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Tumour-initiating cells: challenges
and opportunities for anticancer
drug discovery
Bin-Bing S. Zhou*, Haiying Zhang‡, Marc Damelin*, Kenneth G. Geles*,
Justin C. Grindley* and Peter B. Dirks §
Abstract | The hypothesis that cancer is driven by tumour-initiating cells (popularly
known as cancer stem cells) has recently attracted a great deal of attention, owing to
the promise of a novel cellular target for the treatment of haematopoietic and solid
malignancies. Furthermore, it seems that tumour-initiating cells might be resistant to
many conventional cancer therapies, which might explain the limitations of these agents
in curing human malignancies. Although much work is still needed to identify and
characterize tumour-initiating cells, efforts are now being directed towards identifying
therapeutic strategies that could target these cells. This Review considers recent
advances in the cancer stem cell field, focusing on the challenges and opportunities
for anticancer drug discovery.
Self-renewal
The ability of a cell to
reproduce itself without losing
developmental potential,
characterized by cell divisions
in which differentiation
is blocked in at least one
daughter cell.
*Oncology Discovery,
Wyeth Research, 401 North
Middletown Road, Pearl
River, New York 10965, USA.
‡
Cancer Research, Abbott
Laboratories, 100 Abbott
Park Road, Abbott Park,
Illinois 60064, USA.
§
The Hospital for
Sick Children, 555 University
Avenue, Toronto, Ontario,
M4G 1X8, Canada.
Correspondence to B.-B. S. Z.
and J. C. G.
e-mails: [email protected];
[email protected]
doi:10.1038/nrd2137
Modern anticancer drug discovery began in the midtwentieth century with the observation that cytotoxic
chemotherapeutic agents could be used to target cancers
with high proliferative rates1,2. Since then, the discovery
and development of cancer therapies, initially on the
basis of empirical observations, has become increasingly
dependent on our understanding of human tumour
biology. However, despite advances that have led to the
development of new therapies, treatment options are still
limited for many types of human cancer — particularly
those with undifferentiated phenotypes, such as basal
subtype breast cancer — and prognosis remains poor.
In addition, the ability to manage tumour recurrence
and metastasis following successful initial induction of
remission continues to be a challenge.
The experimental demonstration of tumour-initiating
cells (popularly known as cancer stem cells) in several
human tumours in recent years3–10 supports tumour
hierarchy as a fundamental concept in tumour biology
and promises a new cellular target for anticancer drug
discovery. Although the cancer stem cell hypothesis was
first proposed decades ago (reviewed in Refs 11,12),
many aspects of this hypothesis remain speculative and
are still evolving. A minimal operational definition of
tumour-initiating cells is: those tumour cells that have
the ability to re-grow the tumour from which they were
isolated or identified (fIG. 1), which implies that the
tumour-initiating cells can only be defined experimentally in vivo12. More generally, tumour-initiating cells are
viewed as those cells at the apex of the tumour hierarchy
(BOX 1), which highlights the role of aberrant differentiation in tumorigenesis. Multipotency of lineage differentiation is likely to be a frequent, but not a necessary,
property of tumour-initiating cells.
The term ‘cancer stem cell’ does not imply that the
cell is derived from a normal stem cell13. Depending on
tumour type, the cells originating the tumour can be stem
cells, progenitor cells or differentiated cells, and need not
match the phenotype of the eventual tumour-initiating
cell with respect to self-renewal and differentiation capacity
(see the figure in BOX 1). Nevertheless, the origin of the
tumour-initiating cells could have implications for the
therapeutic window of the strategy that is used to target
them. For example, killing normal stem cells along with
the tumour-initiating cells could lead to a chronic loss
of normal regeneration, whereas destroying the normal
progenitor cells may be less of a long-term problem.
Another important point is that tumour-initiating cells
are not necessarily rare14,15. Moreover, the behaviour and
frequency of tumour-initiating cells could also be influenced by various environmental factors. The fundamental
concept underlying the cancer stem cell hypothesis is not
related to the origin, absolute frequency or particular
activity level (for example, proliferation rate) of these
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Destroy
TICs
?
Differentiate
?
Self
renewal
TICs
Target
niche
Tumour-initiating cell (TIC)
Niche
Tumour progenitor and
differentiated tumour cell
?
Combination
therapy
Figure 1 | The cancer stem cell hypothesis and therapeutic strategies to target
tumour-initiating cells. According to the cancer stem cell hypothesis, tumour cells
Nature Reviews | Drug Discovery
are heterogeneous and only the tumour-initiating cells have the ability to proliferate
extensively, give rise to differentiated cells and form new tumours. There are various
therapeutic strategies that could target tumour-initiating cells. Killing these cells could
be achieved by inhibiting their survival pathways (for example, with inhibitors of
phosphoinositol 3-kinase or interleukin-4-specific monoclonal antibodies), or sensitizing
them to chemotherapeutic agents (for example, with a checkpoint kinase inhibitor).
Some of the survival pathways that are used by tumour-initiating cells could also be used
by the bulk of the tumour, so agents targeting these pathways are expected to kill more
than just tumour-initiating cells. Alternatively, differentiating the tumour-initiating
cells (for example, with bone morphogenetic proteins or CD44-specific monoclonal
antibodies) might be a successful therapeutic strategy, as the bulk of the tumour has
limited proliferation potential. Inhibition of developmental signalling that is involved
in self-renewal (for example, with inhibitors of Wnt, Hedgehog or Notch pathway
signalling) might work by both mechanisms. In addition, anti-angiogenic therapy
might work in part by affecting the vascular niche of tumour-initiating cells. However,
tumour-initiating cells are probably genetically unstable, and a committed progenitor
could regain renewal activity. It might be therapeutically advantageous to combine
agents that target tumour-initiating cells with conventional agents that reduce the
bulk of the tumour and agents that target the niche.
Anoikis
A form of programmed cell
death that is induced in
anchorage-dependent cells
when they become detached
from the surrounding
extracellular matrix.
cells16. Studying cancer cell populations with concepts of
stem cell biology in mind is likely to bring further insight
into molecular drug targets and clinical strategies.
current failure with cancer treatment is not usually
due to a lack of primary response or initial induction
of remission, but to relapse or tumour recurrence after
therapy, in which tumour-initiating cells are thought to
have crucial roles. A major challenge now is to discover
agents and strategies that target cancer and tumour
relapse at their apparent source. Following an overview
of the current status of the cancer stem cell hypothesis,
this article therefore focuses on the challenges and
opportunities for anticancer drug discovery.
Niche
Cells and/or extracellular
matrix components in specific
anatomical locations that
regulate the participation of
the normal stem cells in tissue
generation, maintenance and
repair. In some cases, the
behaviour of tumour-initiating
cells might also be influenced
by interactions with
surrounding cells and matrix.
Cancer stem cells: evidence and controversy
In the mid 1990s, John Dick and colleagues demonstrated the existence of tumour-initiating cells in acute
myeloid leukaemia (AMl) using the non-obese diabetic–
severe combined immunodeficient (NoD–ScID)
mouse model3,17,18 (BOX 2). The purified populations of
leukaemia-initiating cells contained a chromosomal
translocation that was identical to that found in their
progeny, the blast cells. As well as supporting the
clonogenic nature of leukaemia, this important finding
pointed to the organization of leukaemia as a stem cell
hierarchy 19.
Similar studies are much more difficult for solid
tumours. Solid tumours contain both tumour cells and
various stromal cells, and breaking the interactions
between cells in such a tumour might induce anoikis or
change the properties of the tumour cells. Furthermore,
assays that involve injection of cells into a new tissue location in mice may fail to recapitulate the environment of
those tumour cells in the original tumours. Nevertheless,
tumour-initiating cells from solid tumours, including
human breast, brain, colon, pancreatic, liver and ovarian cancer and melanoma, have been successfully isolated using appropriate cell surface markers, including
cD44, cD24, cD133, epithelial cell adhesion molecule
(ePcAM) and ATP-binding cassette sub-family b member 5 (Abcb5)4–10. These tumour-initiating cells can produce phenocopies of the original primary tumours when
transplanted into NoD–ScID mice.
Theoretical and technical questions remain regarding
whether the cells isolated are the true and only tumourinitiating cells that function in the solid tumours of
patients20. current cell sorting protocols are thought to
favour niche-independent tumour-initiating cells (BOX 2),
which may resemble cells in activated or mobilized states
that are selected when transplantation assays are performed on normal stem cells21. certain markers used for
prospective sorting may also select for cells that evade
the immune system22, which could also be a relevant
property for tumour-initiating cells. Nevertheless, a
number of markers used in cell sorting are emerging as
being predictive of disease progression9,23, indicating that
they identify clinically important cell populations.
It was noticed that some experimental mouse models
of leukaemia do not follow the cancer stem cell hypothesis, suggesting that certain human cancers may not
adhere to this model14. However, as with many cell lines
that have lost the hierarchical structure of the primary
leukaemia from which they originated, some experimental mouse models may not accurately reflect the
tumour heterogeneity and pathological environment of
spontaneously occurring human malignancies16. It was
also argued that certain phenotypically distinct populations of human cancers fail to grow when injected into
immunodeficient mice because of differences in the
mouse and human microenvironment 20, so the identification of tumour-initiating cells in mouse models
adds credibility to the cancer stem cell hypothesis24–32.
Furthermore, transgenic models facilitate lineagetracing experiments, which can provide evidence for
tumour initiation and tumour hierarchies without the
limitations and experimental variability of transplantation assays30,31.
Although growing evidence from a wide range of
systems favours the existence of tumour-initiating cells,
there may nevertheless be diversity of tumour hierarchy
in different patients and varying degrees to which different tumour-initiating cells are stem cell-like. Many cancers might contain subpopulations of tumour-initiating
cells, but some could contain common tumorigenic cells,
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Box 1 | Tumour hierarchy and clonal evolution
Clonal evolution
Normal tissue
Stem cell
Tissue or
tumour
hierarchy
Mutations
Initial tumour
Tumour-initiating cell
Advanced tumour
Mutations
Target tumourinitiating cells
Multipotent
progenitor
Progenitor
Differentiated cells
Mutations?
EMT?
Tumour progenitors and
differentiated tumour cells
Target bulk of the tumour cells
For decades, tumour initiation and development has been regarded as a multistep process that is reflected by
the progressive genetic alterations that drive the transformation of normal human cells into highly malignant derivatives202.
As cancers arise only after multiple mutagenic events, long-lived cells are probably the most
capable
of supporting
such
Nature
Reviews
| Drug Discovery
cumulative changes. Based on genetic variations already observed in the tumour-initiating cell populations from different
tumours76,203 and genetic mutations in developmental pathways in different cancers34,38, it has been proposed that
progressive genetic alterations might occur at the level of tumour-initiating cells. The clonal progression to cancer could
operate through the ‘stem cell compartment’ (see the figure), as already shown in leukaemia-initiating cells204.
In normal tissues, the heterogeneity of cells reflects a hierarchical programme of differentiation in which multiple mature
cell types are derived from a common multipotent stem cell through intermediate progenitors. Heterogeneous populations
of cancer cells at various differentiation stages could be the result of both acquired mutations and aberrant but hierarchical
differentiation programmes (see the figure). Cancer is both a proliferation and a differentiation disease, and the ‘clonal
evolution’ and ‘cancer stem cell’ models might not be mutually exclusive, as initially thought. Owing to genetic instability,
the tumour-initiating cells isolated from a clinically detectable tumour would probably have a substantially different
genetic profile from the initial transformed cells that originated the tumour (see the figure). Also, as chemotherapeutic
agents are often mutagenic, the phenotype and frequency of the tumour-initiating cells in relapsed tumours are expected
to be distinct from those of early-stage lesions. In practice, combination treatment involving both traditional therapies and
therapies that target tumour-initiating cells will probably be required to ablate all cancer cells, particularly as a genetically
unstable tumour cell will present a ‘moving target’. EMT, epithelial–mesenchymal transition.
with little hierarchical organization15. controversy aside,
the cancer stem cell hypothesis provides a novel framework to study cellular heterogeneity, aberrant differentiation and tumour–host interactions in many cancers.
Framework for understanding cancer properties
The cancer stem cell hypothesis does not contradict the
established clonal evolution view of cancer, but instead
suggests a key role for tumour cell hierarchy in tumour
evolution and highlights the importance of an aberrant
differentiation programme in tumorigenesis (BOX 1). one
popular explanation for tumour heterogeneity is genetic
instability, which is a common feature of cancers according to comprehensive cancer genomic analysis 33–35.
conversely, a large part of the apparent morphological
and phenotypic heterogeneity can be explained by aberrant differentiation, and epigenetic changes in tumours
could also be dynamic and unstable36,37 (see the figure in
BOX 1). The behaviour of tumour-initiating cells could be
further modulated by tumour–host interactions. In this
section, we consider some of the key characteristics of
tumour-initiating cells.
Tumour-initiating cells diverge from their cells of origin
with increased self-renewal capacity. Although the
origins of tumour-initiating cells may vary, they share
several properties with normal stem cells, especially the
extensive capability of self-renewal12. If the cancer stem
cell hypothesis is correct, the evolution of the tumour is
largely the history of changes in self-renewing tumourinitiating cells (BOX 1), and self-renewal pathways might
be more conserved than surface markers among tumourinitiating cells.
Although pathways that regulate self-renewal are tightly
controlled in normal stem cells, in tumour-initiating
cells they may be constitutively activated or improperly
regulated through genetic and/or epigenetic changes,
leading to uncontrolled growth38. Several studies show
that bMI1 polycomb ring finger oncogene (BMI1) and
the wnt signalling molecule β-catenin regulate the selfrenewal of haematopoietic stem cells (HScs) and the
proliferation of leukaemia-initiating cells39–41. Actually,
many leukaemia-initiating cells have a higher selfrenewal capacity than normal HScs3. In addition, it was
recently shown that maintenance of cutaneous tumourinitiating cells is dependent on wnt and β-catenin
signalling 42. Hedgehog signalling regulates tumourinitiating cells from multiple myeloma43 and chronic
myeloid leukaemia (cMl)44. Developmental signalling
pathways that regulate normal stem cell self-renewal,
including wnt, Hedgehog and Notch (fIG. 2), have been
shown to be active in numerous human cancers38,45 and
may be broadly important for self-renewal in many cancers. They also have important roles in progenitors and a
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Box 2 | Mouse models for cancer research and drug discovery
In the mid 1980s, the availability of athymic (nu–nu) mice and the subsequent development of immunodeficient mouse
strains with other genetic lesions (including severe combined immunodeficient (SCID) mice, which lack both B and
T cells) allowed the widespread use of human tumour xenografts as mouse models for cancer research and drug
discovery205. Xenografts can be established either by injecting human tumour cell lines or by direct implantation
of patient biopsies into immunodeficient mice. The wide range of tumour cell lines and possibility of ex vivo genetic
or pharmacological manipulation before xenotransplantation made human xenograft tumour models popular tools
for discovering and developing not only cytotoxic agents but also tumour-targeted agents.
A major drawback of these mouse models is that they have limited utility in measuring tumour self-renewal in vivo,
particularly for tumour cells from primary sources. Models that permit efficient engraftment are a prerequisite for
assays that directly measure tumour self-renewal by serial transplantation. Animal models with an increased efficiency of
engraftment have since been developed. The SCID mouse was crossed with the non-obese diabetic (NOD) mouse, and
the NOD–SCID progeny can be engrafted by various tumour types and sustain the serial transplantation that is required
for assessing long-term self-renewal potential3–5. However, NOD–SCID assays still have limited efficiency of engraftment
and so can lead to underestimations of the frequency of human tumour-initiating cells. Notably, the use of more highly
immunocompromised NOD–SCID interleukin-2 receptor γ-chain-null (Il2rg–/–) mice can increase the detection of
tumorigenic melanoma cells by several orders of magnitude15. Although engraftment is improved by implantation into
NOD–SCID Il2rg–/– mice, it is also clear that immune cells have a role in the progression of many tumours. To properly
model certain tumours, disabling the mouse immune system might not be desirable. It may ultimately be necessary to
provide a humanized immune repertoire in mice, perhaps by genetic engineering or haematopoietic cell transplantation.
The limitations of xenotransplantation assays are particularly apparent in analysing tumour-initiating cells from
solid tumours. The current protocol, even with recent improvements15, is thought to favour niche-independent
tumour-initiating cells, which may not be representative of the population of tumour cells that can exhibit stem cell-like
properties in their human tumour environment. Orthotopic models might more faithfully maintain the tumour–host
interaction, giving tumour-initiating cells a greater chance to interact with and obtain stimulation from a mouse
environment. Co-injection with tumour-associated stromal cells might be another approach to improve the clinical
relevance of xenotransplantation, and can resolve some of the issues related to the presence of murine stroma in animal
models (fIG. 3b). Avoiding the transplant altogether by generating genetic mouse models of tumours may better
maintain the tumour–host and tumour-initiating cell–niche interactions30,31. However, owing to the practical difficulty of
modelling the multitude of genetic changes in a tumour, this strategy may be more suited to studying tumour-initiating
cells that have few defined genetic lesions, perhaps resembling tumour-initiating cells from early-stage human disease.
Asymmetrical division
A form of cellular replication
in which a cell renews itself
and generates a more
differentiated progeny.
Symmetrical division
A form of cellular replication
in which a single cell gives rise
to two identical cells.
Epithelial–mesenchymal
transition
A cellular program in normal
development and in cancer
whereby cells of an epithelial
origin acquire the properties of
mesenchymal cells, typically
characterized by loss of cell
adhesion, repression of
e-cadherin expression,
and increased cell motility.
Oncomir
MicroRNA known to
be involved in cancer
and tumorigenesis.
wider impact on the lineage, and could have additional
roles in the regulation of tumour and stromal cells. In this
rapidly expanding field of tumour biology, new routes by
which cells may acquire or enhance self-renewal ability
are regularly being discovered, and self-renewal roles for
classical cancer genes are also emerging. For example, by
gaining certain epigenetic programmes or losing certain
combinations of tumour suppressor genes, progenitor
cells could gain the capacity for self-renewal and become
malignant 46,47.
Self-renewal can be affected by changes in proliferation, differentiation and/or apoptosis. Aberrantly
increased self-renewal might therefore be caused by different mechanisms, such as an increasing proliferation rate
or a shift in the balance of cell division from asymmetrical
division to symmetrical division48. Tumour-initiating cells
from AMl and cMl are mostly quiescent 49,50, whereas
tumour-initiating cells from many solid tumours could
be proliferative51. The tumorigenic breast and pancreatic tumour-initiating cells have cell cycle profiles
that are similar to those of bulk tumour cells, which
do not have tumour-initiating ability, showing it is not
a proliferative advantage of these tumour-initiating
cells over the non-tumorigenic cells that leads to differential engraftment4,8. by contrast, slowly proliferating intestinal stem cells from phosphatase and tensin
homologue (Pten)-deficient mice initiate intestinal
polyposis52. The most important and characteristic
feature of tumour-initiating cells therefore seems to be
their increased self-renewal potential, rather than their
proliferation rate. Indeed, during the clonal evolution
of tumour-initiating cells, there may be stronger selection for increased self-renewal capacity than for higher
proliferation53. In addition, a malignant tumorigenic
cell could have undifferentiated pathological features
associated with increased self-renewal, including metastatic and pro-angiogenic capabilities.
Tumour-initiating cells in metastasis and tumour–host
interactions. A corollary of the cancer stem cell hypothesis is that macroscopic metastases may arise from
migrating or disseminated tumour-initiating cells54. In
patients with breast cancer, tumorigenic cD44+cD24–/low
cells are readily detectable in metastatic pleural effusions, and most early-disseminated cancer cells that
are detected in the bone marrow have a putative breast
tumour-initiating cell phenotype 4,55. Interestingly,
cD133+cXcr4+ tumour-initiating cells that are found
at the invasive front of pancreatic tumours have been
shown to determine the metastatic phenotype of the
individual tumour 56. Another hypothesis is that metastatic tumours originate when cells in a primary epithelial
malignancy undergo an epithelial–mesenchymal transition
(eMT)57. like self-renewal, eMT is regulated by developmental signalling pathways, such as the wnt and Notch
pathways58,59. expression of the oncomir LET7 in a breast
tumour-initiating cell model inhibits self-renewal, eMT
and xenograft metastasis60. More recently, it was noticed
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that cells undergoing eMT and tumour-initiating cells
share many markers and properties61. These findings
reveal the potential plasticity of these tumour cells and
suggest that there could be some common regulatory
programmes underlying eMT and self-renewal (see the
figure in BOX 1).
The behaviour of normal stem cells is tightly
regulated by signals that the cells receive from their
microenvironmental niches, which are provided by
the adjacent cells and/or extracellular matrix components62–64. A niche, while supporting the self-renewal
and maintaining the identity of a stem cell, also controls stem cell number and proliferation. This control
of cell number and proliferation might be a preventative
mechanism against cancer 65. Similarly, it is conceivable
that the tumour microenvironment could also constrain
tumour-initiating cells, as some tumours lie dormant or
develop slowly over decades66. one theory of tumour
dormancy is that tumour-initiating cells are held in
check by a niche–stem cell interaction. cancer could
progress if the niche were expanded or altered through
genetic or epigenetic means65. It has been shown that an
alteration in a HSc niche can lead to myeloproliferative
disease67. Tumour-initiating cells could also preferentially localize to environments that favour proliferation,
such as vascular niches68–70, or even promote the formation of their own niche71. Alternatively, mutations might
render tumour-initiating cells independent of niche
signals, thereby lifting environmental controls on selfrenewal72 and increasing the risk of metastasis.
Why do many therapies fail to eradicate cancers?
Many patients with cancer, particularly those with solid
tumours, either do not respond to existing cancer therapies (including chemotherapeutics, radiotherapy and
tumour-targeted agents) or relapse quickly after initial
remission. Key possible reasons for this failure include
the inherent drug resistance of tumour-initiating cells,
the inefficiency of the treatment and/or the genetic
instability of cancer cells.
It has been suggested that the more aggressive and
refractory cancers contain more tumour-initiating
cells73,74. circumstantial evidence in support of this connection already exists in medical practice. Following
cancer therapy, a patient’s tumour is examined to
assess the effects of treatment. If the tumour contains
only mature cells, the cancer usually does not recur.
However, if a large number of immature cells (probably
including a large proportion of tumour-initiating cells)
are present in the tumour sample, the cancer is likely to
return, and further aggressive treatment is warranted72.
It has been shown that a high frequency of stem cells in
AMl at diagnosis predicts high minimal residual disease after therapy and poor prognosis75. In melanoma,
Abcb5 is a marker for malignant melanoma-initiating
cells, and Abcb5+ tumour cells detected in patients
with melanoma show a primitive molecular phenotype
and correlate positively with clinical melanoma progression9. In breast cancer, cD44+ cell-specific genes
included many known stem cell markers and correlated with decreased patient survival76. Furthermore,
chemotherapy treatment increased the percentage of
cD44+cD24–/low tumour cells in patients with breast
cancer 77, consistent with the relative chemoresistance
of these tumour-initiating cells.
Many tumour-initiating cells are thought to be resistant
to chemotherapeutics such as paclitaxel and doxorubicin,
for various reasons including their quiescent or slowly
proliferating nature78, the high expression level of ATPbinding cassette (Abc) drug pumps9,60,79, the intrinsic
high levels of anti-apoptotic molecules11, their relative
resistance to oxidative or DNA damage, and their efficiency of DNA repair 80–82. Although tumour-initiating
cells might commonly be more resistant to therapy
than the bulk of tumours, there could be variation in
sensitivity to therapies among tumour-initiating cells,
as has been shown for normal stem cells21,83.
Similar to traditional anticancer drugs, many novel
tumour-targeted agents were also designed to target
rapidly proliferating cancer cells, so many tumourinitiating cells might also be relatively insensitive to
these agents. For example, imatinib (Gleevec; Novartis)
targets and inhibits the bcr–Abl kinase, which is the
fusion protein product of a chromosomal translocation and is suggested to act as a molecular switch that
promotes proliferation and differentiation of multipotent progenitors in cMl84. bcr–Abl is required for
the survival of proliferating progenitor cells, but not the
quiescent cMl stem cells50,85,86. Therefore, although the
mutations are thought to accumulate in tumour-initiating
cells, their functional effects are mostly manifested further downstream in the tumour hierarchy, leading to
neoplastic proliferation of primitive progenitors. As a
result, imatinib eliminates proliferating, committed leukaemia progenitors, but not primitive, quiescent tumourinitiating cells, and most patients are still positive for the
fusion gene transcripts after treatment 87–89. Alterations
in Ikaros family zinc finger 1 (IKZF1) are thought to
synergize with bcr–Abl to induce lymphoblastic leukaemia and contribute to drug resistance and disease
progression90. Although it is likely that several factors
contribute to the problem, it has been shown that the
drug resistance and disease recurrence that are associated with imatinib treatment of cMl might be avoided
by targeting an essential stem cell maintenance pathway involving Hedgehog 44. As discussed above, there
are other cancer types in which tumour-initiating cells
could be proliferative, and growth signalling pathways
are likely to have important roles in these cells. These
pathways are therefore promising therapeutic targets
in such cancers.
If a patient has a large number of tumour-initiating
cells and only a small number of such cells are required
to regenerate a tumour, then therapy has to be highly
efficient at killing these cells to avoid relapse91. A therapy that kills 95% of cells in a tumour might be considered efficacious based on tumour shrinkage, but may
allow for the survival of sufficient tumour-initiating
cells to cause eventual relapse. Thus, even if tumourinitiating cells are no more resistant to therapy than bulk
tumour cells, they can still be the key to the limitations
of treatment.
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If the cancer stem cell hypothesis is widely applicable, it
probably extends to neoplasms that are amenable to cure,
such as certain germ cell neoplasms (that is, testicular
seminoma) and neuroblastomas. Indeed, chemotherapy
treatments for such tumours often eliminate the undifferentiated cancer cells and produce residual masses that
are benign tumours composed of differentiated cells92.
This suggests that their stem cell components might be
inherently sensitive to chemotherapeutics and unable to
adapt to counteract them. Several recent studies suggested
that the survival benefit of trastuzumab (Herceptin;
Genentech/roche) — a monoclonal antibody specific
for receptor tyrosine protein kinase erbb2 (also known
as Her2) — might relate to its ability to target breast
tumour-initiating cells93,94. However, most patients with
metastatic breast cancer still develop resistance within
1 year of trastuzumab treatment 95, which implies the
genetic and/or epigenetic plasticity of these tumourinitiating cells permit them to evolve as a function of
tumour progression and/or therapeutic challenges.
Targeting tumour-initiating cells might not avoid the
same problems that have been encountered for decades
in treating bulk tumour cell populations: the emergence
of drug resistance and the selection of increasingly
refractory cell types96. Nevertheless, understanding the
molecular basis of tumour-initiating cell behaviour will
allow for the design of new strategies, including combination therapies to counter drug resistance.
Therapeutic opportunities
The cancer stem cell hypothesis provides a rationale for
several therapeutic strategies beyond traditional antiproliferative agents (fIG. 1). Potential approaches to kill
tumour-initiating cells include blocking essential selfrenewal signalling, inhibiting the survival mechanisms
of these cells, or targeting tumour-initiating cell surface
markers through antibody-based cytotoxic approaches.
Another strategy is to induce tumour cell differentiation,
which can potentially be achieved by inhibiting developmental pathways or epigenetic programmes. As many
tumour-initiating cells might depend on a niche to maintain their identity, targeting the niche could be a strategy to
indirectly inhibit or differentiate tumour-initiating cells.
Developmental pathways in self-renewal and differentiation. As many tumour cells are thought to have an
aberrant differentiation programme and deregulated
self-renewal could be a key factor in many types of
cancer 38,45, several developmental signalling pathways
have become the recent focus of drug discovery efforts
(fIG. 2; TABLe 1). During embryonic development, there
is considerable crosstalk between these pathways, with
identifiable signalling centres that generate, receive and
integrate several pathways97,98. Tumour-initiating cells
and their niches might similarly operate as signalling
centres, in which multiple developmental pathways are
active and converge to control self-renewal.
wnt signalling has been shown to be required for
self-renewal of tumour-initiating cells in several cancers, including cMl and squamous cell carcinomas41,42.
extracellular wnt inhibitors, including the secreted
Frizzled-related proteins (SFrPs) and Dickkopf proteins
(DKKs), which act at the cell surface to inhibit wnt signalling through its receptors, have been discovered99.
Active derivatives of these antagonists, if designed to have
desirable pharmacokinetic properties, could be developed
into antitumour agents, particularly as they might be able
to suppress wnt signalling in cancer even when the genes
encoding adenomatous polyposis coli protein (APc) or
β-catenin are mutated100. Small-molecule antagonists of
the oncogenic transcription factor TcF–β-catenin protein complex have been reported101,102. Antibodies against
various wnts103,104, Frizzled proteins and wnt coreceptor low-density lipoprotein receptor-related protein 5
(lrP5)–lrP6 are also being explored.
Inhibition of the Hedgehog signalling pathway
is also a viable therapeutic strategy, and antibodies
against Hedgehog and small-molecule inhibitors of the
Hedgehog coreceptor Smoothened homologue (SMo)
have been identified105,106 (TABLe 1). It has been reported
that pharmacological inhibitors of Hedgehog signalling
display efficacy in various animal models, including those
of basal cell carcinoma, medulloblastoma, small-cell
lung cancer and pancreatic cancer 106–110. More recently,
it was shown that inhibition of Hedgehog signalling kills
cMl tumour-initiating cells, impairs the propagation
of bcr–Abl-driven cMl and the growth of imatinibresistant mouse and human cMl44. However, these
agents are not very effective and the results vary among
cellular models, partly because it is difficult to maintain
Hedgehog pathway activity in vitro due to differentiation under conventional culture conditions111,112 and/or
requirements for the presence of stromal cells63,64,113.
Hedgehog ligand expressed by tumour cells can also
activate the Hedgehog pathway in the tumour stromal
microenvironment, illustrating a paracrine requirement
of Hedgehog signalling 114. Such challenges complicate
in vitro screening assays, as discussed below.
Inhibition of Notch expression by antisense nucleic
acid technology or the pharmacological blockade of
the protease γ-secretase, which cleaves Notch (fIG. 2),
has striking antineoplastic effects in Notch-expressing
transformed cells in vitro and in xenograft models115–117.
A γ-secretase inhibitor has been shown to induce goblet
cell differentiation and regress colon adenomas in
mice carrying a mutation of the Apc gene118. Another
γ-secretase inhibitor depletes tumour-initiating cells in
brain tumours119. However, the therapeutic window of
γ-secretase inhibitors is narrow, because of their inhibition of multiple Notch pathways and the possible
effect on normal stem cells. recently, antibodies that
are selective for the Notch ligand Delta-like 4 (Dll4)
were shown to inhibit tumour growth by deregulating
angiogenesis without much of the toxicity related to
γ-secretase inhibition in animal models 120,121. Such
selective targeting of an individual Notch pathway
might provide a viable strategy for impairing niche
function. Antagonist antibodies against individual
Notch receptors are also being explored122. In addition
to developmental signalling pathways, other signalling
pathways could be important for the self-renewal of
different tumour-initiating cells123,124.
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Wnt
DKK
LRP5–6
Wnt
Hh
SFRP
Fz
SMO
antagonist
Notch
γ-secretase
inhibitor
Hh
PTCH
Ligand
SMO
Notch
Axin
APC
β-Cat
GSK3β
CK1α
STK36 Gli
AKT
SUFU
β-cat
TCF–
LEF
Gli
RBPJ
Normal
stem cells
Haematopoietic,
epidermal and intestinal
Haematopoietic,
neural and germline
Haematopoietic,
epidermal and intestinal
Cancer
Colon carcinoma, epidermal tumours
including breast tumour, leukaemia,
squamous cell carcinoma and
tumours of the digestive tract
Medulloblastoma, basal cell
carcinoma, tumours of the
digestive tract, prostate cancer,
leukaemia and multiple myeloma
Leukaemia, breast
tumours, brain tumours
and lung cancer
Figure 2 | signalling pathways that regulate self renewal during normal stem cell development and cancer
transformation. The Wnt signalling pathway is activated by the binding of Wnt ligands to their receptors Frizzled (Fz)
and low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6, leading to the release
of β-catenin
from
Nature
Reviews | (β-cat)
Drug Discovery
the ‘degradation complex’, which consists of adenomatous polyposis coli protein (APC), axis inhibition protein (axin),
glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α). This facilitates the entry of β-catenin into the nucleus,
where it regulates target gene transcription through association with the transcription factor TCF–LEF (lymphoid
enhancer binding factor). Soluble Frizzled-related protein (SFRP) and Dickkopf protein (DKK) are endogenous secreted
antagonists of Wnt signalling. Activation of the Hedgehog (Hh) signalling pathway is initiated by binding of a Hh ligand to
protein patched homolgue (PTCH). This lifts suppression of Smoothened homologue (SMO), activating a cascade that
leads to the translocation of glioma-associated oncogene homologue (Gli) into the nucleus and the activation of target
genes. Serine–threonine protein kinase 36 (STK36: also known as FU) and suppressor of fused homologue (SUFU) act
downstream of PTCH and SMO to regulate Gli. Cyclopamine is a potent antagonist of SMO, and has been used as a tool
compound to study Hh signalling pathways both in vitro and in vivo. The core Notch pathway is activated by interaction
between the Notch ligand (Delta-like or Jagged) on one cell with the Notch receptor on another cell, resulting in two
proteolytic cleavages of the receptor. This mediates the release of the Notch intracellular domain, which enters the
nucleus and interacts with transcription factors including recombination signal binding protein for immunoglobulin κJ
region (RBPJ; also known as CBF1). It has been suggested that alternative pathways involving AKT exist downstream of
Notch activation126,177. Various γ-secretase inhibitors can inhibit Notch cleavage and activation.
There is growing evidence that wnt, Hedgehog and
Notch signalling are likely to interact with one another
and with additional signals, such as bone morphogenetic
proteins (bMPs) and growth factors that are produced
by tumour-initiating cells, the bulk tumour cells or their
microenvironment 125. These signals converge to generate the distinctive features of tumour-initiating cells,
including self-renewal, proliferation, survival and differentiation. It is still unclear how various signalling
pathways interact to maintain self-renewal activity in
different cancers, and whether inhibition of an individual
pathway would differentiate tumour-initiating cells,
inhibit their proliferation or kill them. The results could
depend on the status and interaction of different oncogenic pathways. For example, NoTcH1 regulates the
PTeN–phosphoinositide 3-kinase (PI3K)–AKT pathway in T cell acute lymphoblastic leukaemia (T-All)126.
This means that use of γ-secretase inhibitors alone is
unlikely to have a therapeutic effect in cases of T-All
with deleted PTEN and constitutively activated AKT126.
To selectively kill tumour-initiating cells, it might be
necessary to inhibit multiple pathways in many cases.
conversely, targeting a specific developmental pathway
to promote differentiation could be a more general strategy for eliminating tumour-initiating cells (fIG. 1). It has
been shown that bMPs could induce the differentiation
of cD133+ glioblastoma tumour-initiating cells predominantly to astrocyte-like cells, which markedly attenuated
their tumour-forming ability in a preclinical model127.
The regulation of self-renewal remains poorly
understood and could involve transcriptional networks
and epigenetic programmes that regulate chromatin
accessibility. It has been shown that mixed-lineage
leukaemia (Mll) fusion proteins, which modulate
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Table 1 | Selected agents targeting self-renewal signalling or tumour-initiating cell surface markers
Target or
Agents
target pathway
cancer indication
status
Hedgehog
pathway
5E1, a Sonic Hedgehog
neutralizing mAb
Oesophageal and prostate
cancer and medulloblastoma
Target validation
Cyclopamine, a SMO antagonist
Solid tumours
Target validation
Basal cell carcinoma
Phase I
200
GDC-0449, a SMO antagonist
Basal cell carcinoma and
metastatic colorectal cancer
Phase II
Genentech website
(www.gene.com)
IPI-926
Advanced and/or metastatic
solid tumours
Phase I
Infinity Pharmaceuticals website
(www.ipi.com)
A WNT2-specific mAb
Melanoma and NSCLC
Target validation
103,104
Various TCF–b-catenin inhibitors
Colon cancer
Target validation
101,102
DBZ, a γ-secretase inhibitor
Intestinal adenomas
Target validation
Preclinical
118
MK-0752, a γ-secretase inhibitor
T-ALL and metastatic or
advanced breast cancer
Phase I
DLL4-specific mAbs
Solid tumours
Preclinical
ABCB5
3C2-1D12, an ABCB5-specific mAb
Malignant melanoma
Target validation
CD44
H90, a CD44-specific mAb
Acute myeloid leukaemia
Preclinical
148
P245, a CD44-specific mAb
Breast cancer
Preclinical
201
Adecatumumab, an
EPCAM-specific human mAb
Metastatic breast cancer
Phase II
Edrecolomab, an EPCAM-specific
mAb
Colon cancer
Phase II–III
142
Catumaxomab, a trifunctional
antibody against EPCAM and CD3
Malignant ascites and ovarian
and gastric cancer
Phase II–III
Trion Pharma website
(www.trionpharma.de)
MT110, a bispecific single-chain
antibody against EPCAM and CD3
Lung and gastrointestinal cancer Phase I
Wnt pathway
Notch pathway
EPCAM
references
105
38
Merck website
(www.merck.com)
120,121
9
Micromet website
(www.micromet.de)
Micromet website
(www.micromet.de)
ABCB5, ATP-binding cassette sub-family B member 5; DLL4, Delta-like 4; EPCAM, epithelial cell adhesion molecule; mAb, monoclonal antibody; NSCLC, non-small-cell
lung cancer; SMO, Smoothened homologue;T-ALL, T cell acute lymphoblastic leukaemia; TCF, T-cell factor.
chromatin structure through histone modification, can
reprogramme differentiated myeloid cells and activate
self-renewal in cells with no inherent self-renewal properties46,128. Polycomb group proteins, such as enhancer of
zeste homologue 2 (eZH2), are essential components by
which stem cells reversibly repress genes that are related
to differentiation129. recently, it was reported that hypermethylation of the gene encoding the bMP receptor 1b
in a subset of glioblastoma-initiating cells is linked to
the activity of eZH2 (Ref. 130). eZH2 also affects bMI1mediated suppression of the p16Ink4a–p19Arf locus to avert
growth arrest or apoptosis of stem cells131. Invasive basal
subtype breast cancers significantly overexpress eZH2,
which could lead to downregulation of breast cancer
type 1 susceptibility protein (brcA1)132. The possible
involvement of these epigenetic programmes in different
tumour-initiating cells and their potential as therapeutic
targets are areas of intense study.
Survival mechanisms in tumour-initiating cells. Although
we know little about survival pathways of tumour-initiating
cells in various tumour types, they are potential targets for
killing tumour-initiating cells. certain oncogenic pathways that are distinct from developmental pathways might
have a role in the survival of some tumour-initiating cells,
but have no effect on cell differentiation or multipotency.
In AMl, nuclear factor κb (NF-κb) was found to be
constitutively active in primitive AMl cells (which are
considered leukaemia-initiating cells), but not in normal
HScs123. MG-132, a proteasome inhibitor with potent
NF-κb signalling inhibitory activity, was shown to induce
rapid cell death in cD34+cD38– leukaemia-initiating
cells, but not normal cD34+cD38– cells. An interleukin-4
(Il-4)-specific antibody reduced the viability of both
cD133– and cD133+ colon cancer cells and increased
the efficacy of chemotherapy, suggesting that molecular
pathways that contribute to bulk tumour growth can also
be successfully targeted to sensitize tumour-initiating
cells to cytotoxic therapies133. In addition, activation of
the PTeN–mammalian target of rapamycin (mTor)–
signal transducer and transcription activator 3 (STAT3)
pathway in breast tumour-initiating cells is required for
their viability and maintenance134, and the PI3K pathway
regulates survival of tumour-initiating cells that reside
in the perivascular niche of medulloblastoma70. recent
studies also suggest an important role for erbb2 in
maintaining tumour-initiating cells in breast cancer 93,94,
in addition to its presumed role in bulk tumour cells. It is
unclear how pathways that regulate self-renewal interact
with those that regulate the survival of tumour-initiating
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cells, although it was recently shown that NoTcH1
regulates the PTeN–PI3K–AKT pathway in T-All126 and
erbb2 expression in breast cancer 94.
In glioblastomas, DNA damage responses were shown
to be preferentially activated in glioma-initiating cells
before and after radiation. The radioresistance of cD133+
glioma-initiating cells can be reversed with a specific
inhibitor of checkpoint kinase 1 (cHK1) and cHK2
(Ref. 81). In breast cancer, certain tumour-initiating cells
have lower levels of reactive oxygen species (roS) than
corresponding non-tumorigenic cells. Pharmacological
depletion of roS scavengers in tumour-initiating cells
markedly increases DNA damage and results in radiosensitization82. In addition, cell cycle restriction through
the expression of cyclin-dependent kinase inhibitor 1A
(cDNK1A; also known as p21) limits DNA damage
and maintains the self-renewal of leukaemia-initiating
cells135. These preliminary studies highlight the potential
of inhibiting DNA damage responses136 to overcome the
resistance of tumour-initiating cells to therapy.
Cell surface markers and niche interaction. Markers on
the surface of tumour-initiating cells are also important
targets, particularly for antibody-based therapeutics
(TABLe 1). The surface markers cD34, cD44, cD133
and Abcb5 have been used to identify tumour-initiating
cells from various tumour types3–9. The surface markers
might reflect the cellular origin and history of that
particular tumour-initiating cell, and could be highly
variable among tumour types, patients and even within
the same patient. uniquely among such markers, cD44
could have several roles in tumorigenesis; its expression is induced by oncogenic signals such as β-catenin–
TcF4 and ras–raf–extracellular signal-regulated kinase
(erK) pathways and negatively regulated by the tumour
suppressor p53 (Refs 137,138). Most of these markers are
expressed in normal cells, and so finding a therapeutic
window could be a challenge. However, there seem to
be some phenotypic differences between leukaemiainitiating cells and HScs, including differences in the
expression of THY1 membrane glycoprotein, KIT and
Il-3 receptor-α (Il3rA)139–141. In particular, Il3rA,
which is not present on normal stem cells, is a specific
surface marker for AMl-initiating cells141.
In addition to binding to their targets and inhibiting
target-dependent signalling, most therapeutic monoclonal antibodies (mAbs) interact with components
of the immune system through antibody-dependent
cellular cytotoxicity (ADcc) and/or complementdependent cytotoxicity, and immune responses might
be part of their antitumour mechanisms142. Interestingly,
systemic administration of a mAb directed against
Abcb5, which induces ADcc in Abcb5+ malignantmelanoma-initiating cells, exerts tumour-inhibitory
effects in a melanoma xenograft model9. Some tumourinitiating cells express immune-tolerance markers143,
potentially making them resistant to immune attack.
However, the antitumour activity of these antibodies
can be enhanced by a cytotoxic immunoconjugate
or engineered antibody binding to both tumour and
immune cells 144,145, which might be able to bypass
various immune tolerance mechanisms. Although Abc
transporter activity can result in tumour-initiating cells
being relatively resistant to many conventional therapies, it may make the cells susceptible to alternative
strategies that target cells with effective drug efflux 146.
Agents that target markers on the surface of tumourinitiating cells could also work by affecting the niche
(fIG. 1). endosteal (osteoblastic) niches have been identified for both normal HScs63 and AMl-initiating cells147.
A mAb specific for the adhesion molecule cD44 was
shown to eradicate human AMl-initiating cells in vivo
by blocking the trafficking of leukaemia-initiating cells
to supportive microenvironments, and by altering their
‘stem cell’ fate through differentiation148. endothelial
niches were also revealed for HScs149, and are a possibility for leukaemia-initiating cells. Neural stem cells
are thought to localize to vascular niches150–152, and
brain tumour-initiating cells reside in a perivascular
niche69,70. Moreover, stem cell-like glioma cells have
been shown to promote tumour angiogenesis71, suggesting that these cells might help to create their own
niche. These data indicate that signalling in niche interactions can be bidirectional, with tumour-initiating
cells as both the source and the target.
It is important to emphasize that much of the nicheinteraction data has been obtained from animal models,
and the role of the niche in various human tissues and
cancers is not yet clear. Nevertheless, the association
between tumour-initiating cells and the vasculature
does raise the intriguing possibility that anti-angiogenic
therapy may work in part by affecting the vascular niche
of tumour-initiating cells, and there could be many reasons to combine agents that target tumour-initiating cells
with anti-angiogenic therapy. Strategies that specifically
target other niches of tumour-initiating cells can also
be envisioned.
Therapeutic windows and combination strategies
The potential therapeutic window is always a concern
for any anticancer approach, including those that target
tumour-initiating cells. However, tumour-initiating cells
that exhibit overactive self renewal might be more sensitive to agents that inhibit self-renewal pathways than
normal stem cells, the self-renewal activity of which
depends on the developmental stage and tissue homeostasis, among other things. As combined loss of certain
tumour suppressor genes in progenitor cells can lead to
malignant cells with increased self-renewal activity47, there
might be mechanistic differences between tumourinitiating cells and normal stem cells with respect to
self renewal. Knockout of the PTEN tumour suppressor causes the generation of transplantable leukaemiainitiating cells and the depletion of normal HScs in
mice153,154. Interestingly, rapamycin not only depleted
leukaemia-initiating cells but also restored normal HSc
levels in this model. Although it has not yet been confirmed in the case of human cancer, this finding highlights the potential feasibility of achieving a therapeutic
window in targeting tumour-initiating cells. recently,
wnt–β-catenin signalling was shown to be involved in
the maintenance of cutaneous tumour-initiating cells
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and malignant human squamous cell carcinomas 42,
whereas wnt–β-catenin signalling is not essential for
normal epidermal homeostasis. As discussed below,
the SMo antagonist GDc-0449 was well tolerated in
Phase I clinical trials155, providing preliminary clinical
evidence of therapeutic windows for strategies that target
self-renewal signalling.
Now that markers of tumour-initiating cells, including
Il3rA for AMl, are known to exist 141, the currently
limited repertoire of those that have been identified
will grow as tumour-initiating cell populations from
various systems are further characterized. As many
tumour-initiating cells might originate from progenitor
cells or partially differentiated cells46,47, many of them
are expected to have surface markers distinct from those
of normal stem cells. mAbs that target different epitopes
of the same target could have different therapeutic windows, owing to differential exposure and signalling, as
shown for cD44-specific mAbs148.
Another example relevant to the potential therapeutic
window of strategies targeting tumour-initiating cells is
the observation that the combination of a proteasome
inhibitor (MG-132) with the cytotoxic drug idarubicin
induces rapid and extensive apoptosis of the leukaemiainitiating cell population while leaving normal HScs
viable both in vitro and in vivo123. The inhibition of Notch
signalling by γ-secretase inhibitors is presumed to have
a narrow therapeutic window. However, treatment with
a γ-secretase inhibitor seemed to be better tolerated in
mice when using a pulsed dosing regimen156, suggesting that normal stem cells could recover quickly from
the treatment. Therefore, based on the pharmacokinetic
properties of the agent, the therapeutic window can be
improved by optimizing the dosing regimen, similar to
the adjustment of dosing schedule of cytotoxic agents.
As discussed above, it is thought that several pathways,
including those that regulate self renewal and cell growth,
converge to regulate tumour-initiating cells. If there is a
sufficiently large therapeutic window, targeting a combination of pathways that are uniquely active in tumourinitiating cells will be more effective than inhibiting a
single pathway. It has been reported that simultaneously
blocking the Hedgehog and epidermal growth factor
receptor (eGFr) pathways using cyclopamine and gefitinib resulted in growth arrest, apoptosis and a decrease in
the invasiveness of prostate cancer cells157. The PI3K–AKT
pathway has been reported to regulate Hedgehog signalling in part by controlling protein kinase A-mediated
glioma-associated oncogene homologue (Gli) activity 158,
indicating the potential therapeutic value of combining
Hedgehog antagonists with PI3K–AKT inhibitors.
Many agents that target tumour-initiating cells differentiate these cells, and may need to be combined with
chemotherapeutics to eliminate cells further down the
tumour hierarchy. Inhibition of promyelocytic leukaemia protein (PMl) by arsenic trioxide disrupted the
maintenance of cMl-initiating cells, induced the differentiation and progression through the cell cycle of
these otherwise quiescent tumour cells and sensitized
them to pro-apoptotic stimuli159. In addition, the PI3K
pathway regulates survival of tumour-initiating cells that
reside in the perivascular niche following radiation in
medulloblastoma in vivo, and inhibition of AKT signalling sensitizes these cells to radiation-induced apoptosis70. The PI3K–AKT pathway regulates AbcG2 activity
in glioma-initiating cells160, providing another rationale
for combining PI3K–AKT inhibitors with chemotherapeutics that are substrates of the Abc drug transporter.
Given the emerging role of Notch and Hedgehog in
the tumour stroma and vasculature114,120,121, agents targeting these pathways might have an impact on both
tumour-initiating cells and tumour vasculature; the
effects of these agents on drug delivery should therefore
also be considered161. It is conceivable that challenging
tumour-initiating cells with both targeted agents and
conventional chemotherapy or radiotherapy would not
only be more effective in cell killing, but also delay the
development of drug resistance compared with either
agent alone; however, preclinical evidence for this is not
yet available.
It is important to evaluate different agents and combinations in the context of the tumour hierarchy and with
biomarkers162 (fIG. 3b), to determine their efficacy and
to study the emergence of drug resistance in preclinical
models. It is also crucial to study their effects on stromal
cells and the vasculature, and to follow closely the therapeutic window of different combinations. Hopefully,
well designed combination strategies based on data
from relevant preclinical models can overcome pathway
redundancy, counter drug resistance and help to achieve
long-term remission in a clinical setting.
Drug discovery with tumour-initiating cells
The conventional approach for anticancer drug discovery
is to target cell proliferation rather than self renewal
and/or differentiation, and so is often biased to select
targets with homogeneous expression patterns and
potent compounds that kill the bulk tumour cells. In
addition, some traditional preclinical models may not
reflect clinical complexities such as tumour hierarchy.
Tumour-initiating cells that depend on a niche and
developmental pathways involving paracrine or juxtacrine signalling may demand more sophisticated drug
discovery platforms than the two-dimensional tissue
culture or subcutaneous xenograft models that have
traditionally been used to characterize autonomous
tumour cells and autocrine signalling in cancer. The
large body of evidence in support of the cancer stem cell
hypothesis and the related therapeutic strategies require
adjustments to anticancer drug discovery platforms to
make them more clinically relevant (fIG. 3).
Tumour-initiating cell enrichment and in vitro culture
conditions. Sources from which to isolate tumour-initiating
cells include samples from patients with primary cancer,
primary tumour xenografts and certain cancer cell lines
(fIG. 3). Interestingly, it was observed that, as with primary
tumour cells, some cancer cell lines cultured under conventional conditions have a tumour hierarchy based on established tumour-initiating cell markers10,60,163–165. Although
their relevance to tumour-initiating cells from primary
patient samples still requires further characterization,
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a
Human tumours
Primary xenografts
Cell lines
b
Human tumours
Primary xenografts
Cell lines
Tissue
dissociation
or fragment
Tissue
dissociation
Enrichment
for TICs
Identify TIC population
TIC population No
known?
Yes
Enrichment of TIC population:
• Surface or surrogate markers
• Self-renewal activities
• Optimized TIC culture
Two-dimensional
culture
+ drugs
Plastic or
coated
plate
Semi-solid
media
Matrix
growth factors
Co-culture
with stromal
cells
Orthotopic models
Co-injection models
Simple, high-throughput
assays
Physiological
microenvironment
Human stromal cells,
susceptible to
molecular engineering
Three-dimensional
organotypic culture
Monitor TIC population:
• Surface markers
• Fluorescent reporters
• Enzymatic reporters
• Functional tests
Standard end points
• Proliferation
• Survival
• Colony size
Subcutaneous models
+ drugs
+ drugs
+ drugs
Replate
+ drugs
TIC-focused end points
• Differentiation markers
TIC Lineage EMT
• Self-renewal (for example,
colony number)
Monitor TIC activity and population in vivo:
• Tumorigenicity
• Cell surface markers
• Fluorescent reporters
• Enzymatic reporters
Standard end points
• Tumour volume
or size
• Metastasis
• Survival
TIC-related end points
• Relapse after
chemotherapy
treatment
• Minimal residual
disease
TIC-focused end points
• Tumorigenic activity
of enriched TICs
• TIC, lineage and
EMT markers
• Imaging TICs with
reporters or
biosensors
Identify global and TIC-specific drug effects
Figure 3 | Anticancer drug discovery platforms to target tumour-initiating cells. Sources of tumour-initiating cells (TICs)
for enrichment and characterization include samples from cancer patients, primary tumour xenografts
and certain
cancer
Nature Reviews
| Drug Discovery
cell lines that maintain tumour hierarchy. a | For in vitro studies, enrichment for TICs is likely to be a frequent but not a
necessary step in TIC characterization. TICs can be studied in heterogeneous systems provided their activities can be
monitored by cell surface markers or signalling reporter activities. Even highly purified TICs are expected to gradually
become more differentiated under tissue culture conditions. TICs that are dependent on a niche and paracrine or juxtacrine
signalling might be better maintained in three-dimensional organotypic culture than two-dimensional culture. Drug effects
on TICs can be studied according to standard end points (such as proliferation, survival and colony size) and TIC-focused
end points such as aberrant differentiation (for example, disproportionate presence of markers of TICs, lineage or
epithelial–mesenchymal transition (EMT)) and self renewal. One way to measure self renewal in vitro is to track long-term
colony-initiating cells in colony-forming unit assays by replating in semi-solid media. TICs are defined by in vivo
experiments, and in vitro results always need to be validated in vivo. b | Depending on the life span of the bulk tumour, the
nature of the tumour hierarchy and the niche requirement for that particular tumour, different in vivo models and end points
may be required to study the drug effects on various TICs. For advanced tumours with a large TIC burden, traditional models
with standard end points (for example, tumour regression and metastasis) might be sufficient, whereas for other tumours,
it might be necessary to focus on the tumour-initiating activity through administration of the agents before and following
implantation of enriched or isolated TICs. TIC-related end points also include the effect on relapse after chemotherapeutic
treatment or the ability of residual tumour cells after treatment to re-engraft in in vivo xenograft models. To understand the
effect of the drug on tumour hierarchy, immunochemistry using antibodies against TIC, lineage and EMT markers needs to
be performed. To promote the tumour–host interaction, orthotopic models, subrenal capsule implantation and
subcutaneous co-injection models that mix tumour cells with stromal cells have been explored. It is essential to develop
in vivo imaging capacity to track TICs and to visualize the compartments in which they reside, and some preliminary
progress has been made in this area.
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the finding that certain cancer cell lines may contain a
subpopulation of tumour-initiating cells is important:
cell lines can provide sufficient material for extensive
molecular and signalling profiling of these cells.
There are several ways to enrich for tumour-initiating
cells: using cell sorting techniques to select for combinations of cell-surface markers, sorting to select for a
subpopulation of cells that efflux dyes166,167, or various
serum-free stem cell culture conditions111,168,169 (fIG. 3a).
Although cell sorting for cell surface markers is useful for isolation of tumour-initiating cells that express
established markers81,123, it could be challenging to
use this technique in many other tumours owing to a
lack of markers and/or the requirement for multiple
markers15.
As the expression of the genes encoding Abc drug
transporters such as AbcG2 is a conserved feature of
several stem cell populations from a range of sources, the
efflux of the DNA-binding dye Hoechst 33342 has been
used to identify and enrich for certain types of tumourinitiating cells160,166,167. Theoretically, this method may be
more suitable to enrich for potential tumour-initiating
cells as it is less constrained by tissue specificity than
the cell surface makers discussed above. However, the
kinetics of dye exclusion, a potentially small window for
detecting stem cells that exclude the dye and the toxicity
of various dyes might limit functional analysis of the
enriched cell populations. It is also possible to enrich for
tumour-initiating cells by selecting other surrogate functional properties of stem cells, such as aldehyde dehydrogenase activity 165,170 and chemotherapeutic resistance60.
Serum-free non-adherent culture has been shown
to enrich for and propagate several types of tumourinitiating cells, including those from brain, breast and
colon cancer 7,73,111,168,171. The serum-free ‘sphere’ culture
was developed when it was observed that central nervous system (cNS) cells grown on non-adherent surfaces
form spheroid colonies (neurospheres) that have the
capacity for self renewal and can generate all of the principal cell types of the brain (that is, neurons, astrocytes
and oligodendrocytes)172. conversely, cells in serum-free
adherent culture can also maintain stem cell-like properties if the plates used have a unique surface (for example,
incorporating various nitrogen-containing functional
groups), or are coated with an appropriate matrix and/
or ligand111,169,173.
It is crucial to develop culture methods and conditions that simulate the growth and differentiationinhibitory signalling that is provided by the niche,
particularly for culturing cells from primary tumours.
Serum-free culture conditions that have been established for normal stem cells154,172,174 provide useful
starting points. A new generation of high-throughput
platforms, such as microfabricated arrays of extracellular matrix or other molecules that are involved in
paracrine and juxtacrine signalling, can be used to
identify relevant microenvironmental signals for different tumour-initiating cells175. Successful approaches to
sustaining and expanding normal stem cells in serumfree culture have included stimulation of the wnt,
Hedgehog and/or Notch pathways and inhibition of
the bMP pathway 174,176,177. Appropriate growth factors
must also be provided111,154,174 and favourable microenvironments, such as laminin-coated plates or laminin-rich matrigel, could be necessary or helpful111,169,174.
Metabolic activity and oxygen tension are other variables to consider in the culture of stem cells and tumourinitiating cells178,179.
As well as considering suitable screening end points,
efforts to adapt these stem cell culture systems to
tumour-initiating cells must take into account the distinct
origins and characteristics of these cells. Developmental
pathways that regulate self-renewal in culture may also
provide therapeutic targets, so the balance of exogenous
factors may be crucial for certain screens. unlike normal stem cells, tumour-initiating cells commonly carry
mutations that alter their growth factor dependence or
responses. Further differences in culture requirements
may be expected when the origin or characteristics of
a tumour-initiating cell is progenitor-like. Finally, the
mutational and pathway profiles of tumour-initiating
cells will vary with tumour subtype and grade, and
so culture conditions might have to be optimized
accordingly.
In vitro assays and screening methods. In many cases,
including under sphere conditions, in vitro culture of
tumour-initiating cells is expected to produce mixed
populations of tumour-initiating cells and more differentiated progeny. This presents both a challenge
(isolating the effect of experimental intervention on
tumour-initiating cells) and an opportunity (the ability
to use differentiation as an end point). Monitoring stem
cell markers with immunofluorescence or fluorescent
reporter gene expression is amenable to high-throughput analysis, and both readouts have been successfully
used to screen for novel regulators of self-renewal in
embryonic stem (eS) cells180,181. Differentiation markers have also been used as reporters to screen for small
molecules or genes that drive or inhibit stem cell differentiation182,183. based on such successes, imaging
platforms and other marker-based screens for modulators of tumour-initiating cell behaviour can be readily
envisioned (fIG. 3a). Nevertheless, efficiently and reliably
obtaining quantitative data from images presents design
challenges in terms of the data collection, data handling
and image processing 184. The other challenge is that few
markers and reporter genes have been established for
various tumour-initiating cells and their differentiated
progeny.
ultimately, the complexity of tumour–stroma interactions and tumour–matrix interactions in vivo might
be more accurately reproduced by cultivating mixed cell
populations in three-dimensional organotypic cultures
that can maintain various aspects of in vivo tumour–host
interactions and might enrich for tumour-initiating cells.
A more successful in vitro screening strategy might be to
use mixed three-dimensional organotypic cultures and
quantify and track tumour-initiating cells within them
using various markers or built-in quantitative fluorescent or enzymatic reporters. one challenge in screening three-dimensional cultures is the production of a
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consistent organotypic structure in a high-throughput
fashion, but aggregation methods that were developed
for reliable production of embryoid bodies from eS
cells185 can perhaps be adapted. To quantify specific cell
populations in three-dimensional culture formats, highspeed imaging systems and automated image analysis
methods can be combined186.
Progress has been made with leukaemia-initiating
cells and cNS tumour-initiating cells in terms of in vitro
assay conditions123,169,171, and it has become feasible to
conduct high-throughput in vitro analyses to search
for compounds that differentiate or kill these cells124,169.
In addition, in-depth knowledge of the stem cells and
progenitor cells from haematopoietic and cNS systems
is beginning to allow direct comparison of normal stem
cells and tumour-initiating cells from these tissues123,124,169.
eventually, similar assays for other tumour-initiating cells
will be established and optimized.
Ideal culture conditions should support cancer cell
proliferation in vitro without genotypic alterations and
with the retention of phenotypic behaviour — most
importantly in vivo tumorigenic ability over passages111.
However, even short-term culture can cause differences in
the in vivo repopulation ability of HScs that have identical cell surface markers, emphasizing the caution that is
needed in drawing conclusions from in vitro results21,187.
Alternatively, using genetic manipulations to achieve
a stabilized mesenchymal-like state that captures many
tumour-initiating cell properties can allow high-throughput screening in vitro, which has recently generated novel
leads against tumour-initiating cells188.
Orthotopic model
A system in which tumour cells
are implanted at the site of the
organ of origin.
In vivo tumour models. To evaluate agents that specifically
target tumour-initiating cells (fIG. 3b), it may be necessary
to focus on the tumour-initiating activity through administration of the agents before and following implantation
of tumorigenic cells; the effect on established tumours
might not be as dramatic as in nascent tumours and
could take longer 9. Alternative end points include the
effect on relapse after chemotherapeutic treatment, the
effect on metastasis and the ability of residual tumour
cells after treatment to re-engraft in in vivo xenograft
models. However, the frequency of tumour-initiating
cells in solid tumours seems to be substantially higher
than that of leukaemia-initiating cells in leukaemia3,5,
and recent mathematical analyses have further indicated
that tumour-initiating cells in advanced tumours may
not occur as a small fraction189. Mathematical modelling predicts that if progenitors acquire self-renewal
ability then self-renewing cells can come to dominate
a tumour 53. Agents that target tumour-initiating cells
might therefore show dramatic activity in certain traditional models of advanced tumours that have a large
tumour-initiating cell burden.
The function of a tumour-initiating cell may be
more effectively assessed when the cell is orthotopically
engrafted4,5,190,191, and tumour metastasis to specific organs
can often be reproduced in an orthotopic model (BOX 2). In
many epithelial tumours, an eMT or loss of differentiation
is frequently evident at the invading edge of the tumour
and is likely to mediate cellular detachment and eventual
metastasis57. cells that are undergoing an eMT could
conceivably be the precursors to metastatic tumourinitiating cells, and so eMT markers could be used as
biomarkers for evaluating agents that target tumourinitiating cells in metastasis models. Despite an increasing
awareness of orthotopic models, for some cancer types
(such as colon cancer), performance of an orthotopic
injection can be technically difficult. In these cases,
alternative approaches, including subcutaneous models
with cells suspended in matrigel (or mixed with stromal
cells) and xenograft models featuring subrenal capsule
implantation, have been explored6,7.
In vivo drug discovery screening demands a reproducible, cost-effective system. Tumour-initiating cell
models involving xenotransplantation of primary tumour
cells4,5,17 have limitations for medium- or high-throughput assays due to the intrinsic variation between tumours
and practical challenges of using freshly resected material, but could be a choice for testing candidate agents.
An alternative approach is to generate cancer cell lines
that are enriched with tumour-initiating cells. A highly
malignant breast cancer cell line (SK-3rd) was generated
by sequential in vivo passage in epirubicin-treated NoD–
ScID mice, taking advantage of the chemotherapy resistance of the tumour-initiating cells60. The SK-3rd cell line
is enriched for cells that display all the putative properties
of breast tumour-initiating cells13; moreover, these cells
metastasize and are capable of serial transplantation60.
More recently, glioma-initiating cell lines that are derived
directly from primary malignant gliomas were successfully cultured and expanded under serum-free adherent
culture conditions169. Genetic manipulation of differentiation status can also be used to produce an undifferentiated, tumorigenic character in cancer cell lines188.
These cell lines could be more clinically relevant than
conventional cancer cell lines, although the relevance of
any results obtained in cell lines needs to be confirmed
in primary cancer cells.
To address the limitations of cell lines and primary
tumour cells, primary tumour xenografts that have been
passaged in vivo offer a unique system for the study of
tumour heterogeneity and hierarchy in preclinical models.
Fragments of surgically resected tumour are implanted
directly into immunocompromised mice (orthotopically or subcutaneously). The resulting xenografts are
passaged to new animals and are therefore maintained
exclusively in vivo 192. The cellular architecture and
heterogeneity of a primary tumour xenograft closely
resemble those of the original patient tumour and are
more complex than the corresponding features of traditional cell line xenografts. Therefore, primary tumour
xenografts coupled with appropriate experimental
analysis tools constitute a tractable preclinical model for
effectively evaluating lead compounds and developing
drug combination and biomarker strategies162.
In vivo biomarker and imaging studies. Given that
tumour-initiating cells represent only a subpopulation of
the cells in a tumour and their existence might depend on
a niche, it is desirable to track them and visualize the compartments in which they reside before, during and after
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treatment in vivo. A preliminary understanding of the
effect of experimental intervention on tumour hierarchy
and tumour–host interaction in vivo can be achieved by
immunohistochemistry using antibodies against markers
of tumour-initiating cells, eMT, lineage differentiation
and different stromal cells (fIG. 3b). For tumour models
that have a large tumour-initiating cell burden, gene
signature and microarray analysis can also provide
biomarkers of tumour-initiating cells. Through in vivo
lineage tracking of cultured melanoma cells that are
differentially fluorochome-conjugated, co-xenografted
Abcb5+ melanoma-initiating cells and Abc5– subpopulations have been assayed for their relative contribution to
tumour growth, self renewal and differentiation9, and the
effect of treatment can in principle be similarly studied.
currently, there are no obvious paths by which to pursue
direct imaging, although the use of labelled antibodies
against specific tumour-initiating cell markers might
offer an entry point. To evaluate metastasis mediated by
tumour-initiating cells, non-invasive magnetic resonance
imaging of magnetically labelled tumour-initiating cells,
as has been used for normal stem cells193, could be useful.
It would also be desirable to develop surrogate reporter
genes or other biosensors that will allow for in vivo monitoring of the activities of self-renewal signalling and
tumour-initiating cells194. Targeted delivery of a reporter
gene has already allowed the locations of normal HScs to
be identified and monitored by whole-animal live imaging 195. More markers and reporter genes of tumour stem
cell-like properties need to be established.
Hopefully, the improvement and modification of
anticancer drug discovery platforms in light of the cancer
stem cell hypothesis will improve the clinical relevance of
preclinical assays and models. These models will not only
help us to understand how current chemotherapeutic
and tumour-targeted agents affect different levels of the
tumour hierarchy, but also reveal novel agents that target
tumour-initiating cells. In addition, preclinical studies
using these models might provide data that support
unique clinical combinations and biomarker strategies
for agents that target tumour-initiating cells.
Clinical strategies and outlook
Many aspects of the aberrant differentiation that is associated with poor prognosis in cancer can be best explained
by the cancer stem cell hypothesis. This is underscored
by the fact that, in clinical trials for advanced cancers,
tumour regression often does not translate into clinically
significant increases in patient survival. efficacy against
minimal residual disease, metastasis, delayed relapse and
tumour-free survival are expected to correlate with the
mechanism-based activity of agents that target tumourinitiating cells.
Given that tumour regression might not be the
most relevant early end point, biomarkers for tumourinitiating cells in patients who are receiving cancer
therapy need to be developed. However, translating the
markers that are used to enrich for tumour-initiating
cells into clinical biomarkers is not necessarily straightforward. For example, the cD44+ cD24–/low expression in patients with breast cancer that is detected by
immunohistochemistry does not correlate with event-free
or overall survival196, suggesting that not all cD44+ cD24–/low
cancer cells are tumorigenic and surface markers might
not be as conserved as was originally thought. However,
gene signatures derived by comparing cD44+ cD24–/low
breast cancer cells with normal breast epithelial cells,
which might reflect the epigenetic state of the tumour
cells, are correlated with decreased patient survival76,197.
circulating tumour cells, although extremely rare, are
a potential alternative to invasive biopsies as a source
of tumour tissue for the detection, characterization and
monitoring of tumour-initiating cells from solid tumours.
A microchip technology that is based on microfluidics
was shown to be sensitive and able to detect circulating
tumour cells in almost all of the examined patients with
recurrent carcinomas198.
It is also important to study the relevance to tumourinitiating cells of current biomarkers, such as prostatespecific antigen (PSA) for prostate cancer and mucin
16 (Muc16; also known as cA125) for ovarian cancer,
which are beginning to guide clinical trials and therapy.
both PSA and Muc16 are expressed in differentiated
tumour cells; it is still unclear whether they are surrogate
only to a bulk tumour cell population or to a tumourinitiating cell population as well. If genetic (for example,
gene amplification) or epigenetic (for example, promoter
methylation) changes or multiple changes of related
pathways could be used to predict the dependence of a
tumour-initiating cell on certain oncogenic pathways,
some of the self-renewal signalling molecules could
represent an Achilles’ Heel of cancer. Inhibitors of these
pathways would have considerable antitumour activity
alone199. In this regard, a diagnostic procedure to prescreen patients with such genetic or epigenetic changes
might be essential for drug development.
Among various agents that target self-renewal pathways, small molecules that target the Hedgehog pathway
are in early clinical studies, and have shown promising
results (TABLe 1). The SMo antagonist cyclopamine was
shown to lead to rapid regression of basal cell carcinoma
in all four patients in which it was tested200. In addition,
an orally-administered small-molecule antagonist of
SMo, GDc-0449, has shown limited toxicity and partial
responses in advanced basal cell carcinoma tumours in a
Phase I clinical trial. It is advancing to Phase II trials for
metastatic colorectal cancer and other advanced epithelial tumours155. The majority of patients with basal cell
carcinoma have genetic mutations in Hedgehog pathway mediators38; it is unclear whether GDc-0449 will
be as effective in other tumour types that do not have
such mutations or whether it must be combined with
other agents to show clinical activity. This combination
approach could be further complicated by the emerging
role of Hedgehog signalling in tumour stromal cells114,161.
GDc-0449 and other SMo antagonists (TABLe 1) will
therefore provide an interesting test of clinical strategies
in targeting renewal signalling.
There are several antibodies against cell surface
markers of tumour-initiating cells in clinical studies
(TABLe 1) . ePcAM is highly expressed in numerous
solid tumours, and was recently shown to be expressed
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on tumour-initiating cells from breast, prostate, colon
and pancreatic cancer 4,7,8. ePcAM-specific mAbs have
shown a limited efficacy in clinical trials142, suggesting
that immune tolerance or ADcc stimulated by these
mAbs by itself might not be effective in killing ePcAMoverexpressing tumour cells in clinical settings. To
overcome the limitations of the naked antibodies, catumaxomab was designed to bind to both human ePcAM
(the target on the tumour) and human cD3 (the target
on T cells), bringing cancer cells into proximity with the
immune-system cells that can destroy them. In addition, catumaxomab induces ADcc and is undergoing
advanced study in patients with malignant ascites145.
conversely, mAbs against cD44 can differentiate
tumour-initiating cells and have single-agent activity in
certain preclinical models148,201 (TABLe 1). It remains to be
seen whether these mAbs will show single-agent activity
in clinical settings or whether they will also need to be
coupled with cytotoxic approaches.
Various drug screening platforms that were specifically
designed to target tumour-initiating cells have begun to
identify novel drug leads169,188. whether tumour-initiating
cell-targeted therapies are effective as single agents, in
terms of short- and long-term clinical end points, will
probably depend on the lifespan of the bulk tumour
cells and the symptoms they cause. For tumours in
which most tumour cells are short-lived or the tumourinitiating cell burden is large, continued tumour growth
and maintenance may be highly dependent on the activity
of tumour-initiating cells. Tumours with populations
of proliferating progenitor-like cells (BOX 1) may take a
long time to regress, even if tumour-initiating cells are
destroyed. For some cancers, continued although limited proliferation of bulk tumour cells might be sufficient
to cause irreversible pathological damage. It is necessary to
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Acknowledgements
We thank J. Dick, L. Li, K. Arndt, R. Abraham, J. Rosen and
F. Behbod for discussions and comments on the manuscript.
Competing interests statement
The authors declare competing financial interests: see web
version for details.
DATABASES
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
BMI1 | Pten
UniProtKB: http://www.uniprot.org
ABCB5 | AKT | APC | BRCA1 | b-catenin | CD24 | CD44 | CD133
| CDNK1A | DLL4 | EGFR | EPCAM | ERBB2 | EZH2 | IL3RA |
LRP5 | LRP6 | mTOR | MUC16 | NOTCH1 | PSA | PTEN | SMO |
STAT3
FURTHER INFORMATION
Genentech website: http://www.gene.com
Infinity Pharmaceuticals website: http://www.ipi.com
Micromet website: http://www.micromet.de
Trion Pharma website: http://www.trionpharma.de
All links Are AcTive in The online pDf
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