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IJC
International Journal of Cancer
Complexity of cancer stem cells
Eiji Sugihara1,2 and Hideyuki Saya1,2
2
Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan
Japan Science and Technology Agency, CREST, Tokyo, Japan
Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy-based model in which cancer stem cells
(CSCs) have the ability both to self-renew and to give rise to differentiated tumor cells and are responsible for the overall
organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of
normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of
human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel
therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell
properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this
review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have
revealed the complexity of these cells and their therapeutic implications.
Identification of Cancer Stem Cells
Many tumors consist of phenotypically and functionally
heterogeneous cancer cells. For many years, such heterogeneity
was considered to be explained by the stochastic (clonal evolution) model1 (Fig. 1a), which is based on the notion that all
cancer cells possess tumorigenic potential and can develop
tumor dependent on genetic and/or epigenetic changes. However, more recent studies have suggested a new paradigm: that
tumors show hierarchy, with a subpopulation of cancer cells
having a tumorigenic potential much greater than that of other
cancer cells.2 This subpopulation of cells at the top of the hierarchy comprises cancer stem cells (CSCs), and tumors are
thus thought to manifest a hierarchical organization—consisting of stem cells, progenitors and differentiated cells—similar
to that of normal tissues (Fig. 1a). A CSC has been defined3
as ‘‘a cell within a tumor that possess[es] the capacity to selfrenew and to cause the heterogeneous lineages of cancer cells
Key words: cancer stem cell, cell of origin, plasticity, clonal diversity,
CSC-targeted therapy
Abbreviations: ALDH: aldehyde dehydrogenase; AML: acute
myeloid leukemia; Ara-C: arabinosylcytosine; CML: chronic myeloid
leukemia; CSC: cancer stem cell; EpCAM: epithelial cell adhesion
molecule; GFP: green fluorescent protein; HSC: hematopoietic stem
cell; iCSC: induced cancer stem cell; iPSC: induced pluripotent stem
cell; pre-B-ALL: pre-B acute lymphoblastic leukemia
Grant sponsor: Ministry of Education, Culture, Sports, Science, and
Technology of Japan
DOI: 10.1002/ijc.27961
History: Received 8 Jul 2012; Accepted 15 Nov 2012; Online 26 Nov
2012
Correspondence to: Hideyuki Saya, Division of Gene Regulation,
Institute for Advanced Medical Research, School of Medicine, Keio
University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan,
Tel.: þ81-3-5363-3982, Fax: þ81-3-5363-3982, E-mail: [email protected]
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that comprise the tumor.’’ Similarly, the term ‘‘tumor-initiating
cell’’ has been used to describe a cell with the potential to initiate a tumor. If this term is used to refer to the subpopulation
of cells within an established tumor that gives rise to a new
tumor when transplanted, then tumor-initiating cells are essentially functionally equivalent to CSCs.
In1963, Bruce and Van Der Gaag first documented that
there is a small number of mouse lymphoma cells capable to
form colonies in spleen using same transplantation method
of normal hematopoietic cells.4 Although the existence of
CSCs had been subsequently suggested in mouse myeloma
and acute myeloid leukemia (AML) in earlier studies,5–7
human CSCs were first prospectively identified for AML in
1997 as the CD34þCD38– cell subpopulation by xenotransplantation into immunocompromised (NOD/SCID) mice.8
Since this initial discovery, CSCs have been identified by flow
cytometry-based prospective analyses in a wide variety of
human cancers including those of the breast (CD44þCD24–/
low
cells),9 brain (CD133þ cells),10 prostate (CD44þCD24–
cells),11,12 colon (CD133þ and EpCAMhiCD44þ cells)13 and
pancreas (CD44þCD24þESAþ cells).14 These cell subpopulations are relatively rare and form secondary tumors that recapitulate the heterogeneity and pathology of the original
tumor when transplanted into experimental animals. Although
CSC-markers such as CD44 and CD133 are detected in CSCs
for a broad range of tumors, tissue-specific CSC markers have
been also reported. For instance, ABCB5, an ABC-transporter
protein, has been shown as a CSC-marker for human melanoma and is assumed to be involved in transport of melanin
as well as chemo-therapeutic agents.15,16 A cell adhesion receptor, Integrin a2b1, was reported as a marker for prostate CSCs
in addition to the above markers and implicates prostate
cancer progression.17,18
Some recent studies, however, have revealed that cell surface markers of CSCs are not restricted to those identified
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Complexity of cancer stem cells
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Figure 1. Two models for cancer heterogeneity and methods for isolation of CSCs. (a) The stochastic model and cancer stem cell (CSC)
model are the two major models to account for heterogeneity of tumors. In the stochastic model (left), all tumor cells possess tumorigenic
activity and are the product of clonal evolution through the acquisition of genetic mutations and epigenetic changes. In the CSC model
(right), only a subpopulation of tumor cells possesses high tumorigenic activity, with these cells representing the top of a hierarchical
organization similar to that of normal tissue. CSCs (red) both undergo self-renewal and generate differentiated progeny cells (pink and
yellow) that are no longer able to form tumors. (b) Four methods for the isolation and enrichment of CSCs. CSCs can be prospectively
isolated by cell sorting based on a combination of cell surface markers (I). Given that they possess self-renewal activity, CSCs for leukemia
form colonies in methylcellulose medium whereas those for solid tumors form spheroids in serum-free medium under nonadherent
conditions (II). When tumor cells are exposed to Hoechst 33342 and subjected to ultraviolet irradiation followed by cell sorting, CSCs are
enriched in a cell subpopulation corresponding to a side area (red box) of a flow cytometric plot of Hoechst blue versus red fluorescence
(III). CSCs have high ALDH activity and therefore can be isolated by cell sorting on the basis of their green fluorescence signal when treated
with Bodipy-conjugated aminoacetaldehyde (IV).
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prospectively. For instance, CD133– cells in brain tumors as
well as CD34þCD38þ cells in AML were also shown to possess high tumorigenic activity.19–21 These studies indicate that
CSCs might not be definable simply on the basis of the
expression of cell surface markers, and that methods based
on such markers have limitations with regard to the identification and purification of CSCs.
Methods for Isolation of CSCs
In addition to prospective cell sorting (Fig. 1bI), several
methods have been developed to distinguish CSCs on the
basis of their functional properties.
Colony or spheroid formation assays have thus been
used to enrich CSCs on the basis of their self-renewal activity
(Fig. 1bII). Colony formation assays with methylcellulose
medium were originally applied to analyze the self-renewal and
multilineage differentiation of immature hematopoietic cells.
Consecutive repetition of such assays (replating) can result in
the isolation of subpopulations of hematopoietic cancer cells
with self-renewal ability and also allows determination of
whether genes of interest have the potential to confer selfrenewal activity in non-transformed hematopoietic cells.22,23
Spheroid formation assays were performed originally to isolate
neural stem or progenitor cells and are now often used to evaluate stem-like characteristics of tumor cells. Suspension culture
in medium not containing serum but supplemented with basic
fibroblast growth factor and epidermal growth factor allows the
isolation of tumor cells with self-renewal capacity.24
The side-population assay is often used for isolation of
normal or tumor cells with the ability to exclude certain
drugs (Fig. 1bIII). Similar to normal tissue stem cells, a subpopulation of tumor cells expresses drug transporters, such
as ABCG2, and is thereby thought to be rendered resistant to
anticancer drugs.25 In this assay, tumor cells are treated with
the DNA binding dye Hoechst 33342, which is then subjected
to fluorescence excitation by ultraviolet irradiation. A small
cell subpopulation with no fluorescence emission can be
detected in a side position on the flow cytometric plot. This
‘‘side population’’ has been shown to possess tumorigenic
activity greater than that of other cells after cell sorting and
transplantation.26,27 The side-population assay may therefore
be useful for the identification of CSCs in some tumor types
for which CSC markers have not been discovered.
A method based on the enzymatic activity of aldehyde
dehydrogenase (ALDH) is also widely used to identify and
evaluate CSCs (Fig. 1bIV).28–30 ALDH catalyzes the irreversible conversion of retinaldehyde to retinoic acid and thereby
contributes to cell proliferation and differentiation.31 High
ALDH activity in CSCs is shown to be related to their resistance to chemotherapy.32 CSCs isolated from breast and
ovarian cancers on the basis of both ALDH activity and previously identified CSC markers manifested higher growth and
tumorigenic potential than did those isolated on the basis of
cell surface markers alone.33,34
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Although these methods have been developed based on
the established methods for isolating normal stem cells, some
problems are still argued. For instance, because the sidepopulation assay uses cytotoxic dye (Hoechst 33342), this
method potentially causes problems to judge tumorigenicity
and cell viability between cells in side population and the
other population that imports this dye.35 In addition, each
method cannot cover whole CSCs.36 The combination of
CSC markers and functional assays might thus provide the
basis for more robust and powerful strategies for the identification of bona fide CSCs.
Cells of Origin for CSCs
The cells of origin for CSCs are assumed to be normal stem
cells in most instances, given that the biological characteristics of normal stem cells, including the abilities to undergo
self-renewal and to generate differentiated progenies, are similar to those of CSCs. In some cancer types, CSCs are
enriched in cell subpopulations expressing normal stem cell
markers. Furthermore, stem cells live much longer than differentiated cells, which can provide time for accumulation of
mutations sufficient for transforming them.
In solid tumors, the origin of CSCs has been debated
because of difficulty to monitor the process from the origin
to CSCs. Recently, Clevers and coworkers developed the ingenious system that allows tracing adenoma initiated from
intestinal stem cell marker-positive (Lgr5þ) cells and further
re-traces Lgr5þ cells in established adenoma.37 They found
that low population of Lgr5þ cells in adenoma generates
Lgr5þ cells and all other adenoma cell types.37 This finding
experimentally proved that intestinal stem cells are the cell of
origin for CSCs in intestinal adenoma.
In hematopoietic tumors, studies on the origin for CSCs
have been more extensively performed than those in solid
tumors due to sufficient markers to isolate a variety of cell
types and well-established transplantation methods. In the
case of human AML, hematopoietic stem cells (HSCs) have
been also considered to be the cells of origin for AML stem
cells because the markers identified for the latter are identical
to those for the former.8,38 On the other hand, some studies
have suggested that the cells of origin for mouse AML stem
cells are not only restricted to HSCs but also include progenitor cells committed to the myeloid lineage using bone marrow transplantation (BMT) model.22,39
In the case of B acute lymphoblastic leukemia (B-ALL),
although CSCs were found in cell subpopulations expressing
HSC markers or B cell markers,40–44 the cells of origin for
B-ALL stem cells remain unclear. We recently established a
Myc-induced mouse model of pre-B-ALL based on BMT
model.45 We found that CSCs in this model were restricted
to cells positive for B cell markers (B220þ and CD19þ). We
further found that Myc-transduced HSCs showed tumorigenicity higher than that of corresponding progenitor or committed cells, suggesting that the cells of origin for our mouse
model of pre-B-ALL are HSCs. Thus, despite the cells of
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Complexity of cancer stem cells
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cells induced from Ink4a/Arf-null progenitor B cells manifested extensive apoptosis after treatment with Nutlin-3, an
inhibitor of the p53-targeting ubiquitin ligase Mdm2, whereas
the tumor cells induced from wild-type HSCs did not
undergo Nutlin-3-induced apoptosis, likely because of p53
mutation in these cells (Fig. 2b). These results suggested that
CSCs derived from different cells of origin are differentially
sensitive to Ara-C and Nutlin-3. The human gene locus
of Ink4a and Arf is frequently deleted in malignant types of
B-ALL such as Philadelphia chromosome-positive and recurrent ALL.47 Analysis of Ink4a and Arf expression in such
types of ALL cells might thus in the future provide information relevant to drug selection at the time of diagnosis. The
Mdm2 inhibitor is currently under evaluation for leukemia
treatment in clinical trials.
Thus, although it is often difficult to identify the origin of
human CSCs, mouse cancer models have the potential to
provide important insight into such cells of origin.
Plasticity in the CSC Model
Figure 2. Different cells of origin result in distinct phenotypes for
pre-B-ALL stem cells. (a) HSCs and committed progenitor B cells
are potential cells of origin for a Myc-induced model of pre-B-ALL.
HSC-derived ALL stem cells are sensitive to Ara-C but resistant to
Nutlin-3 (an Mdm2 inhibitor), whereas progenitor B cell-derived
ALL stem cells are sensitive to Nutlin-3 but resistant to Ara-C. (b)
Molecular mechanisms underlying the differential sensitivity of the
two types of ALL stem cells to Nutlin-3. HSC-derived Myc-induced
ALL stem cells constantly acquire p53 mutation, which is unable to
induce apoptosis even if Nutlin-3 inhibits Mdm2. In contrast,
progenitor B cell-derived ALL stem cells are null for Ink4a/Arf but
express wild-type p53, with the result that stabilization of p53 by
Nutlin-3 leads to the induction of apoptosis.
origin being HSCs, B cell marker-positive cells become preB-ALL stem cells during the course of the tumor development, indicating that differentiation status may differ
between cells of origin and CSCs. Moreover, committed progenitor B cells were also able to serve effectively as cells of
origin in this model in the absence of Ink4a and Arf
(Fig. 2a), the expression of which is maintained at low levels
in HSCs.
It is important to identify the cells of origin for tumors in
order to understand the context in which tumor cells
develop, which in turn may provide useful information for
preventive and therapeutic strategies in the clinical setting.46
In our mouse pre-B-ALL model, we compared the therapeutic sensitivity of CSC-enriched cell preparations derived from
tumors with different cells of origin. We found that the
tumor cells induced from Ink4a/Arf-null progenitor B cells
were more resistant to Ara-C treatment compared with those
induced from wild-type HSCs (Fig. 2a). In contrast, tumor
Similar to those for other cancers, CSCs for human melanoma are considered to constitute a rare subpopulation and
have been identified by prospective isolation based on cell
surface markers (including CD20, CD133, ABCG2, ABCB5
and CD271).16,48–50 However, Morrison and coworkers have
questioned whether implantation of human melanoma cells
into NOD/SCID mice might lead to underestimation of the
frequency and tumorigenicity of isolated CSCs with defined
markers, given that these animals still possess natural killer T
cells even though they lack B and T cells of the adaptive
immune system. These researchers therefore used NOD/
SCID/IL-2Rc-null (NSG or NOG) mice, which lack natural
killer T cells and in which both the innate and adaptive
immune systems are therefore compromised,51 to reevaluate
the frequency of cells with tumor-initiating ability and that
express various cell surface markers including previously
identified CSC markers.52,53 They found that 28% of individual melanoma cells tested was able to give rise to melanoma
reminiscent of the original tumor, and that cell subpopulations with any cell surface markers examined could initiate
melanoma irrespective of the patient from which they were
derived. These results suggested that a large proportion of
melanoma cells possess tumorigenic activity and that melanoma does not therefore conform to the hierarchical CSC
model.53
Around the same time, Herlyn and coworkers proposed a
new concept related to the CSC model for melanoma. They
focused on the histone H3 lysine-4 demethylase JARID1B,
which had been found to be expressed in a small subpopulation of human malignant melanoma.54 To monitor the
dynamics of melanoma cells expressing JARID1B, they
manipulated the cells to express the gene for green fluorescent protein (GFP) under the control of the JARID1B promoter and then assessed the proliferation and tumorigenic
activities of GFPþ and GFP– cells. They found that GFPþ
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Figure 3. New concepts of plasticity and clonal diversity in the CSC model. (a) The dynamic stemness model is based on the conventional
CSC model but introduces the concept of dynamic stemness, whereby a non-CSC can acquire stemness and become a CSC. CSCs and nonCSCs thus exhibit plasticity (reversibility) of stemness as indicated by red arrows. (b) The CSC niche is required for maintenance of stem
cell properties and is considered to consist of many factors including stromal cells, blood vessels, extracellular matrix (ECM), growth
factors, cytokines and hypoxia. Exposure of non-CSCs to niche factors may result in their acquisition of stem cell properties. The EMT
may be one process by which non-CSCs acquire stemness. (c) A combination of the CSC model and the stochastic (clonal evolution)
model has been proposed to account for clonal diversity of CSCs. Each CSC clone is thought to evolve through the acquisition of genetic
mutations. Phenotypically and functionally distinct major clones and minor clones may exist in a tumor. Each clone is organized into a
hierarchical structure.
(JARID1Bþ) cells cycled only slowly, possessed a high selfrenewal potential, generated GFP– (JARID1B–) cells and were
enriched in the side population after staining with Hoechst
33342.55 Furthermore, knockdown of JARID1B expression in
melanoma cells by RNA interference resulted in a marked
reduction in tumorigenic activity, suggesting that JARID1Bþ
cells have characteristics as CSCs. Of note, the researchers
also found that JARID1B– cells gave rise to JARID1Bþ cells
during long-term culture and that JARID1B– cells showed
high tumorigenic activity when injected into immunocompromised mice.55 The JARID1B– cells were therefore able to
gain JARID1B expression and to acquire stem cell properties
under certain conditions. This dynamic transition of stem
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cell properties, termed ‘‘dynamic stemness’’, might account
for the concern as to whether melanoma follows the CSC
model. Whereas the CSC model incorporates a stringent hierarchy in which CSCs generate non-CSCs in a unidirectional
manner, the notion of dynamic stemness allows non-CSCs to
gain stemness and to become CSCs, indicative of phenotypic
plasticity between CSCs and non-CSCs (Fig. 3a).
Such phenotypic plasticity may result from dynamic epigenetic changes regulated by signals from the stem cell niche,
a microenvironment that is crucial for the maintenance of
stem cell properties (self-renewal and an undifferentiated
state) and comprises many elements including stromal cells,
blood vessels, extracellular matrix, growth factors and
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cytokines as well as hypoxia (Fig. 3b).56 The epithelial to
mesenchymal transition (EMT) may be one process by which
non-CSCs acquire stemness and may confer phenotypic plasticity on tumor organization in breast cancer (Fig. 3b).57 In
melanoma, given that JARID1B is shown to be a target of
hypoxia-inducible factor 1 (HIF1),58 hypoxia may up-regulate
JARID1B expression and thereby give rise to a pattern of
histone modification that underlies a change in gene transcription required for stem cell function.
It has yet to be clarified how tumors can acquire plasticity
between CSCs and non-CSCs unlike rigid hierarchy between
normal stem cells and differentiated cells. Our speculation is
that plasticity can be acquired during the course of therapies.
Many chemical therapeutic agents and irradiations drastically
destroy environmental status in the tumor tissues and often
cause stress conditions such as reactive oxygen species and/or
hypoxia, leading to formation of de novo microenvironment
(niche) to evoke stem cell properties to non-CSCs similar to
EMT (Fig. 3b). Once non-CSCs acquire the adaptive ability
to become CSCs, tumors can exploit this plasticity in order
to escape and survive from further therapies. Thus, it might
be important to create new strategies to prevent the formation of de novo niche in addition to conventional treatments.
Induced Cancer Stem Cells
Induced pluripotent stem cells (iPSCs) were first established
in 2006 by the ectopic expression of defined factors in mouse
embryonic or tail-tip fibroblasts.59 Research on iPSCs has
had a large impact on various biological fields including
cancer research, given that the process underlying the reprogramming of somatic cells is similar to that responsible for
cancer initiation. The establishment of iPSCs inspired us to
generate induced cancer stem cells (iCSCs) from somatic cells
of various tissues through expression of a set of defined
factors. iCSCs were originally termed as the somatic cells that
have embryonic stem cell-like genes expression with high
tumorigenicity and were induced by three factors (Ras, IjB,
c-Myc).60 We made some minor modifications to the original
concept of iCSCs and proposed that iCSCs are tumor-initiating cells derived from normal somatic cells by induction of
various combinations of defined factors (not only above three
factors) (Figs. 4a and 4b).
To date, iCSCs have been established from normal
somatic cells such as primary tissue stem, progenitor and differentiated cells through retroviral transduction with driver
genes such as Myc or Ras (Fig. 4a). Similar to iPSCs, such
iCSCs have the potential to undergo self-renewal and to generate differentiated progenies, and they form tumors when
transplanted into recipient mice. By using the combinations
of defined factors that reflect oncogenic events found in
human tumors, we have established several types of iCSCs
capable of forming tumors in mice, such as osteosarcoma,61
brain tumors,62 ovarian tumors,63 choriocarcinoma64 and
leukemia-lymphoma.45 Those tumors have been found to
share phenotypic and functional characteristics with the cor-
Complexity of cancer stem cells
responding human cancers (Fig. 4b). For instance, the iCSCs
that initiate osteosarcoma were established from Ink4a- and
Arf-null bone marrow stromal cells ex vivo by retroviral
transduction of c-Myc.61 These osteosarcoma-initiating iCSCs
have the capacity to undergo trilineage differentiation into
adipocytes, osteocytes and chondrocytes and are highly
tumorigenic, giving rise to metastases in multiple organs
including the lung and bone, when transplanted into syngeneic mouse recipients.61
There are several advantages to the study of iCSCs: (i)
The entire process of tumor development can be monitored
comprehensively from the cell of origin to malignant transformation. We found that tissue invasion by and migration
of brain tumor-initiating iCSCs preceded tumor mass formation, which was apparent only a few days after transplantation of the cells into the brain of recipient mice.62 This cell
migration occurred along fiber tracts and was followed by
perivascular infiltration. The cells then proliferated and
finally formed tumors with pathological characteristics similar
to those of human glioblastoma within a 4-week period.62
We were thus able to recapitulate the entire process of
gliomagenesis with the use of iCSCs. (ii) Given that iCSCderived tumors have a short latency period and tumor penetrance is 100% for all types of iCSCs examined,45,61–64 CSCs
can be readily isolated from the iCSC-derived tumors for
analysis of CSC characteristics such as tumorigenicity,
metastasis and drug resistance. For example, with the use of
ovarian cancer-initiating iCSCs, we found that prospectively
purified tumor cells positive for epithelial cell adhesion molecule (EpCAM) had a tumorigenic potential higher than that
of EpCAM– cells,63 suggesting that mouse ovarian CSCs are
present within the EpCAMþ subpopulation of tumor cells in
this model. (iii) Large numbers of CSCs can be readily
prepared from iCSC-derived tumors for examination of the
potential efficacy of anticancer drugs. In contrast, it is difficult
to perform such screening assays with CSCs of human tumors
both because of the limited number of such cells isolatable
from clinical specimens and because of related ethical considerations. (iv) Mice with tumors formed by transplanted iCSCs
can be subjected to preclinical studies of potential new drugs.
We have thus examined and confirmed the efficacy of arabinosylcytosine (Ara-C or cytarabine), a standard treatment for
hematopoietic tumors, in a mouse model of pre-B-ALL; the
effective doses and each amount of Ara-C administered were
similar to those for actual clinical protocols.45
Therapeutic Implications of CSCs
The resistance of CSCs to conventional cancer treatments
such as chemotherapeutic agents and radiation is considered
a formidable problem because remaining CSCs presumably
trigger relapse after treatment termination. Development of
new therapeutic strategies based on the CSC model has therefore become a key goal in the challenge to achieve complete
eradication of cancer. To date, four strategies have been
considered (Fig. 5).
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Figure 4. Generation and study of iCSCs. (a) iCSCs are established by the introduction of defined genes such as Myc and Ras into somatic
cells including stem, progenitor and differentiated cells of a variety of tissues. The iCSCs are able to undergo both self-renewal and
differentiation, and they form tumors when transplanted into recipient mice. The generated tumors contain CSCs, which can be purified
and subjected to further experiments. (b) Types of iCSCs that have been established to date.45,61–64 Abbreviations not defined in text:
NSC, neural stem cell; siRNA, small interfering RNA; SV40, simian virus 40; LBL, lymphoblastic lymphoma.
The first strategy is to attack CSCs directly. CSCs can be
targeted by agents that kill them specifically or that promote
their differentiation into non-CSCs, which in turn will
undergo apoptosis, senescence or terminal differentiation
(Fig. 5a). A transgenic system was used to target only CSC
marker-positive cells for cell death in a mouse model of
AML.65 This approach resulted in the eradication of all AML
cells, suggesting that CSCs are responsible for the overall cellular organization of AML. A cytotoxic antibody specific for
the cell surface molecule TIM-3, which is expressed in human
AML stem cells but not in normal HSCs, was used to
selectively attack AML stem cells and thereby to eradicate
reconstituted human AML cells in xenografted mice.66
Furthermore, a monoclonal antibody to CD47, a cell surface
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protein that protects cells from attack by phagocytes, was
found to induce phagocytosis of AML stem cells by macrophages.67,68 Such studies of antibodies that preferentially
target AML stem cells may provide more general insight into
CSC-targeted therapy based on antibodies with minimal
adverse effects. We recently showed that a splice variant form
of CD44, a well-known CSC marker, interacts with the cystine transporter subunit xCT and effectively eliminates reactive oxygen species through increased cellular synthesis of
reduced glutathione.69 This CD44 variant may play an important role in homeostasis of CSCs by limiting oxidative stress.
Indeed, ablation of CD44 or treatment with an xCT inhibitor
attenuated development of gastric cancer in mice.69 Redox
regulation is therefore a potential target for anti-CSC therapy.
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Figure 5. CSC model-based strategies for cancer therapy. (a) The
direct targeting of CSCs can result in their eradication, leaving only
non-CSCs, which eventually will undergo senescence, apoptosis or
terminal differentiation. Remaining non-CSCs are thought to be
sensitive to the conventional chemo- and radio-therapies. (b)
Promotion of the transition of CSCs from the quiescent to the
proliferative state renders them sensitive to the induction of
apoptosis by conventional anticancer drugs. (c) Targeting of the
niche for CSCs can lead to CSC differentiation and eventual
senescence, apoptosis or terminal differentiation of the resulting
non-CSCs. (d) Inhibition of the transitions from CSCs to non-CSCs
and from non-CSCs to CSCs may result in long-term dormancy of
CSCs and block the generation of new CSCs from non-CSCs,
respectively.
The second strategy for CSC eradication is to promote the
exit of CSCs from the quiescent state and their entry into the
cycling state (Fig. 5b). Quiescent AML stem cells were found
to reside around endosteal regions of bone marrow after
transplantation of human AML cells into NOG mice.70 Stimulation with granulocyte colony-stimulating factor triggered
the entry of these quiescent AML stem cells into the cell
cycle and thereby increased their sensitivity to treatment with
Ara-C. The tumor suppressor proteins PML and FOXO were
found to be critical for maintenance of the dormancy of
chronic myeloid leukemia (CML) stem cells in a mouse
model of CML.71,72 The targeting of either PML or FOXO
(or the upstream transforming growth factor-b signaling
pathway) forced quiescent CML stem cells to proliferate,
Complexity of cancer stem cells
resulting in an increased sensitivity of these cells to treatment
with imatinib,71,72 a selective tyrosine kinase inhibitor of the
BCR-ABL fusion oncoprotein. The combination of treatment
to promote the proliferation of CSCs and conventional anticancer drugs that target proliferating cancer cells is thus a
promising approach to CSC-targeted therapy.
The third strategy is to attack the CSC niche (Fig. 5c). As
mentioned in the previous section, the niche is thought to
provide a supportive environment for maintenance of CSCs
(Fig. 3b).56 Tumor blood vessels are implicated as a major
component of the vascular niche for glioblastoma CSCs.73
The vascular niche is assumed to be formed via angiogenesis
stimulated by vascular endothelial growth factor (VEGF),
which is presumably released from CSCs. Bevacizumab, a
neutralizing antibody to this growth factor, was found to
block endothelial cell migration and tube formation, resulting in suppression of glioma formation, in a xenograft
model.74 Inhibition of angiogenesis is thus a potential
approach to therapy that targets the CSC niche. The chemokine CXCL12 (SDF1) and its receptor CXCR4 have been
implicated in metastasis of breast cancer cells and in homing
of leukemic cells.75 Given that CXCR4 is expressed on both
breast CSCs and leukemic stem cells,75,76 CXCR4 antagonists
such as plerixafor might also prove effective for disruption
of the interaction between CSCs and their niche and therefore lead to a loss of stem cell properties. Further characterization of the components and molecular framework of the
CSC niche should facilitate the development of targeted
therapies.
The fourth strategy is to inhibit the transitions between
CSCs and non-CSCs (Fig. 5d). Blockade of the differentiation
of CSCs into non-CSCs is a conceptual approach to maintain
CSC dormancy and thereby to increase the effectiveness of
direct CSC-targeted therapy (Fig. 5a). The notion of blocking
the de-differentiation of non-CSCs into CSCs derives from
the concept of plasticity in the hierarchical organization of
tumors such as that incorporated into the dynamic stemness
model (Fig. 3a). Similar to normal stem cell differentiation,
the differentiation of CSCs is associated with dynamic epigenetic changes.77 Recent studies using global transcriptome
analysis and next-generation sequencing have revealed aberrant expression or mutation of genes related to epigenetic
regulation in many tumors.78,79 The formation and maintenance of CSCs are therefore thought to require epigenetic
abnormalities resulting from such aberrant expression of
mutation of epigenetic modifiers such as those responsible
for DNA methylation, histone acetylation and histone methylation.78,79 Plasticity in the CSC model might be driven by
changes in the pattern of histone modification that give rise
to changes in gene transcription, rather than by changes in
DNA methylation, which tend to be less dynamic. Although
development of inhibitors of histone-modifying enzymes is
being widely pursued, further studies are required before
novel therapeutic approaches that target the differentiation
and plasticity of CSCs and non-CSCs can be realized.
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Clonal Diversity in the CSC Model
Two independent groups recently showed that CSCs (leukemia-initiating cells) for human B-ALL with the fusion genes
BCR-ABL or ETV6-RUX1 are genetically diverse.80,81 Profiling
of DNA copy number alterations in individual leukemic cells
with the use of global SNP (single nucleotide polymorphism)
arrays or multiplex fluorescence in situ hybridization revealed
the existence of multiple genetically distinct clones that could
initiate leukemia when transferred to immunocompromised
mice. These clones are assumed to evolve by acquiring mutations in a multiple-branching manner rather than in linear
succession. Furthermore, the pattern of clones found at diagnosis was different from that at the stage of overt leukemia
or relapse, suggesting that only certain clones present at diagnosis, presumably those resistant to drug treatment and with
a high tumorigenic activity, were able to survive and evolve.
Some cases of B-ALL are thus likely to be best explained by a
combination of the CSC model and the stochastic (clonal
evolution) model (Fig. 3c).
With regard to solid tumors, CSCs for human colon cancer have also been found to consist of functionally distinct
clones.82 The authors isolated CSCs based on the property to
generate spheroid formation. Using molecular marking
approach with lentiviral vectors, at least three major clones
were found in CSCs: a clone that maintained self-renewal
activity over the long-term in serial transplants, a clone with
limited or no self-renewal activity that was able to form
tumors only in primary mouse recipients and a clone with
delayed tumorigenic activity that became apparent in secondary or tertiary recipients. Of note, the clone with long-term
self-renewal activity disseminated to bone marrow and was
alone responsible for metastasis.82 These findings thus suggest
the existence of clonal diversity that conforms to a hierarchical organization originated from CSCs, with the clones being
functionally and phenotypically distinct presumably as a
result of differential acquisition of mutations (Fig. 3c).
Although it remains to be determined whether diversity of
CSCs is common or restricted to a small number of tumor
types, the possible existence of distinct CSC clones with differential sensitivities to antitumor therapies should be kept in
mind and warrants further investigation. Characterization of
individual CSC clones may shed light on common mechanisms underlying the maintenance of stem cell properties in
all clones and thereby inform the development of new CSCtargeted therapies.
Conclusions
Ever since the first experimental identification of CSCs, the
CSC model has been a major topic of debate as a result of
uncertainties concerning the properties of these cells such as
their defining cell surface markers, frequency and plasticity.83 However, we believe that the introduction of the CSC
concept has resulted in important advances in cancer
research. For instance, the CSC-based hierarchical model
has provided a better understanding of the tumor heterogeneity. In addition, the goal of development of anticancer
drugs will change from reduction of tumor size to targeting
of subpopulations of tumor cells with a high tumorigenic
potential. Future studies of CSCs will need to expand
beyond the xenograft approach, in which cells derived from
human tumors are transplanted into immunocompromised
mice, and include the development and characterization of
mouse tumor models that recapitulate aspects of tumor heterogeneity and the microenvironment in order to provide
further insight into the complexity of CSCs observed in
actual human tumors.
Acknowledgements
The work in the authors’ laboratory was supported by grants from
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan (to E.S. and H.S.). The authors declare no potential conflicts
of interest.
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