- Email: [email protected]
Cancer Stem Cells in Solid Tumors: An Overview
Cancer Stem Cells in Solid Tumors: An Overview Catherine Adell O’Brien, MD,* Antonija Kreso, BSc,† and John E. Dick, PhD*,† It has long been appreciated that significant functional and morphologic heterogeneity can exist within the individual cells that comprise a tumor. Increasing evidence indicates that many solid tumors are organized in a hierarchical manner in which tumor growth is driven by a small subset of cancer stem cells (CSCs) or tumor-initiating cells. Although these cells represent a small percentage of the overall tumor population, they are the only cells capable of initiating and driving tumor growth. Emerging evidence indicates that these cells are also resistant to chemotherapy and radiation therapy, which has led to much speculation and interest surrounding the potential clinical applicability of CSCs. Semin Radiat Oncol 19:71-77 © 2009 Elsevier Inc. All rights reserved.
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ancer is characterized by the excessive and uncontrolled growth of abnormal cells, which can invade and destroy other tissues.1 From this description, one may infer that all cancer cells within a tumor are equally abnormal and therefore possess equal capacity to initiate and sustain the tumor. However, it has long been recognized that malignant cells within the same tumor display morphologic, proliferative, and functional heterogeneity.2,3 Two models have been proposed to explain tumor heterogeneity: the stochastic and hierarchical models. Both models predict that only a small number of cells within a tumor have the capacity to initiate and sustain tumor growth. However, the stochastic model proposed that all cells within a tumor are biologically homogenous and therefore have equal capacity to regenerate the tumor. The stochastic events that govern tumor-initiating capacity can be intrinsic, such as a requirement for appropriate levels of critical transcription factors, or extrinsic, such as a requirement for the appropriate microenvironment and immune response. In contrast, the hierarchical model (also referred to as the cancer stem cell model) suggested that only a subset of tumor cells possesses the capacity to regenerate the tumor.4 – 6 According to the hierarchical model, it should be possible to isolate tumor cells into fractions that are tumor initiating and non-tumor initiating, where only the former are capable of sustaining tumor growth. The tumorinitiating cells, also referred to as CSCs, are defined by their capacity for self-renewal, potential to develop into any cells in the tumor, and proliferative capacity to drive continued expan-
*Department of General Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada. †Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada. Address reprint requests to Dr. C.A. O’Brien, Toronto General Hospital, 200 Elizabeth St., Room EN 220, Toronto, Ontario M5G2C4. E-mail: [email protected]
1053-4296/09/$-see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.semradonc.2008.11.001
sion of the tumor population.7 The stochastic model states that all cells within a tumor possess equal potential to drive tumor growth, making it impossible to prospectively isolate the tumorinitiating fraction (Fig 1). The idea that human cancers are organized in a hierarchical manner with a CSC being at the apex resembles in many ways the organization of many highly regenerating normal tissues, such as the hematopoietic and intestinal systems.5 However, it is important to emphasize that although the CSC hypothesis states that there is a stem-like cell that maintains the tumor, it does not suggest that the CSCs are derived from normal stem cells. This so-called “cell of origin” question, although interesting and important, is distinct from the CSC hypothesis. Much more experimental work is required to address this question and is outside the scope of this review. In recent years, the concept of CSCs or tumor-initiating cells has ignited a great deal of interest in large part because of the potential clinical implications associated with these cells. Simply stated, the CSC hypothesis suggests that the route to fully eradicating a tumor will require the use of agents that target the root of the cancer or CSCs. The research on CSCs in solid tumors remains at a very early stage, and the question of clinical relevance remains unanswered at this time. Preliminary work has fueled much excitement in the field; however, a great deal of work remains to be done both at the level of understanding the basic biology of CSC and their clinical relevance.
The CSC Model and Leukemia Although the concepts surrounding tumor heterogeneity, cancer stem cells, and the merits of the stochastic or hierarchical models have a long history extending for many decades, conclusive proof required 2 important technological developments: cell sorting and reliable and quantitative in 71
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AML, and these can be categorized by differing capacities for self-renewal and clonal longevity.10 Clonal tracking remains the gold standard assay in CSC biology; however, AML remains the only human malignancy in which a thorough study of clonal tracking has been performed.
The CSC Model and Solid Tumors Figure 1 Two models were described to explain the heterogeneity of tumor cells. The first being the stochastic model, which states that all cells within a tumor have the potential to regenerate the tumor; however, this is determined by entry into the cell cycle, a low probability stochastic event. According to this model, it would not be possible to prospectively isolate the tumorigenic fraction. The alternative explanation for tumor heterogeneity is the hierarchical or cancer stem cell model, which postulates that tumors possess a subset of CSCs that are the only cells capable of initiating and maintaining tumor growth. According to this model, it should be possible to prospectively isolate CSCs (tumorigenic cells) from the bulk of the tumor.
vivo xenograft assays. Cell sorting became widely available in the 1980s and was applied to the purification of normal hematopoietic stem cells (HSC). Reliable human normal and leukemia stem cell xenotransplantation assays were first developed in the late 1980s. Initial studies on acute myelogenous leukemia (AML) performed by Dick et al in the 1990s showed that primary human AML cells could be fractionated based on the CD34⫹CD38⫺ cell surface phenotype, and, although these cells represented a small percentage of the total leukemic cells, these were the only cells that could give rise to leukemic growth in a severe combined immunodeficient (SCID) mouse model and could be serially transplanted.8,9 Moreover, the leukemia that arose was an exact phenocopy of the human leukemia from which it was derived, thereby showing that the CD34⫹CD38⫺ fraction was able to both self-renew and give rise to more differentiated progeny.8 This work provided the first conclusive evidence that a rare leukemic stem cell (LSC) existed in human AML (based on a repopulation assay) and that this cell could reestablish the entire hierarchy, thereby ruling out the stochastic model in favor of the hierarchical or CSC model for AML. The development of quantitative assays to assess the frequency of LSCs represented an invaluable tool for performing this work; however, the ultimate test is to be able to study the biological activity of individual LSCs. The development of in vivo clonal tracking assays provided a means to accomplish this goal. Hope et al10 used lentivirus vector-mediated clonal tracking and the non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation system to assay LSCs in AML at a clonal level. Their study was the first to provide direct evidence for the self-renewal of individual LSC in human AML.10 Furthermore, by applying a clonal assay to their system, they were able to show that there is heterogeneity in the self-renewal properties within the LSC pool; thus, functionally there is more than 1 type of LSC in
One decade after the identification of AML tumor-initiating cells, Al Hajj et al11 tested the same principles in a solid tumor. They started by generating single-cell suspensions from human breast cancer specimens (largely pleural effusion samples) and injected the cells into the mammary fat pad of NOD/SCID mice. By using flow cytometry–assisted cell sorting, they isolated a CD44⫹CD24⫺ subset of breast cancer cells; although these represented a small proportion of the total population of breast cancer cells, they exclusively possessed the tumor-initiating capacity. In 8 out of the 9 samples tested, the CD44⫹CD24⫺ cells gave rise to xenografts that were an exact phenocopy of the patient’s tumor from which they were derived. The xenografts generated could be serially passaged, showing their capacity for self-renewal while also giving rise to nontumorigenic daughter cells.11 In the ensuing 6 years since the publication of the breast cancer study, a number of articles have been published investigating the existence of CSC in a wide range of solid malignancies. The solid tumors that have been shown to possess a CSC subset include brain,12 colon,13–15 head and neck,16 pancreas,17,18 lung,19 melanoma,20 mesenchymal,21 and hepatic tumors22 (Table 1).
Table 1 Markers Used to Enrich for Solid Tumor CSCs CD133 Brain Glioblastoma Medulloblastoma Colon Pancreas Lung CD44 Breast (Edinburgh, Scotland) Head and neck Colon Pancreas CD90 Hepatocellular ABC proteins Melanoma Functional markers Side Population Mesenchymal ALDH Breast Colon
(CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD44ⴙCD24ⴚ) (CD44ⴙ) (CD44ⴙEpCAMⴙ) (CD44ⴙCD24ⴙ ESAⴙ.) (CD90ⴙ) (ABCB5ⴙ)
(SPⴙ)46 (ALDHⴙ) (CD44ⴙEpCAMⴙALDH1ⴙ)
CSCs in solid tumors Singh et al12 published the identification of CSC in human brain tumors based on the identification of the cell surface antigen CD133 (human prominin-1), a 5 transmembrane domain glycoprotein. The biological function of CD133 is not well understood, it was originally identified as a cell surface antigen present on CD34⫹ hematopoietic23 and neural stem cells.24,25 In a NOD/SCID xenograft model, only the injection of CD133⫹ brain tumor cells resulted in xenograft formation, with as few as 100 CD133⫹ cells giving rise to a xenograft that phenotypically and histologically recapitulated the patient tumor from which it was derived. In contrast, injection of up to 105 CD133⫺ cells did not result in xenograft formation. Importantly, viable CD133⫺ brain cancer cells could be detected long after transplantation, arguing that the microenvironment was supportive of these cells but they did not possess the capacity to regenerate the tumor. The xenografts that arose from the CD133⫹ cells could be serially transplanted, thereby showing the ability to self-renew, a central tenet of stem cell biology.12 Since the initial publication on brain cancer, CD133 has been studied as a marker of the CSC population in a variety of other cancers, including colon,13,14,26 pancreas,18 and lung cancer.19 By using model systems very similar to the one used in the brain cancer study, a number of groups were able to show that the tumor-initiating capacity was enriched in the CD133⫹ cancer cells compared with their CD133⫺ counterparts. It is important to appreciate that although CD133 can enrich for CSC, no one has shown that every CD133⫹ cell represents a CSC.12,13 In our colon cancer work, we performed limiting dilution assays on unfractionated, CD133⫹, and CD133⫺ colon cancer cells, allowing us to estimate the CSC frequency for each fraction. The pooled CSC frequency for our cohort of 17 patients was calculated to be approximately 1 in 56,000 in the unfractionated cells, whereas the activity was significantly enriched in the CD133⫹ fraction where there was approximately 1 CSC for every 262 CD133⫹ colon cancer cells. In contrast, the CD133⫺ cells did not give rise to xenografts, with 1 exception at the highest cell dose.13 Therefore, the purification based on CD133 expression provides a significant enrichment for CSC activity; however, more work is required to identify new markers that can be used in combination with CD133 to further purify the population. CD133 expression has also been identified as a CSC marker in human pancreatic cancer. Interestingly, the authors used an orthotopic NOD/SCID xenograft model that could reliably produce metastatic disease. By using this model, they were able to phenotypically distinguish the CSCs that could initiate metastatic disease based on the cell surface expression of CD133 and CXCR4. Both the CD133⫹CXCR4⫺ and the CD133⫹CXCR4⫹ fractions could initiate disease at the primary site; interestingly, only the CD133⫹CXCR4⫹ CSCs were capable of initiating metastatic disease.18 These findings show that heterogeneity exists within the pancreatic CSC subset, and further work is required to determine if the same organizational structure exists within other tumors where different subtypes of CSC give rise to metastatic versus primary lesions. Furthermore, this may also provide insight into why particular cancers preferentially spread to specific organs.27
73 Since the initial publication that the CD44⫹CD24⫺ expression profile identified breast tumor-initiating cells,11 a number of studies have been published using CD44, a widely expressed adhesion molecule,28 –30 either alone in combination with other markers to identify the CSC subset in a variety of tumors including colon (ESA⫹ CD44⫹),15,31 pancreas (ESA⫹CD44⫹CD24⫹),17 and head and neck (CD44⫹).16 Interestingly, both the pancreatic and head and neck studies looked at expression profiles in their CSC (CD44⫹) versus non-CSC (CD44⫺) fractions. Prince et al16 found a preferential expression of bone marrow insertion-1 (BM-1) in the CD44⫹ subset compared with the nontumorigenic fraction. Bone marrow insertion-1 is a member of the polycomb family and has a well-established role in self-renewel,32,33 thereby providing biological evidence to support the functional difference between the CD44⫹ and CD44⫺ cells. Similar results were published by Li et al17 in the pancreatic CSC model in which they identified a differential expression of sonic hedgehog in the ESA⫹CD44⫹CD24⫹ CSCs as compared with the nontumorigenic fraction.17 Hedgehog pathway activation occurs in a significant number of primary human pancreatic carcinomas and is believed to be an early mediator of pancreatic cancer tumorigenesis.34,35 The finding that sonic hedgehog was preferentially upregulated in the CSC fraction lends further support to the idea that the CSC fraction is fundamentally different from their non-CSC progeny. These studies were among the first to look at expression differences in CSC and non-CSC fractions of solid tumors. In the future, this will represent an important area of investigation if we are to develop a deeper understanding of the biology and as a result target the pathways that are crucial for CSC survival. Another approach being used to identify tumorigenic fractions is the use of the side population (SP) assay.21 This assay exploits the high levels of expression of cell membrane adenosine triphosphate (ATP)-binding cassette (ABC) transporter proteins that provide a cell with the ability to efflux chemotherapeutic drugs and certain dyes, such as Hoechst 33342.36 Wu et al21 studied the SP⫹ and SP⫺ cells in 23 primary musculoskeletal tumors ranging from benign to high-grade sarcomas and showed that tumor-initiating capacity existed exclusively within the SP⫹ population. Tumors arising from SP⫹ cells recapitulated the heterogeneity of the parent tumor from which they were derived and were able to be serially passaged showing a self-renewal capacity.21 The authors were also able to correlate the percentage of SP cells with the aggressiveness of the musculoskeletal tumor, with more aggressive tumors possessing a higher percentage of SP⫹ cells.21 Further support for the role of ABC proteins in CSC function was published by Schatton et al20 in human melanoma. Instead of studying the SP⫹ cells, they looked at the expression of one of the chemoresistance-mediating ABC proteins (ABCB5) on the surface of melanoma cells. The ABCB5⫹ melanoma cells were enriched for tumor-initiating cells as compared with their ABCB5⫺ counterparts and were capable of being serially transplanted, showing their capacity for selfrenewal.20 The authors took this work a step further by targeting the ABCB5⫹ cells with systemic administration of a monoclonal antibody capable of inducing antibody-dependent
74 cell-mediated cytotoxicity. In vivo administration of the antibody commenced 14 days after tumor injection into NOD/SCID mice, and treatment was continued for 21 days. At the end of the treatment period, the mean tumor volume in the mice that received the ABCB5 antibody was 32.7 mm3 ⫾ 9.4 mm3 as compared with the control antibody and untreated groups, which had mean tumor volumes of 226.6 mm3 ⫾ 53.8 mm3 and 165.4 mm3 ⫾ 36.9 mm3, respectively.20 Therefore, not only does expression of the chemoresistance mediator ABCB5 enrich for melanoma CSCs, but antibodies targeted against surface markers on these cells can result in decreased tumor growth in a xenograft model. In summary, a variety of methods are being used to enrich for tumor-initiating activity, including phenotypic and functional markers. The current direction is to identify new markers and combinations of markers that will further purify the CSC fraction. The ultimate goal is to be able to purify the population such that the injection of 1 cell can generate a tumor that recapitulates the heterogeneity of the parent tumor. Another goal is the ability to track solid tumors at a clonal level that will provide information about the self-renewal capacity and functional heterogeneity of CSC as well as providing information about the hierarchical organization of solid tumors.
Clinical Relevance The identification and functional isolation of CSCs represents the first step of what remains a very young field in the area of solid tumor biology. The interest focused on the field stems, in large part from the potential clinical promise of studying the CSC subset. The identification of these cells has led cancer researchers to question whether these are the cells most responsible for disease recurrence and metastases. If this is proven to be the case, it would change how future adjuvant therapies are developed. Instead of the current standard of practice in which adjuvant agents are chosen for their activity against the bulk of the tumor cells, the focus would shift to identifying agents that specifically target the CSC subset. Preliminary evidence exists to indicate that the CSC subset are preferentially resistant to the effects of both radiation37 and chemotherapy17,26 when compared with the non-CSC fraction within the same tumor. Furthermore, there is evidence that the CSC subset may be responsive to differentiation therapies, which in the future may represent a new way in which to approach the treatment of solid tumors.38
The CSC Model and Chemotherapy There have been a number of studies published in xenograft models showing chemotherapy resistance of the CSC fraction. One such study was published by Hermann et al18 who treated both CSC (CD133⫹) and non-CSC (CD133⫺) pancreatic cancer cells with the standard chemotherapy agent gemcitabine. The CD133⫹ cells showed a preferential drug resistance to gemcitabine treatment as compared with the
C.A. O’Brien et al CD133⫺ cells from the same tumor. After 5 days of in vitro treatment with gemcitabine, the percentage of CD133⫹ cells had increased approximately 50-fold, from 1.5% to 47%. Furthermore, the authors performed an in vivo study in which they evaluated xenografts derived from mice that had received either gemcitabine or vehicle treatment biweekly for 21 days. The tumors treated with gemcitabine displayed a profound enrichment of CD133⫹ cells.18 These preliminary results suggest that the CSC fraction is resistant to conventional chemotherapy. However, these results must be verified in the clinical setting with human patients. Initial evidence for chemoresistance in colon CSC was published by Todaro et al26 who analyzed the cell viability of primary colon cancer cells after treatment with oxaliplatin and/or 5-fluorouracil (5-FU). The CD133⫺ cells showed a high sensitivity to both in vitro and in vivo drug treatment. In contrast, the CD133⫹ cells derived from the same tumors were largely inert to the effects of chemotherapy.26 Furthermore, the authors showed that CD133⫹ colon cancer cells use interleukin-4 in an autocrine manner as protection against chemotherapeutic agents. By treating mice with a neutralizing antibody against interleukin-4, they were able to render the previously resistant CD133⫹ colon cancer cells sensitive to chemotherapeutic treatment with oxaliplatin and/or 5-FU, thereby preventing any enrichment in the CSC fraction.26 This represented one of the first studies to both document chemoresistance of CSC and investigate a potential mechanism for this resistance. More recently, another article investigated the role of chemoresistance in colon CSC after treatment with cyclophosphamide or irinotecan in an NOD/SCID mouse model.31 The results indicated that after treatment with chemotherapy residual tumors are enriched for cells with the CSC phenotype, and, when the cells were tested in an in vivo assay, they were shown to have increased tumorigenic cell frequency. Resistance to cyclophosphamide has been suggested to result from high aldehyde dehydrogenase (ALDH) activity.39 Interestingly, when the colon cancer cells were studied for ALDH1 activity, it was determined that the CSC fraction (ESA⫹ CD44⫹) possessed most of the ALDH activity. By using short hairpinRNA (shRNA) against ALDH1 or chemical-mediated inhibition (diethylaminobenzaldehyde), they were able to decrease the ALDH1 activity in the CSC subset and subsequently render the cells sensitive to treatment with cyclophosphamide. The inhibition of ALDH1 activity did not have any effect on the CSC resistance to irinotecan treatment indicating that the mechanism of resistance is drug dependent.31 Not only is ALDH1 a potentially useful marker, it also represents a functional pathway that appears to have a crucial role in rendering the colon CSC resistant to treatment with cyclophosphamide. Currently, there are very few studies that attempt to correlate CSC populations with clinical outcomes. One such study has tested the effect of conventional chemotherapy versus lapatinib (an epidermal growth factor receptor/HER2 pathway inhibitor) on the CD44⫹CD24⫺ breast cancer cell population in the setting of a neoadjuvant clinical trial for locally advanced breast cancer patients.40 In this study, treat-
CSCs in solid tumors ment with conventional chemotherapy (docetaxel or doxorubicin and cyclophosphamide) led to an increased percentage of CD44⫹CD24⫺ cells and increased mammosphere formation efficiency (MFSE). Moreover, the tumorigenic cells were functionally evaluated after chemotherapy and were shown to possess enhanced self-renewal capacity in vitro and in vivo by MFSE and xenograft formation, respectively. In contrast, combined treatment with lapatinib and conventional chemotherapy prevented any enrichment in the CD44⫹CD24⫺ population that was observed after chemotherapy treatment alone. This observation may in part explain the survival benefit conferred by treatment with lapitinib.40 Albeit preliminary, this experimental clinical trial represents the essential proof of principle approach that needs to be taken to translate the clinical relevance of CSCs. The work by Li et al,40 represents an excellent framework for future studies, starting with the fact that they tested tissue at multiple time points, including pre- and post-chemotherapy treatment. Of equal importance, the authors did not only measure changes in the percentage of CD44⫹CD24⫺ breast cancer cells, they also functionally tested the tumorigenicity of these cells both pre- and post-treatment by using in vitro (MFSE) and in vivo xenograft assays.
The CSC Model and Radioresistance The evidence for the radioresistance of the CSC subset stems mainly from work performed in brain tumors. Using a xenograft model of glioma, Bao et al37 found that the CD133⫹ CSC fraction was significantly increased after irradiation, resulting in tumors that possessed an increased percentage of CD133⫹ cells relative to the parent tumor from which they were derived. These findings were consistent whether the cells were irradiated in vitro, before injection, or post-implantation into NOD/SCID mice. The enrichment in the CSC fraction correlated with these cells having a survival advantage after irradiation as compared with their non-CSC counterparts (CD133⫺ cells). It was determined that the increased survival of the CD133⫹ cells after irradiation stemmed from their ability to activate the DNA damage response more readily than their CD133⫺ counterparts. The use of a small molecule inhibitor against the Chk1/2 kinases (members of the DNA damage and replication checkpoint) rendered the CD133⫹ cells radiosensitive and eliminated their survival advantage over their CD133⫺ counterparts.37 These findings suggest that the DNA damage response plays an important role in the survival of glioma CSCs. However, future studies are required to confirm these findings in a clonogenic assay.41
The CSC Model and Differentiation Therapy By its nature, a hierarchy model implies that cancer cells are capable of maturation, albeit abnormal. Thus, another avenue that is being investigated is the identification of methods that can induce differentiation of the CSC population. In a
75 glioblastoma xenograft model, researchers showed that transient in vitro exposure or in vivo treatment of established tumors with bone morphogenic protein 4 (BMP4) abolished the ability of transplanted glioblastoma cells to establish intracerebral xenografts. The exposure of glioblastoma cells to BMP4 resulted in a reduction in proliferation and increased expression of markers of neural differentiation. Interestingly, there was no observed effect on cell viability as determined by rates of cell death and apoptosis. There was an observed decrease in the size of the CD133⫹ pool by approximately 50% that correlated with the observed decrease in clonogenic ability.38 The results from this study show that the CSC fraction may still possess the ability to respond to biological cues for maturation that may in the future lead to new non-toxic therapies being developed to drive differentiation of malignant tissues. More recently, another study showed that there is a subset of glioblastoma CSCs that do not undergo differentiation in response to BMP4 treatment. The inability to respond to BMP4 treatment was the result of EZH2-dependent epigenetic silencing of BMP receptor 1B, a state normally found in early embryonic neural stem cells. Forced expression of BMP receptor 1B, either by transgene expression or demethylation of the promoter, restored the differentiation capacity of the glioblastoma CSCs and induced the loss of their tumorigenicity in response to BMP4 treatment.42 This elegant work takes the concept of CSC a step further by attempting to better understand the operative and aberrant differentiation pathways in glioblastoma CSCs. It also stresses the importance of understanding the biology of the CSC fraction and developing an appreciation for the biological diversity that exists within the subset that we currently lump together as the CSC fraction. If novel therapeutic approaches are to be successful, there is an enormous amount of understanding that must be achieved so that treatments can be chosen based on a solid understanding of the aberrant biology that underlies the cells that drive tumor formation.
Current Challenges and Future Directions The existence of CSCs is changing how we think of solid tumor biology; however, it is crucial to emphasize that much more work is needed to better understand how these cells function to initiate and sustain tumor growth. It is equally important to acknowledge the controversies that exist in the field, the main one being how universal the CSC model is and whether some tumors exist where there is no hierarchy and every cell is a CSC. A related question is whether the xenogenic assay system may select for a cell subset that is more capable of surviving and generating tumors in NOD/SCID mice. One way in which to address these questions is to identify CSC fractions within mouse models of cancer that would allow for syngeneic transplantation studies to be performed in immune-competent mice. However, this is also potentially wrought with problems because many of the murine models of cancer involve the generation of transgenic
76 mice, and it is becoming clear from recent work that the hierarchical structure of cells within these tumors can be very different than what we see in human cancers.43 Some examples have been reported in which the CSC frequency is very high, indicating that not all murine tumors rigidly follow the CSC model. However, other murine cancer models do possess a hierarchical organization with a CSC subset at the apex. One such example being the MOZ-TIF retroviral transduction/transplantation model, in which the leukemia CSC frequency was calculated to be 1 in 1 ⫻ 104 leukemic cells.44 More recently, it was shown in a Pten deletion model of AML that only 1 out of every 6 ⫻ 105 leukemic cells was able to reconstitute the disease in transplantation experiments.45 These studies show that even in syngeneic transplantation studies in selected murine models, hierarchies do exist and only a small percentage of the cancer cells is capable of initiating and sustaining the disease. In the future, these concepts must also be tested in mouse models of solid tumor formation, thereby providing proof of concept in a syngeneic setting. These models would also provide an opportunity to perform studies that cannot be done in xenograft assays, such as examining the relationship between the immune system and CSCs. The ultimate question remains whether targeting these cells will prove to be functionally relevant in the context of treating human cancers. There remains a significant amount to be learned about tumor-initiating cells before being able to fully exploit their existence clinically. Currently, these cells can be enriched for tumorigenic activity; however, there is a need to further purify the cells to be able to better understand their unique biology. It is only through developing a better understanding of the biology that we will be able to exploit the pathways used by these cells and thereby be able to successfully eradicate them in patients.
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sistance by preferential activation of the DNA damage response. Nature 444:756-760, 2006 Piccirillo SG, Vescovi AL: Bone morphogenetic proteins regulate tumorigenicity in human glioblastoma stem cells. Ernst Schering Found Symp Proc 5:59-81, 2006 Sladek NE, Kollander R, Sreerama L, et al: Cellular levels of aldehyde dehydrogenases (ALDH1A1 and ALDH3A1) as predictors of therapeutic responses to cyclophosphamide-based chemotherapy of breast cancer: A retrospective study. Rational individualization of oxazaphosphorine-based cancer chemotherapeutic regimens. Cancer Chemother Pharmacol 49:309-321, 2002 Li X, Lewis MT, Huang J, et al: Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100:672-679, 2008 Baumann M, Krause M, Hill R: Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8:545-554, 2008
77 42. Lee J, Son MJ, Woolard K, et al: Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cells 13:69-80, 2008 43. Kelly PN, Dakic A, Adams JM, et al: Tumor growth need not be driven by rare cancer stem cells. Science 317:337, 2007 44. Huntly BJ, Shigematsu H, Deguchi K, et al: MOZ-TIF2, but not bcr-abl, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cells 6:587-596, 2004 45. Yilmaz OH, Valdez R, Theisen BK, et al: Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441:475-482, 2006 46. Leemhuis T, Yoder MC, Grigsby S, et al: Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and rhodamine 123. Exp Hematol 24:1215-1224, 1996
C
ancer is characterized by the excessive and uncontrolled growth of abnormal cells, which can invade and destroy other tissues.1 From this description, one may infer that all cancer cells within a tumor are equally abnormal and therefore possess equal capacity to initiate and sustain the tumor. However, it has long been recognized that malignant cells within the same tumor display morphologic, proliferative, and functional heterogeneity.2,3 Two models have been proposed to explain tumor heterogeneity: the stochastic and hierarchical models. Both models predict that only a small number of cells within a tumor have the capacity to initiate and sustain tumor growth. However, the stochastic model proposed that all cells within a tumor are biologically homogenous and therefore have equal capacity to regenerate the tumor. The stochastic events that govern tumor-initiating capacity can be intrinsic, such as a requirement for appropriate levels of critical transcription factors, or extrinsic, such as a requirement for the appropriate microenvironment and immune response. In contrast, the hierarchical model (also referred to as the cancer stem cell model) suggested that only a subset of tumor cells possesses the capacity to regenerate the tumor.4 – 6 According to the hierarchical model, it should be possible to isolate tumor cells into fractions that are tumor initiating and non-tumor initiating, where only the former are capable of sustaining tumor growth. The tumorinitiating cells, also referred to as CSCs, are defined by their capacity for self-renewal, potential to develop into any cells in the tumor, and proliferative capacity to drive continued expan-
*Department of General Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada. †Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada. Address reprint requests to Dr. C.A. O’Brien, Toronto General Hospital, 200 Elizabeth St., Room EN 220, Toronto, Ontario M5G2C4. E-mail: [email protected]
1053-4296/09/$-see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.semradonc.2008.11.001
sion of the tumor population.7 The stochastic model states that all cells within a tumor possess equal potential to drive tumor growth, making it impossible to prospectively isolate the tumorinitiating fraction (Fig 1). The idea that human cancers are organized in a hierarchical manner with a CSC being at the apex resembles in many ways the organization of many highly regenerating normal tissues, such as the hematopoietic and intestinal systems.5 However, it is important to emphasize that although the CSC hypothesis states that there is a stem-like cell that maintains the tumor, it does not suggest that the CSCs are derived from normal stem cells. This so-called “cell of origin” question, although interesting and important, is distinct from the CSC hypothesis. Much more experimental work is required to address this question and is outside the scope of this review. In recent years, the concept of CSCs or tumor-initiating cells has ignited a great deal of interest in large part because of the potential clinical implications associated with these cells. Simply stated, the CSC hypothesis suggests that the route to fully eradicating a tumor will require the use of agents that target the root of the cancer or CSCs. The research on CSCs in solid tumors remains at a very early stage, and the question of clinical relevance remains unanswered at this time. Preliminary work has fueled much excitement in the field; however, a great deal of work remains to be done both at the level of understanding the basic biology of CSC and their clinical relevance.
The CSC Model and Leukemia Although the concepts surrounding tumor heterogeneity, cancer stem cells, and the merits of the stochastic or hierarchical models have a long history extending for many decades, conclusive proof required 2 important technological developments: cell sorting and reliable and quantitative in 71
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72
AML, and these can be categorized by differing capacities for self-renewal and clonal longevity.10 Clonal tracking remains the gold standard assay in CSC biology; however, AML remains the only human malignancy in which a thorough study of clonal tracking has been performed.
The CSC Model and Solid Tumors Figure 1 Two models were described to explain the heterogeneity of tumor cells. The first being the stochastic model, which states that all cells within a tumor have the potential to regenerate the tumor; however, this is determined by entry into the cell cycle, a low probability stochastic event. According to this model, it would not be possible to prospectively isolate the tumorigenic fraction. The alternative explanation for tumor heterogeneity is the hierarchical or cancer stem cell model, which postulates that tumors possess a subset of CSCs that are the only cells capable of initiating and maintaining tumor growth. According to this model, it should be possible to prospectively isolate CSCs (tumorigenic cells) from the bulk of the tumor.
vivo xenograft assays. Cell sorting became widely available in the 1980s and was applied to the purification of normal hematopoietic stem cells (HSC). Reliable human normal and leukemia stem cell xenotransplantation assays were first developed in the late 1980s. Initial studies on acute myelogenous leukemia (AML) performed by Dick et al in the 1990s showed that primary human AML cells could be fractionated based on the CD34⫹CD38⫺ cell surface phenotype, and, although these cells represented a small percentage of the total leukemic cells, these were the only cells that could give rise to leukemic growth in a severe combined immunodeficient (SCID) mouse model and could be serially transplanted.8,9 Moreover, the leukemia that arose was an exact phenocopy of the human leukemia from which it was derived, thereby showing that the CD34⫹CD38⫺ fraction was able to both self-renew and give rise to more differentiated progeny.8 This work provided the first conclusive evidence that a rare leukemic stem cell (LSC) existed in human AML (based on a repopulation assay) and that this cell could reestablish the entire hierarchy, thereby ruling out the stochastic model in favor of the hierarchical or CSC model for AML. The development of quantitative assays to assess the frequency of LSCs represented an invaluable tool for performing this work; however, the ultimate test is to be able to study the biological activity of individual LSCs. The development of in vivo clonal tracking assays provided a means to accomplish this goal. Hope et al10 used lentivirus vector-mediated clonal tracking and the non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation system to assay LSCs in AML at a clonal level. Their study was the first to provide direct evidence for the self-renewal of individual LSC in human AML.10 Furthermore, by applying a clonal assay to their system, they were able to show that there is heterogeneity in the self-renewal properties within the LSC pool; thus, functionally there is more than 1 type of LSC in
One decade after the identification of AML tumor-initiating cells, Al Hajj et al11 tested the same principles in a solid tumor. They started by generating single-cell suspensions from human breast cancer specimens (largely pleural effusion samples) and injected the cells into the mammary fat pad of NOD/SCID mice. By using flow cytometry–assisted cell sorting, they isolated a CD44⫹CD24⫺ subset of breast cancer cells; although these represented a small proportion of the total population of breast cancer cells, they exclusively possessed the tumor-initiating capacity. In 8 out of the 9 samples tested, the CD44⫹CD24⫺ cells gave rise to xenografts that were an exact phenocopy of the patient’s tumor from which they were derived. The xenografts generated could be serially passaged, showing their capacity for self-renewal while also giving rise to nontumorigenic daughter cells.11 In the ensuing 6 years since the publication of the breast cancer study, a number of articles have been published investigating the existence of CSC in a wide range of solid malignancies. The solid tumors that have been shown to possess a CSC subset include brain,12 colon,13–15 head and neck,16 pancreas,17,18 lung,19 melanoma,20 mesenchymal,21 and hepatic tumors22 (Table 1).
Table 1 Markers Used to Enrich for Solid Tumor CSCs CD133 Brain Glioblastoma Medulloblastoma Colon Pancreas Lung CD44 Breast (Edinburgh, Scotland) Head and neck Colon Pancreas CD90 Hepatocellular ABC proteins Melanoma Functional markers Side Population Mesenchymal ALDH Breast Colon
(CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD133ⴙ) (CD44ⴙCD24ⴚ) (CD44ⴙ) (CD44ⴙEpCAMⴙ) (CD44ⴙCD24ⴙ ESAⴙ.) (CD90ⴙ) (ABCB5ⴙ)
(SPⴙ)46 (ALDHⴙ) (CD44ⴙEpCAMⴙALDH1ⴙ)
CSCs in solid tumors Singh et al12 published the identification of CSC in human brain tumors based on the identification of the cell surface antigen CD133 (human prominin-1), a 5 transmembrane domain glycoprotein. The biological function of CD133 is not well understood, it was originally identified as a cell surface antigen present on CD34⫹ hematopoietic23 and neural stem cells.24,25 In a NOD/SCID xenograft model, only the injection of CD133⫹ brain tumor cells resulted in xenograft formation, with as few as 100 CD133⫹ cells giving rise to a xenograft that phenotypically and histologically recapitulated the patient tumor from which it was derived. In contrast, injection of up to 105 CD133⫺ cells did not result in xenograft formation. Importantly, viable CD133⫺ brain cancer cells could be detected long after transplantation, arguing that the microenvironment was supportive of these cells but they did not possess the capacity to regenerate the tumor. The xenografts that arose from the CD133⫹ cells could be serially transplanted, thereby showing the ability to self-renew, a central tenet of stem cell biology.12 Since the initial publication on brain cancer, CD133 has been studied as a marker of the CSC population in a variety of other cancers, including colon,13,14,26 pancreas,18 and lung cancer.19 By using model systems very similar to the one used in the brain cancer study, a number of groups were able to show that the tumor-initiating capacity was enriched in the CD133⫹ cancer cells compared with their CD133⫺ counterparts. It is important to appreciate that although CD133 can enrich for CSC, no one has shown that every CD133⫹ cell represents a CSC.12,13 In our colon cancer work, we performed limiting dilution assays on unfractionated, CD133⫹, and CD133⫺ colon cancer cells, allowing us to estimate the CSC frequency for each fraction. The pooled CSC frequency for our cohort of 17 patients was calculated to be approximately 1 in 56,000 in the unfractionated cells, whereas the activity was significantly enriched in the CD133⫹ fraction where there was approximately 1 CSC for every 262 CD133⫹ colon cancer cells. In contrast, the CD133⫺ cells did not give rise to xenografts, with 1 exception at the highest cell dose.13 Therefore, the purification based on CD133 expression provides a significant enrichment for CSC activity; however, more work is required to identify new markers that can be used in combination with CD133 to further purify the population. CD133 expression has also been identified as a CSC marker in human pancreatic cancer. Interestingly, the authors used an orthotopic NOD/SCID xenograft model that could reliably produce metastatic disease. By using this model, they were able to phenotypically distinguish the CSCs that could initiate metastatic disease based on the cell surface expression of CD133 and CXCR4. Both the CD133⫹CXCR4⫺ and the CD133⫹CXCR4⫹ fractions could initiate disease at the primary site; interestingly, only the CD133⫹CXCR4⫹ CSCs were capable of initiating metastatic disease.18 These findings show that heterogeneity exists within the pancreatic CSC subset, and further work is required to determine if the same organizational structure exists within other tumors where different subtypes of CSC give rise to metastatic versus primary lesions. Furthermore, this may also provide insight into why particular cancers preferentially spread to specific organs.27
73 Since the initial publication that the CD44⫹CD24⫺ expression profile identified breast tumor-initiating cells,11 a number of studies have been published using CD44, a widely expressed adhesion molecule,28 –30 either alone in combination with other markers to identify the CSC subset in a variety of tumors including colon (ESA⫹ CD44⫹),15,31 pancreas (ESA⫹CD44⫹CD24⫹),17 and head and neck (CD44⫹).16 Interestingly, both the pancreatic and head and neck studies looked at expression profiles in their CSC (CD44⫹) versus non-CSC (CD44⫺) fractions. Prince et al16 found a preferential expression of bone marrow insertion-1 (BM-1) in the CD44⫹ subset compared with the nontumorigenic fraction. Bone marrow insertion-1 is a member of the polycomb family and has a well-established role in self-renewel,32,33 thereby providing biological evidence to support the functional difference between the CD44⫹ and CD44⫺ cells. Similar results were published by Li et al17 in the pancreatic CSC model in which they identified a differential expression of sonic hedgehog in the ESA⫹CD44⫹CD24⫹ CSCs as compared with the nontumorigenic fraction.17 Hedgehog pathway activation occurs in a significant number of primary human pancreatic carcinomas and is believed to be an early mediator of pancreatic cancer tumorigenesis.34,35 The finding that sonic hedgehog was preferentially upregulated in the CSC fraction lends further support to the idea that the CSC fraction is fundamentally different from their non-CSC progeny. These studies were among the first to look at expression differences in CSC and non-CSC fractions of solid tumors. In the future, this will represent an important area of investigation if we are to develop a deeper understanding of the biology and as a result target the pathways that are crucial for CSC survival. Another approach being used to identify tumorigenic fractions is the use of the side population (SP) assay.21 This assay exploits the high levels of expression of cell membrane adenosine triphosphate (ATP)-binding cassette (ABC) transporter proteins that provide a cell with the ability to efflux chemotherapeutic drugs and certain dyes, such as Hoechst 33342.36 Wu et al21 studied the SP⫹ and SP⫺ cells in 23 primary musculoskeletal tumors ranging from benign to high-grade sarcomas and showed that tumor-initiating capacity existed exclusively within the SP⫹ population. Tumors arising from SP⫹ cells recapitulated the heterogeneity of the parent tumor from which they were derived and were able to be serially passaged showing a self-renewal capacity.21 The authors were also able to correlate the percentage of SP cells with the aggressiveness of the musculoskeletal tumor, with more aggressive tumors possessing a higher percentage of SP⫹ cells.21 Further support for the role of ABC proteins in CSC function was published by Schatton et al20 in human melanoma. Instead of studying the SP⫹ cells, they looked at the expression of one of the chemoresistance-mediating ABC proteins (ABCB5) on the surface of melanoma cells. The ABCB5⫹ melanoma cells were enriched for tumor-initiating cells as compared with their ABCB5⫺ counterparts and were capable of being serially transplanted, showing their capacity for selfrenewal.20 The authors took this work a step further by targeting the ABCB5⫹ cells with systemic administration of a monoclonal antibody capable of inducing antibody-dependent
74 cell-mediated cytotoxicity. In vivo administration of the antibody commenced 14 days after tumor injection into NOD/SCID mice, and treatment was continued for 21 days. At the end of the treatment period, the mean tumor volume in the mice that received the ABCB5 antibody was 32.7 mm3 ⫾ 9.4 mm3 as compared with the control antibody and untreated groups, which had mean tumor volumes of 226.6 mm3 ⫾ 53.8 mm3 and 165.4 mm3 ⫾ 36.9 mm3, respectively.20 Therefore, not only does expression of the chemoresistance mediator ABCB5 enrich for melanoma CSCs, but antibodies targeted against surface markers on these cells can result in decreased tumor growth in a xenograft model. In summary, a variety of methods are being used to enrich for tumor-initiating activity, including phenotypic and functional markers. The current direction is to identify new markers and combinations of markers that will further purify the CSC fraction. The ultimate goal is to be able to purify the population such that the injection of 1 cell can generate a tumor that recapitulates the heterogeneity of the parent tumor. Another goal is the ability to track solid tumors at a clonal level that will provide information about the self-renewal capacity and functional heterogeneity of CSC as well as providing information about the hierarchical organization of solid tumors.
Clinical Relevance The identification and functional isolation of CSCs represents the first step of what remains a very young field in the area of solid tumor biology. The interest focused on the field stems, in large part from the potential clinical promise of studying the CSC subset. The identification of these cells has led cancer researchers to question whether these are the cells most responsible for disease recurrence and metastases. If this is proven to be the case, it would change how future adjuvant therapies are developed. Instead of the current standard of practice in which adjuvant agents are chosen for their activity against the bulk of the tumor cells, the focus would shift to identifying agents that specifically target the CSC subset. Preliminary evidence exists to indicate that the CSC subset are preferentially resistant to the effects of both radiation37 and chemotherapy17,26 when compared with the non-CSC fraction within the same tumor. Furthermore, there is evidence that the CSC subset may be responsive to differentiation therapies, which in the future may represent a new way in which to approach the treatment of solid tumors.38
The CSC Model and Chemotherapy There have been a number of studies published in xenograft models showing chemotherapy resistance of the CSC fraction. One such study was published by Hermann et al18 who treated both CSC (CD133⫹) and non-CSC (CD133⫺) pancreatic cancer cells with the standard chemotherapy agent gemcitabine. The CD133⫹ cells showed a preferential drug resistance to gemcitabine treatment as compared with the
C.A. O’Brien et al CD133⫺ cells from the same tumor. After 5 days of in vitro treatment with gemcitabine, the percentage of CD133⫹ cells had increased approximately 50-fold, from 1.5% to 47%. Furthermore, the authors performed an in vivo study in which they evaluated xenografts derived from mice that had received either gemcitabine or vehicle treatment biweekly for 21 days. The tumors treated with gemcitabine displayed a profound enrichment of CD133⫹ cells.18 These preliminary results suggest that the CSC fraction is resistant to conventional chemotherapy. However, these results must be verified in the clinical setting with human patients. Initial evidence for chemoresistance in colon CSC was published by Todaro et al26 who analyzed the cell viability of primary colon cancer cells after treatment with oxaliplatin and/or 5-fluorouracil (5-FU). The CD133⫺ cells showed a high sensitivity to both in vitro and in vivo drug treatment. In contrast, the CD133⫹ cells derived from the same tumors were largely inert to the effects of chemotherapy.26 Furthermore, the authors showed that CD133⫹ colon cancer cells use interleukin-4 in an autocrine manner as protection against chemotherapeutic agents. By treating mice with a neutralizing antibody against interleukin-4, they were able to render the previously resistant CD133⫹ colon cancer cells sensitive to chemotherapeutic treatment with oxaliplatin and/or 5-FU, thereby preventing any enrichment in the CSC fraction.26 This represented one of the first studies to both document chemoresistance of CSC and investigate a potential mechanism for this resistance. More recently, another article investigated the role of chemoresistance in colon CSC after treatment with cyclophosphamide or irinotecan in an NOD/SCID mouse model.31 The results indicated that after treatment with chemotherapy residual tumors are enriched for cells with the CSC phenotype, and, when the cells were tested in an in vivo assay, they were shown to have increased tumorigenic cell frequency. Resistance to cyclophosphamide has been suggested to result from high aldehyde dehydrogenase (ALDH) activity.39 Interestingly, when the colon cancer cells were studied for ALDH1 activity, it was determined that the CSC fraction (ESA⫹ CD44⫹) possessed most of the ALDH activity. By using short hairpinRNA (shRNA) against ALDH1 or chemical-mediated inhibition (diethylaminobenzaldehyde), they were able to decrease the ALDH1 activity in the CSC subset and subsequently render the cells sensitive to treatment with cyclophosphamide. The inhibition of ALDH1 activity did not have any effect on the CSC resistance to irinotecan treatment indicating that the mechanism of resistance is drug dependent.31 Not only is ALDH1 a potentially useful marker, it also represents a functional pathway that appears to have a crucial role in rendering the colon CSC resistant to treatment with cyclophosphamide. Currently, there are very few studies that attempt to correlate CSC populations with clinical outcomes. One such study has tested the effect of conventional chemotherapy versus lapatinib (an epidermal growth factor receptor/HER2 pathway inhibitor) on the CD44⫹CD24⫺ breast cancer cell population in the setting of a neoadjuvant clinical trial for locally advanced breast cancer patients.40 In this study, treat-
CSCs in solid tumors ment with conventional chemotherapy (docetaxel or doxorubicin and cyclophosphamide) led to an increased percentage of CD44⫹CD24⫺ cells and increased mammosphere formation efficiency (MFSE). Moreover, the tumorigenic cells were functionally evaluated after chemotherapy and were shown to possess enhanced self-renewal capacity in vitro and in vivo by MFSE and xenograft formation, respectively. In contrast, combined treatment with lapatinib and conventional chemotherapy prevented any enrichment in the CD44⫹CD24⫺ population that was observed after chemotherapy treatment alone. This observation may in part explain the survival benefit conferred by treatment with lapitinib.40 Albeit preliminary, this experimental clinical trial represents the essential proof of principle approach that needs to be taken to translate the clinical relevance of CSCs. The work by Li et al,40 represents an excellent framework for future studies, starting with the fact that they tested tissue at multiple time points, including pre- and post-chemotherapy treatment. Of equal importance, the authors did not only measure changes in the percentage of CD44⫹CD24⫺ breast cancer cells, they also functionally tested the tumorigenicity of these cells both pre- and post-treatment by using in vitro (MFSE) and in vivo xenograft assays.
The CSC Model and Radioresistance The evidence for the radioresistance of the CSC subset stems mainly from work performed in brain tumors. Using a xenograft model of glioma, Bao et al37 found that the CD133⫹ CSC fraction was significantly increased after irradiation, resulting in tumors that possessed an increased percentage of CD133⫹ cells relative to the parent tumor from which they were derived. These findings were consistent whether the cells were irradiated in vitro, before injection, or post-implantation into NOD/SCID mice. The enrichment in the CSC fraction correlated with these cells having a survival advantage after irradiation as compared with their non-CSC counterparts (CD133⫺ cells). It was determined that the increased survival of the CD133⫹ cells after irradiation stemmed from their ability to activate the DNA damage response more readily than their CD133⫺ counterparts. The use of a small molecule inhibitor against the Chk1/2 kinases (members of the DNA damage and replication checkpoint) rendered the CD133⫹ cells radiosensitive and eliminated their survival advantage over their CD133⫺ counterparts.37 These findings suggest that the DNA damage response plays an important role in the survival of glioma CSCs. However, future studies are required to confirm these findings in a clonogenic assay.41
The CSC Model and Differentiation Therapy By its nature, a hierarchy model implies that cancer cells are capable of maturation, albeit abnormal. Thus, another avenue that is being investigated is the identification of methods that can induce differentiation of the CSC population. In a
75 glioblastoma xenograft model, researchers showed that transient in vitro exposure or in vivo treatment of established tumors with bone morphogenic protein 4 (BMP4) abolished the ability of transplanted glioblastoma cells to establish intracerebral xenografts. The exposure of glioblastoma cells to BMP4 resulted in a reduction in proliferation and increased expression of markers of neural differentiation. Interestingly, there was no observed effect on cell viability as determined by rates of cell death and apoptosis. There was an observed decrease in the size of the CD133⫹ pool by approximately 50% that correlated with the observed decrease in clonogenic ability.38 The results from this study show that the CSC fraction may still possess the ability to respond to biological cues for maturation that may in the future lead to new non-toxic therapies being developed to drive differentiation of malignant tissues. More recently, another study showed that there is a subset of glioblastoma CSCs that do not undergo differentiation in response to BMP4 treatment. The inability to respond to BMP4 treatment was the result of EZH2-dependent epigenetic silencing of BMP receptor 1B, a state normally found in early embryonic neural stem cells. Forced expression of BMP receptor 1B, either by transgene expression or demethylation of the promoter, restored the differentiation capacity of the glioblastoma CSCs and induced the loss of their tumorigenicity in response to BMP4 treatment.42 This elegant work takes the concept of CSC a step further by attempting to better understand the operative and aberrant differentiation pathways in glioblastoma CSCs. It also stresses the importance of understanding the biology of the CSC fraction and developing an appreciation for the biological diversity that exists within the subset that we currently lump together as the CSC fraction. If novel therapeutic approaches are to be successful, there is an enormous amount of understanding that must be achieved so that treatments can be chosen based on a solid understanding of the aberrant biology that underlies the cells that drive tumor formation.
Current Challenges and Future Directions The existence of CSCs is changing how we think of solid tumor biology; however, it is crucial to emphasize that much more work is needed to better understand how these cells function to initiate and sustain tumor growth. It is equally important to acknowledge the controversies that exist in the field, the main one being how universal the CSC model is and whether some tumors exist where there is no hierarchy and every cell is a CSC. A related question is whether the xenogenic assay system may select for a cell subset that is more capable of surviving and generating tumors in NOD/SCID mice. One way in which to address these questions is to identify CSC fractions within mouse models of cancer that would allow for syngeneic transplantation studies to be performed in immune-competent mice. However, this is also potentially wrought with problems because many of the murine models of cancer involve the generation of transgenic
76 mice, and it is becoming clear from recent work that the hierarchical structure of cells within these tumors can be very different than what we see in human cancers.43 Some examples have been reported in which the CSC frequency is very high, indicating that not all murine tumors rigidly follow the CSC model. However, other murine cancer models do possess a hierarchical organization with a CSC subset at the apex. One such example being the MOZ-TIF retroviral transduction/transplantation model, in which the leukemia CSC frequency was calculated to be 1 in 1 ⫻ 104 leukemic cells.44 More recently, it was shown in a Pten deletion model of AML that only 1 out of every 6 ⫻ 105 leukemic cells was able to reconstitute the disease in transplantation experiments.45 These studies show that even in syngeneic transplantation studies in selected murine models, hierarchies do exist and only a small percentage of the cancer cells is capable of initiating and sustaining the disease. In the future, these concepts must also be tested in mouse models of solid tumor formation, thereby providing proof of concept in a syngeneic setting. These models would also provide an opportunity to perform studies that cannot be done in xenograft assays, such as examining the relationship between the immune system and CSCs. The ultimate question remains whether targeting these cells will prove to be functionally relevant in the context of treating human cancers. There remains a significant amount to be learned about tumor-initiating cells before being able to fully exploit their existence clinically. Currently, these cells can be enriched for tumorigenic activity; however, there is a need to further purify the cells to be able to better understand their unique biology. It is only through developing a better understanding of the biology that we will be able to exploit the pathways used by these cells and thereby be able to successfully eradicate them in patients.
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