorigenic, thereby
implying that expression of the demethylase indicates the CSC state.
Notably, the authors showed that JARID1B expression fluctuates;
even when starting from a single cell, JARID1B+ cells could arise
from cells that did not express JARID1B and vice versa. The authors
concluded that tumor maintenance may be viewed as a dynamic
process mediated by temporarily distinct subpopulations of cancer
cells33. In a related example, Settleman and his colleagues detected a
subpopulation of reversibly drug-tolerant cells in human cancer cell
lines34. These cells showed >100-fold reduced drug sensitivity, which
required expression of the histone demethylase JARID1A. The drugtolerant phenotype was transiently acquired and lost at low frequencies by individual cells34.These examples indicate that plasticity of
the CSC state should be given serious consideration. Only if the CSC
phenotype is a stable trait will it be advantageous to selectively target
CSCs as a cancer treatment.
How good is the xenograft assay? Currently, only a single assay exists
that defines human CSCs. As described above, it involves xenotransplantation of sorted cancer cells into immunodeficient mice. What
does this assay score? A critical view suggests that the sorted and transplanted human cancer cell has been challenged by various experimental
manipulations and subsequently ends up in a context that is dramatically different from the original tumor niche. A cell that manages to
colonize a mouse tissue must by definition be highly robust, but there
is no direct evidence to state that cellular behavior after xenotransplantation indeed reflects the hierarchical position of the same cell
within the original tumor. In addition to the cellular stress induced by
the experimental procedure, two experimental caveats are inherent to
xenografting: the species barrier and the transplantation setting.
Species barriers may impede certain crucial niche functions such as
interactions between adhesion molecules or between growth factors
and their receptors. And even a rather trivial phenomenon such as
the delivery of essential iron by the transferrin–transferrin receptor system occurs at 1% efficiency across the mouse-human species
barrier35. Some of these complications may be mitigated by providing human growth factors36 or human stromal elements37, or by
orthotopic transplantion rather than transplantation under the skin
or kidney capsule38. Indeed, when the species barrier is avoided by
transplanting mouse tumor cells into fully histocompatible recipient
mice, the efficiency of engraftment can be dramatically improved39–41.
Yet, encouragingly, it is often still possible to show preferential
engraftment of selected tumor cells for certain mouse leukemias40–42,
for mouse breast cancers43–45 and for mouse squamous cell carcinomas10,46. Preferential engraftment of potential CSCs was not observed
in some other mouse leukemia models, however, leading to the conclusion in the latter models that CSCs do not exist39,47.
nature medicine VOLUME 17 | NUMBER 3 | MARCH 2011
Although the cross-species barrier seems to affect the outcome
quantitatively but often not qualitatively, this may not be true for the
technique of transplantation. As pointed out by Morrison and his
colleagues, transplantation of any stem cell can reveal the potential
of the stem cell under the particular assay conditions, but it cannot
reveal the actual fate of the transplanted cell in its original tissue or
tumor31 . This may seem trivial, but multiple well-documented examples illustrate the dramatic change in stem cell behavior induced by
transplantation. For instance, quiescent hair bulge stem cells will generate only hair, but not epidermis or sebaceous glands, as assessed by
Cre-mediated genetic lineage tracing. Upon transplantation, however,
the same cells will readily generate all three components of the mouse
epidermis48. A noteworthy recent example of stem cell plasticity is
provided by Barrandon and his colleagues, who show that endodermal
thymic epithelial cells can adopt the fate of ectodermal hair follicle
stem cells when exposed to an inductive skin microenvironment49.
At present, no direct evidence exists that unmanipulated solid
tumors harbor CSCs, or, in other words, cells with self-renewing
properties that fuel long-term tumor expansion. The radiolabeling
techniques that historically allowed a primitive version of stem and
progenitor cell tracing in bone marrow and leukemias are no longer
ethically acceptable in humans, as they involve exposure to radiation.
To complement the xenotransplantation assay, it is of central importance that assays be developed that can show the functional presence
of CSCs within the primary tumor. The development of such assays
that would avoid cell isolation and transplantation may require the
identification of single, definitive marker genes for CSCs. Based on
such markers, knock-in mouse models or viral-tagging strategies may
be developed to allow genetic lineage tracing. It is currently not obvious how such technologies can be developed to allow the definitive
identification in situ of CSCs in humans.
What are bona fide CSC markers? The original studies on leukemia
stem cells rested heavily on the well-understood stem-progenitor
hierarchy of healthy bone marrow. Markers such as CD34 and CD38
had been extensively validated in the identification of normal hemato­
poietic stem cells and were obvious choices for the definition of
­leukemia stem cells22,23. Unfortunately, stem cells and the developmental hierarchy are poorly characterized in most tissues that develop solid
cancers. As a consequence, few if any definitive stem cell markers are
available to the CSC researcher of solid tumors. Indeed, Al-Hajj et al.24
chose the CD24 and CD44 marker combination, as these markers
showed heterogeneous expression in breast tumors, and not because
they were known to define breast epithelial stem cells. Another
commonly used marker in CSC studies, CD133 (also known as prominin), has been implied as a normal stem cell marker, but recent
evidence has revealed that it is widely expressed in many organs 50.
Probably as a consequence, there has been considerable disagreement over the usefulness and reproducibility of particular ­markers
for different tumor types. For instance, CD133 was used in the ­initial
studies on colon cancer27,28 and brain cancer CSCs25,51 but was contested in subsequent studies on colon26,50 and brain cancer52–55.
Similarly conflicting results were reported with the marker CD271 in
melanoma32,56. Morrison and his colleagues recently pointed out an
additional complication30,32; they showed for a large series of popular
CSC markers that sorted marker-positive and marker-negative populations readily regenerate the original pattern of marker expression.
The sobering conclusion is that current CSC markers (Table 1) are
primarily chosen as robust, heterogeneously expressed FACS markers
that allow the faithful sorting of marker-positive and marker-­negative
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review
© 2011 Nature America, Inc. All rights reserved.
populations; however, they are not selected on the basis of a deep
understanding of the underlying stem cell biology of the pertinent
tissue from which the cancer originates.
Validation of important premises and promises of the CSC concept
Are CSCs rare? The original data on AML showed that CSCs in this
tumor type were exceedingly rare22,23. This has led to the commonly
held premise that CSCs should always be rare. The inverse logic states
that a tumor containing high numbers of transplantable cells does not
adhere to the CSC model. Transplantation of sorted populations of
human CSCs typically involves the injection of thousands of cells into
a single mouse to result in tumor growth. It has been reasoned—but
never proven—that subsequent tumor growth indicates the presence of a single CSC in the injected cell sample. On the basis of this
premise, CSC frequency is essentially a calculated value. Strasser and
his colleagues reported in 2007 (ref. 39) that leukemias of mouse origin transplanted into histocompatible recipients showed a very high
frequency of CSCs of at least one in ten (ref. 39). They suggested that
the low frequency of tumor-initiating cells observed in xenotransplantation studies may only reflect the limited ability of human tumor cells
to adapt to growth in a xenoenvironment (as discussed above), and
presented these findings as evidence against the CSC concept. Similar
observations of high-efficiency engraftment of mouse leukemia cells
were reported, for instance, by Williams et al.47 in 2007.
Studies on human melanoma provide an illustration of the complexity of determining CSC frequency. In an initial study, a frequency
for CSCs in human melanoma of <1 per million cells was reported57.
Morrison and his colleagues did an insightful experiment in which
they showed that the apparent frequency of tumor-initiating cells
in the same human tumor type was strongly dependent on experimental xenograft conditions. Sequential adaptations to their original
assay led to a protocol in which one of every four unsorted, singly
transplanted human melanoma cells could grow out into a tumor, an
improvement of more than 105-fold over the originally determined
frequency30. Boiko et al.56 provided an additional twist to this story.
They used CD271 as a marker and reported a melanoma CSC frequency between 2.5% and 41%. Even more recently, this observation
was again challenged by Morrison and his colleagues; they showed
that CD271+ and CD271− single primary melanoma cells from similar
subjects as used by Boiko et al.56 can both initiate tumors at frequencies of at least one in four30.
It should be concluded that the frequency at which CSCs are present
within a tumor may be irrelevant to deciding if a tumor adheres to the
CSC model. But when CSCs compose the bulk of a tumor, it becomes
less relevant to distinguishing those cells from the bulk population of
cancer cells when considering targeted therapy.
Are CSCs dormant? Transient and long-term quiescence, the ­latter
also termed dormancy, are generally believed to be fundamental
attributes of adult stem cells58,59. On the basis of this premise, stem
cells are often identified by their propensity to retain DNA labels
much longer than their rapidly proliferating offspring. Dormancy may
be a crucial mechanism for the resistance of CSCs to anti-­proliferative
chemotherapy. Moreover, if indeed CSCs occur in a dormant state,
this would explain the appearance of local recurrence or distant
metastasis after long lag periods.
Most likely because of technical challenges, the existence of dormant
CSCs in human tumors has not been directly explored. It is actually
unknown whether CSCs that are dormant in a primary tumor can be
detected by the xenograft assay as it is currently performed. Indeed,
316
Table 1 Surface markers used for the identification of CSCs
Marker
Expression in healthy tissue
CD19
CD20
CD24
Broadly on B lymphocytes
B cell malignancies
Broadly on B lymphocytes
Melanoma
Broadly on B cells; neuroblasts Pancreas/lung cancer, negative
on breast cancer
Hematopoietic and endothelial Hematopoietic malignancies
progenitors
Multiple stages of B and T cells Negative on AML
Broadly on many tissues
Breast/liver/head and neck/
pancreas cancer
T cells, neurons
Liver cancer
Proliferative cells in multiple
Brain/colorectal/lung/liver
organs
cancer
Panepithelial marker
Colorectal cancer, pancreatic
cancer
Keratinocyte progenitors
Melanoma
CD34
CD38
CD44
CD90
CD133
EpCAM/ESA
ABCB5
Marks cancer stem cells in
ABCB5, ATP-binding cassette transporter B5; EpCAM, epithelial cell adhesion
molecule; ESA, epithelial-specific antigen. Table is adapted from ref. 29.
Al Hajj et al.24 did not note cell cycle differences between tumorigenic
and nontumorigenic breast cancer cells. The xenograft is typically read
within months after transplantation, which may restrict CSC detection to only the most robustly proliferating cells. It is notable that
increasing numbers of tumors are scored when transplanted mice are
monitored over longer time periods30. In an indirect attempt to probe
dormancy of breast CSCs, Di Fiore and his colleagues derived a gene
signature for normal quiescent mammary gland stem cells present
within cultured mammospheres on the basis of their ability to retain
the lipophilic dye PKH26. When applied to breast cancers, this gene
signature correlated with CSC behavior60. The definitive identification
of quiescent CSCs in primary tumors will—at the least—require adaptations to current assay conditions, ideally allowing the transplantation
of a single candidate CSC with a defined quiescence status.
Are CSCs therapy resistant? It is often suggested that CSCs are resistant to therapy in the same way that normal stem cells are protected
against insult; these protections include, for example, mechanisms
such as quiescence, expression of ABC drug pumps, high expression of antiapoptotic proteins and resistance to DNA damage61. Some
groups have started to explore if CSCs are indeed more resistant to
therapy than their progeny. For instance, CD133-expressing glioma
cells survive ionizing radiation better relative to CD133− tumor cells51.
CD44highCD24low breast cancer CSCs appear intrinsically resistant to
conventional chemotherapy62 and ionizing radiation63. And chronic
myeloid leukemia (CML) is sustained by leukemic stem cells that are
relatively resistant to the drug imatinib64,65. Nevertheless, the phenomenon of intrinsically therapy-resistant CSCs cannot be generalized, as, for instance, the undifferentiated cells that drive testicular
germ cell tumors are more sensitive to radiation or cisplatin therapy
than their differentiated cellular progeny66. Tumor cells that escape
therapy, however, may not be endowed with intrinsic therapy resistance; rather, they may simply be the stochastic ‘winners’ of the tumor
cell–killing process. On the contrary, when intrinsic differences in
the sensitivity of cancer cells to therapy do exist, these may also be
determined genetically rather than by epigenetic differences67.
Recent clinical studies have begun to monitor the prevalence of CSCs
during chemotherapy. Li et al.62 studied subjects undergoing neoadjuvant chemotherapy. Exactly as predicted by the CSC concept, the
cells without CSC markers were killed by the chemotherapy, resulting
in tumor regression, but cells with CSC markers appeared ­resistant,
VOLUME 17 | NUMBER 3 | MARCH 2011 nature medicine
as their relative numbers increased in the
residual tumor. Along these lines, Dick and his
colleagues identified leukemia-initiating cells
in T-lineage acute lymphoblastic leukemia
(T-ALL) disease and relapse and showed that
leukemia-initiating cells persist following dexamethasone treatment in high-risk T-ALL68.
Clearly, more clinical studies are required
to assess how responses to therapy correlate
with CSC biomarkers. In particular, it will be
of interest to determine if CSC markers correlate better with clinical outcomes than bulk
tumor properties do.
Cancer stem cell
Epithelium
1
2
Benign
lesion
Stem cell
(progenitor or
differentiated cell)
Malignant
tumor
Malignant clone
Metastatic
cell
3
Blood
vessel
4
Are CSCs responsible for metastasis and
relapse? Directly related to the therapy resistance of CSCs is the hypothetical causal role
of CSCs in tumor relapse, be it at local or at distant sites. This issue is
very difficult to address experimentally. An extreme interpretation of
the xenograft assay is that it actually scores cells that are particularly
good at colonizing foreign sites; it would thus identify these cells as
metastasis ‘champions’.
Some studies have attempted to address whether CSCs are involved
in metastasis. In the invasive front of pancreatic tumors, a distinct
subpopulation of CD133+CXCR4+ cancer stem cells was identified
that determines the metastatic phenotype of the individual tumor69.
Tumorigenic pancreas carcinoma cell lines were then used to show
that depletion of CD133+CXCR4+ cells virtually abrogated the metastatic potential of the cell lines without affecting their tumorigenic
potential69. These results can be interpreted to suggest that a subset
of CSCs is responsible for metastasis. Along these lines, tumorigenic
CD44+CD24−/low cells are readily detectable in metastatic pleural fluid
in subjects with breast cancer; in addition, breast cancer cells found in
bone marrow show a putative breast CSC phenotype24,70. In sum, studies
that suggest a role of CSCs in relapse are very limited in number,
and they largely provide indirect evidence for this notion. Additional
experimental approaches will need to be developed before it can be
concluded that CSCs are responsible for local relapse and metastasis.
nature medicine VOLUME 17 | NUMBER 3 | MARCH 2011
Katie Vicari
Figure 2 A theoretical synthesis of the clonal
evolution and CSC concepts. Top to bottom:
clonal evolution drives tumor progression. (1) The first oncogenic mutation (lightning
arrow) occurs in a stem cell (or, alternatively,
in a progenitor or even a differentiated cell) of
a healthy epithelium, resulting in the growth
of a genetically homogeneous benign lesion.
(2) The second hit targets one of the cells in
the benign lesion, which leads to the growth
of a more malignant and invasive clone within
the primary tumor. (3) A third hit in a cell
within the malignant subclone causes further
transformation, visualized as entry into a blood
vessel for distant metastasis. Genetically
independent subclones can coexist within the
tumor. (4) A final mutational hit leads to tumor
being entirely taken over by cells that behave as
cancer stem cells. Shown, left to right: at each
stage of this clonal evolution process, tumors
and subclones within tumors contain some cells
that behave as CSCs. The final hit (4) causes
all cells to behave as CSCs, rendering the CSC
concept meaningless at this stage.
Clonal evolution
© 2011 Nature America, Inc. All rights reserved.
review
Are CSCs potential targets of new therapeutic strategies? The CSC
concept promises the development of therapeutic strategies beyond
traditional antiproliferative agents. Potential approaches to kill CSCs
may exploit the survival mechanisms (such as dormancy) of these
stem-like cells. As discussed above, the existence of dormant CSCs in
human tumors has not been directly explored. Nevertheless, Ishikawa
and his colleagues have recently provided evidence for the presence
of quiescent AML stem cells within the bone marrow of mice transplanted with human AML cells71. The quiescent state of these cells
could be broken: granulocyte colony–stimulating factor treatment
induced cell cycle entry and increased the sensitivity of the AML stem
cells to chemotherapy. Moreover, cultured CD34+ stem and progenitor cells isolated from subjects positive for BCR-ABL1 (breakpoint
cluster region–c-abl oncogene-1, non-receptor kinase) CML contain
quiescent cells that are resistant to imatinib, a small molecule targeting the constitutively active BCR-ABL kinase in CML72. Indeed, most
people with CML respond very well to imatinib. Nevertheless, even
people showing apparently complete responses to treatment are not
cured, as discontinuation of imatinib treatment frequently leads to
relapse of the disease. This may be due to in vivo equivalents of the
imatinib-resistant, quiescent CML stem cells observed in culture72.
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© 2011 Nature America, Inc. All rights reserved.
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Other avenues of targeting CSCs may not survive close scrutiny.
The markers that have been used so far to define CSCs constitute
unlikely candidates for antibody therapy given that they are usually
broadly expressed in healthy tissue. Moreover, in many cases, CSCs
can only be identified by marker combinations; this obviates their use
as therapy targets. Also, the oncogenic changes in CSCs do not represent unique vulnerabilities of these cells relative to their offspring, as
the oncogenic mutation is present in every cell of the CSC hierarchy.
In the case of the oncogenic BCR-ABL1 translocation in CML, the
CSCs actually seem less sensitive to the targeting agent imatinib72
than the bulk of the tumor. An opportunistic approach, unbiased by
theory, to uncover known or new compounds that target CSCs will
involve high-throughput screening. To that end, CSCs will have to
be characterized in great depth and assays with high specificity will
need to be developed.
If CSC-targeting therapeutic strategies will reach the clinic, new
clinical trial designs will have to be developed to assess the efficacy of
these therapies. Tumor regression, a currently used clinical endpoint,
will be inadequate when CSCs constitute only the minority of the cells
within a solid tumor. Indicators of effective stem cell targeting will
need to be developed and agreed on with the regulatory authorities.
Epilogue: are CSCs and clonal evolution mutually exclusive?
To date, the CSC field has treated tumors as genetically homogeneous
entities, by and large ignoring the fact that the observed tumor heterogeneity may result from underlying genetic differences. However, it is
well known that most solid tumors show extensive genomic instability73. Moreover, genetic defects in a large variety of molecules that
are involved in the maintenance of the integrity of the genome are
well-known drivers of oncogenesis74. Even in a disease like CML, so
clearly driven by stem cells, clonal evolution can be seen at work when
imatinib is administered: the malignancy becomes tumor-resistant
through the emergence of clones that carry mutations in the target
of imatinib, the BCR-ABL1 fusion gene75. And the progression of
CML into ALL blast crisis is caused by the emergence of subclones
that harbor inactivating lesions in the cyclin-dependent kinase
inhibitor 2A (CDKN2A, also known as ARF) gene in addition to the
BCR-ABL1 translocation76. The evidence for clonal evolution in the
pathogenesis of cancer is so overwhelming that it appears inescapable
that all models should be integrated with it.
The recent rapid advances in DNA sequencing are now allowing
the global analysis of genomic changes of cancer cells 77,78. These
analyses have confirmed many previously known common genetic
alterations in cancer, and they have also revealed some new common mutations as well as unexpectedly large numbers of rare mutations. As a next step, this technology can be applied to chart genetic
heterogeneity within individual tumors as well as between primary
tumors and their local recurrences and metastases79,80. It should thus
be possible to map, in both space and time, the genetic evolution
of a tumor. Two recent studies on pancreatic cancer have done just
that81,82, reporting that genetic heterogeneity exists within primary
tumors and among metastasis-initiating cells, and that metastasic
behavior requires driver mutations beyond those needed within
primary tumors. The analysis even allowed a reconstruction of the
timeline of tumor progression82.
Whereas these genetic analyses focus primarily on ‘hard-wired’
DNA changes in the genome of the cancer cell, the CSC concept has
introduced another valuable perspective to the biology of the cancer
cell. Even though cancer cells may genetically be the same, they may
still occupy different positions in a differentiation hierarchy, thereby
318
mirroring the physiological hierarchy of the tissue of origin. Moreover,
a single tumor may contain multiple cancer stem cell clones that are
genetically distinct, but that will always have a common ancestor: the
cell of origin of the tumor that sustained the first oncogenic mutation. Thus, the assays and insights that have been brought into cancer
research by the CSC field may be layered over the genetic data (Fig. 2).
In conjunction with a successful synthesis with insights derived from
clonal evolution, the CSC concept may fulfill its promise.
Acknowledgments
The author thanks E. Verheyen for discussions.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
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