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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.
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: firstname.lastname@example.org; email@example.com 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
806 | o cTober 2009 | VoluMe 8 © 2009 Macmillan Publishers Limited. All rights reserved
have been successfully isolated using appropriate cell surface markers. Killing these cells could be achieved by inhibiting their survival pathways (for example. Hedgehog or Notch pathway signalling) might work by both mechanisms. In addition. Furthermore. pancreatic. so the identification of tumour-initiating cells in mouse models adds credibility to the cancer stem cell hypothesis24–32. the blast cells. tumour-initiating cells are probably genetically unstable. certain markers used for prospective sorting may also select for cells that evade the immune system22. 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. brain. According to the cancer stem cell hypothesis. However. VoluMe 8 | o cTober 2009 | 807 © 2009 Macmillan Publishers Limited. a number of markers used in cell sorting are emerging as being predictive of disease progression9. cells16. including cD44. current failure with cancer treatment is not usually due to a lack of primary response or initial induction of remission. which could also be a relevant property for tumour-initiating cells. Following an overview of the current status of the cancer stem cell hypothesis. 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. or sensitizing them to chemotherapeutic agents (for example. as the bulk of the tumour has limited proliferation potential. In some cases. It was noticed that some experimental mouse models of leukaemia do not follow the cancer stem cell hypothesis. Although growing evidence from a wide range of systems favours the existence of tumour-initiating cells. cD24. Niche Cells and/or extracellular matrix components in specific anatomical locations that regulate the participation of the normal stem cells in tissue generation. Many cancers might contain subpopulations of tumour-initiating cells. this article therefore focuses on the challenges and opportunities for anticancer drug discovery. Nevertheless. cD133. with inhibitors of Wnt. As well as supporting the NATure reVIewS | Drug Discovery . give rise to differentiated cells and form new tumours. with a checkpoint kinase inhibitor). there may nevertheless be diversity of tumour hierarchy in different patients and varying degrees to which different tumour-initiating cells are stem cell-like. Cancer stem cells: evidence and controversy In the mid 1990s. in which tumour-initiating cells are thought to have crucial roles. and a committed progenitor could regain renewal activity. The purified populations of leukaemia-initiating cells contained a chromosomal translocation that was identical to that found in their progeny. colon. A major challenge now is to discover agents and strategies that target cancer and tumour relapse at their apparent source. with bone morphogenetic proteins or CD44-specific monoclonal antibodies) might be a successful therapeutic strategy. However. with inhibitors of phosphoinositol 3-kinase or interleukin-4-specific monoclonal antibodies). Nevertheless. tumour-initiating cells from solid tumours. liver and ovarian cancer and melanoma. 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. epithelial cell adhesion molecule (ePcAM) and ATP-binding cassette sub-family b member 5 (Abcb5)4–10. current cell sorting protocols are thought to favour niche-independent tumour-initiating cells (BOX 2). as with many cell lines that have lost the hierarchical structure of the primary leukaemia from which they originated. differentiating the tumour-initiating cells (for example.18 (BOX 2). 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. Solid tumours contain both tumour cells and various stromal cells. indicating that they identify clinically important cell populations. this important finding pointed to the organization of leukaemia as a stem cell hierarchy 19.REVIEWS clonogenic nature of leukaemia. transgenic models facilitate lineagetracing experiments. including human breast. but some could contain common tumorigenic cells. Furthermore. 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. anti-angiogenic therapy might work in part by affecting the vascular niche of tumour-initiating cells. some experimental mouse models may not accurately reflect the tumour heterogeneity and pathological environment of spontaneously occurring human malignancies16. These tumour-initiating cells can produce phenocopies of the original primary tumours when transplanted into NoD–ScID mice. so agents targeting these pathways are expected to kill more than just tumour-initiating cells.23.17. 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. which may resemble cells in activated or mobilized states that are selected when transplantation assays are performed on normal stem cells21. the behaviour of tumour-initiating cells might also be influenced by interactions with surrounding cells and matrix. Inhibition of developmental signalling that is involved in self-renewal (for example. which can provide evidence for tumour initiation and tumour hierarchies without the limitations and experimental variability of transplantation assays30. suggesting that certain human cancers may not adhere to this model14. There are various therapeutic strategies that could target tumour-initiating cells. tumour cells Nature Reviews | Drug Discovery are heterogeneous and only the tumour-initiating cells have the ability to proliferate extensively. maintenance and repair. but to relapse or tumour recurrence after therapy. Anoikis A form of programmed cell death that is induced in anchorage-dependent cells when they become detached from the surrounding extracellular matrix.31. Some of the survival pathways that are used by tumour-initiating cells could also be used by the bulk of the tumour. Alternatively. All rights reserved Destroy TICs Self renewal Differentiate 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. and breaking the interactions between cells in such a tumour might induce anoikis or change the properties of the tumour cells. Similar studies are much more difficult for solid tumours.
it has been proposed that progressive genetic alterations might occur at the level of tumour-initiating cells. As cancers arise only after multiple mutagenic events.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. Although the origins of tumour-initiating cells may vary. The clonal progression to cancer could operate through the ‘stem cell compartment’ (see the figure). especially the 808 | o cTober 2009 | VoluMe 8 extensive capability of self-renewal12.38. Owing to genetic instability. Based on genetic variations already observed in the tumour-initiating cell populations from different tumours76. which is a common feature of cancers according to comprehensive cancer genomic analysis 33–35. with little hierarchical organization15. EMT. Tumour-initiating cells diverge from their cells of origin with increased self-renewal capacity. Hedgehog signalling regulates tumourinitiating cells from multiple myeloma43 and chronic myeloid leukaemia (cMl)44. All rights reserved . many leukaemia-initiating cells have a higher selfrenewal capacity than normal HScs3. 2).nature. Although pathways that regulate self-renewal are tightly controlled in normal stem cells. and the ‘clonal evolution’ and ‘cancer stem cell’ models might not be mutually exclusive.45 and may be broadly important for self-renewal in many cancers. The behaviour of tumour-initiating cells could be further modulated by tumour–host interactions. and self-renewal pathways might be more conserved than surface markers among tumourinitiating cells. Hedgehog and Notch (fIG. In normal tissues. combination treatment involving both traditional therapies and therapies that target tumour-initiating cells will probably be required to ablate all cancer cells. leading to uncontrolled growth38. controversy aside. Developmental signalling pathways that regulate normal stem cell self-renewal. particularly as a genetically unstable tumour cell will present a ‘moving target’.203 and genetic mutations in developmental pathways in different cancers34. If the cancer stem cell hypothesis is correct. they share several properties with normal stem cells. the cancer stem cell hypothesis provides a novel framework to study cellular heterogeneity. the evolution of the tumour is largely the history of changes in self-renewing tumourinitiating cells (BOX 1). have been shown to be active in numerous human cancers38. including wnt. one popular explanation for tumour heterogeneity is genetic instability. as initially thought.REVIEWS Box 1 | Tumour hierarchy and clonal evolution Clonal evolution Normal tissue Stem cell Tissue or tumour hierarchy Multipotent progenitor Progenitor Mutations? EMT? Mutations Initial tumour Tumour-initiating cell Advanced tumour Mutations Target tumourinitiating cells Differentiated cells Tumour progenitors and differentiated tumour cells Target bulk of the tumour cells For decades. a large part of the apparent morphological and phenotypic heterogeneity can be explained by aberrant differentiation. 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 derivatives202.37 (see the figure in BOX 1). 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. as chemotherapeutic agents are often mutagenic. conversely. In this section. 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). it was recently shown that maintenance of cutaneous tumourinitiating cells is dependent on wnt and β-catenin signalling 42. 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. 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). aberrant differentiation and tumour–host interactions in many cancers. In addition. the phenotype and frequency of the tumour-initiating cells in relapsed tumours are expected to be distinct from those of early-stage lesions. 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). Actually. in tumour-initiating cells they may be constitutively activated or improperly regulated through genetic and/or epigenetic changes. as already shown in leukaemia-initiating cells204. long-lived cells are probably the most capable of supporting such Nature Reviews | Drug Discovery cumulative changes. and epigenetic changes in tumours could also be dynamic and unstable36. Cancer is both a proliferation and a differentiation disease. epithelial–mesenchymal transition. They also have important roles in progenitors and a www. In practice. we consider some of the key characteristics of tumour-initiating cells. Framework for understanding cancer properties The cancer stem cell hypothesis does not contradict the established clonal evolution view of cancer. Also.
Xenografts can be established either by injecting human tumour cell lines or by direct implantation of patient biopsies into immunodeficient mice. is thought to favour niche-independent tumour-initiating cells. Animal models with an increased efficiency of engraftment have since been developed. In addition. For example. and most early-disseminated cancer cells that are detected in the bone marrow have a putative breast tumour-initiating cell phenotype 4. progenitor cells could gain the capacity for self-renewal and become malignant 46.55.47. even with recent improvements15. Avoiding the transplant altogether by generating genetic mouse models of tumours may better maintain the tumour–host and tumour-initiating cell–niche interactions30. Oncomir MicroRNA known to be involved in cancer and tumorigenesis. To properly model certain tumours. and could have additional roles in the regulation of tumour and stromal cells. owing to the practical difficulty of modelling the multitude of genetic changes in a tumour. such as the wnt and Notch pathways58.REVIEWS Box 2 | Mouse models for cancer research and drug discovery In the mid 1980s. such as an increasing proliferation rate or a shift in the balance of cell division from asymmetrical division to symmetrical division48. rather than their proliferation rate.50. The limitations of xenotransplantation assays are particularly apparent in analysing tumour-initiating cells from solid tumours. NOD–SCID assays still have limited efficiency of engraftment and so can lead to underestimations of the frequency of human tumour-initiating cells. a malignant tumorigenic cell could have undifferentiated pathological features associated with increased self-renewal. eMT and xenograft metastasis60. differentiation and/or apoptosis. More recently. giving tumour-initiating cells a greater chance to interact with and obtain stimulation from a mouse environment. The most important and characteristic feature of tumour-initiating cells therefore seems to be their increased self-renewal potential. 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. Asymmetrical division A form of cellular replication in which a cell renews itself and generates a more differentiated progeny. by gaining certain epigenetic programmes or losing certain combinations of tumour suppressor genes. disabling the mouse immune system might not be desirable. It may ultimately be necessary to provide a humanized immune repertoire in mice. Although engraftment is improved by implantation into NOD–SCID Il2rg–/– mice. typically characterized by loss of cell adhesion. The current protocol. 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. Notably. In this rapidly expanding field of tumour biology. this strategy may be more suited to studying tumour-initiating cells that have few defined genetic lesions. expression of the oncomir LET7 in a breast tumour-initiating cell model inhibits self-renewal. The tumorigenic breast and pancreatic tumour-initiating cells have cell cycle profiles that are similar to those of bulk tumour cells. A major drawback of these mouse models is that they have limited utility in measuring tumour self-renewal in vivo. The SCID mouse was crossed with the non-obese diabetic (NOD) mouse. wider impact on the lineage.59. A corollary of the cancer stem cell hypothesis is that macroscopic metastases may arise from migrating or disseminated tumour-initiating cells54. showing it is not a proliferative advantage of these tumour-initiating cells over the non-tumorigenic cells that leads to differential engraftment4. perhaps resembling tumour-initiating cells from early-stage human disease.8. Epithelial–mesenchymal transition A cellular program in normal development and in cancer whereby cells of an epithelial origin acquire the properties of mesenchymal cells. which do not have tumour-initiating ability. including metastatic and pro-angiogenic capabilities. during the clonal evolution of tumour-initiating cells. and increased cell motility. Interestingly. repression of e-cadherin expression. Models that permit efficient engraftment are a prerequisite for assays that directly measure tumour self-renewal by serial transplantation. it was noticed VoluMe 8 | o cTober 2009 | 809 NATure reVIewS | Drug Discovery © 2009 Macmillan Publishers Limited. it is also clear that immune cells have a role in the progression of many tumours. In patients with breast cancer. slowly proliferating intestinal stem cells from phosphatase and tensin homologue (Pten)-deficient mice initiate intestinal polyposis52.31. 3b). 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. However. particularly for tumour cells from primary sources. by contrast. and self-renewal roles for classical cancer genes are also emerging. Tumour-initiating cells from AMl and cMl are mostly quiescent 49. like self-renewal. which lack both B and T cells) allowed the widespread use of human tumour xenografts as mouse models for cancer research and drug discovery205. eMT is regulated by developmental signalling pathways. whereas tumour-initiating cells from many solid tumours could be proliferative51. perhaps by genetic engineering or haematopoietic cell transplantation. Orthotopic models might more faithfully maintain the tumour–host interaction. 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. tumorigenic cD44+cD24–/low cells are readily detectable in metastatic pleural effusions. 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. which may not be representative of the population of tumour cells that can exhibit stem cell-like properties in their human tumour environment. Another hypothesis is that metastatic tumours originate when cells in a primary epithelial malignancy undergo an epithelial–mesenchymal transition (eMT)57. Co-injection with tumour-associated stromal cells might be another approach to improve the clinical relevance of xenotransplantation. Indeed. Self-renewal can be affected by changes in proliferation. Tumour-initiating cells in metastasis and tumour–host interactions. there may be stronger selection for increased self-renewal capacity than for higher proliferation53. and can resolve some of the issues related to the presence of murine stroma in animal models (fIG. All rights reserved . Aberrantly increased self-renewal might therefore be caused by different mechanisms. However. new routes by which cells may acquire or enhance self-renewal ability are regularly being discovered. Symmetrical division A form of cellular replication in which a single cell gives rise to two identical cells.
even if tumourinitiating cells are no more resistant to therapy than bulk tumour cells.79. As discussed above. but not primitive. 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. there are other cancer types in which tumour-initiating cells could be proliferative. and Abcb5+ tumour cells detected in patients with melanoma show a primitive molecular phenotype and correlate positively with clinical melanoma progression9. Following cancer therapy. However. Similarly. their relative resistance to oxidative or DNA damage. For example. the inefficiency of the treatment and/or the genetic instability of cancer cells. the high expression level of ATPbinding cassette (Abc) drug pumps9. Thus. imatinib eliminates proliferating. chemotherapy treatment increased the percentage of cD44+cD24–/low tumour cells in patients with breast cancer 77. Therefore.nature. Many tumour-initiating cells are thought to be resistant to chemotherapeutics such as paclitaxel and doxorubicin. thereby lifting environmental controls on selfrenewal72 and increasing the risk of metastasis.74. imatinib (Gleevec. if a large number of immature cells (probably including a large proportion of tumour-initiating cells) are present in the tumour sample. they can still be the key to the limitations of treatment. leading to neoplastic proliferation of primitive progenitors. or even promote the formation of their own niche71. so many tumourinitiating cells might also be relatively insensitive to these agents. their functional effects are mostly manifested further downstream in the tumour hierarchy. radiotherapy and tumour-targeted agents) or relapse quickly after initial remission. as has been shown for normal stem cells21. but may allow for the survival of sufficient tumour-initiating cells to cause eventual relapse. Furthermore. A niche. These pathways are therefore promising therapeutic targets in such cancers. a patient’s tumour is examined to assess the effects of treatment. and further aggressive treatment is warranted72. Novartis) targets and inhibits the bcr–Abl kinase. one theory of tumour dormancy is that tumour-initiating cells are held in check by a niche–stem cell interaction. committed leukaemia progenitors. as some tumours lie dormant or develop slowly over decades66. mutations might render tumour-initiating cells independent of niche signals. such as vascular niches68–70. 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. there could be variation in sensitivity to therapies among tumour-initiating cells. the cancer usually does not recur. It has been shown that an alteration in a HSc niche can lead to myeloproliferative disease67. consistent with the relative chemoresistance of these tumour-initiating cells. Key possible reasons for this failure include the inherent drug resistance of tumour-initiating cells. which are provided by the adjacent cells and/or extracellular matrix components62–64. cD44+ cell-specific genes included many known stem cell markers and correlated with decreased patient survival76. Although tumour-initiating cells might commonly be more resistant to therapy than the bulk of tumours. In breast cancer. the intrinsic high levels of anti-apoptotic molecules220.127.116.11. 810 | o cTober 2009 | VoluMe 8 .REVIEWS that cells undergoing eMT and tumour-initiating cells share many markers and properties61. and their efficiency of DNA repair 80–82.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. quiescent tumourinitiating cells.83. Similar to traditional anticancer drugs. This control of cell number and proliferation might be a preventative mechanism against cancer 65. also controls stem cell number and proliferation. the cancer is likely to return. The behaviour of normal stem cells is tightly regulated by signals that the cells receive from their microenvironmental niches. Alternatively. A therapy that kills 95% of cells in a tumour might be considered efficacious based on tumour shrinkage. cancer could progress if the niche were expanded or altered through genetic or epigenetic means65. then therapy has to be highly efficient at killing these cells to avoid relapse91. All rights reserved Why do many therapies fail to eradicate cancers? Many patients with cancer. As a result. although the mutations are thought to accumulate in tumour-initiating cells. but not the quiescent cMl stem cells50. 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. while supporting the self-renewal and maintaining the identity of a stem cell. www. bcr–Abl is required for the survival of proliferating progenitor cells. and most patients are still positive for the fusion gene transcripts after treatment 87–89. Tumour-initiating cells could also preferentially localize to environments that favour proliferation. particularly those with solid tumours. In melanoma. for various reasons including their quiescent or slowly proliferating nature78. it is conceivable that the tumour microenvironment could also constrain tumour-initiating cells. many novel tumour-targeted agents were also designed to target rapidly proliferating cancer cells. either do not respond to existing cancer therapies (including chemotherapeutics. 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. 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). Although it is likely that several factors contribute to the problem. Abcb5 is a marker for malignant melanoma-initiating cells. If the tumour contains only mature cells. 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. and growth signalling pathways are likely to have important roles in these cells. It has been suggested that the more aggressive and refractory cancers contain more tumour-initiating cells73. circumstantial evidence in support of this connection already exists in medical practice.
receive and integrate several pathways97. targeting the niche could be a strategy to indirectly inhibit or differentiate tumour-initiating cells. could be developed into antitumour agents. other signalling pathways could be important for the self-renewal of different tumour-initiating cells123. including those of basal cell carcinoma.98. During embryonic development. or targeting tumour-initiating cell surface markers through antibody-based cytotoxic approaches.42. Potential approaches to kill tumour-initiating cells include blocking essential selfrenewal signalling.102. Antagonist antibodies against individual Notch receptors are also being explored122. recently.94. including cMl and squamous cell carcinomas41. Several recent studies suggested that the survival benefit of trastuzumab (Herceptin. medulloblastoma. illustrating a paracrine requirement of Hedgehog signalling 114. Inhibition of Notch expression by antisense nucleic acid technology or the pharmacological blockade of the protease γ-secretase. Another strategy is to induce tumour cell differentiation.64. there is considerable crosstalk between these pathways.112 and/or requirements for the presence of stromal cells63. 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. Frizzled-related proteins (SFrPs) and Dickkopf proteins (DKKs). including combination therapies to counter drug resistance.104. In addition to developmental signalling pathways. which can potentially be achieved by inhibiting developmental pathways or epigenetic programmes. TABLe 1). 2. Tumour-initiating cells and their niches might similarly operate as signalling centres. have been discovered99. which cleaves Notch (fIG. 1). Another γ-secretase inhibitor depletes tumour-initiating cells in brain tumours119. However. small-cell lung cancer and pancreatic cancer 106–110. with identifiable signalling centres that generate.45. which act at the cell surface to inhibit wnt signalling through its receptors. Developmental pathways in self-renewal and differentiation. Hedgehog ligand expressed by tumour cells can also activate the Hedgehog pathway in the tumour stromal microenvironment. 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. It has been reported that pharmacological inhibitors of Hedgehog signalling display efficacy in various animal models. has striking antineoplastic effects in Notch-expressing transformed cells in vitro and in xenograft models115–117. wnt signalling has been shown to be required for self-renewal of tumour-initiating cells in several cancers. the therapeutic window of γ-secretase inhibitors is narrow. 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. All rights reserved Therapeutic opportunities The cancer stem cell hypothesis provides a rationale for several therapeutic strategies beyond traditional antiproliferative agents (fIG. 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. Such challenges complicate in vitro screening assays. testicular seminoma) and neuroblastomas. This suggests that their stem cell components might be inherently sensitive to chemotherapeutics and unable to adapt to counteract them. chemotherapy treatments for such tumours often eliminate the undifferentiated cancer cells and produce residual masses that are benign tumours composed of differentiated cells92. it probably extends to neoplasms that are amenable to cure. Nevertheless. 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. However. Antibodies against various wnts103. Such selective targeting of an individual Notch pathway might provide a viable strategy for impairing niche function. understanding the molecular basis of tumour-initiating cell behaviour will allow for the design of new strategies. in which multiple developmental pathways are active and converge to control self-renewal. it was shown that inhibition of Hedgehog signalling kills cMl tumour-initiating cells. Indeed. as discussed below. such as certain germ cell neoplasms (that is. VoluMe 8 | o cTober 2009 | 811 © 2009 Macmillan Publishers Limited. several developmental signalling pathways have become the recent focus of drug discovery efforts (fIG. 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 mutated100.113. and antibodies against Hedgehog and small-molecule inhibitors of the Hedgehog coreceptor Smoothened homologue (SMo) have been identified105. Small-molecule antagonists of the oncogenic transcription factor TcF–β-catenin protein complex have been reported101. these agents are not very effective and the results vary among cellular models. Frizzled proteins and wnt coreceptor low-density lipoprotein receptor-related protein 5 (lrP5)–lrP6 are also being explored. because of their inhibition of multiple Notch pathways and the possible effect on normal stem cells.REVIEWS If the cancer stem cell hypothesis is widely applicable. However. if designed to have desirable pharmacokinetic properties. most patients with metastatic breast cancer still develop resistance within 1 year of trastuzumab treatment 95.124. 2). inhibiting the survival mechanisms of these cells. As many tumour-initiating cells might depend on a niche to maintain their identity. More recently.106 (TABLe 1). impairs the propagation of bcr–Abl-driven cMl and the growth of imatinibresistant mouse and human cMl44. Inhibition of the Hedgehog signalling pathway is also a viable therapeutic strategy.121. Active derivatives of these antagonists. extracellular wnt inhibitors. partly because it is difficult to maintain Hedgehog pathway activity in vitro due to differentiation under conventional culture conditions111. including the secreted NATure reVIewS | Drug Discovery .
survival and differentiation. 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. To selectively kill tumour-initiating cells. It has been shown that mixed-lineage leukaemia (Mll) fusion proteins. This lifts suppression of Smoothened homologue (SMO). breast tumours. tumours of the digestive tract. leukaemia and multiple myeloma Haematopoietic. There is growing evidence that wnt. It has been suggested that alternative pathways involving AKT exist downstream of Notch activation126. epidermal and intestinal Colon carcinoma. The results could depend on the status and interaction of different oncogenic pathways. It has been shown that bMPs could induce the differentiation of cD133+ glioblastoma tumour-initiating cells predominantly to astrocyte-like cells. For example. 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. leukaemia. axis inhibition protein (axin). inhibit their proliferation or kill them. Hedgehog and Notch signalling are likely to interact with one another and with additional signals. basal cell carcinoma. including self-renewal. 812 | o cTober 2009 | VoluMe 8 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. resulting in two proteolytic cleavages of the receptor.REVIEWS Wnt DKK LRP5–6 Wnt Fz SFRP Hh PTCH SMO Notch Axin APC β-Cat GSK3β CK1α STK36 Gli SUFU AKT Hh SMO antagonist Notch Ligand γ-secretase inhibitor β-cat TCF– LEF Gli RBPJ Normal stem cells Cancer Haematopoietic. epidermal tumours including breast tumour. activating a cascade that leads to the translocation of glioma-associated oncogene homologue (Gli) into the nucleus and the activation of target genes. and has been used as a tool compound to study Hh signalling pathways both in vitro and in vivo. glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α). Soluble Frizzled-related protein (SFRP) and Dickkopf protein (DKK) are endogenous secreted antagonists of Wnt signalling. such as bone morphogenetic proteins (bMPs) and growth factors that are produced by tumour-initiating cells. These signals converge to generate the distinctive features of tumour-initiating cells.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. proliferation. which modulate www. targeting a specific developmental pathway to promote differentiation could be a more general strategy for eliminating tumour-initiating cells (fIG. squamous cell carcinoma and tumours of the digestive tract Haematopoietic. it might be necessary to inhibit multiple pathways in many cases. which enters the nucleus and interacts with transcription factors including recombination signal binding protein for immunoglobulin κJ region (RBPJ.nature. also known as CBF1). brain tumours and lung cancer Figure 2 | signalling pathways that regulate self renewal during normal stem cell development and cancer transformation. Cyclopamine is a potent antagonist of SMO. The regulation of self-renewal remains poorly understood and could involve transcriptional networks and epigenetic programmes that regulate chromatin accessibility.177. This facilitates the entry of β-catenin into the nucleus. neural and germline Medulloblastoma. where it regulates target gene transcription through association with the transcription factor TCF–LEF (lymphoid enhancer binding factor). and whether inhibition of an individual pathway would differentiate tumour-initiating cells. 1). which consists of adenomatous polyposis coli protein (APC). NoTcH1 regulates the PTeN–phosphoinositide 3-kinase (PI3K)–AKT pathway in T cell acute lymphoblastic leukaemia (T-All)126. conversely. prostate cancer. It is still unclear how various signalling pathways interact to maintain self-renewal activity in different cancers. epidermal and intestinal Leukaemia. All rights reserved . leading to the release of β-catenin| (β-cat) from Nature Reviews Drug Discovery the ‘degradation complex’. 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. which markedly attenuated their tumour-forming ability in a preclinical model127. This mediates the release of the Notch intracellular domain. Various γ-secretase inhibitors can inhibit Notch cleavage and activation. Activation of the Hedgehog (Hh) signalling pathway is initiated by binding of a Hh ligand to protein patched homolgue (PTCH). the bulk tumour cells or their microenvironment 125.
Invasive basal subtype breast cancers significantly overexpress eZH2. a bispecific single-chain antibody against EPCAM and CD3 cancer indication Oesophageal and prostate cancer and medulloblastoma Solid tumours Basal cell carcinoma Basal cell carcinoma and metastatic colorectal cancer Advanced and/or metastatic solid tumours Melanoma and NSCLC Colon cancer Intestinal adenomas T-ALL and metastatic or advanced breast cancer Solid tumours Malignant melanoma Acute myeloid leukaemia Breast cancer Metastatic breast cancer Colon cancer Malignant ascites and ovarian and gastric cancer status Target validation Target validation Phase I Phase II Phase I Target validation Target validation Target validation Preclinical Phase I Preclinical Target validation Preclinical Preclinical Phase II Phase II–III Phase II–III references 105 38 200 Genentech website (www. epithelial cell adhesion molecule.102 118 Merck website (www.94.121 9 148 201 Micromet website (www. but not in normal HScs123. 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. NSCLC. NATure reVIewS | Drug Discovery but have no effect on cell differentiation or multipotency. Delta-like 4. recent studies also suggest an important role for erbb2 in maintaining tumour-initiating cells in breast cancer 93. can reprogramme differentiated myeloid cells and activate self-renewal in cells with no inherent self-renewal properties46. In AMl.micromet.128. in addition to its presumed role in bulk tumour cells. are essential components by which stem cells reversibly repress genes that are related to differentiation129. a trifunctional antibody against EPCAM and CD3 MT110.de) 142 Trion Pharma website (www.de) Lung and gastrointestinal cancer Phase I ABCB5. EPCAM. a Sonic Hedgehog neutralizing mAb Cyclopamine.trionpharma. a SMO antagonist IPI-926 Wnt pathway Notch pathway A WNT2-specific mAb Various TCF–b-catenin inhibitors DBZ. was shown to induce rapid cell death in cD34+cD38– leukaemia-initiating cells. 130). MG-132. T-cell factor.com) 120.com) Infinity Pharmaceuticals website (www. suggesting that molecular pathways that contribute to bulk tumour growth can also be successfully targeted to sensitize tumour-initiating cells to cytotoxic therapies133. 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.T-ALL. a CD44-specific mAb Adecatumumab. an EPCAM-specific human mAb Edrecolomab. Although we know little about survival pathways of tumour-initiating cells in various tumour types. It is unclear how pathways that regulate self-renewal interact with those that regulate the survival of tumour-initiating VoluMe 8 | o cTober 2009 | 813 © 2009 Macmillan Publishers Limited. a γ-secretase inhibitor MK-0752. nuclear factor κb (NF-κb) was found to be constitutively active in primitive AMl cells (which are considered leukaemia-initiating cells). DLL4. they are potential targets for killing tumour-initiating cells. and the PI3K pathway regulates survival of tumour-initiating cells that reside in the perivascular niche of medulloblastoma70. Smoothened homologue. eZH2 also affects bMI1mediated suppression of the p16Ink4a–p19Arf locus to avert growth arrest or apoptosis of stem cells131.REVIEWS Table 1 | Selected agents targeting self-renewal signalling or tumour-initiating cell surface markers Target or Agents target pathway Hedgehog pathway 5E1. an EPCAM-specific mAb Catumaxomab. ATP-binding cassette sub-family B member 5. TCF.gene. T cell acute lymphoblastic leukaemia. monoclonal antibody. a SMO antagonist GDC-0449. Polycomb group proteins. Survival mechanisms in tumour-initiating cells.com) 103.micromet. An interleukin-4 (Il-4)-specific antibody reduced the viability of both cD133– and cD133+ colon cancer cells and increased the efficacy of chemotherapy. non-small-cell lung cancer. but not normal cD34+cD38– cells. a proteasome inhibitor with potent NF-κb signalling inhibitory activity. recently.104 101. a CD44-specific mAb P245. an ABCB5-specific mAb H90. In addition.merck. All rights reserved .de) Micromet website (www. 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.ipi. mAb. SMO. a γ-secretase inhibitor DLL4-specific mAbs ABCB5 CD44 EPCAM 3C2-1D12. such as enhancer of zeste homologue 2 (eZH2). chromatin structure through histone modification. certain oncogenic pathways that are distinct from developmental pathways might have a role in the survival of some tumour-initiating cells.
Although Abc transporter activity can result in tumour-initiating cells being relatively resistant to many conventional therapies. potentially making them resistant to immune attack. which induces ADcc in Abcb5+ malignantmelanoma-initiating cells. is a specific surface marker for AMl-initiating cells141. and could be highly variable among tumour types. certain tumour-initiating cells have lower levels of reactive oxygen species (roS) than corresponding non-tumorigenic cells.145. which is not present on normal stem cells. there seem to be some phenotypic differences between leukaemiainitiating cells and HScs. the antitumour activity of these antibodies can be enhanced by a cytotoxic immunoconjugate or engineered antibody binding to both tumour and immune cells 144. wnt–β-catenin signalling was shown to be involved in the maintenance of cutaneous tumour-initiating cells www. Agents that target markers on the surface of tumourinitiating cells could also work by affecting the niche (fIG. 1). Therapeutic windows and combination strategies The potential therapeutic window is always a concern for any anticancer approach. with tumour-initiating cells as both the source and the target. endothelial niches were also revealed for HScs149. it may make the cells susceptible to alternative strategies that target cells with effective drug efflux 146.138). including differences in the expression of THY1 membrane glycoprotein. endosteal (osteoblastic) niches have been identified for both normal HScs63 and AMl-initiating cells147. However. These preliminary studies highlight the potential of inhibiting DNA damage responses136 to overcome the resistance of tumour-initiating cells to therapy. Markers on the surface of tumour-initiating cells are also important targets.REVIEWS cells. and by altering their ‘stem cell’ fate through differentiation148. although it was recently shown that NoTcH1 regulates the PTeN–PI3K–AKT pathway in T-All126 and erbb2 expression in breast cancer 94. In particular. particularly for antibody-based therapeutics (TABLe 1).70. cD44. In glioblastomas. Some tumourinitiating cells express immune-tolerance markers143. the self-renewal activity of which depends on the developmental stage and tissue homeostasis. KIT and Il-3 receptor-α (Il3rA)139–141. All rights reserved various immune tolerance mechanisms. and brain tumour-initiating cells reside in a perivascular niche69.154. this finding highlights the potential feasibility of achieving a therapeutic window in targeting tumour-initiating cells. also known as p21) limits DNA damage and maintains the self-renewal of leukaemia-initiating cells135. exerts tumour-inhibitory effects in a melanoma xenograft model9. However. patients and even within the same patient. In breast cancer. Strategies that specifically target other niches of tumour-initiating cells can also be envisioned. Pharmacological depletion of roS scavengers in tumour-initiating cells markedly increases DNA damage and results in radiosensitization82. 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. tumour-initiating cells that exhibit overactive self renewal might be more sensitive to agents that inhibit self-renewal pathways than normal stem cells. most therapeutic monoclonal antibodies (mAbs) interact with components of the immune system through antibody-dependent cellular cytotoxicity (ADcc) and/or complementdependent cytotoxicity. However. cell cycle restriction through the expression of cyclin-dependent kinase inhibitor 1A (cDNK1A. 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.nature. Cell surface markers and niche interaction. Most of these markers are expressed in normal cells. systemic administration of a mAb directed against Abcb5. which might be able to bypass 814 | o cTober 2009 | VoluMe 8 © 2009 Macmillan Publishers Limited. including those that target tumour-initiating cells. 81). These data indicate that signalling in niche interactions can be bidirectional. and immune responses might be part of their antitumour mechanisms142. Interestingly. DNA damage responses were shown to be preferentially activated in glioma-initiating cells before and after radiation.com/reviews/drugdisc . The surface markers might reflect the cellular origin and history of that particular tumour-initiating cell. In addition. In addition to binding to their targets and inhibiting target-dependent signalling. cD133 and Abcb5 have been used to identify tumour-initiating cells from various tumour types3–9. recently. and so finding a therapeutic window could be a challenge. among other things. Knockout of the PTEN tumour suppressor causes the generation of transplantable leukaemiainitiating cells and the depletion of normal HScs in mice153. cD44 could have several roles in tumorigenesis. It is important to emphasize that much of the nicheinteraction data has been obtained from animal models. 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. Nevertheless. Il3rA. there might be mechanistic differences between tumourinitiating cells and normal stem cells with respect to self renewal. and the role of the niche in various human tissues and cancers is not yet clear. suggesting that these cells might help to create their own niche. rapamycin not only depleted leukaemia-initiating cells but also restored normal HSc levels in this model. As combined loss of certain tumour suppressor genes in progenitor cells can lead to malignant cells with increased self-renewal activity47. and there could be many reasons to combine agents that target tumour-initiating cells with anti-angiogenic therapy. Neural stem cells are thought to localize to vascular niches150–152. Although it has not yet been confirmed in the case of human cancer. The radioresistance of cD133+ glioma-initiating cells can be reversed with a specific inhibitor of checkpoint kinase 1 (cHK1) and cHK2 (Ref. Moreover. stem cell-like glioma cells have been shown to promote tumour angiogenesis71. and are a possibility for leukaemia-initiating cells. The surface markers cD34. uniquely among such markers. Interestingly.
treatment with a γ-secretase inhibitor seemed to be better tolerated in mice when using a pulsed dosing regimen156. preclinical evidence for this is not yet available. as with primary tumour cells. 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. 3). however. primary tumour xenografts and certain cancer cell lines (fIG. Given the emerging role of Notch and Hedgehog in the tumour stroma and vasculature114. and may need to be combined with chemotherapeutics to eliminate cells further down the tumour hierarchy. suggesting that normal stem cells could recover quickly from the treatment. providing another rationale for combining PI3K–AKT inhibitors with chemotherapeutics that are substrates of the Abc drug transporter. In addition. the SMo antagonist GDc-0449 was well tolerated in Phase I clinical trials155. It is also crucial to study their effects on stromal cells and the vasculature. are known to exist 141. 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. 3). the therapeutic window can be improved by optimizing the dosing regimen. counter drug resistance and help to achieve long-term remission in a clinical setting. the currently limited repertoire of those that have been identified will grow as tumour-initiating cell populations from various systems are further characterized. Now that markers of tumour-initiating cells. owing to differential exposure and signalling.163–165. it is thought that several pathways. 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. well designed combination strategies based on data from relevant preclinical models can overcome pathway redundancy. Sources from which to isolate tumour-initiating cells include samples from patients with primary cancer. whereas wnt–β-catenin signalling is not essential for normal epidermal homeostasis. 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. As many tumour-initiating cells might originate from progenitor cells or partially differentiated cells46. the effects of these agents on drug delivery should therefore also be considered161. including Il3rA for AMl. As discussed below. It has been reported that simultaneously blocking the Hedgehog and epidermal growth factor receptor (eGFr) pathways using cyclopamine and gefitinib resulted in growth arrest. Hopefully.47. agents targeting these pathways might have an impact on both tumour-initiating cells and tumour vasculature. converge to regulate tumour-initiating cells. and so is often biased to select targets with homogeneous expression patterns and potent compounds that kill the bulk tumour cells. 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. If there is a sufficiently large therapeutic window. Therefore. All rights reserved reside in the perivascular niche following radiation in medulloblastoma in vivo. to determine their efficacy and to study the emergence of drug resistance in preclinical models. and inhibition of AKT signalling sensitizes these cells to radiation-induced apoptosis70. as shown for cD44-specific mAbs148. targeting a combination of pathways that are uniquely active in tumourinitiating cells will be more effective than inhibiting a single pathway. indicating the potential therapeutic value of combining Hedgehog antagonists with PI3K–AKT inhibitors. Tumour-initiating cell enrichment and in vitro culture conditions. mAbs that target different epitopes of the same target could have different therapeutic windows. apoptosis and a decrease in the invasiveness of prostate cancer cells157.REVIEWS and malignant human squamous cell carcinomas 42. providing preliminary clinical evidence of therapeutic windows for strategies that target self-renewal signalling. However. based on the pharmacokinetic properties of the agent. VoluMe 8 | o cTober 2009 | 815 . including those that regulate self renewal and cell growth. As discussed above. Many agents that target tumour-initiating cells differentiate these cells. The inhibition of Notch signalling by γ-secretase inhibitors is presumed to have a narrow therapeutic window.120. it was observed that. many of them are expected to have surface markers distinct from those of normal stem cells. and to follow closely the therapeutic window of different combinations. Although their relevance to tumour-initiating cells from primary patient samples still requires further characterization. but also delay the development of drug resistance compared with either agent alone. 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. 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 vivo123.121. 3b). In addition. Interestingly. some cancer cell lines cultured under conventional conditions have a tumour hierarchy based on established tumour-initiating cell markers10. The PI3K–AKT pathway regulates AbcG2 activity in glioma-initiating cells160. the PI3K pathway regulates survival of tumour-initiating cells that NATure reVIewS | Drug Discovery © 2009 Macmillan Publishers Limited. similar to the adjustment of dosing schedule of cytotoxic agents. It is important to evaluate different agents and combinations in the context of the tumour hierarchy and with biomarkers162 (fIG.60. some traditional preclinical models may not reflect clinical complexities such as tumour hierarchy.
Sources of tumour-initiating cells (TICs) for enrichment and characterization include samples from cancer patients. 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. subrenal capsule implantation and subcutaneous co-injection models that mix tumour cells with stromal cells have been explored. 816 | o cTober 2009 | VoluMe 8 © 2009 Macmillan Publishers Limited. enrichment for TICs is likely to be a frequent but not a necessary step in TIC characterization. Drug effects on TICs can be studied according to standard end points (such as proliferation. 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. lineage and EMT markers • Imaging TICs with reporters or biosensors TIC Lineage EMT • Self-renewal (for example.nature.com/reviews/drugdisc . survival and colony size) and TIC-focused end points such as aberrant differentiation (for example. and in vitro results always need to be validated in vivo. orthotopic models. tumour regression and metastasis) might be sufficient. the nature of the tumour hierarchy and the niche requirement for that particular tumour.REVIEWS a Human tumours Primary xenografts Cell lines b Human tumours Primary xenografts Cell lines Tissue dissociation Tissue dissociation or fragment 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 Subcutaneous models Simple. To understand the effect of the drug on tumour hierarchy. To promote the tumour–host interaction. immunochemistry using antibodies against TIC. b | Depending on the life span of the bulk tumour. lineage and EMT markers needs to be performed. high-throughput assays Orthotopic models Physiological microenvironment Co-injection models Human stromal cells. a | For in vitro studies. 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. It is essential to develop in vivo imaging capacity to track TICs and to visualize the compartments in which they reside. whereas for other tumours. TICs are defined by in vivo experiments. susceptible to molecular engineering Two-dimensional culture Plastic or coated plate Matrix growth factors Semi-solid media Co-culture with stromal cells Three-dimensional organotypic culture + drugs Replate + drugs + drugs + drugs Monitor TIC population: • Surface markers • Fluorescent reporters • Enzymatic reporters • Functional tests Standard end points • Proliferation • Survival • Colony size TIC-focused end points • Differentiation markers + drugs 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. 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. primary tumour xenografts and certainDiscovery Nature Reviews | Drug cancer cell lines that maintain tumour hierarchy. lineage or epithelial–mesenchymal transition (EMT)) and self renewal. different in vivo models and end points may be required to study the drug effects on various TICs. traditional models with standard end points (for example. Even highly purified TICs are expected to gradually become more differentiated under tissue culture conditions. disproportionate presence of markers of TICs. and some preliminary progress has been made in this area. All rights reserved www. For advanced tumours with a large TIC burden. TICs can be studied in heterogeneous systems provided their activities can be monitored by cell surface markers or signalling reporter activities. colony number) Identify global and TIC-specific drug effects Figure 3 | Anticancer drug discovery platforms to target tumour-initiating cells.
such as microfabricated arrays of extracellular matrix or other molecules that are involved in paracrine and juxtacrine signalling. could be necessary or helpful111. including those from brain.183.171. efficiently and reliably obtaining quantitative data from images presents design challenges in terms of the data collection.123. In vitro assays and screening methods. the efflux of the DNA-binding dye Hoechst 33342 has been used to identify and enrich for certain types of tumourinitiating cells160. can be used to identify relevant microenvironmental signals for different tumour-initiating cells175. cells in serum-free adherent culture can also maintain stem cell-like properties if the plates used have a unique surface (for example.173. Theoretically. As well as considering suitable screening end points. and so culture conditions might have to be optimized accordingly.111. imaging platforms and other marker-based screens for modulators of tumour-initiating cell behaviour can be readily envisioned (fIG. 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. Successful approaches to sustaining and expanding normal stem cells in serumfree culture have included stimulation of the wnt. efforts to adapt these stem cell culture systems to tumour-initiating cells must take into account the distinct origins and characteristics of these cells. Monitoring stem cell markers with immunofluorescence or fluorescent reporter gene expression is amenable to high-throughput analysis. 3a).REVIEWS 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. astrocytes and oligodendrocytes)172.167. It is also possible to enrich for tumour-initiating cells by selecting other surrogate functional properties of stem cells. data handling and image processing 184. or various serum-free stem cell culture conditions111. Finally. the mutational and pathway profiles of tumour-initiating cells will vary with tumour subtype and grade. 3a).154.170 and chemotherapeutic resistance60. neurons. in vitro culture of tumour-initiating cells is expected to produce mixed populations of tumour-initiating cells and more differentiated progeny.169 (fIG.73. sorting to select for a subpopulation of cells that efflux dyes166. The other challenge is that few markers and reporter genes have been established for various tumour-initiating cells and their differentiated progeny. including under sphere conditions. Nevertheless.176.168.179. incorporating various nitrogen-containing functional groups). particularly for culturing cells from primary tumours.169. 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.174 provide useful starting points.169. Hedgehog and/or Notch pathways and inhibition of NATure reVIewS | Drug Discovery © 2009 Macmillan Publishers Limited. In many cases. Further differences in culture requirements may be expected when the origin or characteristics of a tumour-initiating cell is progenitor-like. tumour-initiating cells commonly carry mutations that alter their growth factor dependence or responses. so the balance of exogenous factors may be crucial for certain screens. ultimately. the kinetics of dye exclusion. Metabolic activity and oxygen tension are other variables to consider in the culture of stem cells and tumourinitiating cells178. conversely.166. 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 VoluMe 8 | o cTober 2009 | 817 . breast and colon cancer 7.177. based on such successes.174. or are coated with an appropriate matrix and/ or ligand111. 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. unlike normal stem cells. However.181. Differentiation markers have also been used as reporters to screen for small molecules or genes that drive or inhibit stem cell differentiation182.167.172. Although cell sorting for cell surface markers is useful for isolation of tumour-initiating cells that express established markers81. 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. Appropriate growth factors must also be provided111. Serum-free non-adherent culture has been shown to enrich for and propagate several types of tumourinitiating cells. All rights reserved the bMP pathway 174. There are several ways to enrich for tumour-initiating cells: using cell sorting techniques to select for combinations of cell-surface markers. A new generation of high-throughput platforms. such as laminin-coated plates or laminin-rich matrigel. such as aldehyde dehydrogenase activity 165. 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. It is crucial to develop culture methods and conditions that simulate the growth and differentiationinhibitory signalling that is provided by the niche. 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. 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).168.174 and favourable microenvironments. Serum-free culture conditions that have been established for normal stem cells154. and both readouts have been successfully used to screen for novel regulators of self-renewal in embryonic stem (eS) cells180. Developmental pathways that regulate self-renewal in culture may also provide therapeutic targets.
In vivo drug discovery screening demands a reproducible. To address the limitations of cell lines and primary tumour cells. Given that tumour-initiating cells represent only a subpopulation of the cells in a tumour and their existence might depend on a niche. during and after www. Despite an increasing awareness of orthotopic models. tumorigenic character in cancer cell lines188. using genetic manipulations to achieve a stabilized mesenchymal-like state that captures many tumour-initiating cell properties can allow high-throughput screening in vitro. In addition.REVIEWS consistent organotypic structure in a high-throughput fashion. However. Genetic manipulation of differentiation status can also be used to produce an undifferentiated.169.5. In vivo tumour models. Therefore. The resulting xenografts are passaged to new animals and are therefore maintained exclusively in vivo 192.169. which has recently generated novel leads against tumour-initiating cells188. taking advantage of the chemotherapy resistance of the tumour-initiating cells60. and it has become feasible to conduct high-throughput in vitro analyses to search for compounds that differentiate or kill these cells124. for some cancer types (such as colon cancer). 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. and so eMT markers could be used as biomarkers for evaluating agents that target tumourinitiating cells in metastasis models. the effect on metastasis and the ability of residual tumour cells after treatment to re-engraft in in vivo xenograft models. have been explored6. the frequency of tumour-initiating cells in solid tumours seems to be substantially higher than that of leukaemia-initiating cells in leukaemia3.190.5. it may be necessary to focus on the tumour-initiating activity through administration of the agents before and following implantation of tumorigenic cells. More recently.191. Progress has been made with leukaemia-initiating cells and cNS tumour-initiating cells in terms of in vitro assay conditions123. 818 | o cTober 2009 | VoluMe 8 . 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. but aggregation methods that were developed for reliable production of embryoid bodies from eS cells185 can perhaps be adapted. However. Fragments of surgically resected tumour are implanted directly into immunocompromised mice (orthotopically or subcutaneously). To quantify specific cell populations in three-dimensional culture formats.com/reviews/drugdisc © 2009 Macmillan Publishers Limited. An alternative approach is to generate cancer cell lines that are enriched with tumour-initiating cells. the effect on established tumours might not be as dramatic as in nascent tumours and could take longer 9. these cells metastasize and are capable of serial transplantation60. To evaluate agents that specifically target tumour-initiating cells (fIG. moreover. glioma-initiating cell lines that are derived directly from primary malignant gliomas were successfully cultured and expanded under serum-free adherent culture conditions169. Tumour-initiating cell models involving xenotransplantation of primary tumour cells4. performance of an orthotopic injection can be technically difficult. including subcutaneous models with cells suspended in matrigel (or mixed with stromal cells) and xenograft models featuring subrenal capsule implantation. The function of a tumour-initiating cell may be more effectively assessed when the cell is orthotopically engrafted4. alternative approaches. These cell lines could be more clinically relevant than conventional cancer cell lines.17 have limitations for medium. A highly malignant breast cancer cell line (SK-3rd) was generated by sequential in vivo passage in epirubicin-treated NoD– ScID mice. 3b). 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. even short-term culture can cause differences in the in vivo repopulation ability of HScs that have identical cell surface markers.7.187. highspeed imaging systems and automated image analysis methods can be combined186. but could be a choice for testing candidate agents.nature. Alternatively. The SK-3rd cell line is enriched for cells that display all the putative properties of breast tumour-initiating cells13. In these cases.124. cells that are undergoing an eMT could conceivably be the precursors to metastatic tumourinitiating cells. cost-effective system. 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. All rights reserved Orthotopic model A system in which tumour cells are implanted at the site of the organ of origin. Alternative end points include the effect on relapse after chemotherapeutic treatment. and tumour metastasis to specific organs can often be reproduced in an orthotopic model (BOX 2).171. eventually.or high-throughput assays due to the intrinsic variation between tumours and practical challenges of using freshly resected material. In many epithelial tumours.169. 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. 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. emphasizing the caution that is needed in drawing conclusions from in vitro results21. 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. primary tumour xenografts that have been passaged in vivo offer a unique system for the study of tumour heterogeneity and hierarchy in preclinical models.5. it is desirable to track them and visualize the compartments in which they reside before. In vivo biomarker and imaging studies. although the relevance of any results obtained in cell lines needs to be confirmed in primary cancer cells.
although extremely rare. Through in vivo lineage tracking of cultured melanoma cells that are differentially fluorochome-conjugated. Inhibitors of these pathways would have considerable antitumour activity alone199. efficacy against minimal residual disease. a diagnostic procedure to prescreen patients with such genetic or epigenetic changes might be essential for drug development. GDc-0449 and other SMo antagonists (TABLe 1) will therefore provide an interesting test of clinical strategies in targeting renewal signalling. small molecules that target the Hedgehog pathway are in early clinical studies. delayed relapse and tumour-free survival are expected to correlate with the mechanism-based activity of agents that target tumourinitiating cells. 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.REVIEWS treatment in vivo. preclinical studies using these models might provide data that support unique clinical combinations and biomarker strategies for agents that target tumour-initiating cells. such as prostatespecific antigen (PSA) for prostate cancer and mucin 16 (Muc16. It is also important to study the relevance to tumourinitiating cells of current biomarkers. translating the markers that are used to enrich for tumour-initiating cells into clinical biomarkers is not necessarily straightforward. has shown limited toxicity and partial responses in advanced basal cell carcinoma tumours in a Phase I clinical trial. co-xenografted Abcb5+ melanoma-initiating cells and Abc5– subpopulations have been assayed for their relative contribution to tumour growth. biomarkers for tumourinitiating cells in patients who are receiving cancer therapy need to be developed. characterization and monitoring of tumour-initiating cells from solid tumours. which are beginning to guide clinical trials and therapy. In this regard. This is underscored by the fact that. self renewal and differentiation9. ePcAM is highly expressed in numerous solid tumours. in clinical trials for advanced cancers. it is still unclear whether they are surrogate only to a bulk tumour cell population or to a tumourinitiating cell population as well.197. 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. 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. lineage differentiation and different stromal cells (fIG. Hopefully. circulating tumour cells. More markers and reporter genes of tumour stem cell-like properties need to be established. the cD44+ cD24–/low expression in patients with breast cancer that is detected by NATure reVIewS | Drug Discovery . also known as cA125) for ovarian cancer. However. It is advancing to Phase II trials for metastatic colorectal cancer and other advanced epithelial tumours155. metastasis. non-invasive magnetic resonance imaging of magnetically labelled tumour-initiating cells. which might reflect the epigenetic state of the tumour cells. Among various agents that target self-renewal pathways. 3b). gene amplification) or epigenetic (for example. currently. There are several antibodies against cell surface markers of tumour-initiating cells in clinical studies (TABLe 1) . For example. 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. and was recently shown to be expressed VoluMe 8 | o cTober 2009 | 819 © 2009 Macmillan Publishers Limited. GDc-0449.161. This combination approach could be further complicated by the emerging role of Hedgehog signalling in tumour stromal cells114. If genetic (for example. To evaluate metastasis mediated by tumour-initiating cells. and the effect of treatment can in principle be similarly studied. In addition. gene signatures derived by comparing cD44+ cD24–/low breast cancer cells with normal breast epithelial cells. an orally-administered small-molecule antagonist of SMo. gene signature and microarray analysis can also provide biomarkers of tumour-initiating cells. All rights reserved 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. some of the self-renewal signalling molecules could represent an Achilles’ Heel of cancer. suggesting that not all cD44+ cD24–/low cancer cells are tumorigenic and surface markers might not be as conserved as was originally thought. as has been used for normal stem cells193. tumour regression often does not translate into clinically significant increases in patient survival. eMT. there are no obvious paths by which to pursue direct imaging. In addition. immunohistochemistry does not correlate with event-free or overall survival196. The SMo antagonist cyclopamine was shown to lead to rapid regression of basal cell carcinoma in all four patients in which it was tested200. are a potential alternative to invasive biopsies as a source of tumour tissue for the detection. These models will not only help us to understand how current chemotherapeutic and tumour-targeted agents affect different levels of the tumour hierarchy. although the use of labelled antibodies against specific tumour-initiating cell markers might offer an entry point. For tumour models that have a large tumour-initiating cell burden. Given that tumour regression might not be the most relevant early end point. 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. could be useful. The majority of patients with basal cell carcinoma have genetic mutations in Hedgehog pathway mediators38. are correlated with decreased patient survival76. However. but also reveal novel agents that target tumour-initiating cells. and have shown promising results (TABLe 1). 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. both PSA and Muc16 are expressed in differentiated tumour cells. 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.
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