Novel Properties of SP cells in STS, and How They May Be Targeted to Develop Potential Therapies

Transcription

1 Novel Properties of SP cells in STS, and How They May Be Targeted to Develop Potential Therapies by Chang Ye Yale Wang A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto Copyright by Chang Ye Yale Wang 2010

2 Novel Properties of SP cells in STS, and How They May Be Targeted to Develop Potential Therapies Abstract Chang Ye Yale Wang Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2010 Tumours contain heterogeneous cell populations. A population enriched in tumour-initiating potential has been identified in soft-tissue sarcoma (STS) by the isolation of "side population" (SP) cells. In this study, we compared the gene expression profiles of SP and non-sp cells in STS and identified Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, using primary tumor xenografts in NOD-SCID mice as our experimental model, we used the Hh blocker Triparanol and the Notch blocker DAPT to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour-initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies. ii

3 Table of Contents Table of Contents... iii List of Figures... vi List of Tables... vii List of Abbreviations... viii Chapter 1 Introduction Cancer stem cells in soft-tissue sarcoma and their importance for cancer therapy Abstract Soft-tissue Sarcoma Pathological and Etiologic Features Molecular Features Treatment Malignant Fibrous Histiocytoma (MFH) Tumour Initiation Cells Overview TICs in Solid Tumours General Properties of TICs Important Molecular Pathways in TICs Interactions between Molecular Pathways TIC and Resistance to Therapies Model for TIC Controversies Side Population Cells SP Cells in Normal Tissues iii

4 1.4.2 SP Cells in Human Cancer Molecular Features of SP Cells in Human Cancer Implication of SP cells in Cancer Therapies Criticisms SP Cells in Mesenchymal Neoplasms Summary and Conclusion Hypothesis and Rationales References Chapter Novel Properties of SP cells in STS, and How They May Be Targeted to Develop Potential Therapies Abstract Introduction Results Microarray Profiling Show that SP Cells in MFH Exhibit Up-regulated Hedgehog and Notch Signalling Real-time PCR Confirmation that SP Cells in MFH Exhibit Up-regulated Hedgehog and Notch Signalling Inhibition of Hedgehog and Notch Signalling Depletes the Abundance of SP cells in MFH Xenografts Inhibition of Hedgehog and Notch Signalling Depletes the Abundance of SP cells in MFH Xenografts Inhibition of Hedgehog and Notch Signalling Reduces the Size of MFH Xenografts Inhibition of Hedgehog and Notch Signalling Affects the Rate of Apoptosis and Proliferation in Xenografts of STS Tumours Inhibition of Hh and Notch Signalling Reduces the Re-transplantation Rate of MFH Xenografts Discussion Material and method iv

5 2.5.1 Primary Tumours Establishing Initial Xenograft from Primary Tumours Flow Cytometry RNA Extraction Real time-rt-pcr Gene profiling Pathology In vivo Blockade of Hedgehog and Notch Signalling Establishing Secondary Xenograft from Treated Tumours References Chapter Summary and Future Directions Summary Future Directions References v

6 List of Figures Figure 1-1: The two models for cancer development Figure 1-2: The Notch signalling Pathway Figure 1-3: The Hedgehog signalling Pathway Figure 1-4: Side population cells isolated using flow-cytomentry Figure 2-1: Expression comparison of downstream transcriptional targets of Hedgehog and Notch pathways in SP cells and NSP cells isolated from primary MFH tumours using real-time PCR Figure 2-2: Expression of downstream target genes in Hh and Notch signalling pathways after drug treatment Figure 2-3: Proportion of SP cells in xenografted tumours after treatment with the drug Triparanol and DAPT Figure 2-4: Weight and size of xenografted tumours after treatment with the drug Triparanol and DAPT Figure 2-5: Histology and immunochemical staining of tumour sections after treatment with the drug Triparanol and DAPT Figure 2-6: Cellularity, apoptosis rate, and proliferation rate in xenografted tumours posttreatment Figure 2-7: The rate of tumour re-growth upon secondary transplantation using cells harvested from the initial treatment vi

7 List of Tables Table 2-1: Top 10 differentially regulated molecular pathways in SP cells (Genespring) Table 2-2: Up-regulation of key genes in th Hh pathway as shown in the microarray Table 2-3: Top 10 differentially regulated molecular pathways in SP cells (Using Ingenuity Pathway Analysis Program) vii

8 List of Abbreviations ABC APC AF AML CFU-F CML CSC DMSO FAP FBS FIF HSC LSC MPC MSC OS SP SMO TIC ATP binding cassette adenomatous polyposis coli aggressive fibromatosis acute myeloid leukemia colony forming unit-fibroblastic chronic myelogenous leukemia cancer stem cell dimethyl sulfoxide familial adenomatous polyposis fetal bovine serum familial infiltrative fibromatosis hematopoietic stem cell leukemic stem cell mesenchymal progenitor cells mesenchymal stem cell osteosarcoma side population smoothened tumor initiating cell viii

9 Chapter 1 Introduction 1 Cancer stem cells in soft-tissue sarcoma and their importance for cancer therapy 1.1 Abstract Like many other tumour types, soft-tissue sarcomas (STS) contain heterogeneous cell populations. While no marker is yet known to identify the tumour initiating cells in STS, a population enriched for tumour initiating potential has been identified in STS by the isolation of "side population" (SP) cells, which has the capacity to extrude dyes such as Hoechst It is likely that they behave as relatively quiescent stem-like cells, while the remainder of tumour cells acts as rapid proliferating transient amplifying cells. Because traditional cancer therapies often focus on eliminating rapidly proliferating cells, the persistence of SP cells is likely responsible for relapse and failed response to therapy. 1

10 1.2 Soft-tissue Sarcoma Soft-tissue sarcoma (STS) is defined as a malignant neoplasm or cancer that arises in mesenchymal tissues other than bone, which include muscle, fat, and other fibrous supporting structures Pathological and Etiologic Features About 50 subtypes of STS have been defined according to the type of tissue they resemble. 1 Most widely used grading systems for STS normally takes into account the degree of differentiation, the mitotic count, and the extent of necrosis; these factors are often employed along with other important determinants, such as the depth and size of the tumor to estimate the prognosis.. 2, 3 While often depending the type and site of the tumor and other factors, the fiveyear overall survival rates for stages I, II, III, and IV of STS are respectively around 90, 70, 50, and 10 to 20 percent. 4 Over the past decades, the overall incidence of soft-tissue sarcoma has been increasing, perhaps as a result of improved recognition and diagnosis, and the increase in Kaposi's sarcoma associated with AIDS. 5 Kaposi's sarcoma is only one of the few STS subtypes that have a identifiable association with certain cause, as most soft-tissue sarcomas are sporadic Molecular Features Certain genetic factors are associated with STS: for example, children with hereditary retinoblastoma (germ-line mutation in the RB1 tumor-suppressor gene) and patients with the Li Fraumeni syndrome (germ-line mutation in the p53 tumor-suppressor gene) both carry an exceptionally high risk of osteosarcoma and soft-tissue sarcoma, 6, 7 The classification and characterization of soft-tissue sarcomas is evolving, as immunohistochemical and other molecular techniques become more accessible. 1 The study of formalinfixed, paraffin-embedded samples have led to an improved understanding of some of the underlying molecular mechanisms in STS, as well as some common genetic aberrations in certain tumor subtypes: for example, specific translocations resulting in new fusion genes have 2

11 been consistently identified in many sarcomas such as synovial sarcoma, clear cell sarcoma, alveolar soft-part sarcoma, etc., and the mutational analysis of gastrointestinal stromal tumors identified the KIT gene to have a major effect on treatment response and survival. 8 By considering such molecular and genetic features, STS may be broadly classified into two major categories: those with specific molecular alterations and simple karyotypes via reciprocal translocation or specific point mutation, and those with nonspecific genetic alterations and complex karyotypes via the accumulation of many genetic changes. 9 For STS with clearly defined genetic alterations, many of them involve chromosomal translocation. However, despite the successful cloning of such translocation and identification of the specific fusion genes, attempts in generating transgenic models for these mutations have been difficult For example, in the attempt to clone the Pax3-Fkhr fusion gene, which is observed commonly in rabdomyosarcoma, it was revealed that tumours only form when the fusion gene is conditionally expressed in late muscle progenitors specifically. 10 A similar example was observed when generating a transgenic model for the SYT-SSX fusion protein frequently observed in synovial sarcoma: synovial sarcomas formed when the fusion protein was conditionally expressed in myoblast via a Cre/loxP cystem driven by Myf5. 12 These pieces of evidence seem to suggest that it may be crucial for the molecular events that lead to the development of STS to happen in certain precursor cell or stem cell. Many prevalent STS such as malignant malignant fibrous histiocytoma and skeletal chondrosarcoma belong to the category of STS with complex karyotype and nonspecific genetic alterations. 9 One feature that is common among these sarcomas is the aberrant checkpoint function of the p53 pathway. Despite its prevalent occurrence, among this category of sarcomas, p53 pathway alterations have relatively weak prognostic value, possibly suggesting that inactivation of p53 serves as a necessary early event in tumour development. 13, 14 Some recent studies have shown that p53 is important in promoting quiescence in hematopoietic stem cells and in suppressing self-renewal of adult neural stem cells. 15, 16 Combined with the observation that the suppression of p53 promotes the generation of induced pluripotent stem cell generation, 17 it seem that the acquisition of stem-cell like properties such as evasion of senescence and ability to self-renew may be a crucial event during early stages of tumourgenesis. 3

12 1.2.3 Treatment Surgery The most commonly employed treatment method for STS is surgical removal with wide margins, sometimes in conjunction with radiation therapy. 18 In STS, the tumour cells often penetrates beyond the visible tumour mass, so a wide (2-3cm) resection margin is removed in surgery whenever possible to avoid local recurrences. 19 Such treatment is often curative for localized STS, and the 5-year survival rate for stage I and stage II STS are 86% and 72%. 1 However, the rate decreases to 52% for stage III (regional nodal metastasis) STS and 10-20% (distant metastasis) for stage IV STS Radiotherapy Radiotherapy is generally considered for intermediate to high grade STS, and less often considered for low grade STS. 4 Radiation can be delivered to the patient either as external-beams or brachytherapy, the implanting of radioactive seeds. 9 A study completed in 1992 showed that additional brachytherapy after surgery decreases the chance for local disease recurrence. 20 The data was especially interesting in that, for patients with high-grade tumours, despite greatly improving the 10-year local disease-free survival (90% compared to 63% for the control group), brachytherapy produced no difference in the overall survival. 20 A similar study using externalbeam radiotherapy also confirmed that, while radiotherapy was effective in reducing the local recurrence rate, it had little effect on overall survival Chemotherapy Compared to surgery and radiotherapy, which aim to improve the local control of tumours, chemotherapy is often utilized to achieve systematic control. 1 Some STS types have shown to be sensitive to chemotherapy; for example, adjuvant combination chemotherapy dramatically improves the overall survival of Ewing's sarcoma patients, from under 10 percent without the introduction of systemic treatment to greater than 60 percent. 22 However, meta-analysis suggest that, although adjuvant chemotherapy improves STS patient s progression-free survival, they had little effect on their overall survival. 23 Drugs used in the chemotherapy of STS include Cyclophosphamide and ifosfamide, vincristine, doxorubicin, dactinomycin, and etoposide. 1 4

13 Although chemotherapy provides a palliative option for most patients with unresectable or metastatic disease, it remains unclear whether it improves overall survival. 24 Therefore, while still widely used, the overall benefit of chemotherapy is highly controversial Targeted Molecular Therapy With the advancement of molecular biology, the availability of therapies that target specific molecular targets has been increasing. The best example for this is the targeting of the KIT protein using the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumours. 26 Clinical trials have shown that the response rate to the drug is over 60 percent, 27 and tightly associated with specific genetic changes present in the disease mutation in exon 11 of c-kit is associated with a response rate of 85 percent, whereas mutations in other gene such as PDGFRA are associated with a much lower rate. 8 Moreover, such progress is not just limited to gastrointestinal stromal tumours. For example, the epidermal growth factor receptor inhibitor gefitinib is currently being tested in a phase II trial of patients with synovial sarcoma. 28, 29, the vascular endothelial growth factor-neutralizing antibody bevacizumab is currently being tested in patients with Kaposi s sarcoma, 30 and blockade of the hedgehog signalling pathway has been shown to reduce the size of engrafted chondrosarcoma. 31 Although relatively simple disease models such as gastrointestinal stromal tumours provides strong support for the investigation of molecular mechanisms that underlie STS, developing targeted molecular therapy for the majority of STS, such as malignant fibrous histiocytoma, which features complex karyotypes and nonspecific genetic alterations, will likely be more difficult. Hence, as discussed previously, the clue that the acquisition of stem cell-like properties such as evasion of senescence and ability to self-renew may be crucial for tumourgenesis suggest that the investigation of properties in specific tumour cells responsible for the initiation and maintenance of tumour growth may yield valuable insights for understanding and developing effective targeted therapies against STS Malignant Fibrous Histiocytoma (MFH) MFH is a common, aggressive, soft-tissue sarcoma of adulthood. 32 Because cultured explants of these tumours exhibited some of the characteristics of histiocytes in that they developed several short cytoplasmic processes, moved in an ameboid fashion, and contained abundant granular 5

14 cytoplasm, they were thought to be histiocytic in origin, but capable of developing the structural and functional characteristics of fibroblasts. 33, 34 Consequently, the term malignant fibrous histiocytoma was developed, popularized and formally incorporated into classification schemes and routine diagnostic of sarcomas Gradually, as more cases of MFH have been observed and documented, the term MFH has come to include several subtypes: storiform-pleomorphic, myxoid, inflammatory, giant cell rich and angiomatoid Between the eighties to the nineties, MFH, especially the storiform-pleomorphic and myxoid variants became recognized as the most common soft tissue sarcoma of adulthood MFH cases were mostly found in middle-aged to elderly adults, and present themselves as large, high grade, and biologically aggressive tumours. 43, 48 They often located at muscles of the proximal extremities and retroperitoneum; however, they could be found anywhere in the body, including the superficial soft tissues, viscera, and the skeleton Most MFHs arises de novo; however, a minority developed as a result of long-term radiation. 52 The survival rates for MFH have improved very little in the past decades, and surgical management remains the cornerstone of therapy for MFH. 43, 53 While adjuvant radiotherapy and chemotherapies have been sometimes attempted, they have been found to have very little improvement on overall survival. 21 Like the majority of STS, belong to the category of STS with complex karyotype and nonspecific genetic alterations. 9 Immnohistochemistry shows that MFH frequently stain for antigens such as vimentin, actin, CD-68, and alpha-1-antitrypsin, alpha-1-antichymotrypsin, and variably stain for antigens such as smooth muscle actin, desmin, keratin, epithelial membrane antigen, S-100 protein, and neurofilament Most of these antigens are commonly found in other tumors, such as leiomyosarcoma, liposarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor, sarcomatoid carcinoma, and melanoma, but was not found to be specific to histiocytes, suggesting that MFH is not a true histiocytic neoplasm. 58, 59 As a result, some authors begin to argued that MFH "constitutes a heterogeneous, non-cohesive collection of poorly differentiated tumors" and is "a term that has become a meaningless, diagnosis of convenience". 60, 61 By the mid to late 1990s, only a small group of undifferentiated pleomorphic sarcomas with no definable line of differentiation was cautiously and reluctantly considered to be MFH 62, 63 represent, and the myxoid variant was being preferentially classified as myxofibrosarcoma. 6

15 However, in 2000 and 2001, several series in combination reported that the ultrastructural and immunohistochemical indicates MFH actually has unique features and does not represent other tumour types in most cases Moreover, the fact that such a tumour exists and may even represent one of the most common groups of sarcoma should not be surprising, because fibroblasts are the most common mesenchymal cells in the body and benign and malignant neoplasms tend to recapitulate their normal tissue counterparts. 32 The observation that welldefined sarcomas of other cell lineages may contain areas identical to MFH does not negate the existence of pure pleomorphic fibroblastic and myofibroblastic sarcomas. 32 It actually points to MFH as an excellent model for high-grade sarcomas, by the fact many all high-grade STS seem to have the mechanism to drives them toward express a fibroblastic/myofibroblastic phenotype. 1.3 Tumour Initiation Cells Overview As have been discussed, inter-tumour variation is a common phenomenon that depends on the patient s genetic disposition, as well as the tumour s origin and location among many other factors. It is less well-known, however, that the phenomenon of intra-tumour variation is also a very common occurrence in STS and many other cancer types, possibly because in most cases cancers cells are thought to be clonal in their origin. 66 It is now a well recognized fact that most tumours contain distinct subpopulations of cells that differ in their morphological, biochemical, genetic, and karyotypic characteristics. 67 The precise mechanism responsible for this heterogeneity is far from certain. Currently, there have been two distinct hypotheses: the clonal evolution and the cancer stem cell models. 67, 68 According to the clonal evolution hypothesis, or the stochastic model, each cell in a tumour is initially biologically equivalent but is shaped differently by either random genetic or epigenetic changes or the influence of microenvironment as the tumour develops. 68 On the other hand, the cancer stem cell hypothesis, or the hierarchical model, suggests that tumours are similar to normal tissues and that the tumour growth is sustained by the cancer stem cells at the top of the hierarchy, and heterogeneity arises in a specific and well-organized way. 68 Throughout many years of heated debates, many important pieces of evidence have been accumulated by each side, but neither side have been proven or disproven. These hypotheses are illustrated in Figure

16 Figure 1-1: The two models for cancer development. The clonal evolution hypothesis, or the stochastic model, states that each cell in a tumour is initially biologically equivalent but is shaped differently by either random genetic or epigenetic changes or the influence of microenvironment as the tumour develop, so each cell in a tumour have similar responsibilities in initiating and sustaining tumour growth. On the other hand, the cancer stem cell hypothesis, or the hierarchical model, suggests that tumours are similar to normal tissues and that the tumour growth is sustained by the cancer stem cells at the top of the hierarchy, and heterogeneity arises in a specific and well-organized way. 8

17 While the concept of cancer stem-cell is still up in the air, the intra-tumour heterogeneity in tumour cells ability to initiate and sustain tumour growth is widely recognized. The fact that not all tumour cells have the ability to initiate tumour growth was first observed in the 1960 s, when 35 patients were implanted with 1 billion of their own cancer cells and only 7 out of the 35 implantations actually initiated tumour formation. 69 The first concrete evidence for intra-tumour heterogeneity in tumour formation came in mid 1990s, when John Dick and colleagues demonstrated the existence of tumour-initiating cells (TICs) on acute myeloid leukaemia (AML) using non-obese diabetic severe combined immune-deficient (NOD-SCID) mouse model: 70 where most human AML cells have limited proliferative capacity, John Dick s team was able to isolate a rare population of AML-initiating cells (1 per 250,000 AML cells), marked by the expression of CD34 and the lack of CD38 at the cell surface, that home to the bone marrow and proliferate extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukemic cell morphology similar to that seen in the original patients. 71 The he major challenge for current cancer treatment does not usually lie in the lack of primary response or initial induction of remission but rather often lies in relapses and recurrence of tumour after the treatment. In this sense, TICs with their superior ability to initiate and sustain tumour growth logically become an interesting group of cells to target for the development of tumour therapies. As will be discussed below, there are evidences that suggest that TICs are not only crucial in metastasis and tumour-host interactions but also play important roles in resistance to radiation and chemotherapies. Therefore, investigating the characteristics and behaviours of TICs provide a unique opportunity for further understanding the mechanisms of cancer development and for developing more effective novel therapeutics TICs in Solid Tumours After the initial identification of TICs in AML, such population of cells that are enriched in their ability to initiate tumour formation have also been indentified in various solid tumours. The first 9

18 evidence for TIC tumour was found in breast cancer: Al-Hajj et al. demonstrated using primary patient tumours that a relatively rare population of CD44(+)CD24(-/low) cells has the ability to initiate tumour formation when as few as 100 of such cells were injected into immune-deficient mice, whereas the rest of the tumour cells failed to form tumours even when as much as tens of thousands of cells were injected. 72 The study also demonstrated the important fact that, in contrast with the rest of the tumour cells, the TIC subpopulation in unique in that could be serially passaged to recapitulate the complex cell composition of the original tumour: xenografted tumours generated from these cells contained additional CD44(+)CD24(- /low)lineage(-) tumorigenic cells as well as the phenotypically diverse mixed populations of other non-tumorigenic cells present in the initial tumour. 72 Soon after the demonstration of a specific tumourgenic population in breast tumours, TICs have been identified in many types of solid tumours including, colon, pancreatic, liver and ovarian cancer and melanoma, isolated by their unique expression profile of cell-surface marker including CD44, CD24, CD133, epithelial cell adhesion molecule (EPCAM) and ATP-binding cassette sub-family B member 5 (ABCB5) As was expected, these tumour-initiating cells were able to recapitulate the complex morphology and cytological heterogeneity of the original primary tumours when xenografted into NOD SCID mice. 70 The consistent isolation TIC across many different types of TIC and their similar behaviours points to possible molecular and genetic similarities and again points to the likely importance and universality of TICs in cancer General Properties of TICs As TICs are defined by their ability to re-grow and recapitulate the morphology of the initial tumour from which they were isolated, self-renewal is undoubtedly a key characteristic associated with TICs, and that is also a big reason why TICs are often associated with the term cancer stem cells (CSCs). Studies have shown that TICs in leukaemia cells often have a higher self-renewal capacity than normal Hematopoietic stem cells (HSCs). 79 In colorectal cancer, the tumourigenesis in the intestinal epithelium seems to be initially caused by the hyper-self-renewal of intestinal-crypt cells, followed by the accumulation of additional mutations that confer 80, 81 malignancy. 10

19 Curiously, TICs in AML and CML tumours are relatively quiescent, in a manner similar to normal stem cells in their normal resting state. 82, 83 In fact, it was demonstrated as early as in the 1970 s that, in AML the majority of leukemic blasts in vivo were post-mitotic and needed to be continuously replenished from a relatively small proliferative fraction, and that only a small fraction (~ 5%) of leukemic blasts was rapidly cycling in vivo; 84 however, a more careful examination reveals that there are 2 proliferative fractions a larger, fast cycling subset with a 24-hour cycle time and a smaller, slow cycling fraction with a dormancy estimated to last from weeks to months, and the slow cycling fraction was actually generating the fast cycling fraction and responded to the depletion of the leukemia cell by going into rapid expansion. 85 This piece of data serves well to explain why the putative TIC populations isolated using various markers could sometimes be more or less proliferative when compared to the bulk tumour cells the marker may often label a mixture of both the fast and slow cycling fractions. The fact that TICs may be less proliferative compared to the bulk tumour cells suggests that the ability of these cells to initiate tumour formation is probably not due to their proliferative advantage of over the rest of the bulk tumour cells. Consequently, rather than their ability to proliferate, the most determining characteristic of TICs seems to be their increased self-renewal potential. For instance, the slowly proliferating intestinal stem cells from phosphatase and tensin homologue(pten)-deficient mice are the ones responsible for initiate intestinal polyposis. 86 As cells capable of initiate de novo tumour formation, it is reasonable to hypothesize that macroscopic metastases may arise from migrating or disseminated TICs. 87 So far, there have been some strong evidences that support that: for example in patients with breast cancer, tumorigenic CD44+CD24-/low cells can be readily detected in metastatic pleural effusions, and most earlydisseminated cancer cells that are detected in the bone marrow have a putative breast tumourinitiating cell phenotype. 72, 88 Another example would be the demonstration of CD133+CXCR4+ TICs that are found at the invasive front of pancreatic tumours have been shown to determine the metastatic phenotype of the individual tumour. 89 However, it is important to note, while TICs may possess certain stem-cell-like properties such as self-renewal and may be in certain aspect parallel to the concept of CSCs, the term TIC is only defined by its unique ability to re-grow and recapitulate the morphology of the initial 11

20 tumour from which they were isolated. Moreover, the concept of TIC is principally helpful for the understanding of the sustained growth and maintenance of a developing neoplasm, but do not directly entail queries regarding their point of origin or whether they phenotypically resembles a normal stem cell. In fact, it is generally accepted that the origins of TICs may vary, and while they may exploit several important mechanisms that are typically unique to stem cells, certain characteristic and behaviours of TICs may be fundamentally different from a stem cell in the classical sense Important Molecular Pathways in TICs Many recent molecular studies have shown that signalling pathways that regulate normal stemcell self-renewal are also important in the development of various tumours. Several key signalling pathways have been indicated in these studies, including the WNT pathway, the BMI1 pathways, the PTEN pathway, the hedgehog (Hh) pathway, and the Notch pathway WNT The Wnt pathway is associated with many types of stems cells. In blood, Wnt signalling promotes the self-renewal and proliferation of HSCs. 91 In skin, Wnt has been shown to be responsible for stem cell activation and expansion in skin. 92, 93 In intestine, activated Wnt signalling induces crypt cell proliferation. 94, 95. Mechanistically, Wnt signaling is activated when Wnt binds its receptor Frizzled and inhibits a negative complex formed by adenomatous polyposis coli (APC) and glycogen synthase kinase-3β, which controls the phosphorylation and subsequent degradation of β-catenin, and when this happens β-catenin can then enter the nucleus and interact with TCF/LEF family transcription factors to promote downstream gene expressions. 96 Abnormal activation of the Wnt signalling pathway causes cells to receive continuous signals for proliferation due to accumulation of β-catenin in the nuclei, thereby leading to the development of APC in intestine, hair follicle tumors in skin, and leukemia in bone marrow In skin, activation of a tamoxifen-induced β-catenin transgene led to hair follicle tumours resembling trichofolliculomas. 101 In colon cancer, it was shown that Tcf4 drives the same genetic programme in colorectal cancer cells as in crypt stem and progenitor cells. 95 Moreover, in vivo induced activation of Wnt cascade populated the entire villi with crypt-like cells within two days, 12

21 and many of the induced crypt markers were previously identified in human colorectal cancer cell lines. 102 In leukemia, granulocyte macrophage progenitors (GMPs) from chronic myelogenous leukaemia patients and blast crisis cells from patients resistant to therapy display activated Wnt signalling. 97 Moreover, the inhibition of β-catenin decreases the re-plating capacity of leukaemic cells in vitro, suggesting that the leukaemia precursors are dependent on Wnt signalling for growth and renewal; conversely, over-activation of Wnt signalling endows GMPs, which normally have limited self-renewal capacity, with stem-cell-like properties of long-term renewal Bmi-1 B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), was identified as an oncogene that cooperates with c-myc in the initiation of lymphoma. 103, 104 It is a core member of the the polycomb repressive complex 1 (PRC1) complex, which acts as an important epigenetic gene silencers The Bmi-1 protein contains a conserved RING finger domain in its N- terminal end and a central helix-turn-helix-turn-helix-turn motif, which is required for inducing 106, 110, 111 telomerase activity and immortalization of human epithelial cells. Increasing evidences suggest that Bmi-1 plays an important role in the self-renewal and differentiation of human stem cells. Bmi-1 is highly expressed in adult and fetal mouse and adult HSCs, and by knocking out its expression, the number of HSCs becomes greatly reduced postnatally while the fetal liver acquired from Bmi-1-/- mice could only transiently contribute to hematopoiesis. 112 Furthermore, down-regulation of Bmi-1 expression in neural stem cells leads to their decreased ability to proliferate and self-renew both in vivo and in vitro, 113 while upregulation of Bmi-1 expression promotes self-renewal by transcriptional repression of INK4a and ARF. 114 Bmi-1 expression is frequently up-regulated in human neoplasm, such as lung cancer, ovarian cancer, acute myeloid leukemia, nasopharyngeal carcinoma, breast cancer, and neuroblastoma, which indicates that Bmi-1 might play an important role in tumourgenesis Lessard et al. have showed that the stem cell number in the peripheral leukemia cells from Bmi-1 wild-type mice was significantly higher than that in Bmi-1-knockout mice along with a higher number of apoptotic cells and decreased colony-forming abilities In human medulloblastoma 13

22 cell lines, knocking-down of Bmi-1 causes inhibition of proliferation, loss of clonogenic survival, as well as declining tumour formation in vivo. 122 Moreover, the cooperation of Bmi-1 with c-myc has been shown to induce telomerase activity and down-regulate p16ink4a and p19arf expression; allowing cells to bypass senescence and immortalizes them It is generally hypothesized that in tumour genesis Bmi-1 mainly acts by promoting telomerase activity or by helping the cells bypass cell cycle arrestin the G1 and G2/M thereby preventing 124, 125 apoptosis PTEN In humans, PTEN encodes a lipid phosphatase and is an important tumour suppressor gene. 126 It is a key regulator for cell proliferation and survival by negatively regulating the phosphoinositide 3-kinase (PI-3K) signalling pathway by keeping the levels of PIP-3 low Activation of PI3-kinase activates proto-oncogene serine/threonine kinase Akt, which acts as a well-established survival factor and a negative regulator of apoptosis. 130 In the hematopoietic system, PTEN ise frequently associated to leukemogenesis, and recently it was shown that PTEN plays a critical role in controlling HSC proliferation and differentiation. 131, 132 Experiments have shown that following PTEN depletion in the HSC compartment, mice develop myeloproliferative disorder followed by acute leukemia development. 131, 132 In prostate, PTEN deletion leads to expansion of the Sca-1+ subpopulation, in which the regenerative capacity of prostate stem/progenitor cells has been shown to reside. 133, 134 In the skin deleting PTEN in CK5+ basal cells resulted in epithelial hyperplasia and 135, 136 spontaneous tumour formation. Moreover, PTEN deficiency was also shown to enhance neural stem/progenitor cell selfrenewal by promoting G0-G1 cell cycle transition, 137 and the in the intestine resulted in altered cellular differentiation and an increase of intestinal stem cells in the crypt, along with an increase in nuclear β-catenin. 86 Germ-line PTEN mutations can lead to development of multiple benign tumours and an increased susceptibility to breast and thyroid malignancies, 138 and mice heterozygous for PTEN-inactivating mutations display hyperplastic-dysplastic features and a 138, 139 high incidence of spontaneous tumours of various histological origins. 14

23 On the other hand, restoration of wild-type PTEN in several PTEN / cell lines, including glioblastoma and breast and renal carcinoma reduced cell growth and induced apoptosis. 140 In most cases, complete inactivation of PTEN is associated with late-stage, more aggressive, and usually metastatic tumours, and as a result, loss of this gene is often regarded a late event in the transformation process Notch To date, four vertebrate Notch genes have been identified: Notch-1-4, along with five mammalian ligands: Dll-1, Dll-3, Dll-4, Jagged-1 and Jagged The pathway is illustrated in Figure 1-2. Upon binding of its ligand, Notch is cleaved, releasing Notch intracellular domain (NCID) through a cascade of proteolytic cleavages by the metalloprotease tumor necrosis factorα-converting enzyme (TACE) and γ-secretase. 141 The released extracellular domain is transendocytosed by the ligand-expressing cell and the intracellular domain is translocated into the nucleus for transcriptional activation of Notch target genes including the Hairy enhance of split-1 (Hes-1), Hey-1, Hey-2, Nuclear factor-κb (NF-κB), Cyclin D1, c-myc, etc. 141 Notch signalling is crucial to the function of a variety of mammalian stem cells such as embryonic, hematopoietic, neural, intestinal, and epithelial stem cells Recently, it was shown that Notch signalling promotes the undifferentiated cell state in various stem and precursor cell populations, via its downstream genes such as Hes-1 and Hey , 151 In zebra fish, Notch induction promotes progenitors to remain in quiescence, whereas blockade of Notch massively reinitiates their division and subsequent commitment toward proliferative differentiation. 152 Moreover, in human cells, it was shown that was essential for quiescence of non-dividing cells to be reversible, as it prevented both premature senescence and inappropriate differentiation, 153 and the Notch inhibitor DAPT was able to inhibit the expression of HES1 and prevent the reversibility of quiescence. 153 Clinically, Notch gene is also often abnormally activated in many human malignancies such as cervical, lung, colon, head and neck, renal carcinoma, acute myeloid, Hodgkin and Large-cell lymphomas and pancreatic cancer In breast cancer and prostate cancer, activated Notch signalling was found to be associated with poor prognosis. 168, 169 Furthermore, activated Notch signalling has been shown to contribute to mammary carcinogenesis by 15

24 deregulating the self-renewal of normal mammary stem cells, 146 and the self-renewal TIC population in breast cancer can be stimulated in a Notch-dependent by induction of Jagged-1, whereas inhibitors of the Notch signalling can block such effect. 170 Farnie et al. further confirmed that the CD44+/CD24 TIC population in breast cancer exhibits up-regulated Notch genes expression. 171 The populating of TIC in glioma cell line, identified by the isolation of side population (SP) cells, also display elevated level of Notch Interestingly, these cells are chemo-resistant possibly because of the high expression levels of drug-transporter proteins such as ABCG Over-expression of Notch-1 in glioma cells promoted the growth and the formation of neurosphere-like colonies, 149, 173 and the blockade Notch reduced the CD133+ TIC fraction by almost 5-fold and totally abolished all SP cells. 174 In leukemia, Jagged-2, a Notch ligand, was found to be over-expressed in the TIC fraction, and the gamma-secretase inhibitor DAPT was able to inhibits the colony formation ability of the TIC fraction

25 Figure 1-2: The Notch signalling Pathway. Notch receptors are expressed on the cell surface as heterodimeric proteins. The signalling of the pathway is triggered upon ligand-receptor interaction (1) which induces two sequential proteolytic cleavages (2), the first in the extracellular domain mediated by TACE, and the second within the transmembrane domain mediated by γ-secretase. This second cleavage allows the release (3) and translocationv(4) of the intracellular domain of Notch (NICD) into the nucleus where it associates with other transcription factors to displace co-repressors and initiate transcriptional activation (5). 17

26 SHH The Hedgehog (Hh) signalling is well conserved in vertebrates and acts as a critical mediator of segmental patterning during embryonic development by regulating the proliferation, migration, and differentiation of target cells. 176, 177 Postnatally, organs such as the central nervous system and the lung, rely on continued Hh signaling for tissue homeostasis and repair following injury Hh pathway can be activated by the binding of Sonic (SHh), Desert (DHh), or Indian Hedgehog (IHh), to the receptor Patched (Ptch1). Following Hh ligand binding to Ptch, the repression of Smoothened (Smo) is released, and the expression and post-translational processing of the Gli zinc-finger transcription factors is modulated. 181 The major transcriptional target of Hh pathway include Gli1, Ptch1, and hedgehog interacting protein (Hip). 181 The pathway is illustrated in Figure 1-3. The first link between Hh signalling and tumour formation was in discovered in Gorlin's syndrome, as its familial predisposition to basal-cell carcinomas (BCCs), medulloblastomas and rhabdomyosarcomas has been found to be closely associated to Ptch mutations Moreover, a more careful examination showed that the large majority of sporadically occurring BCCs and one-third or more of all human medulloblastoma involve hyper-activated Hh signalling Experiments in mice confirmed that over-expression of Shh, Gli1, Gli2 or constitutively active Smo in the skin produces BCC-like lesions in histological appearance and expression of phenotypic markers In animal models, Ptch+/- mice produced a similar phenotype to patients with Gorlin's syndrome with an abnormally high frequency of cerebellar and muscle tumours. 191, 192 Furthermore, when such mice are treated with Hh blocking agents such as cyclopamine, the spontaneously developed tumours were completely eliminated Hh blockade has also been shown to inhibit the growth of mouse medulloblastoma cells in both 197, 198 culture and in mouse allograft models. In solid tumours, studies have found excessive Hh signalling in bone/cartilage tumours such as endochondroma, osteochondroma and chondrosarcoma. 31, 199 Additionally, Hh blockade 18

27 have been shown to be effective in inhibiting the growth of small-cell lung cancer, pancreatic 179, cancer, and prostate cancer in their respective xenograft models. Hh signalling also serves important function in progenitor and stem cells, as well as certain TIC populations in various tumours. Human cells highly enriched for hematopoietic progenitors (CD34+Lin-CD38-) exhibited increased self-renewal in response to Sonic hedgehog signalling in vitro, 205 and knocking out Smo in normal HSCs markedly decreased their regenerative capacity with a similar effects in leukaemia stem cells. 206 In multiple myelomam Hh signalling is crucial in maintaining its TIC population, 207 and in chronic myelogenous leukemia(cml), when Hh signal was blocked, fewer cells expressed the surface marker signatures for the TIC population, while stimulating Hh signalling increased the number of these cells and accelerated disease progression. 206 Moreover, cyclopamine, a Hh blocker, has been shown to be effective in reducing the incidence and relapse rate of CML in animal models. 208 For neural cells, hedgehog has been shown to enhance proliferation and inhibits differentiation of central nervous system (CNS), and thereby contribute to CNS tumorigenesis. 209 In Glioma and Colon cancer, Hh signalling has been shown to be essential for sustained tumour growth and the maintenance and self-renewal of the TIC population. 210, 211 For breast tissues, of Hh signalling play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells: it increased the number of mammosphere-initiating stem/progenitor cell and mammosphere size, but was inhibited when these cells were induced to differentiate, and in the CD44+CD24 /lowlin population of human breast cancer cells, which are highly enriched in tumour initiation potential, Hh signalling was highly active

28 Figure 1-3: The Hedgehog signalling Pathway. At cell membrane, SHH ligand binds to the Patched-1 (PTCH1) receptor(1) to inhibits the activity of PTCH1. In the absence of ligand, PTCH1 inhibits Smoothened (SMO), a downstream protein in the pathway (2), which negatively regulate the activity of GLI. Therefore, the binding of SHH relieves SMO inhibition, leading to activation of the GLI transcription factors (3), which then translocate into the nucleus (4) and initiatethe transcription of hedgehog target genes(5). 20

29 1.3.5 Interactions between Molecular Pathways Recently, it has been demonstrated that Bmi-1 is a downstream gene in the Hedgehog pathway: Bmi-1 expression is up-regulated up to six times when the Hedgehog pathway is activated and significantly downregulated when the Hedgehog pathway is blocked by sirna. 175, 212, 213 Moreover, both Gli1 and Gli2 over-expressing mammospheres display higher Bmi-1 expression levels, and knocking down of Bmi-1 could significantly hinder the effects of Hedgehog activation on both primary and secondary mammosphere formation, suggested that Bmi-1 is responsible for mediating the effects of Hedgehog signalling pathway on promoting the 175, 212, 213 self-renewal of mammary stem cells or progenitor cells. Emerging evidence shows that the loss of PTEN function leads to activation of WNT and the active form of β-catenin, thereby promoting self-renewal: homozygous Pten deletion in the mammary epithelium leads to precocious mammary gland development and breast cancer formation, similar to phenotypes associated with WNT activation, 214 and crossing WNT transgenic mice with Pten+/ mice accelerated tumour development, suggesting cross-talks between the WNT and PTEN/PI3K pathways. 215 In leukemia models, the activation WNT pathway is known to be essential in the self-renewal of HSCs and leukemic TICs, demonstrated by its necessity in the replating potential of these cells in vitro, 97 and PTEN inactivation in HSCs has been demonstrated to serves as the first hit by activating the PI3K/AKT pathway, conferring survival and proliferative advantages, and promoting genomic instability, followed by the further activation of β-catenin. 216 Interestingly, simultaneously deleting Pten and one allele of β-catenin in HSCs impaired leukemic TIC formation/self-renewal and T-lineage acute lymphoblastic leukemia (T-ALL) development caused by PTEN loss. 216 NOTCH signalling has also been shown to interact with PTEN. In human T-ALL, NOTCH pathway activation leads to reduced PTEN expression in human T-ALL via the transcriptional expression of HES-1 and c-myc. 217, 218, Moreover, in PTEN-null leukemia model, although no alterations in NOTCH1 signalling are detected, a recurring chromosome translocation was found to over-express c-myc the TIC fraction and CD3+ leukemic blasts, suggesting PTEN inactivation and c-myc may be functionally downstream of NOTCH pathway 21

30 activation. 216 Some recent data have suggested that the expression of Hes1, a principal effector of the Notch pathway, can be stimulated by Hedgehog signalling in both C3H/10T1/2 mesodermal and MNS70 neural cells, in which Smoothened function was necessary. 219 Additionally, the coordination between WNT and the TGF-β pathway component such as Noggin and BMPs has been show to be crucial in activating stem cells in skin and intestine. 220, 221 The observation that both WNT activation and over-expression of Noggin, which inhibits TGF-β signalling, leads to intestinal polyposis further supports this link The evidence seem to hint toward when the balance in stem-cell homeostasis is disrupted, stem cells may proliferate without restraint and lead to neoplasm, such as the loss of BMP signalling or abnormal activation 97, 100, 220, 224 of Wnt signalling TIC and Resistance to Therapies Normal progenitor or stem cells, including HSCs, characteristically express drug-resistance proteins, such as the MDR1 and ABC transporters, which may serve as mechanisms that protect them from chemotherapy insult and induction of apoptosis. 225, , 228 Interestingly, the more aggressive and refractory cancers that are resistant to radiation and chemo therapies often contain a higher proportion of TICs. 229, 230.For example, high abundance of stem-like TIC cells in AML at diagnosis predicts high minimal residual disease after therapy and poor prognosis, 231 and in breast cancer, chemotherapy treatment increased the percentage of CD44+CD24-/low TICs in patients, consistent with the relative chemo-resistance of these cells. 232 The failure of tradition therapies in improving overall survival for cancers such as STS may be due to the fact that despite eliminating most of cells and inducing the tumour shrinkage, sufficient TICs were left behind to cause eventual relapse. Recently, there have been many strong evidence to suggest that TICs are often more resistant to therapy than bulk tumour cells Chemotherapy In leukemia, TICs display chemotherapy resistance relative to the bulk tumour cells that are more differentiated. 233 In colon cancer, the CD133+ TIC fraction showed preferential resistance to Oxaliplatin and 5-fluorouracil, two standard chemotherapy regimens against colon cancer. 234 In ovarian cancers, the TICs are highly resistant to cisplatin or paclitaxel treatment, 235 and in glioblastoma the TICs have been shown to be resistant to a wide array of chemotherapeutic 22

31 agents. 236 In pancreatic cancer, a subpopulation that has been found to functionally resemble stem cells exhibit a strong resistance to gemcitabine both in vitro and in vivo. 89 In hepatocellular carcinoma, the TIC fraction also showed resistance to chemotherapy. 237 Moreover, TICs often express molecular metabolic mediators like aldehyde dehydrogenase 1 (ALDH1) that confer resistance to cyclophosphamide in normal stem cells. 238 ALDH1 activity is amplified in leukemic and breast TICs and is associated with a poor prognosis. 239, 240 Intriguingly, TICs have also demonstrated an ability to resist the effect of many novel anticancer drugs with specific molecular targets, such as imatinib, which targets and inhibits the BCR ABL kinase. 241 Imatinib eliminates proliferating, committed leukaemia progenitors, but not primitive, quiescent tumour-initiating cells. 83, 242, 243 However, the drug resistance and disease recurrence associated with imatinib treatment of leukemia can be quenched by targeting stem cell maintenance pathway involving Hedgehog Radiation Therapy Hematopoietic stem cells lacking p53 have been frequently linked to leukemic TICs, and interestingly they are resistant to radiation induced apoptosis. 244 In gliomas, it was shown that the abundance of TICs was dramatically increased by irradiation and that irradiated TICs have survival advantages relative to the non-tic population. 245 Moreover, the same study showed that radiation-resistant tumours displayed an increased percentage of CD133+ TICs and radiation failed to affect the ability of TICs to regrow tumours. 245 Medulloblastoma TICs that express CD133 are more resistant to radiation than the CD133- cells. 246 In breast cancer cell line, it was also shown that cells expressing TIC markers are resistant to radiation. 247, 248. This study also showed a possible role of the Notch signalling on radiation-resistance, as Jagged-1 expression and level of activated Notch-1 were both stimulated after radiation. 248 The Hedgehog-Gli1 pathway has also been found to be involved in TIC-mediated tumour recurrence after radiation therapy. 210 In soft-tissues, human mesenchymal stem cells have been shown to be resistant to irradiation, providing a possible link to STS. 249 Therefore, it is often suggested that, because most cytotoxic therapies used for cancer targets highly proliferative tumour cells, whereas TICs are relatively often quiescent, TICs would be relatively resistant to such treatment leading to further relapses and metastasis. 250 It is also 23

32 thought that the expression of ATP-binding cassette (ABC) pumps, 74, 226, 251 their relative resistance to oxidative or DNA damage and their efficiency of DNA repair, 245, 252, 253, also contribute to the fact that TICs are commonly more resistant to therapy than the bulk of tumours Model for TIC Isolation of TIC TIC rich fractions are often isolated in three ways: cell sorting by the expression of various cellsurface markers, cell sorting by the cell s ability to efflux certain dyes, 172, 254 or using cell behaviours in serum-free culturing conditions The isolation method using specific cell surface markers is often the most consistent and clear-cut, such the isolation of CD133+ population in brain, 258 prostate, 259 and colon cancer, 260 CD44+CD24-/low population in breast cancer 261, and the CD34+/CD38- in leukemia 262 However, the challenge lies in that many other tumours still lack a set of validated markers that are preferentially expressed in the TIC fraction. For the dye efflux method, the DNA-binding dye Hoechst has often been used to identify and enrich for TICs in certain tumours, as the expression of the genes encoding ABC drug transporters such as ABCG2 is often a conserved feature in stem-like populations in both tumour and normal tissues. 172, 254, 263, 264 Ideally, this isolation method for TIC should be very consistent and reproducible since it is not constrained by tissue specificity as in the case of cell surface makers, but the delicate and complex kinetics of dye exclusion along with the toxicity of various dyes limit the application of such method. 70 An alternative of dye exclusion is the selection of other surrogate functional properties of stem cells, such as aldehyde dehydrogenase 240, 265 activity. The third method of serum-free non-adherent culture is developed from the observation that central nervous system (CNS) cells grown on non-adherent surfaces form spheroid colonies (neurospheres), 266, 267 and studies have demonstrated the validity of this TIC isolation method 229, 240, 255, 257, 268 brain, breast and colon cancer. 24

33 Xenograft Models Although there are certain ways to observe the behaviours of TICs in vitro, the most direct way to study the tumour initiating properties of these cells is by allowing them to generate tumours in an in vivo setting that resembles how tumours normally develop and grow. This is often done by implanting tumorigenic cells into immune-deficient mice. 74 These models can be used to investigate relapses after chemotherapeutic treatment and the process of metastasis. 269, 270 Orthotopic implantations are often preferred, but when it become too technically difficult in cases such as colon cancer, subcutaneous implantation with cells suspended in matrigel (or mixed with stromal cells) and xenograft models featuring subrenal capsule implantation can 75, 76 often be used to implace of the orthotopic implantation. TIC models involving xeno-transplantation of primary tumour cells in vivo provide a reproducible, cost-effective system for meeting drug screening and discovery demands. 271 In such models, fragments of fresh surgically resected tumour are implanted directly into immunecompromised mice either at its orthotopic site or subcutaneously. The resulting xenografts can be serially passaged into new animals and maintained exclusively in vivo. 272 Studies have shown that the cellular architecture and heterogeneity of a serially passaged primary tumour xenograft closely resemble those of the original patient tumour and are more complex than the corresponding features of traditional cell line xenografts. 264 Hence, these xenograft models could serve a excellent preclinical models for cancer research, as much of the complex features in the primary human tumour is kept intact. Moreover, the possibility of serial transplantations at different cell numbers enables researchers to compare the tumour cells potential for tumour initiation after various treatments aimed to target the TIC population. Although there are still challenges due to the intrinsic high variation between individual tumours and the practical difficulty in obtaining and utilizing freshly resected tissues, it remains the ideal choice for testing candidate agents. 70 By the same token, compared to the implantation of cell lines, the use of primary tumour xenografts offer great advantages in providing a more complete picture of tumour heterogeneity and hierarchy that is distinct in each patient. 25

34 In summary, primary tumour xenografts coupled with appropriate experimental analysis tools serves as an excellent in vivo model for evaluating therapeutic method aimed to impede cancer growth and eliminate their progression and recurrences Controversies There have been a lot of controversies surrounding TIC populations, largely due to the link between TIC and cancer stem cells (CSC); the two terms are often used interchangeably. The first concrete evidence for CSC was found in 1997, when a rare (0.2%) CD34+CD38- population from AMLwas demonstrated to be both necessary and sufficient to transfer the disease from patients to NOD-SCID mice. 79 This study utilizes the most widely accepted assay to validate a candidate cancer stem cell subpopulation: tumour transplantation and serial transplantation into immune-compromised mice to verify if a specific population of cells can recapitulate the heterogeneity of the primary tumour. 79, 273 Using similar approaches, putative CSC populations have also been detected in a variety of solid tumours, such as prostate cancer, 274 squamous cell carcinoma, 275 brain cancer, 78 liver cancer, 73 breast cancer, 72 and colon cancer. 75 However, such observations has been challenged by the possibility that nontransplantable cell population might simply lack a critical feature for growth in the foreign microenvironment, such as a cytokine receptor responsive to mouse factors or a chemokine receptor that attracts the cells to a nurturing niche; rather than being uniquely responsible for tumour growth, the transplantable population may simply have inadvertently acquired features that endow those cells with the ability to survive in the foreign milieu. 276 The observation of a specific transplantable cell population does not imply that other tumour cells do not contribute to tumour maintenance and growth. This argument is supported by the fact that, in spontaneous mouse leukemia and lymphomas, the frequency of transplantable tumour cells is quite high, and that, for melanoma, while transplantation into NOD/SCID indicate that only rare human cancer cells ( %) form tumours, transplantation into a more immune-compromised mice strain reveals that in single-cell transplants 27% of unselected melanoma cells formed tumours. 281 Moreover, it has been suggested that cell sorting selectively favour free-moving niche-independent tumour-initiating cells, which may resemble cells in activated or mobilized states that are selected when transplantation assays are performed on normal stem cells, 282 and certain markers used for TIC identification may select for cells that evade the immune system

35 Improvement to the sensitivity and reliability of CSC detection can be achieved via depletion of residual immune activity, direct injection of cells into their natural niche, and supplementing/transgenic expression of human cytokines. 284 However, there are many inherent problems in these approaches: for example, over-depletion of the recipient immune activity may allow even non-cancer donor cells to grow into a tumour mass and the local concentration human cytokines is often finely controlled within specific cell compartment and almost impossible to mimic. 285 Nevertheless, there have been other lines of evidences that support the CSC hypothesis. For example, although it is generally accepted that the evidence for CSC in solid tumour is less advanced due to the fact that no CSC population in solid tumours has yet been highly purified, 276 the ability of the putative CSC populations to repeatedly and closely recapitulate previous morphology of heterogeneous tumours provide strong support for the hierarchical CSC hypothesis. 284, 286 Clinically, a number of markers used in isolating CSCs are emerging as being predictive of disease progression. 74, 287 It was also demonstrated that, in AML, the majority of leukemic blasts in vivo were post-mitotic and needed to be continuously replenished from a relatively small proliferative fraction, and that only a small fraction (~ 5%) of leukemic blasts was rapidly cycling in vivo. 84 A more careful examination reveals that there are 2 proliferative fractions a larger, fast cycling subset with a 24-hour cycle time and a smaller, slow cycling fraction with a dormancy estimated to last from weeks to months; the slow cycling fraction was actually generating the fast cycling fraction, and respond to depletion of the leukemia cell by going into rapid expansion. 85 Furthermore, the identification of tumour-initiating cells in transgenic mouse models that develops intrinsic neoplasm adds credibility to the cancer stem cell hypothesis Considering both sides of argument, there does not exist a definite proof for CSC hypothesis, yet it is quite clear that within tumour there exist a unique population of cells that is more adept at transplantation, can be consistent isolate, and can repeatedly and closely recapitulate previous morphology of heterogeneous tumours. This population is likely to process certain interesting properties that may lead to a better understanding of tumourgenesis. In some recent literatures, this population has been linked to resistance to radiation and chemotherapies. 237, 245, 246, Therefore, it may be helpful to take a step back, and simply 27

36 consider them as a subset of tumour cells that possesses potent tumour initiating potential, i.e. tumour initiating cells (TIC). Moreover, when discussing TICs, it is important to focus on its functional properties, rather than their cell of origin; the self-renewal of TIC does not reflect the derivation of the cell from normal stem cells, and TICs may not behave in a similar fashion to normal stem cells other than sustaining and maintaining the tumour tissues. Consequently, when selecting methods to study TICs, it is important to select assays that properly capture and read out the functionality of those cells rather than simply looking for the expression of static surface markers. Moreover, it is important to keep in mind that TIC itself may represent a heterogeneous population with varying different properties. 1.4 Side Population Cells Hoechst is a fluorescent dye that has an absorption wavelength of 350nm in the UV range, and two emission wavelength of 450nm and 675 nm, in the blue and red region respectively. 297 This dye binds to the AT-rich regions of the minor groove of DNA, and the emission intensity is dependent on many factors involved in DNA structure such as chromatin structure, DNA content and position of the cell within the cell cycle. 298, 299 The dye is universally taken up by all cells, but certain cells have an enhanced ability to efflux the dye; such cells were first identified in the mouse bone marrow and they were referred to as side population (SP) cells as they fell to the side of the bulk of the positively stained cells in FACS analysis plots. 300 This is illustrated in Figure

37 Figure 1-4: Side population cells isolated using flow-cytomentry. Hoechst is a fluorescent dye that has an absorption wavelength of 350nm in the UV range, and two emission wavelength of 450nm and 675 nm, in the blue and red region respectively. The dye is universally taken up by all cells, but certain cells have an enhanced ability to efflux the dye; such cells were first identified in the mouse bone marrow and they were referred to as side population cells as they fell to the side of the bulk of the positively stained cells in FACS analysis plots. The dye efflux of SP cells can be inhibited by Verapamil, resulting in all cells becoming stained with the dye. 29

38 1.4.1 SP Cells in Normal Tissues When SP cells were first identified in the mouse bone marrow, they were observed to be highly enriched for long term repopulating cells. 300 Since that discovery, SP cells have been identified in a variety of tissues : including skin, 304, 305 lung, 306, 307 liver, 308 heart, 308 brain, 309 mammary gland 310 and skeletal muscle 311. In normal tissues, SP cells express high levels stem cell markers such as Sca-1 and CD34 and possess multi-potent differentiation potential. 300 Moreover, SP cells isolated from mouse bone marrow and muscle share transcriptome signatures, while under-expressing genes representing their tissue specific functions. 312 Interestingly,, SP cells were shown to have more cells in G1/G0 than non-sp cells. 297 The mechanism that confers the dye-efflux ability of SP cell is conventionally thought to be the expression of ATP binding cassette protein (ABC) transporters. 226 Forced expression of these membrane transporters promote massive expansion of murine stem cells. 313, 314 However, Mdr1a/b/Bcrp1 triple knockout mice were observed to viable and still retained some SP cells in the bone marrow. 315 Hence, it is likely that there is either a redundancy in transporter function and/or the mechanism in which the SP phenotype is not determined is solely by the expression a few transporter proteins. 297 Interestingly, SP cells from neuroblatsomas had increased expression of C-kit/CD117 and had lowered expression of AC133/CD71 and CD56, a pattern is similar to the cellular phenotype of neural crest progenitor cells. 254 Overall, in adult tissues, the many stem-like features of SP cells seem to suggest that they may possibly behave in a way similar to regular stem cells SP Cells in Human Cancer SP cells have been identified in a large variety of cancer cell lines with an abundance between 0% to 20%. 254, 264, In cell lines where they are present, SP cells have been shown to be responsible for the maintenance and the tumour-initiation In the C6 glioma cell line, only SP cells was able to regenerate both SP and non-sp fractions. 317 The same study also showed that only SP cells had the ability to form neurospheres, a hallmark of neuronal stem cells. 317 Along with similar report in the MCF-7 breast cancer cell line, 172, the evidence seem to suggest that in cell lines SP cells have an increased capacity for self-renewal and are responsible for the 30

39 replenishing of the bulk cells, just like stem cells in normal tissues. However, it should be noted, that not all cell lines contain SP cells. In primary tumours, SP cells have been identified in glioblastoma, 254 the ascites of ovarian cancers, 322 and in a wide range of mesenchymal neoplasms. 264 The fact that SP cells are present in primary osteosarcomas but not SaOS and U2OS osteosarcoma cell lines seem to suggest that cell lines are only crude representations for primary tumours in vivo nature of tumours, due to genetic alterations or unnatural culturing conditions. 297 In xenograft models using immune-deficient mice, the SP fraction in hepatocellular, 320 lung, 318 gastric, 319 and nasopharyngeal carcinoma 321 was shown to be highly enriched for the capacity to initiate tumour formation compared bulk tumour cells Molecular Features of SP Cells in Human Cancer In line with their stem-cell like behaviours, compared to its non-sp counterpart, SP cells have been shown to have increased expression of genes that are involved in the regulation of stem cell function. SP cells from both colon carcinoma and breast carcinoma cell lines have elevated expression of genes involved in the WNT signalling pathway. 319 As discussed, the WNT pathway along with a few others have been shown to be important in normal stem cells and TICs. In MCF-7 cells, SP cells exhibited an up-regulation in genes belonging to PI3K/AKT pathway, and the PI3K inhibitor LY was able to deplete the percentage of SP cells and result in decreased in vitro colony formation and in vivo tumour formation. 323 Recent data from prostate cancer, breast cancer and multiple myeloma have demonstrated that the Hh pathway is essential in the maintenance of SP cells, and the blockade of Hh pathway by small molecules such as cyclopamine induces a depletion in the abundance of SP cells. 207, 261, 324 Similarly, in embryonal brain tumours, the inhibition of Notch pathway lead to the depletion of the stem-like SP cells and blocks tumour engraftment. 174 Gene profiling experiment in thyroid and small-cell lung cancer cell lines also showed that both the Notch and Hh pathway was up-regulated in the SP cells. 316, 325 Recent work have suggested that in breast and colon cancers a relatively small number of signalling pathways are disrupted at a high frequency while a large number of pathways are disrupted at a low frequency. 326 Therefore, identifying signalling pathways that are consistently up-regulated in SP cells of STS using high-throughput methods, such as micro-array 31

40 and mass-spectrometry, may prove to be a useful strategies for understanding the progression of STS and developing potential therapies Implication of SP cells in Cancer Therapies In primary mesenchymal tumours, there exists a positive correlation between tumour grade and the percentage of SP cells present. 264 Moreover, there have been many studies that focus on the link between SP cells and chemo-resistance, as ABC transporter proteins are highly expressed SP cells and have been shown to efflux lipophilic chemotherapeutic agents such as doxorubicin. 327 Leukemic side population cells, which are enriched for CSCs, have an amplified ability to pump chemotherapeutic drugs like daunorubicin and mitoxantrone out of the cell, and stem-like neuroblastoma cells displayed a similar ability to pump mitoxantrone, resulting in increased cell survival Specifically, the ABC transporters BCRP and MDR1 have been implicated in specifically expelling chemotherapeutic agents from cells and thus may mediate chemo-resistance when expressed by SP cells: MDR1 has been shown to remove vinblastine 328 and paclitaxel 329, while BCRP prevents accumulation of imatinib mesylate, 330 topotecan 331 and methotrexate 332. Furthermore, studies on radiation resistance also show that SP cells possess a survival advantage when subjected to intense radiation Criticisms There exist several arguments concerning the use of Hoechst dye exclusion as a means of isolating TIC cells. First, because as Hoechst dye binds to DNA and is toxic to the cells, the TIC properties of SP cells versus the rest of the cells may simply be due to a difference in viability. Moreover, great experimental variation exist in the analysis of SP cells, as subtle changes in temperature, dye concentration, staining time, and cellular concentration could all greatly affect the outcome of the flow-cytometry analysis. By the same token, the gating strategy by utilized by each individual labs in isolate SP cells via flow-cytometry is never consistent due to the individual heterogeneity of each tumour. 333 Although efflux blockers such as verapamil, are used as a control and ensure the capture of true SP cells, this methodology does not always yield ideal results as positively stained cells can remain within the negative or SP gate. It has been shown that different regions of the SP fraction possess different properties, with the cells in the lowest 32

41 quadrant have increased stem-like characteristics when compared to cells in the upper quadrant. 333 Nevertheless, there have been evidences that rebut against such criticisms. Recent study by Wu et al. showed that in primary mesenchymal tumours non-sp cells were able to form tumours in the primary round of injections, but failed to engraft after secondary transplantation; likely due to lack of sustained self-renewal. The initial tumour formation, however, provide strong evidence that non-sp cells are indeed viable after retaining Hoechst dye. 264 Moreover, in MCF-7 breast carcinoma cells it was shown that the in vivo tumorigenic potential and in vitro colony forming potential of unstained non-sp cells were similar to those that have been positively stained for Hoechst dye. 172 Ultimately, it is unlikely that SP cells is composed of solely and entirely TICs; it is more likely, that SP either represent a small portion of TICs or vice versa. After all, it is important to keep in mind that all isolation strategies have their shortfalls and perhaps the combination of different isolation methods are required to enhance the consistency and purity of TIC isolation SP Cells in Mesenchymal Neoplasms As discussed previously, STS is a type of mesenchymal neoplasms that occurs in all connective tissues except bone. In primary mesenchymal tumours, it was shown that the SP cells were enriched for tumour initiating potential, and only SP cells have the capacity to initiate tumours and recapitulate the original morphology of the primary tumours upon serial transplantation in NOD/SCID mice. 264 This study also showed that, in biopsy samples from surgical resections, tumours with relatively higher percentages of SP cells were those of higher grades. Moreover, cells derived from non-sp tumours have increased DNA content when compared to cells derived from SP tumours, possibly suggesting that the non-sp fraction contain a population of transiently amplifying cells that have the ability to initially form tumours through rapid proliferation, whereas only the SP cells have the ability for sustained self-renewal and proliferation. Consequently, despite being about the initiate tumour upon the initial transplantation in large number, the majority of the tumour cells, the non-sp fraction, do not have the capacity to selfrenew and therefore cannot sustain tumour initiation upon serial transplantation. 33

42 1.5 Summary and Conclusion Overall, the information gathered in this large section indicates that SP cells in STS represent a population of clinically relevant cells that not only are enriched for tumour initiating potential, but also play important roles in tumour progression, tumour maintenance, metastasis, relapses and resistance to cancer therapy. Therefore, further knowledge of this population in STS will be important for the understanding of the disease and the advancement effective treatment against STS. More specifically, the identification of specific molecular features in SP cells of STS may provide revealing insights regarding the biology of the process of tumour development and provide precise and effective ways of eliminating soft tissue neoplasm from its root. 34

43 1.6 Hypothesis and Rationales The goal of this research is to investigate novel molecular features in the SP fraction of STS, more specifically MFH tumours, and how such features may be targeted to provide potential therapies for these tumours. We hypothesize that certain pathways are differentially regulated in SP cells vs. NSP cells, and the specific targeting of such pathways may lead to a depletion of tumour initiating SP cells, resulting in retarded tumour growth and a reduced success rate of tumourigenesis upon serial transplantation. To examine these hypotheses, the following questions will be addressed: 1) Which molecular signalling pathways are activated in SP cell versus the non-sp cells which make up a majority of the bulk tumour cells? As mentioned previously, certain molecular pathways are activated in SP and TICs. By isolating SP and non-sp cells form fresh primary MFH, the RNA expression profile of each population can be compared using microarray analysis. The pathways that are preferentially activated in SP cells may serve as possible candidate targets upon the verification of microarray result via qpcr. 2) Can the targeting of such pathways lead to a depletion in the abundance of SP cells? Primary tumour can be established subcutaneously into immune-deficient mice to establish the initial xenografts. The mice then can be treated with small molecules that blocks candidate pathways to investigate the changes in SP abundance upon treatment. 3) Does the targeting of candidate pathways and the depletion of SP abundance lead to reduced tumour growth and tumourigenic potential upon further transplant? The treated tumour can be analyzed to observe difference in growth and gene expressions. They can also be further implanted for secondary transplantation to 35

44 examine whether the depletion of tumourigenic SP cells translate into the change in formation efficiency of bulk tumour cells upon secondary transplantation. The molecular features of SP cells in STS, and more specifically MFH, is still unknown at this point. This study would provide some elucidation as to the molecular signalling that is signifies these cells and enables them to possess enhanced tumourigenic abilities and sustained selfrenewal. This important information will aid in the understanding of the putative TIC fraction in STS and provide methods that can specifically targeted against them. Moreover, this study can provide revealing insights regarding tumour-initiation and provide clues that may lead to the development of efficacious therapeutic treatments against STS clinically. 36

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69 Chapter 2 2 Novel Properties of SP cells in STS, and How They May Be Targeted to Develop Potential Therapies 2.1 Abstract Tumours contain heterogeneous cell populations. A population enriched in tumour-initiating potential has been identified in soft-tissue sarcoma (STS) by the isolation of "side population" (SP) cells. In this study, we compared the gene expression profiles of SP and non-sp cells in STS and identified Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, using primary tumor xenografts in NOD-SCID mice as our experimental model, we used the Hh blocker Triparanol and the Notch blocker DAPT to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour-initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies. 61

70 2.2 Introduction Soft-tissue sarcoma (STS) is defined as a malignant neoplasm or cancer that arises in mesenchymal tissues other than bone, which include muscle, fat, and other fibrous supporting structures. 1 With STS may be broadly classified into two major categories: those with specific molecular alterations and simple karyotypes, and those with nonspecific genetic alterations and complex karyotypes via the accumulation of many genetic changes. 2 The majority of prevalent STS such as malignant malignant fibrous histiocytoma (MFH) and skeletal chondrosarcoma belong to the latter category. 2 In fact, MFH is the most common form of soft tissue sarcoma identified in adult patients. 3-8 The survival rates for STS, as well as for MFH, have improved very little in the past decades, and surgical management remains the cornerstone of therapy. 3, 9 While adjuvant radiotherapy and chemotherapies have been sometimes attempted, they have been found to have very little improvement on overall survival. 10 In most type of tumours, including STS, intra-tumour cytological heterogeneity is a common phenomenon. 11 It is well recognized that tumours contain distinct subpopulations of cells that differ in their morphological, biochemical, genetic, karyotypic characteristics, as well as the ability for tumour initiation. 12 The first concrete evidence for tumour-initiating cells (TICs) came in mid 1990s, when John Dick and colleagues demonstrated the existence a rare population of CD34+CD38- cells in acute myeloid leukaemia (AML) that were able to selfrenew and recreate pattern of dissemination and leukemic cell morphology similar to that seen in 13, 14 the original patients. TICs have been also been identified in many types of solid tumours including breast, colon, pancreatic, liver and ovarian cancer and melanoma In primary mesenchymal tumours, a putative TIC population has been identified by the isolation of SP cells using their ability to efflux Hoechst dye. 21 The study showed that SP cells were enriched for tumour initiating potential, and were the only cell fraction that had an enhanced capacity to initiate tumours and the ability to recapitulate the original morphology of the primary tumours upon serial transplantation in NOD/SCID mice. 21 It was also found in this study that, in biopsy samples from surgical resections, tumours with relatively higher percentages of SP cells were those of higher grades. 62

71 TICs and SP cells have been frequently linked to resistance to radiation and chemotherapies. In neural and breast cancers the more aggressive and refractory cancers that are resistant to radiation and chemotherapies often contain a higher proportion of TICs. 22, 23. In colon cancer, TIC fraction showed preferential resistance to Oxaliplatin and 5-fluorouracil, 24 and in glioblastoma the TICs have been shown to be resistant to a wide array of chemotherapeutic agents. 25 Similar observations have been made in ovarian cancers 26, pancreatic cancer 27, hepatocellular carcinoma 28, and leukemia In SP cells, the highly expressed dye effluxing ABC transporter have been shown to efflux chemotherapeutic agents such as doxorubicin 32, vinblastine 33, paclitaxel 34, imatinib mesylate 35, topotecan 36 and methotrexate 37. In the case of radiation therapy, studies in breast cancer cell lines and glioblastomas have shown that TICs are highly resistant to irradiation , 40 ; in fact, the study done using breast cancer cell line utilized SP isolation as one of the way to isolate TICs, and the SP cells demonstrated a strong SP cells a survival advantage when subjected to intense radiation. 39 Therefore, the SP populations clearly process certain interesting properties that may lead to a better understanding of tumourigenesis and the development of potential therapeutics. In this study, we compared the microarray gene expression profiles of SP and non-sp cells in STS tumours and identified the Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, we used the Hh blocker Triparanol 41 and the Notch blocker DAPT 42 to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies. 63

72 2.3 Results Microarray Profiling Show that SP Cells in MFH Exhibit Upregulated Hedgehog and Notch Signalling To examine whether certain pathways may be differentially regulated in SP cells versus non-sp cells, which represent the bulk of tumour cells, we compared the gene expression profiles of SP and non-sp cells in four primary STS tumours each from different patient. Tumour A1 was from an eighty-four years old female patient with a tumour mass initially indentified around the proximal lower limb and inguinal region; the tumour was excised from the quadriceps and diagnosed as a grade 2 MFH on a three-grade scale. Tumour A2 was from an eighty-one years old male patient with a tumour mass initially indentified around the proximal lower limb and inguinal region; the tumour was excised from the abductor and diagnosed as a grade 2 MFH on a three-grade scale. Tumour A3 was from a sixty-seven years old male patient with a tumour mass initially indentified around the proximal lower limb and inguinal region; the tumour was excised from the abductor and diagnosed as a grade 3 MFH on a three-grade scale. Tumour A4 was from a fifty-seven years old male patient with a tumour mass initially indentified around the proximal upper limb including the scapula; the tumour was excised from the axilla and diagnosed as a grade 3 MFH, more specifically a myofibrosarcoma subtype, on a three-grade scale. Upon surgical excision, these tumours were dissociated mechanically and enzymatically into individual cells. Via Hoechst dye staining and flow-cytometry, these primary tumour cells were sorted into distinct side population and non-side population fractions. Total RNA is extracted from an equal number of SP and NSP cell from each primary tumour. cdna from each sample was generated from the isolated total RNA and hybridized onto Affymetrix Human Genome EukGE-WS2v4 gene chips against the same reference cdna library. After initial processing of the raw data, using the Genespring GX software, the expression of SP cells from each tumour was normalized against the expression of the corresponding non-sp cells. A gene list was constructed by selecting genes that were regulated in the same direction (SP vs. NSP) in all sample pairs with a fold change greater than This list was examined using Genespring GX significant pathway function to identify differentially regulated pathways, by the degree of enrichment within the list for example, if there are 20,000 gene probes in total and pathway A 64

73 include 200 gene, then within a random list of 2,000 genes we would expect a representation of 20 genes from pathway A on average; if within a selected list of 2,000 differentially regulated genes, 50 genes are from pathway A, then pathway A is identified as a potentially differentially regulated pathway due to its higher than average congregation of differentially regulated genes. The top ten differentially regulated pathways identified within this list of differentially regulated genes from five primary STS tumours are listed in Table 2-1. This technique is known to be somewhat promiscuous. However, a sensitive method is needed to make sure that important clues are not missed in the screening process. To screen out false positives, qpcr verifications will be performed on the candidate pathway to confirm their legitimacy. As discussed previously, two of the top ten differentially regulated pathways have been significantly implicated as key molecular features of TICs: the Notch pathway, and the TGF-β pathway. The relationship between TIC and TGF-β pathway is rather indirect through its interplay with the Wnt pathway, as the coordination between WNT and the TGF-β pathway component such as Noggin and BMPs has been show to be crucial in stem cells in skin and intestine, as well as in TICs. 43,44 Moreover, it is difficult to isolated the specific role of TGF-β pathway in tumour development, as the corresponding activation of the Wnt pathway often provide the more direct link between specific molecular aberrations and tumourigenesis. 43,45-49 On the other hand, the Notch and Hh pathway have both been repeatedly implicated to play crucial role in the functioning and maintenance of TICs In the list compiled using Genespring (Table 2-1), the Notch pathway was among the top 10 pathways implicated. Moreover, upon close inspection, three key genes in the Hedgehog pathway, which is also known to be crucial in the functioning and maintenance of TICs : Gli1, Ptch1, and HHIP. Consequently, with readily available and proven small molecular blockers blockers, 41,42 we identified these two pathways as the ideal candidates for the targeting of SP cell in STS. The list of differentially regulated genes were also analyzed via a similar program: Ingenuity Pathway Analysis Program to detect pathways that are significantly enriched within the list. The top 10 pathways are listed in Table 2-3. As can be observed, this program is even more promiscuous (as shown by the number of pathways with P<0.05) and the result it returned were quite different from the one derived using Genespring GX, a reflection of the fact that the gene list for each pathways were defined differently within different programs. 65

74 Table 2-1: Top 10 differentially regulated molecular pathways in SP cells (Genespring) Top 10 differentially regulated molecular pathways in SP cells, identified by enriched presence of molecular pathways within the compiled list of differentially regulated genes, using Genespring GX11 Number of Number of Number of Pathways Gene Entities Specified in Entities Matched to the EukGE- Entities Matched to the Compiled P-value the Pathway WS2v4 Array Gene List TGFBR E-4 IL IL IL IL KitReceptor EGFR NOTCH Wnt Alpha6Beta4Integrin The complete array data have been submitted to the NCBI GEO database and can be found at: 66

75 Table 2-2: Up-regulation of key genes in th Hh pathway as shown in the microarray Three key genes in the Hedgehog pathway: Gli1, Ptch1, and HHIP are up-regulated in at least three out of the four samples. All values are expressed as fold-change, normalized by dividing the expression of SP cells over the expression of the corresponding NSP cells. Genes Sample A1 Sample A2 Sample A3 Sample A4 HHIP GLI PTCH The complete array data have been submitted to the NCBI GEO database and can be found at: 67

76 Table 2-3: Top 10 differentially regulated molecular pathways in SP cells (Using Ingenuity Pathway Analysis Program) Top 10 differentially regulated molecular pathways in SP cells, identified by enriched presence of molecular pathways within the compiled list of differentially regulated genes, using Ingenuity Pathways P-value FAK Signaling ILK Signaling Integrin Signaling RhoA Signaling HMGB1 Signaling PTEN Signaling Relaxin Signaling FLT3 Signaling in Hematopoietic Progenitor Cells IL-9 Signaling mtor Signaling Pathway Analysis Program The complete array data have been submitted to the NCBI GEO database and can be found at: 68

77 2.3.2 Real-time PCR Confirmation that SP Cells in MFH Exhibit Upregulated Hedgehog and Notch Signalling Hh pathway can be activated by the binding of ligand to the receptor Patched (Ptch1). Following that, the repression of Smoothened (Smo) is released, and the expression and post-translational processing of the Gli zinc-finger transcription factors is modulated. 64 The major transcriptional target of Hh pathway include Gli1, Ptch1, and hedgehog interacting protein (HHIP). 64 In solid tumours, studies have found excessive Hh signalling in bone/cartilage tumours such as endochondroma, osteochondroma and chondrosarcoma. 41,65 In many tumour types, Hh signalling is crucial in maintaining its TIC population. 57,58,61-63 In Notch signalling, upon binding of its ligand, Notch is cleaved, releasing Notch intracellular domain (NCID) through a cascade of proteolytic cleavages by TACE and γ- secretase. 66 The released intracellular domain is translocated into the nucleus for transcriptional activation of Notch target genes including the Hairy enhance of split-1 (Hes-1), Hey-1, Hey-2, Nuclear factor-κb (NF-κB), etc. 66 Active Notch signalling stimulated by ligands Jagged-1 and 2 have been found to stimulate the self-renewal of TIC populations in breast cancer and leukemia. 51,67 The expression of its downstream genes such as Hes-1, Hey-1, and Hey-2 promote the undifferentiated cell state in various stem and precursor cell populations, 68,69 and are required for the quiescence of non-dividing cells to be reversible. 42 Therefore, to validated that the downstream transcriptional activities are indeed differentially regulated in SP cells compared to non-sp cells, we examined the expression level of three Hh target genes (Ptch1, HHIP, and Gli-1) and three Notch target genes (Hes-1, Hey-1, and Hey-2) in SP and non-sp cells harvested from five distinct STS tumours using real-time PCR. Tumour B1 was from a forty-eight years old male patient with a tumour mass initially indentified around the proximal upper limb including the scapula; the tumour was excised from the scapular girdle and diagnosed as a grade 2 MFH on a three-grade scale. Tumour B2 was from a seventy-three years old female patient with a tumour mass initially indentified around the proximal lower limb including the buttock and inguinal region; the tumour was excised from the buttock and diagnosed as a grade 3 MFH on a three-grade scale. Tumour B3 was from a fiftytwo years old male patient with a tumour mass initially indentified around the distal lower limb; the tumour was excised from the posterior leg and diagnosed as a grade 3 MFH on a three-grade 69

78 scale. Tumour B4 was from an eighty-two years old male patient with a tumour mass initially indentified around the proximal lower limb; the tumour was excised from the quadriceps and diagnosed as a grade 3 MFH on a three-grade scale. Tumour B5 was from a fifty-three years old female patient with a tumour mass initially indentified around the proximal lower limb; the tumour was excised from the quadriceps and diagnosed as a grade 1 MFH on a three-grade scale. These tumours were dissociated mechanical and enzymatic into individual cells upon surgical excision, and sorted into distinct SP and non-sp fractions via Hoechst dye staining and flow-cytometry. Total RNA is extracted from an equal number of SP and NSP cells, and specific Taqman primer were used to measure the expression level of each gene upon the generation of cdna from the isolated RNA. As shown in Figure 2-1, with respect to the transcriptional activity of the Hh target genes, side population cells showed approximately a 3-fold up-regulation in the expression of Ptch1 and HHIP, and a 4-fold upregulation in expression of Gli-1 (Figure 2-1A), suggesting that the downstream transcriptional activity of Hh pathway is indeed more active in the SP cells. With respect to the transcriptional activity of the Notch target genes, side population cells showed approximately a 4-fold up-regulation in the expression of Hes-1, Hey-1, and Hey-2 (Figure 2-1B), suggesting that the downstream transcriptional activity of Hh pathway is also more active in the SP cells. The real-time PCR data are in-line with the micro-array data, further supporting these two pathways as the ideal candidates for the targeting of SP cell in STS tumours. 70

79 Figure 2-1: Expression comparison of downstream transcriptional targets of Hedgehog and Notch pathways in SP cells and NSP cells isolated from primary MFH tumours using realtime PCR. (A) Expression of downstream transcriptional targets of Hedghog pathway, including Ptch1, HHIP and Gli1. (D) Expression of downstream transcriptional targets of Notch pathway, including HES1, HEY1, and HEY2. The expression values for SP cells are represented in the graphs after being normalized to the expression value of NSP cells. Each data point represents the expression value from one distinct tumour. The wide horizontal bar in the middle represent the geometric mean, and the error bars represent the 95% confidence interval. The dotted lines at y=1 represent the relative expression value of non-sp cells after normalization. Asterisks denote P<0.05 analyzed via two-tailed paired Student s t-test. 71

80 2.3.3 Inhibition of Hedgehog and Notch Signalling Depletes the Abundance of SP cells in MFH Xenografts We established xenografts in NOD/SCID mice from five different primary STS tumours. Tumour C1 was from an eighty years old male patient with a tumour mass initially indentified and excised from around the paraspinal region; the tumour was diagnosed as a grade 3 MFH on a three-grade scale. Tumour C2 was from a fifty-five years old female patient with a tumour mass initially indentified around the proximal lower limb including the buttock and inguinal region; the tumour was excised from the quadriceps and diagnosed as a grade 3 MFH on a three-grade scale.tumour C3 was from a seventy-two years old male patient with a tumour mass initially indentified around the proximal upper limb including the scapula; the tumour was excised from the deltoid and diagnosed as a grade 3 MFH on a three-grade scale. Tumour A3 (same tumour was used in the microarray study) was from a sixty-seven years old male patient with a tumour mass initially indentified around the proximal lower limb including the buttock and inguinal region; the tumour was excised from the adductor and diagnosed as a grade 3 MFH on a threegrade scale. Tumour C4 (same tumour was used in the microarray study) was from a fifty-seven years old male patient with a tumour mass initially indentified around the proximal upper limb including the scapula; the tumour was excised from the axilla and diagnosed as a grade 3 MFH, more specifically a myofibrosarcoma subtype, on a three-grade scale. After the tumours have been allowed to establish in the host animal and become visible, they were treated with a hedgehog blocker (Triparanol at 400mg/kg), a Notch blocker (DAPT at 10mg/kg), or vehicle only as the negative control. After three weeks of treatment, the mice were sacrificed and the tumours were harvested with some of the tumour tissues snap-frozen in liquid nitrogen for RNA extraction. Total RNA was extracted from the frozen tissue, and specific Taqman primer were used to measure the expression level of each gene upon the generation of cdna from the isolated RNA. Quantitative real-time PCR analysis showed that Triparanol successfully inhibited the expression of downstream transcription targets in the Hedgehog signalling pathway, Ptch-1, HHIP, and Gli-1 gene, all by more than 60 percent (Figure 2-2A), and that DAPT successfully inhibited the expression of downstream transcription targets in the Notch pathway, Hes-1 and Hey-2 gene, by more than 60 percent, and Hey-1 gene by more than 80 percent (Figure 2-2B). 72

81 Figure 2-2: Expression of downstream target genes in Hh and Notch signalling pathways after drug treatment Expression of downstream target genes in Hh and Notch signalling pathways after respective blockade using the drug Triparanol and DAPT. (A) Triparanol treatment significantly downregulated the expression of Ptch1 and Gli1. (B) DAPT treatment significantly down-regulated the expression of HES1, HEY1, and HEY2. All expression values in the graph are normalized to the expression level in the corresponding control group. Each data point represents the expression value from one distinct tumour. The wide horizontal bar in the middle represent the geometric mean, and the error bars represent the 95% confidence interval. The dotted lines at y=1 represent the relative expression value of the control group cells after normalization. Asterisks denote P<0.05 analyzed via two-tailed paired Student s t-test 73

82 2.3.4 Inhibition of Hedgehog and Notch Signalling Depletes the Abundance of SP cells in MFH Xenografts To examine whether the inhibition Hh and Notch pathways could effectively target SP cells, we performed SP analysis on cells obtained from the harvested tumours post-treatment. SP analysis is based on the cell s the ability to efflux Hoechst dye. When analyzed via flowcytometry, the dye can be excited by ultra-violet light around 350nm and emits lights in both the blue-wavelength region around 450nm and in the red-wavelength region around 675nm. Because such cells with superior efflux abilities do not become completely stained, they fall to the lower left quadrant side of the stained bulk cells upon flow-cytometry analysis. As a negative control, cells were also incubated in the presence of verapamil, a chemical inhibitor of the ABC protein family of transporters, which inhibits the efflux of Hoechst, so that all cells become stained. Of the five different primary xenografts established in NOD/SCID mice, distinct SP fractions were detected four of them. Moreover, with the addition of verapamil, the presence of SP cells was abolished, indicating that dye efflux via ABC transporters was occurring in SP cells (Figure 2-3A). Furthermore, the data demonstrate that, on average, the abundance of SP cells was significantly reduced by approximately 10-fold after treatment with Triparanol and 3-fold after treatment with DAPT when compared to the respective vehicle controls, except for the one particular xenograft line which lacked the presence of SP cells altogether (Figure 2-3B). This result suggests that the inhibition of Hh and Notch pathways were effective in specifically targeting SP cells and depleting their abundance. This is consistent with literature. In the case of Hh signalling, in multiple myeloma, glioma, and colon cancer, Hh is crucial in maintaining the TIC population, 57,61,62 and in CML, when Hh signal was blocked, fewer cells expressed the surface marker signatures for the TIC population. 58 In breast cancer, Hh signalling increases the number of mammosphere-initiating stem/progenitor cell and mammosphere size, and is highly active in the CD44+CD24 /lowlin TICs. 63 As for Notch signalling, over-expression of Notch-1 in glioma cells promoted the growth and the formation of neurosphere-like colonies, 54,55 and the blockade of Notch reduced the CD133+ TIC fraction by almost 5-fold and totally abolished all SP cells. 56 In leukemia, DAPT was able to inhibits the colony formation ability of the TIC fraction

83 Hoechst Blue Figure 2-3: Proportion of SP cells in xenografted tumours after treatment with the drug Triparanol and DAPT. (A) A representative sample from the four distinct xenograft lines established from primary MFH tumour showing the depletion in SP cells after Triparanol and DAPT treatment. (B) Relative proportion of SP cells after Triparanol and DAPT treatment, all measurements normalized to control. The graph is shown as the arithmetic mean accompanied by error bars representing the 95% C.I; asterisks denote P<0.05 analyzed via two-tailed paired Student s t-test. Control Triparanol DAPT B Hoechst Red 75

84 2.3.5 Inhibition of Hedgehog and Notch Signalling Reduces the Size of MFH Xenografts To examine whether the inhibition of Hh and Notch pathways could impart an effect on the overall tumour growth we weighed the harvested tumours after three weeks of treatment. As shown in figure 2-4, both Triparanol and DAPT treatments significantly reduced the weight of the xenografted tumours. Compared to the vehicle control, the growths of xenografted tumours were reduced by approximately 60 percent after Triparanol treatment and by approximately 40% after DAPT treatment (Figure 2-4B). As can be observed from the pictures taken, the sizes of the tumour were also strikingly smaller, especially in the Triparanol treated group (Figure 2-4A). To exclude the possibility that such effects could be due to a cytotoxic impact, tumour free NOD/SCID mice were treated with each of the drugs. After three weeks, the weight of the mouse in the Triparanol treated group reduced very little, while the weight in the DAPT treated group actually increased a little bit (Figure 2-4C). Moreover, because the treatment only started after the tumours have been allowed to establish in the host animal and become visible, the treatment should have had little effect on the engraftment process. This is in-line with many findings recorded in literature. Clinically, large majority of sporadically occurring basal-cell carcinomas and one-third or more of all human medulloblastoma involve hyper-activated Hh signalling, and Hh blockade has also been shown to inhibit the growth of mouse medulloblastoma cells in both culture and in mouse allograft models. 74,75 In solid tumours, studies have found excessive Hh signalling in bone/cartilage tumours such as endochondroma, osteochondroma and chondrosarcoma, 41,65 and Hh blockade have been shown to be effective in inhibiting the growth of small-cell lung cancer, pancreatic cancer, chondrosarcoma, and prostate cancer in their respective xenograft models. 41,76-81 As for Notch pathway, clinically it is often abnormally activated in many human malignancies such as cervical, lung, colon, head and neck, renal carcinoma, acute myeloid, Hodgkin and Large-cell lymphomas and pancreatic cancer In breast cancer and prostate cancer, activated Notch signalling was found to be associated with poor prognosis. 96,97 Furthermore, activated Notch signalling has been shown to contribute to mammary carcinogenesis, 50 and promote the growth and formation of neurosphere-like colonies in glioma cells. 54,55 76

85 Figure 2-4: Weight and size of xenografted tumours after treatment with the drug Triparanol and DAPT (A) Three representative samples from the five distinct xenograft lines established from primary MFH tumour showing the reduction in tumour size after Triparanol and DAPT. (B) Relative tumour weight after Triparanol and DAPT treatment, all measurements normalized to the control group. (B) Relative mouse weight after Triparanol and DAPT treatment, all measurements normalized to the control group. All graph are shown as the arithmetic mean accompanied by error bars representing the 95% C.I; asterisks denote P<0.05 analyzed via two-tailed paired Student s t-test. 77

86 2.3.6 Inhibition of Hedgehog and Notch Signalling Affects the Rate of Apoptosis and Proliferation in Xenografts of STS Tumours To investigate the cytological changes imparted by the inhibition Hh and Notch pathways that may underlie the reduced tumour growth, we examined the histological and immuno-histological features of the xenografted tumours after three weeks of treatment, using paraffin-embedded tissues that have been preserved at the time of harvesting with formalin fixation. We performed hematoxylin and eosin (H&E) staining to look at the general cytological features of the treated tumours. In this method, oxidized haematoxylin colours the nuclei blue, while eosin colors other structures in various shades of red and pink. 98 To examine the apoptotic rate in the treated tumour tissues, we performed terminal deoxynucleotidyl transferase dutp nick end labelling (TUNEL) staining, which is a common method that detects DNA fragmentation that results from apoptotic signalling cascades. 99 To examine the proliferation rate in the treated tumour tissues, we performed immuno-histological staining of Ki-67 protein, which serves as a proven cellular marker for proliferation. Ki-67 is strictly associated with cell proliferation, as it is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0). 100 H&E staining revealed that both Hh and Notch blockade significantly decreased the cellularity in treated tumours (Figure 2-5A and 2-6A). The TUNEL staining showed that, although both Hh (~2% apoptotic cells) and Notch blockade (~2% apoptotic cell) induced a significantly higher rate of apoptosis compared to the control (very few ~1% apoptotic cell), the effect was unlikely to be biologically significant due to the small absolute number (Figure 2-5B and 2-6B). The Ki-67 staining showed that both Hh (~5 fold decrease) and Notch blockade (~60% decrease) significantly decreased the proliferation in treated tumours. This could be the reason for the decreased tumour growth as shown previously due to the accumulation of tumour growth overtime. These observations are consistent with literatures. Hh blockade have been observed to induce apoptosis in small intestine epithelium, rhabdo-myosarcoma, and prostate cancer, 81,101,102 as well as inhibit proliferation in chondrosarcoma, prostate cancer, hepatocelluar and basal cell carcinoma. 41,98,103,104 Similarly, Notch blockade has also been suggested to induce apoptosis in myeloma and prostate cancer, 105,106, as well as inhibit proliferation in neuroendocrine tumours, prostate cancer, and colorectal cancers

87 Figure 2-5: Histology and immunochemical staining of tumour sections after treatment with the drug Triparanol and DAPT A) H&E staining (10X magnification) showing that Hh and Notch blockade decreases cellularity in treated tumours. B) TUNEL staining (20X magnification) showing Hh but not Notch blockade increases the apoptosis rate in treated tumours, brow colour indicates apoptotic cells. C) Ki-67 staining (20X magnification) showing that that Hh and Notch blockade decreases the proliferation in treated tumours, brow colour indicates proliferating cells. 79

88 Figure 2-6: Cellularity, apoptosis rate, and proliferation rate in xenografted tumours posttreatment. A) H&E staining showing that Hh and Notch blockade decreases cellularity in treated xenograft tumours. B) TUNEL staining showing Hh but not Notch blockade dramatically increases the apoptosis rate in treated xenograft tumours. C) Ki-67 staining showing that both Hh and Notch blockade slightly decreases the proliferation in treated xenograft tumours. All graph are shown as the arithmetic mean accompanied by error bars representing the 95% C.I; asterisks denote P<

89 2.3.7 Inhibition of Hh and Notch Signalling Reduces the Retransplantation Rate of MFH Xenografts Because SP cells have been shown to be enriched for tumour initiation potential and our data have shown that both Triparanol and DAPT are effective in targeting and depleting SP cells, it is logical then to test whether the Triparanol and DAPT can affect the tumour re-initiation rate upon secondary transplantation after the initial treatment. Therefore, some of the harvested cells from each treatment group of the five distinct xenograft lines were re-transplanted into NOD/SCID mice at the concentration of 1000 cells per site without further treatment. The tumour growth in these mice are monitored closely to examine the effect of Triparanol and DAPT treatment on subsequent tumourigenesis, which may serve as a reflection of the effectiveness of these drugs at depleting tumour initiating cells and preventing future relapses. So far, secondary transplantation data from three distinct xenograft lines established from primary tumours (tumour C1, C2, and C3) showed that both Notch and Hh blockade were effective in depleting the tumour initiating potential of the treated tumour cells upon secondary transplantation without further treatment (Figure 2-7). This observation is coherent with the depletion of SP cells post-treatment and the previous observation that SP cells in mesenchymal neoplasm are enriched in tumour initiating potentials. 21 This finding is also coherent with literatures. Notch signalling is essential for quiescence of non-dividing cells to be reversible, and DAPT has been shown to prevent the reversibility of quiescence. 42 In glioma cells, activation of Notch signalling promoted the growth and the formation of neurosphere-like colonies, 54,55 and in leukemia, DAPT was able to inhibits the colony formation ability of the TIC fraction. 67 As for Hh signalling, knocking out Smo in leukaemia stem cells markedly decreased their regenerative capacity. 58 In chronic myelogenous leukemia (CML), blockade of Hh signals depleted the TIC population, while stimulating Hh signalling increased the number of these cells and accelerated disease progression. 58 Moreover, cyclopamine, a Hh blocker, has been shown to be effective in reducing the incidence and relapse rate of CML in animal models. 59 In breast tissues, Hh signalling plays important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells: it increased the number of mammosphere-initiating stem/progenitor cell and mammosphere size, but was inhibited when these cells were induced to differentiate

90 DAPT Triparanol Ctrl Figure 2-7: The rate of tumour re-growth upon secondary transplantation using cells harvested from the initial treatment a) The rate of tumourigenesis upon secondary transplantation by the implanting of treated xenograft tumour cells into a new batch of NOD-SCID mice, error bars represent 95% confidence interval b) Photograph illustrating the reduction in tumour-initiating efficiency induced by triparanol and DAPT treatment. A B 82