Identification and Characterization of Cancer Stem Cells in Mouse Medulloblastoma and Glioma

Ryan Jackson Ward

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto

Cancer Stem Cells in Mouse Brain Tumours

Ryan Jackson Ward

Doctor of Philosophy

Department of Laboratory Medicine and Pathobiology University of Toronto

2010 Abstract

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that ii selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

iii

Table of Contents

Table of Contents ...... iv

List of Abbreviations ...... viii

List of Tables ...... xi

List of Figures ...... xii

List of Supplemental Figures ...... xiv

List of Supplemental Tables ...... xv

Chapter 1 ...... 1

1 Introduction ...... 1

1.1 Brain Tumours ...... 1

1.1.1 Medulloblastoma ...... 1

1.1.2 Hedgehog Signalling & Human Medulloblastoma ...... 3

1.1.3 Human Glioma ...... 7

1.1.4 Mouse Glioma ...... 10

1.2 Developmental Neurobiology ...... 12

1.2.1 Cerebellar Development ...... 15

1.2.2 Forebrain Development ...... 16

1.3 Mouse and Human Neural Stem Cells ...... 17

1.4 Cancer Stem Cells ...... 19

1.4.1 The Cancer Stem Cell Hypothesis ...... 19

1.4.2 Leukemia Stem Cells ...... 22

1.4.3 Cancer Stem Cells in Solid Malignancies ...... 23

1.4.4 Brain Tumour Stem Cells ...... 23

1.4.5 The Cancer Stem Cell Controversy ...... 24

1.5 The Cell-of-origin: Cancerous Stem Cells versus Cancer Stem Cells ...... 25

1.6 Hypothesis, Potential Significance and Specific Aims of this Ph.D. Thesis ...... 27

1.6.1 Specific Aims ...... 27

Chapter 2 ...... 28

2 Multipotent CD15+ Cancer Stem Cells in Patched-1 Deficient Mouse Medulloblastoma ...... 28

2.1 Abstract ...... 28

2.2 Introduction ...... 29

2.3 Materials & Methods ...... 31

2.3.1 Mouse Husbandry and Tumour Processing ...... 31

2.3.2 Flow Cytometry, Cell Sorting and In vivo Injections ...... 31

2.3.3 Intracerebellar Ganciclovir Infusion ...... 32

2.3.4 Tissue Culture, DNA, RNA and Protein Analysis ...... 32

2.3.5 Immunocytochemistry & Immunohistochemistry ...... 33

2.4 Results ...... 34

2.4.1 Rare, Phenotypically Primitive and Multipotent Ptc1+/- MB Cells can be Propagated In Vitro...... 34

2.4.2 Ptc1+/-p53-/- MB Cell Lines Initiate the Growth of Phenotypically Representative Tumours In Vivo...... 37

2.4.3 Ptc1+/- MB Cell Lines Maintain Activated Hedgehog and Notch Signalling Pathways...... 40

2.4.4 Loss of Ptc1 Heterozygosity, or WT RNA Expression, is not required for Ptc1+/- MB Development...... 42

2.4.5 CD15 Enriches for Proliferative Cells In Vitro and Tumourigenic Cells In Vivo ...... 44

2.5 Discussion ...... 48

Chapter 3 ...... 60

3 Percoll Density Centrifugation Separates Functionally Distinct CD15+ Patched-1 Mouse Medulloblastoma Cells...... 60

3.1 Abstract ...... 60

3.2 Introduction ...... 61 v

3.3 Materials and Methods ...... 63

3.4 Results ...... 65

3.5 Discussion ...... 70

Chapter 4 ...... 73

4 Cellular, Molecular and Chemical Profile of Clinically Representative Cancer Stem Cells from a Chemical-Genetic Mouse Model of Glioma...... 73

4.1 Abstract ...... 73

4.2 Introduction ...... 74

4.3 Materials & Methods ...... 76

4.3.1 Mouse Husbandry, Mating Strategies & Carcinogen Administration ...... 76

4.3.2 Orthotopic Injections of Freshly Dissociated Tumours and Established Cell Lines ...... 76

4.3.3 Fluorescent Activated Cell Sorting of Mouse Gliomas, Establishment of Glioma Stem Cell Lines In Vitro and Differentiation Assays ...... 76

4.3.4 Histology, Immunocytochemistry and Intracellular Flow Cytometry ...... 77

4.3.5 Microarray Analysis ...... 77

4.3.6 BIOMOL Chemical Library Screen of Mouse and Human Glioma Stem Cell Lines ...... 78

4.4 Results ...... 79

4.4.1 Chemical mutagenesis of p53-deficient embryonic mice generates glioma, as well as other tumour types...... 79

4.4.2 ENU Administration to Ptc1+/- Mice Generates High Incidence Medulloblastoma...... 82

4.4.3 Tissue Specific p53-Deletion and Chemical Mutagenesis Generates High Incidence Glioma in the Absence of Other Tumour Types...... 82

4.4.4 ENU+NC+p53f/- Gliomas are Tumourigenic and Demonstrate Functional Heterogeneity In Vivo...... 85

4.4.5 CD15 Enriches for Clonogenic Mouse Glioma Cells In Vitro ...... 87

4.4.6 Mouse Tumour Stem Cell Lines Are Readily Established and Demonstrate Unique Phenotypes and Properties In Vitro...... 89

4.4.7 Mouse Glioma NS Cell Lines are Tumourigenic when Low Cell Densities are Injected In Vivo...... 94

4.4.8 Microarray Expression Analysis of Mouse Glioma NS Cells Reveals a p53- Deficient- and Glioma-Associated Expression Signature...... 97

4.4.9 Identification of a Misregulated Homeobox Gene Regulatory Network in both Mouse and Human Glioma Stem Cells...... 99

4.4.10 Chemical Screening of Human and Mouse Glioma Stem Cell Lines Identifies Selective Pharmacological Inhibitors...... 102

4.5 Discussion ...... 104

4.6 Supplemental Figures ...... 111

Chapter 5 ...... 123

5 General Discussion ...... 123

5.1 The Cancer Stem Cell Hypothesis: a Lingering Debate...... 123

5.2 The Medulloblastoma Cancer Stem Cell Phenotype: Differences in Perspective, Methodology or Both? ...... 124

5.3 The Cell-of-Origin of Brain Tumours: Elucidated, but Completely Unresolved...... 125

5.4 Brain Cancer: A Disease of Misplaced Identity? ...... 126

5.5 Future Experimental Directions ...... 127

6 Summary and Conclusion ...... 130

Supplemental Methods ...... 131

References ...... 133

vii

List of Abbreviations

Abl - c-abl oncogene 1 CGCP - Cerebellar granule cell precursor

Act - Activator Ci - Cubitus interruptus

Akt - v-akt murine thymoma viral oncogene CKI - Casein kinase I homolog CNS - Central nervous system AML - Acute myeloid leukemia CNSC - Cerebellar neural stem cell APAF - Apoptotic Peptidase Activating Factor Cos2 - Costal-2

APC - Adenomatosis polyposis coli Cre - Cre-recombinase

AraC - Cytarabine CSC - Cancer stem cell

Arx - Aristaless related homeobox Dhh - Desert Hedgehog

Bax - Bcl2-associated X protein Dlx - Distal-less homeobox

BCC - Basal cell carcinoma e - embryonic day

Bcr - Breakpoint cluster region Ebf3 - Early B-cell factor 3 bFGF - Basic fibroblast growth factor EGF - Epidermal growth factor

Bmi1 - Bmi1 polycomb ring finger EGFR - Epidermal growth factor receptor oncogene EGL - External granule layer BMP - Bone morphogenic protein EMU - N-methyl-N-nitrosourea BRAF - v-raf murine sarcoma viral oncogene homolog B1 Emx - Empty spiracles homeobox

BTSC - Brain tumour stem cell ENU - N-ethyl-N-nitrosourea

CBP - CREB-binding protein ENU+NC+p53f/- - Nestin-Cre+;p53flox/-

CD133 - AC133/CD133/Prominin1 FACS - Fluorescent activated cell sorting

CD15 - Lewis X/Stage Specific Embryonic FBS - Foetal bovine serum Antigen 1/CD15 Fezf - Fez family zinc finger CDK - Cyclin dependent kinase FGF - Fibroblast growth factor CDKN - Cyclin-dependent kinase inhibitor FL - Full length viii

Fu - Fused MAPK - mitogen-activated protein kinase

GBM - Glioblastoma multiforme Mash - Achaete-scute complex homolog

Gbx - Gastrulation brain homeobox Math - Mouse atonal homolog

GCV - Ganciclovir MB - Medulloblastoma

GFAP - Glial fibrillary acidic protein MDM2 - Mdm2 p53 binding protein homolog GFP - Green fluorescent protein MHB - Midbrain-hindbrain boundary Gli - GLI family zinc finger MLH - mutL homolog GM - Glioma MPNST - Malignant peripheral nerve sheath GO - Gene ontology tumour

GSK3β - Glycogen synthase kinase-3β Msh - Musashi homolog

Gxh - GS homeobox mTOR - mechanistic target of rapamycin

Hh - Hedgehog Myc - v-myc myelocytomatosis viral oncogene homolog Hox - Homeobox NBCCS - Nevoid basal cell carcinoma H-Ras - v-Ha-ras Harvey rat sarcoma viral syndrome oncogene homolog NC - NC+p53f/- HTP - High-throughput NF - Neurofibromin IC - Inhibitory concentration Ngn - Neurogenin IDH - Isocitrate dehydrogenase NOD - Non-obese diabetic IGL - Internal granule layer NSC - Neural stem cell Ihh - Indian Hedgehog NSG - NOD/SCID-gamma IR - γ-irradiation Olig - Oligodendrocyte lineage transcription Irx - Iroquois homeobox factor

KEGG - Kyoto Encyclopaedia of Genes and Otx - Orthodenticle homeobox Genomes p - post-natal day Lmo1 - LIM domain only 1 p53 - tumour protein 53 LOH - Loss of heterozygosity Pax - Paired box gene LSC - Leukemia stem cell ix

PDGFRA - Alpha-type platelet-derived SGZ - Subgranule Zone growth factor receptor Shh - Sonic Hedgehog PDGFRA - Platelet-derived growth factor receptor alpha polypeptide Shox - Short stature homeobox

PERP - p53 apoptosis effector related to SL-IC - SCID leukemia-initiating cell PMP-22 Smo - Smoothened PI - Propidium iodide SmoA1, A2 or M2 - Activated Smoothened PI3K - Phosphoinositide-3-kinase Sox - SRY-box containing gene PKA - Protein kinase A SSEA-1 - Stage Specific Embryonic PMS2 - Postmeiotic segregation increased 2 Antigen 1

PNET - Primitive neuroectodermal tumours SuFu - Suppressor of fused

PNS - Peripheral nervous system SVZ - Subventricular Zone

PSB - Pallial–subpallial boundary TIC - Tumour initiating cell

Ptc - Patched TK - Thymidine kinase

PTEN - Phosphatase and tensin homolog TNFRS10b - Tumour Necrosis Factor Receptor Superfamily, Member 10b Rb - Retinoblastoma TPC - Tumour propagating cell Rbl1 - Retinoblastoma-like 1/p107 v-Src - v-src sarcoma (Schmidt-Ruppin A-2) Rep - Repressor viral oncogene homolog

Rh - Rhombomere VZ - Ventricular zone

RMS - Rostral migratory stream WHO - World Health Organization

Runx2 - Runt-related transcription factor 2 Wnt - Wingless/Int-1

SCID - Severe combined immune-deficient WT - Wild-type

SCZ - Subcallosal zone WWOX - WW domain-containing oxidoreductase SEZ - subependymal zone ZLI - Zona limitans intrathalamica

x List of Tables

Table 1-1 ...... 9

Table 2-1 ...... 47

Table 4-1 ...... 80

Table 4-2 ...... 86

Table 4-3 ...... 95

List of Figures

Figure 1-1 ...... 5

Figure 1-2 ...... 14

Figure 1-3 ...... 21

Figure 2-1 ...... 35

Figure 2-2 ...... 39

Figure 2-3 ...... 41

Figure 2-4 ...... 43

Figure 2-5 ...... 46

Figure 3-1 ...... 64

Figure 3-2 ...... 67

Figure 3-3 ...... 68

Figure 3-4 ...... 69

Figure 4-1 ...... 81

Figure 4-2 ...... 84

Figure 4-3 ...... 88

Figure 4-4 ...... 90

Figure 4-5 ...... 93

Figure 4-6 ...... 96

Figure 4-7 ...... 98

xii

Figure 4-8 ...... 101

Figure 4-9 ...... 103

xiii

List of Supplemental Figures

Supplemental Figure 2-1 ...... 52

Supplemental Figure 2-2 ...... 53

Supplemental Figure 2-3 ...... 54

Supplemental Figure 2-4 ...... 55

Supplemental Figure 2-5 ...... 56

Supplemental Figure 2-6 ...... 57

Supplemental Figure 2-7 ...... 58

Supplemental Figure 2-8 ...... 59

Supplemental Figure 4-1 ...... 111

Supplemental Figure 4-2 ...... 112

Supplemental Figure 4-3 ...... 113

Supplemental Figure 4-4 ...... 114

Supplemental Figure 4-5 ...... 115

Supplemental Figure 4-6 ...... 116

Supplemental Figure 4-7 ...... 117

Supplemental Figure 4-8 ...... 118

xiv

List of Supplemental Tables

Supplemental Table 4-1 ...... 119

Supplemental Table 4-2 ...... 120

Supplemental Table 4-3 ...... 121

Supplemental Table 4-4 ...... 122

xv 1

Chapter 1 1 Introduction 1.1 Brain Tumours

Malignant brain tumours are amongst the most aggressive and therapeutically refractory cancers in humans [1, 2]. Different brain tumours affect children and young adults (0-14 and 15-29 years of age, respectively) versus adults (>30 years of age), the former thought to involve the transformation of proliferating cells in the developing brain, the latter thought to be a consequence of slowly accumulating genetic insults over the lifetime of the individual [1, 2]. Amongst children and young adults, the most common brain tumours are low grade astrocytomas, ependymomas, and embryonal tumours including medulloblastoma (MB) and primitive neuroectodermal tumours (PNET) [2]. Of these tumours, MBs are the second most common paediatric malignancy, after leukemia, and the most common cause of cancer-related death [1-3]. In adults, the most frequent brain tumour is glioma (GM), and within this tumour classification glioblastoma multifome (GBM) is the most common as well as the most aggressive [1, 2].

1.1.1 Medulloblastoma

MBs are World Health Organization (WHO) grade IV primary brain tumours occurring in the cerebellum of children and young adults, and are predominantly of neuronal phenotype [1, 4]. The majority of MBs (~75%) arise deep within the cerebellum and project into the fourth ventricle with the remaining 25% occurring in the cerebellar hemispheres [1, 4]. Clinical treatment for MB includes surgical resection, chemotherapy and radiation-therapy with high (>80%) 10-year survival rates achieved in patients presenting with non-metastatic disease, however, patients with metastatic disease do not generally demonstrate such favourable outcomes with 10-year survival rates of ~40% [5].

Medulloblastomas can be classified as classic, large cell or desmoplastic based on histopathological characteristics [1, 4]. Classic MB is characterized by sheets of densely packed hypo-cytoplasmic cells, the majority of which demonstrate expression of the neuronal marker synaptophysin, but with some cells expressing glial fibrillary acidic protein (GFAP) indicating a

capacity for glial differentiation in these tumours [1]. Desmoplastic MBs are characterized by the presence of reticulin-circumscribed differentiated nodules surrounded by densely packed highly proliferative cells. Unlike classic MB, desmoplastic MB most often occurs in the cerebellar hemispheres, is well contained and is associated with a good clinical outcome in some cases without aggressive therapy [1, 2, 4]. Finally, large cell MB represents less than 5% of all clinical cases and will not be further discussed.

The causes of sporadic medulloblastoma are largely unknown, however, some environmental and genetic influences have now been identified. Maternal consumption of nitrosamine- containing foods, such as hot dogs and cured meats, is correlated to an increased risk of brain tumours in children though its association with MB may only be weak [6, 7]. Occupation-related parental pesticide exposure was shown to be associated with an increased relative risk of MB in children [8], as was gestational exposure to heat such as parental use of saunas or electric blankets [9].

Recent high resolution genotyping of >200 primary sporadic MB samples identified recurrent genetic amplifications and deletions common within the disease. These aberrations include losses on chromosomes 6, 8, 9q, 10q, 11, 16q and 17p and gains on chromosomes 1q, 7 and 17q, with the consequences of deleting regions containing known tumour suppressor proteins such as cyclin-dependent kinase inhibitor 2A (CDKN2A/Ink4A-Arf) and WW domain-containing oxidoreductase (WWOX), and/or amplifying regions containing known proto-oncogenes such as v-myc myelocytomatosis viral oncogene homolog (MYC) and Alpha-type platelet-derived growth factor receptor (PDGFRA) [10]. 10% of Li Fraumeni syndrome patients develop MB, and 16% of sporadic MBs harbour tumour protein 53 (p53) mutations demonstrating a role for p53 signalling in MB development [1, 11]. MB is the predominant (~80%) brain tumour type in Turcot syndrome patients with germline mutations in the adenomatosis polyposis coli (APC) gene, and mutations within Wingless/Int-1 (WNT) signalling pathway members APC and β- catenin are observed in 13% of sporadic MBs [12, 13]. Finally, the role of the Hedgehog signalling pathway in MB has been intensively studied given its activation in clinical samples and experimental models of the disease.

1.1.2 Hedgehog Signalling & Human Medulloblastoma

The Hedgehog signalling pathway was first elucidated in drosophila after the identification of the segment polarity genes hedgehog (Hh), patched (Ptc), and smoothened (Smo), and the observations regarding their function in the development of larval body segments and adult appendages [14-17]. In all organisms studied Ptc is the receptor for Hh. In the absence of Hh ligand, Ptc functions to inhibit the activity of Smo [18-21]. In drosophila, the major effector of Hh signalling is the zinc-finger transcription factor Cubitus interruptus (Ci) [22, 23]. In the absence of Hh ligand the majority of Ci is associated with Suppressor of fused (SuFu) and is sequestered in the cytoplasm. Some Ci is phosphorylated by a protein complex comprising the scaffold protein Costal-2 (Cos2), protein kinase A (PKA), casein kinase I (CKI), Glycogen synthase kinase-3β (GSK3β) and the serine/threonine kinase Fused (Fu) [24, 25]. Phosphorylation of Ci by this complex leads to proteosomal processing and removal of its co- activator binding domain generating a transcriptionally repressive N-terminal protein fragment (Ci repressor, CiRep) which can translocate to the nucleus and repress expression of Hh target genes [26, 27]. Activation of the signalling pathway occurs when Hh binds Ptc, derepressing its effect on Smo and allowing Smo to move to the plasma membrane after phosphorylation by PKA, GSK3β and CKI [28, 29]. The Cos2/PKA/CKI/GSK3β/Fu complex then associates with the SuFu/Ci complex releasing full length Ci (Ci activator, CiAct) which localizes to the nucleus and interacts with the co-activator CREB-binding protein (CBP) to activate transcription of Hh target genes [30, 31].

Hedgehog signalling in mammalian cells (Figure 1.1) is similar to that in drosophila, but with some important differences. Three mammalian Hedgehog ligands exist, Indian (Ihh), Desert (Dhh) and Sonic hedgehog (Shh), capable of interacting with two Patched receptors (Ptc1 and Ptc2), all of which are expressed in tissue specific patterns [32-34] . Three Ci orthologues are known, GLI family zinc finger (Gli) 1-3, which were identified as amplified sequences in glioma cells. Like Ci, Gli2 and Gli3 undergo limited proteolysis to generate transcriptional repressors (Rep) in the absence of Hh, and all Gli proteins act as full length activators (Act) when the pathway is stimulated [23, 35-37]. Signal transduction in mammalian cells is dependent on localization of Hh pathway components to the primary cilium, and disrupting localization can ablate pathway activity [38, 39]. In mammalian cells Ptc1 receptors inhibit Smo by an unknown catalytic mechanism thought to involve Smo sequestration away from primary cilia by an

indirect interaction between the two proteins [40, 41]. When stimulated by Shh Ptc1 protein levels are decreased and its inhibitory effect on Smo is removed, allowing Smo to localize to the plasma membrane and interact with the Cos2 (Kif7 in mammals)/Fu/kinase/SuFu/Gli protein complexes [40, 41]. Gli proteins associated with the SuFu and Cos2 protein complexes then localize to the nucleus as transcriptional activators and induce the expression of Hh target genes (Figure 1.1) [37].

Interest regarding the involvement of the hedgehog signalling pathway in cancer was first generated when mutations in the Ptc1 gene were found in sporadic basal cell carcinomas (BCC), and in tumours occurring in Gorlin’s syndrome/nevoid basal cell carcinoma syndrome (NBCCS) patients [42, 43]. 5-10% of Gorlin’s syndrome patients develop MB of the desmoplastic variant and harbour germline mutations in the PTC1 gene, and 10% of sporadic desmoplastic MBs demonstrate activated Hh signalling due to truncating mutations of SUFU [44-46]. Together these clinical observations indicate that aberrations within the Hh signalling pathway are important for the development of MB, though principally for tumours of the desmoplastic phenotype [44, 47].

Figure 1-1 The Hedgehog signalling pathway in mammalian cells. Left: In the absence of Hh the pathway is inactive and Ptc1 inhibits the localization of Smo away from the primary cilia via an unknown indirect mechanism. Activated Gli transcription factors (GliAct) are sequestered in the cytoplasm by interactions with SuFu and Kif7(Cos2)/Fu. Some Gli2 and Gli3 undergo limited proteolysis after phosphorylation by GSK3β/PKA/CKI to generate transcriptional repressors (GliRep) which inhibit the expression of target genes. Right: Hh ligand binds Ptc1, derepressing its effect on Smo. Smo localizes to the membrane of primary cilia, is phosphorylated by GSK3β/PKA/CKI and interacts with the Kif7/Fu/SuFu complex allowing full length GliAct to translocate to the nucleus and activate expression of Hh target genes.

Hedgehog Signalling and Mouse Medulloblastoma

Systemic, homozygous disruption/deletion of Ptc1 (Ptc1-/-) in mice is lethal at embryonic day (e) 9.0 with the presence of neural tube defects, however, heterozygosity (Ptc1+/-) generates viable mice that recapitulate many features of Gorlin’s syndrome. For example, Ptc1+/- mice develop spontaneous MBs at a reproducible frequency of 5-30% by 12 weeks of age, as well as spontaneous basal cell carcinomas and rhabdomyosarcomas [48, 49]. The frequency of MB occurring in these mice can be augmented to near 100% by systemic p53-deficiency (p53-/-), exposure of newborn pups to γ-irradiation (IR) or by tissue-specific Ptc1-/- [50-54]. Interestingly, an age-dependent relationship is observed between MB incidence and IR or targeted Ptc1-/-, as irradiated Ptc1+/- pups demonstrate increased MB frequencies of >80% and ~50% when exposed at post-natal day (p) 1 and p4 respectively, but not when irradiated at p10 or later [51, 52]. Tissue-specific deletion of Ptc1 by tamoxifen-inducible Cre-recombinase (Cre) generates MB in 100% of mice when tamoxifen is administered from e14.5 to p8, after which point MBs are rarely observed [53]. Finally, an alternative strategy to generate mouse MB by Hh signalling activation is via forced expression of a constitutively active Smo (SmoA1, A2 or M2), with SmoA1+/+ and SmoA1+/- mice developing MB at a frequencies of ~90% and ~50%, respectively [54-56].

Initial characterization of Ptc1+/- MBs indicated that spontaneous tumours were of the desmoplastic variant [57]. MB induction by Ptc1+/- and IR or p53-/- however, was reported to generate tumours of the classic phenotype [51, 57, 58]. Ptc1+/- MB cells were first thought to express full length, wild-type (WT) Ptc1 mRNA from their remaining WT allele, suggesting that Ptc1 haploinsufficiency was sufficient for tumourigenesis [59]. This observation was challenged when allele-specific PCR analysis demonstrated that loss heterozygosity (LOH) occurred in preneoplastic cerebellar lesions and in MBs suggesting that Ptc1 LOH was obligate for MB development in these mice [60, 61]. More recently however, careful analysis of mouse MBs indicates that alternative Ptc1 splice variants are expressed in Ptc1+/- MB, but Ptc1 protein is never detected suggesting that loss of protein expression, rather than LOH, is the fundamental step for MB development in these mice [62].

1.1.3 Human Glioma

Gliomas comprise a heterogeneous group of primary brain tumours of glial (astrocytic or oligodendroglial) phenotype occurring in the cerebrum of patients. GBMs are WHO grade IV brain tumours and arise de novo (primary GBM), or after disease progression from a lower grade astrocytoma (secondary GBM). Primary GBMs make up the majority of cases (~90%), develop very rapidly and occur in older patients (mean, 62 years of age) [1]. Secondary GBMs typically occur in younger individuals (mean, 45 years of age) with a average interval of 4-5 years for disease progression from lower grade astrocytoma to GBM [1]. Despite intensive therapy including surgical resection, radiation and systemic temozolomide chemotherapy, the median survival of GBM patients remains at 14.6 months [63].

The association between environmental factors and an augmented risk of glioma is controversial, with studies supporting or challenging the potential causes [64]. There is no clear association between electromagnetic field exposure (from large or small sources such as semiconductor and data storage device facilities, or cell phone use, respectively) and glioma [64-67]. An increased risk of developing glioma was reported for individuals with high pesticide, vinyl chloride and nitrosamine exposure, however a cautious interpretation of these results is warranted as many studies find relative risks of less than two, with large 95% confidence intervals [64, 68-70]. Despite evidence that high dose IR can induce gliomas in primates, clinical IR of the head and neck for diagnostic purposes does not appear to be an important risk factor for human glioma, nor does there appear to be an association with previous head trauma [71, 72].

Large scale genotyping of human GBM samples identified the most frequently deleted, mutated or amplified genes in GBMs [73, 74]. CDKN2A, CDKN2B, p53, phosphatase and tensin homolog (PTEN), neurofibromin 1 (NF1), retinoblastoma 1 (RB1) and isocitrate dehydrogenase 1 (IDH1) were all inactivated by homozygous deletion or mutation in a high percentage (>10%) of sporadic tumours (Table 1-1). Conversely, the epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), PDGFRA and Mdm2 p53 binding protein homolog (MDM2) were all activated by mutation or sequence amplification (Table 1-1) [73, 74]. Importantly, when global pathway analysis was performed, GBMs demonstrated mutations, deletions and/or amplifications in signalling components within the phosphoinositide-3-kinase (PI3K), p53 or RB

8 pathways in 88%, 87%, and 78% of all samples, respectively, and 74% of tumours harboured aberrations in all three pathways [74].

Gene Alteration % Reference

CDKN2A Homozygous Deletion or Mutation 49-50 [73, 74]

CDKN2B Homozygous Deletion or Mutation 47 [74]

EGFR Mutation or Amplification 37-45 [73, 74]

p53 Homozygous Deletion or Mutation 35-40 [73, 74]

PTEN Homozygous Deletion or Mutation 30-36 [73, 74]

NF1 Homozygous Deletion or Mutation 15-18 [73, 74]

CDK4 Amplification 14-18 [73, 74]

MDM2 Amplification 14 [74]

PDGFRA Amplification 13 [74]

RB1 Homozygous Deletion or Mutation 11-12 [73, 74]

IDH1 Mutation 11 [73] Table 1-1 The most frequently observed genetic aberrations occurring in human GBM samples, as determined by genotyping of >200 samples.

These results are complimentary to studies of brain tumours from patients with known germline mutations. Approximately 50% of all brain tumours occurring Li Fraumeni syndrome patients are gliomas, manifesting early in life at a mean age of 34 years [1, 75]. Neurofibromatosis type 1 and type 2 patients harbour mutations in the NF1 and NF2 genes, and develop gliomas of the optic nerve and spinal cord, respectively. Cowden/Lhermitte-Duclos disease patients have germline mutations in PTEN and develop brain tumours, though mostly benign and within the cerebellum. Finally Turcot syndrome patients carrying mutations in the DNA mismatch repair genes mutL homolog 1 or 2 (MLH1, MLH2), or postmeiotic segregation increased 2 (PMS2) develop gliomas through a mechanism thought to involve the inactivation of NF1 [1].

1.1.4 Mouse Glioma

Many rodent models of glioma have been reported the past 50 years, more recently based on the clinically relevant genetic aberrations mentioned above. In general however, experimental gliomas have been induced by one of two strategies: induction of brain tumours by random carcinogenesis or by targeted disruption of tumour suppressor genes and/or forced expression of oncogenes.

Older strategies, first reported in the 1950 and 1960s, involved administration of carcinogens to rodents by inhalation, venous injection, intracranial implantation or by in utero transplacental exposure [76-81]. Nitrosamine compounds, such as N-ethyl-N-nitrosourea (ENU) and N- methyl-N-nitrosourea (EMU), are alkylating agents which generate random point mutations in DNA predominantly by adenine to thymine nucleobase transition [82]. High incidence glioma occurring in the brain and spinal cord was first reported in rats receiving repeated intravenous injections of EMU [78], and from in utero ENU or EMU exposure [79, 80, 83]. These tumours were propagated in the brains and/or subcutaneous flanks of recipients in vivo, and in serum- containing cultures in vitro, representing the first in vitro and in vivo experimental model of glioma. These cell lines are still in use today, for example the C6 rat glioma cell line from which cancer stem cells (CSCs) are reportedly identified was generated from an EMU-induced rat glioma, originally described in 1968 [84-88]. Histopathological characterization of EMU/ENU- induced rat brain tumours revealed that carcinogen exposure induced a phenotypically diverse group of brain tumours, including low and high grade astrocytomas, mixed gliomas of astrocytic and oligodendral phenotype, and meningiosarcomas [84]. Careful analysis by light and electron

microscope revealed that abnormal clusters of proliferating, undifferentiated cells could be seen by 8 weeks of age in the subependymal layer of ENU-exposed rats, and that the most frequent location to observe clusters/microtumours at this early time point was the superior lateral angle of the forebrain ventricles [89-91]. Interestingly, the authors of this report suggested that mutant brain stem cells were the origin of the disease, despite the generally held belief of the time that the postnatal brain was postmitotic [89, 90, 92]. More recently, pre-neoplastic neural stem cells (NSC) isolated from the p0 or p30 forebrain of ENU-treated rats showed deletions spanning the CDKN2A locus and, unlike their normal counterparts, grew adherent to plastic and not as spheres when propagated in stem cell conditions [93].

Pre- or post-natal exposure of WT mice to nitrosamine carcinogens rarely generates tumours of the central or peripheral nervous system (CNS, PNS respectively), rather WT mice treated with ENU develop multiple cancers of the liver, lungs, thymus and/or spleen [94]. The biological half-life of ENU is short (~15 min) and cancer-susceptibility may reflect the ability of cells to clear alkylated-DNA at the time of ENU administration. In support of this idea, an inverse relationship is reported between the levels of O6-alkylguanine-DNA alkyltransferase activity in tissues and the occurrence of ENU-induced tumours. In particular, enzymatic activity in the brains of rats is very low when compared to the brains of mice [95-97]. Microarray analysis identified p53 and its transcriptional targets as upregulated immediately after transplacental ENU administration, and in utero treatment of p53-/- mice generates gliomas in 70% of animals consistent with the idea that ENU-mediated tumourigenesis relates to a cells inability to recognize and repair damaged DNA [98, 99]. Leukemias, lymphomas, sarcomas and lung tumours are also observed at high frequency in ENU-treated p53-/- mice, owing to systemic deletion of the gene and whole body carcinogen exposure. Finally, no association was reported between the developmental timing of in utero ENU administration and the incidence or phenotype of the resulting brain tumours, though the sample size studied was limited (n=17 mice analyzed) [99].

More recent experiments have utilized targeted disruption/deletion of tumour suppressor genes and/or forced expression of oncogenes to generate high incidence glioma in mice. Germline p53-deficiency/inactivation in humans is associated with high incidences of breast, bone, brain, blood and soft tissue cancers [100]. This phenotype is shared by systemic p53-/- mice, and 80% of CNS-selective p53-/- mice develop gliomas late in life (50% survival, 43 weeks) [101, 102].

CNS-specific NF1-/- mice demonstrate increased glial lineage proliferation and abnormal neuronal differentiation in vivo, and like NF1-deficient patients, optic gliomas [103, 104]. NF1- deletion in the PNS generates neurofibromas, or malignant peripheral nerve sheath tumours (MPNSTs) when combined with Ink4A-/- or p53-/- [105]. Expression of a constitutively active EGFR in the mouse CNS is itself not gliomageneic, however when combined with a Ink4a-/- and/or PTEN-/- genotype, high grade gliomas resembling GBMs are induced [106, 107]. Similarly Ink4a-/-, and brain specific PTEN-/- and p107-/- mice do not develop gliomas despite the observation that mice of all these gentotypes display increased neural stem cell self-renewal and proliferation [108-112]. However, high grade gliomas in mice are often induced by combining neural-specific p53-/-, Ink4a-/-, NF1-/-, Rb-/- and/or PTEN-/- genotypes, with the specific combination governing the phenotype and behaviour of the resulting tumours [102, 106-108, 110, 113-115].

Other mouse models of glioma are reported [116], many of them dependent on the forced expression of constitutively active Ras and Akt proteins, analogous to mutations rarely (<5%) implicated with the human disease [73, 74, 117-120]. Though the Ras-Raf-MAPK signalling pathway is activated in human GBM samples, it is primarily due to growth factor receptor activation via mutation or amplification of EGFR and PDGFR [74, 121-124]. The most frequently deleted/mutated Ras-pathway protein in glioma is v-raf murine sarcoma viral oncogene homolog B1 (BRAF), which is found aberrant almost exclusively in low grade/pilocytic astrocytomas, and not in GBMs [125-128]. Nonetheless, activated BRAF, when expressed in combination with Ink4a-/- or activated Akt, generates glioma in mice [129]. Finally, with the goal of generating brain tumours from low/single cell infections, in vivo injections of lentivirus encoding H-RasV12 and activated Akt into the brain of p53+/- mice also generates aggressive brain tumours of glial phenotype [118].

1.2 Developmental Neurobiology

A basic understanding of how the mammalian brain develops is fundamental to understanding the origins and molecular mechanisms governing gliomas and medulloblastomas. In the developing embryo, the neuroectoderm is induced by the notochord to give rise to the neural plate by TGFβ-signalling inhibition mediated by chordin, noggin and follistatin [130, 131]. The neural plate folds to become the neural tube and undergoes regionalization along its dorsoventral

and anteriorposterior axes, due mainly to the positional and temporal effects of hedgehog and bone morphogenic protein (BMP) ligands in the former, and in addition to retinoid, fibroblast growth factor (FGF) and Wnt ligands in the latter [130, 131]. The neural tube is thus specified to generate the prosencephalon, mesencephalon and rhombencephalon, the future forebrain, midbrain and hindbrain/spinal cord respectively (Figure 1.2) [130, 131].

Figure 1-2 A- During development the neural tube is regionalized to generate the prosencephalon, mesencephalon and rhombencephalon. The prosencephalon is further regionalized to generate the telencephalon and diencephalon. Similarly, the hindbrain is subdivided into seven rhombomeres, and Hox family genes are expressed throughout the rhombencephalon in rhombomere specific patterns (dashed line). Fgf8 and Irx2 expression induces the development of the cerebellum in Rhombomere 1 by Gbx2 expression and inhibiting Otx2 expression. The zona limitans intrathalamica is established by the anterior (A) expression of Fezf and Arx and the posterior (P) expression of Irx1,3,5 proteins. The pallial-subpallial boundary is established by the dorsal (D) expression of Pax6, Emx1 and Ngn2 and the ventral (V) expression of Mash1, Dlx and Gxh2. B, Top- The cerebellum develops from two germinal zones, the ventricular zone which produces Purkinje neurons (PN) and Bergman glia (BG) and the rhombic lip. GFAP+ rhombic lip NSCs give rise to Olig2+ cells which generate Math1+ CGCPs in the EGL. B, Bottom- CGCPs proliferate in the EGL, which is clearly seen in the p7 cerebellum, however CGCPs migrate inwards to become postmitotic neurons of the IGL by p21. LV, lateral ventricle; Aq, aqueduct; CB, cerebellum; EGL, external granule layer; IGL, internal granule layer, ML, molecular layer; M, medial; L, lateral.

1.2.1 Cerebellar Development

The rhombencephalon is further regionalized into seven rhombomeres (Rhs) which give rise to distinct structures of the hindbrain and spinal cord [132]. Rhombomere specification is mediated by the coordinated expression of numerous transcription factors within each region, with distinct lineages specified in each rhombomere segment and in the absence of cellular migration between adjacent rhombomeres (Figure 1.2A) [132]. Of these transcription factors, the homeobox (Hox) family transcription factors have been particularly well studied with individual Hox genes demonstrating rhombomere restricted expression patterns [133, 134]. The cerebellum arises from the rostral portion of Rh1, influenced by Wnt and Fgf molecules expressed by the isthmus organizer at the midbrain-hindbrain boundary (MHB) [133, 135, 136]. In the chick, the location of cerebellar induction is dependent on the expression of Fgf8. The activation of MAPK signalling by FGF8 and the phosphorylation/activation of homeobox transcription factor Iroquois 2 (Irx2) induces the expression of Gastrulation brain homeobox 2 (Gbx2) and Fgf8, and inhibits the expression of Orthodenticle homeobox 2 (Otx2) [137]. Misexpression of Irx2 and Fgf8, and activation of this gene regulatory network in the midbrain is sufficient to induce ectopic cerebellar development [137]. The cerebellum itself is generated from two anatomically distinct germinal zones: the cerebellar ventricular zone (VZ) which becomes the roof of the fourth ventricle in the mature brain, and the rhombic lip (Figure 1.2B) [138-140]. The cerebellar VZ generates radially migrating postmitotic Purkinje neurons, interneurons and glia. In contrast, the rhombic lip gives rise to cerebellar granule cell precursors (CGCPs) which migrate over the surface of the developing cerebellum, proliferate in response to Shh secreted by Purkinje cells to generate the external granule layer (EGL), and then migrate internally to produce mature granule neurons of the internal granule layer (IGL) [138, 140, 141]. This migration and maturation is now matched with lineage data suggesting that, beginning at e12.5 in the mouse, Nestin+GFAP+ rhombic lip NSCs give rise to Oligodendrocyte lineage transcription factor 2 (Olig2)+/ Paired box gene 6 (Pax6)+ CGCPs which persist until just before birth, and then proliferative mouse Atonal homolog 1 (Math1)+ CGCP which persist postnatally in the EGL, but are depleted to extinction by p21 [53, 54, 60, 140]. Finally, in the postnatal cerebellum NSCs reside in the cortex and white matter, and were prospectively isolated from the p7 cerebellum using the cell surface marker AC133/CD133/Prominin1 (CD133) [142, 143].

1.2.2 Forebrain Development

By e11 in mouse development the prosencephalon is comprised of two parts, the telencepahlon which gives rise to the cerebrum, and the diencephalon which generates the thalamus, hypothalamus and the posterior portion of the pituitary gland (Figure 1.2A) [144]. Similar to the MHB there exists at least two specification boundaries in the prosencephalon, the pallial– subpallial boundary (PSB) which separates the future cortex from the lateral ganglionic eminencies, and the zona limitans intrathalamica (ZLI) positioned at the interface between the thalamic and the prethalamic primordia [132]. The controls of segmentation in the forebrain are less clearly understood compared to the hindbrain due to the observation that lineage traced cells cross presumptive boundaries [132]. Nonetheless, some of the molecular mediators which establish or mark boundaries of forebrain segmentation have now been characterized. The PSB is established by the dorsal expression of Pax6, Empty spiracles homeobox 1 (Emx1), and Neurogenin 2 (Ngn2) in the pallium, and the ventral expression of Achaete-scute complex homolog 1 (Mash1), Distal-less homeobox (Dlx) and GS homeobox 2 (Gxh2) in the subpallium [145-147]. In Xenopus, the ZLI is established by mutual antagonism between Irx genes (predominantly Irx1 and 3), Fez family zinc finger 1 (Fezf1) and Aristaless related homeobox (Arx), with the anterior border of Irx protein expression abutting the posterior border of Fezf1 and Arx expression. Transcriptional inhibition of Irx proteins by Fezf1 and Arx is direct, with Arx acting as a transcriptional repressor of Irx proteins to limit their rostral expression [148].

Embryonic forebrain NSCs generate neurons, oligodendrocytes and astrocytes, while self- renewing to maintain a multipotent population of NSCs that persists into adulthood [149, 150]. This phenomenon was first appreciated in the 1960’s when tritiated-thymidine labelled rat forebrains revealed the presence of newly born glia and neurons, contradicting the long prevailing “no new neuron” hypothesis [92, 151-154]. Despite subsequent electron microscope confirmation of these results, including co-localization of tritiated nuclei and mitotic figures, the assertion that neurogenesis occurred in the adult vertebrate brain was largely dismissed [92, 155- 158]. This conclusion was accepted when rare, proliferating, multipotent adult mouse NSCs were expanded in vitro as floating spheroid clusters, termed neurospheres [150]. Neurosphere cells were characterized as expressing the neuroepithelial marker Nestin, but not neuronal cell markers neurofilament, neuron-specific enolase or GFAP. Subsequent experiments demonstrated that neurospheres could be expanded in serum-free media in vitro, could

differentiate to generate cells of all three brain lineages in vitro, and retained their capacity to engraft and functionally incorporate when injected into the brains of recipient mice [150, 159, 160]. Similarly, human neural stem cells were prospectively identified using the cell surface marker CD133, could be propagated long-term in vitro in serum-free media and could functionally engraft the brains of immune-compromised mice [161, 162].

1.3 Mouse and Human Neural Stem Cells

In the adult mouse brain NSCs exist within the subependymal zone (SEZ)/subventricular zone (SVZ) of the lateral ventricles of the forebrain, the subgranule zone (SGZ) of the dentate gyrus, and the subcallosal zone (SCZ) which lies between the hippocampus and the corpus callosum [163-169]. Neurogenesis is also reported to occur in the cortex after injury, though the multi- potentiality of these proliferating cells is unknown suggesting that unipotent neuronal progenitors, rather than NSCs, may reside in the cortex [170]. SVZ neural stem cells are relatively quiescent GFAP+ cells which give rise to neuroblasts that migrate along the rostral migratory stream (RMS) to the olfactory bulb where they integrate as olfactory interneurons [166, 171, 172]. These GFAP+ neural stem cells were termed type ‘B’ astrocytes by the Alvarez- Buylla laboratory after their electron microscope and functional characterization of the subependymal layer. Type ‘B’ astrocytes give rise to type ‘C’ intermediate cells which proliferate to produce the migrating type ‘A’ neuroblasts [168]. Functional studies demonstrated that ablation of the type A and C cells by cytarabine (AraC) treatment induced ‘B’ cells into the cell cycle to repopulate the cellular hierarchy, and ablating proliferating GFAP+ NSCs, either in vitro or in vivo, virtually abolished sphere forming capacity confirming the GFAP+ phenotype of SVZ NSCs in the adult mouse [168, 173].

Unlike in the mouse, human forebrain NSCs exist within an astrocyte ribbon separated from the ependymal layer by a hypocellular gap [174, 175]. These neural stem cells, as well as those located in the subpial zone of the cerebral cortex and the subgranular zone of the hippocampus, are characterized by their expression of GFAP-delta, a protein isoform generated by alternative splicing [175]. The destiny of forebrain NSC progeny is still a matter of debate, with conflicting reports regarding the existence of a functional RMS within the human brain [174, 176]. Nonetheless, NSCs within the adult human forebrain have been identified, though their functional role in the normal and diseased brain is not clearly understood [177].

The neurosphere culture system has been an invaluable tool for the interrogation and characterization of NSCs. NSCs from many mammals have now been propagated in vitro in serum-free conditions containing saturating concentrations of EGF and/or bFGF [178-182]. Neurospheres seeded at low densities (< 10 cells/μl) were once thought to be of clonal origin however the observation of sphere fusion, even when growing neurospheres from cells seeded at low density, cautions against assuming a clonal lineage unless spheres are derived from a single cell plated in isolation [183]. Endogenous factors secreted in neurosphere media can greatly enhance sphere formation clearly illustrating the effect of culturing cells at high density or in conditioned media [184, 185]. Nonetheless, analysis by limiting dilution allows an interpretive and quantitative analysis of a culture’s clonality. Limiting dilution analysis is performed by plating a series of wells in replicate, with each series differing in starting cell-density by half that of the former. After a defined period of time (typically 7-10 days), the number of wells which do not contain at least one proliferating neurosphere is calculated and plotted against cell density. Regression analysis is used to calculate the density of cells at which 37% of wells are empty (37% intercept). By Poisson distribution the 37% intercept corresponds to the statistical density at which one sphere will be observed in each well, and serves to quantify clonality in the absence of plating multiple single cells and calculating the percentage of wells bearing single spheres [181].

The neurosphere assay has enabled the discovery of the molecular mechanisms governing stem proliferation and self renewal. For example, EGF, FGF, Hh, Notch, Wnt and PI3K signalling pathways have all been interrogated in vitro and in vivo and found to be important for neural stem cell proliferation, self-renewal and/or maintenance [108, 181, 186-192]. Bmi1 polycomb ring finger oncogene (Bmi-1), Cdkn1a (p21), Cdkn1b (p27), retinoblastoma-like 1/p107 (Rbl1), and p53 are all negative regulators of neural stem cell self renewal, and in their absence SVZ proliferation and neurosphere formation is enhanced in vivo and in vitro, respectively [112, 193- 197]. Finally, neural stem cells grown as spheres are now being used in high-throughput chemical screens to identify inhibitors and activators of proliferation and/or survival, and numerous neurotransmission pathways are now implicated in neural stem cell maintenance potentially relating to lineage specification [198, 199].

The neurosphere culture system is not without some limitations. Immunocytochemical and functional studies demonstrate that neurospheres comprise a diverse repertoire of cells including

a sub-fraction of Nestin+ NSCs, and other cells that differentiate to express markers of mature astrocytes and neurons [200-202]. Introduction of exogenous genes into cells within a neurosphere can be problematic, and mechanical and/or enzymatic dissociation can compromise cell viability [201, 202]. Alternatively, NSCs can be propagated as an adherent monolayer grown in serum-free media and anchored to plastic by a poly-ornithine, gelatine and/or laminin matrix [201, 203, 204] . Human and mouse neural stem cells cultured as adherent monolayers, termed ‘NS’ cells, demonstrate the hallmark properties of neural stem cells in vitro, can functionally incorporate in vivo, and are thought to comprise a more highly purified population of NSCs amenable for biological and drug discovery purposes [201, 203, 204].

Embryonic and adult mouse neural stem cells isolated from the forebrain and cerebellum are commonly characterized as Nestin+, GFAP+, SRY-box containing gene 2 (Sox2)+, Musashi homolog 1 and 2 (Msh1/2)+, and Olig2+, and green fluorescent protein (GFP) driven from these promoters identifies/enriches for neural stem cells [54, 142, 150, 168, 173, 205-211]. Finally, prospective identification of neural stem cells can be achieved by fluorescent activated cell sorting (FACS) for the cell surface markers CD133 and Lewis X/Stage Specific Embryonic Antigen 1/CD15 (CD15) from the WT mouse forebrain and hindbrain, and for CD133 and CD15/CD29HI/CD24LO from human brain samples [142, 161, 212-216].

1.4 Cancer Stem Cells

1.4.1 The Cancer Stem Cell Hypothesis

Human cancers are a morphologically and functionally heterogeneous population of cells [217- 223]. Specifically, a subpopulation of tumour cells from some cancers has the capacity to regenerate a tumour and sustain its growth when injected into immune-compromised mice [224]. This phenomenon is exemplified in a set of controversial experiments performed in the 1950s and 1960s where patients were injected with increasing numbers of their own explanted cancer cells [225, 226]. Even when a million ovarian and breast cancer cells were implanted subcutaneously only 50% of injections developed into a palpable nodule [226]. At least two models of cancer growth can explain the increased number of cells required to initiate tumours in vivo (Figure 1.3). The first, termed the stochastic model, assumes that every cancerous cell has the capacity to extensively proliferate and regenerate a tumour, however the probability of this event for any given cell is low [227]. This model predicts that isolating discrete populations of cancer

20 cells cannot enrich for tumour initiating capacity versus the bulk population, due to the assumption that all cells have an equal, albeit low, probability of regenerating a tumour. In contrast, the cancer stem cell model assumes that only a subset of cells within the tumour population has the capacity to initiate and sustain tumour growth [227]. This assumption implies functional heterogeneity within the pool of cells that make up a tumour, and suggests that isolating this cellular sub-fraction can purify a potently tumourigenic population of cells. The development of an in vivo model in which to test the tumour initiating ability of discrete cellular populations, coupled with techniques of phenotypic or functional cell purification, has allowed the identification of CSCs from an ever increasing list of primary human tumours, including human leukemia, breast, and brain, lung, colon, pancreas, head-and-neck, skin, prostate and mesenchymal cancers [218-220, 222, 228-237]. CSCs are defined functionally as a potently tumourigenic, self-renewing population in vivo with the ability to differentiate and generate mature cell types reflective of the original tumour phenotype. Importantly, the definition and characterization of a CSC has no implications regarding its tumour cell-of-origin [224].

Figure 1-3 Stochastic versus Cancer Stem Cell Hypothesis of Tumour Development and Maintenance: At least two models of tumour development exist- The Stochastic Model (left) predicts that all cells (coloured circles) within a heterogeneous tumour population (top) can regenerate and maintain tumour growth in vivo, however the likelihood of this event for any given cell is very small (thin arrow) or non-existent. Conversely, the Cancer Stem Cell Model (right) suggests that cancer stem cells (CSC, red) have the capacity to regenerate and maintain a tumour in vivo. In contrast, the bulk heterogeneous tumour population is not tumourigenic, while cancer stem cells have potent tumour-initiating capacity (thick arrow).

1.4.2 Leukemia Stem Cells

In 1997, a serially transplantable population of human leukemia cells was enriched for tumour initiating ability [218]. Of principle importance was the development of an experimental system in which to test the repopulation capacity of haematopoietic and leukemic human cells when injected in mice. Previously it had been demonstrated that cells from acute myeloid leukemia (AML) patients exhibiting the haematopoietic stem cell surface phenotype CD34+/CD38- could engraft severe combined immune-deficient (SCID) mice to generate a leukemia similar to that of the original patient [228]. Injected cells proliferated and repopulated the bone marrow of mice, however the leukemia-initiating frequency was determined to be 1 engraftment per 250,000 cells and leukemias could not be serially transplanted into secondary SCID recipients [228]. The use sub-lethally irradiated non-obese diabetic/SCID (NOD/SCID) mice proved a better in vivo model and leukemia initiating cells, termed SCID leukemia-initiating cells (SL-ICs) or leukemia stem cells (LSCs), were consistently and exclusively found in the CD34+CD38- sub-fraction of cells [218]. In all AML patient samples tested, this sub-fraction comprised only 0.02% to 2% of the total leukemic population. Further, when limiting numbers of AML cells were injected into primary NOD/SCID recipients, as few as 5x103 CD34+CD38- cells were able to initiate leukemic growth while as a many as 5x105 CD34+CD38+ cells failed to produce tumours. Bone marrow analysis of the CD34+CD38- engrafted mice demonstrated that LSCs had the ability to proliferate extensively and produce CD45+, CD38+ and CD33+ cells indicating their capacity to differentiate and acquire lineage markers to regenerate the original AML disease in vivo. LSCs could also initiate the growth of leukemias in secondary recipients demonstrating their ability to proliferate extensively, self-renew, and serially transplant the disease [218]. Further characterization of LSCs revealed the existence of a functional hierarchy within the LSC population. Clonal analysis of GFP marked LSCs isolated from the bone marrow of primary, secondary, and tertiary transplanted NOD/SCID mice demonstrated the existence of clones with different self-renewal capacities within the same CD34+CD38- AML sample [217]. Both short-term LSC clones, which were only identified in the primary recipient, and long-term LSC clones, which could be identified in secondary and tertiary recipients, were observed. Fewer clones were observed in tertiary NOD/SCID mice suggesting that the self-renewal capacity required for a clone to repopulate primary, secondary and tertiary recipients is limited to a small percentage of the LSC population [217]. Therefore, in the same way that the bulk tumour population is functionally

heterogeneous, the LSC population demonstrates functional differences suggesting a hierarchy in the CSC population.

1.4.3 Cancer Stem Cells in Solid Malignancies

By applying a similar experimental protocol to the study of solid tumours, CSCs have now been prospectively identified from an ever increasing number of solid malignancies (as outlined above) and are actively sought from all tumour types.

Isolating breast cancer cells based on a CD44+CD24-/lowLineage- cell surface phenotype enriched for the tumour initiating capacity of primary tumours and metastatic pleural effusions [219]. Injection of limiting numbers of cells into the upper mammary fat pads of NOD/SCID mice revealed that as few as 1x103 uncultured CD44+CD24-/lowLineage- cells could regenerate a tumour in recipient mice, while injection of 1x105 CD44+CD24+Lineage- cells failed to initiate tumour growth. Tumourigenic breast cancer cells were serially transplanted and analysis of tumours arising in NOD/SCID mice by flow cytometry revealed the presence of a heterogeneous population of cells similar in phenotype to that of the original tumour, though no differences in cell cycle were observed between tumourigenic and non-tumourigenic cells to explain the differences in in vivo growth ability [219]. These results constituted the first identification of a CSC population in solid tumours that could self-renew, proliferate extensively and differentiate to regenerate the phenotypically heterogeneous tumour when injected into mice.

1.4.4 Brain Tumour Stem Cells

Similar experiments performed with adult and paediatric brain tumours led to the identification brain tumour stem cells (BTSCs) [220, 229, 238-240]. Culturing mechanically and/or enzymatically dissociated brain tumours in serum-free conditions containing EGF and bFGF gives rise to neurospheres which demonstrate properties and phenotypes similar to those of normal neural stem cells; notably they can proliferate as self-renewing, serially passageable spheres; differentiate into neurons, astrocytes, or oligodendrocytes when cultured in the absence of growth factors (with or without the presence of serum); and can regenerate tumours when injected sub-cutaneously or intracranially into immune-compromised mice [229, 238-240]. Like normal human neural stem cells, CD133+ tumour cells demonstrated a capacity to proliferate and generate neurospheres in vitro, while CD133- cells became adherent and did not proliferate [229,

241]. CD133+ brain tumour cells made up the minority of the primary tumour population, ranging in percentage from 3.5% to 46%, and with an increased fraction generally correlated with higher tumour grade [220, 229]. Intracranial injection of as few as 100 CD133+ cells regenerated a serially transplantable copy of the original tumour in the brains of NOD/SCID mice, a result which was consistent for MBs, ependymomas, and gliomas from both paediatric and adult patients [220, 242]. More recently the CD15+, and autofluorescenthigh populations of freshly dissociated GBMs were shown to be enriched for BTSCs in vitro and in vivo [243, 244]. Characterization of BTSCs suggests that they are relatively resistant to IR and traditional chemotherapies with suggestions that they are a likely source for disease recurrence and/or progression [245, 246]. Moreover, many of the same molecular pathways that govern self- renewal, survival and/or differentiation of normal neural stem cells are also important molecular mediators within BTSCs. In vitro or in vivo BMP treatment of BTSCs results in reduced proliferation, tumourigenicity and increased neural differentiation [247]. Hh signalling regulates the self-renewal of GBM BTSCs and chemical or genetic inhibition of the pathway reduces tumour growth in vivo [248]. Notch signalling is reported to mediate radio-resistance of BTSCs, along with their capacity to form neurospheres in vitro and tumours in vivo [249, 250]. Finally, like normal neural stem cells, BTSCs can be cultured as NS cell lines, growing adherent to plastic on a poly-L-ornithine/laminin matrix, and are potently tumourigenic with as few as 100 cells capable of initiating tumours that demonstrate patient specific characteristics [201, 202, 251].

1.4.5 The Cancer Stem Cell Controversy

The discovery of CSCs attracted public attention and some criticisms, particularly with respect to the methodology used to identify human CSCs [252-254]. Of chief concern is the technical aspect of injecting purified populations of human cells into immune-compromised mice, with critics arguing that these methods may simply identify a population of cells capable of engrafting the tissue of another organism, rather than identifying the cells driving the disease in situ [254, 255]. This hypothesis was fuelled by the observation that in vivo tumour propagation was not mediated by rare CSCs in mouse leukemias and lymphomas. When 10 unsorted cells isolated from either Eμ-myc B-cell mouse lymphoma, Eμ-N-Ras T-cell mouse lymphoma or PU.1-/- mouse AML were transplanted into nonirradiated congenic recipients, tumours arose in almost all mice suggesting that mouse leukemia/lymphoma was not organized in a cellular hierarchy

[255]. More recently, roughly 30% of single sorted melanoma cells were shown to be tumourigenic in the more immune-permissive NOD/SCID-gamma (NSG) mice, deficient for the IL2 receptor gamma chain, contradicting a previous report identifying melanoma CSCs [237, 256]. Accordingly, the validity of identifying human CSCs as determined by tumour formation in rodents is debated, and it remains possible that some human cancers do not contain a subpopulation of tumour initiating cells [257]. However, the identification of CSCs in other mouse tumour models argues in favour of the cancer stem cell hypothesis, and supports the conclusion that some human cancers, including brain tumours, are organized as a cellular hierarchy (as discussed further in Chapters 2-5).

1.5 The Cell-of-origin: Cancerous Stem Cells versus Cancer Stem Cells

Of critical importance is the distinction between CSCs and cancerous stem cells. Though their name may suggest that CSCs are derived from normal stem cells, the definition of a CSC is independent of its cellular origins [224]. CSCs display the stem cell properties of self-renewal and differentiation. Together with similarities in cell surface phenotype it is attractive to think of normal stem cells as the cellular source of cancer. It is also possible that oncogenic transformation occurs in a progenitor cell, or differentiated cell. Perhaps the most obvious argument against stem cells as the source of malignant disease is the fact that both differentiated epithelial cells and fibroblasts can be transformed in vitro and in vivo to generate cells that exhibit all the hallmarks of cancer [258]. The minimal requirement for human fibroblast transformation and in vivo growth is the co-expression of SV40 large T antigen and activated Ras [259]. Further, targeting differentiated epithelial cells with various oncogenes (ie. v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (v-Src), v-Ha-ras Harvey rat sarcoma viral oncogene homolog (H-Ras), v-myc myelocytomatosis viral oncogene homolog (Myc) or v- akt murine thymoma viral oncogene homolog 1 (Akt)) can produce transformed cell types with in vivo growth capacity [260]. Therefore, differentiated cells have malignant capacity if coordinated expression of oncogenes and/or viral proteins is forced.

Another possibility is that progenitor cells serve as the cell-of-origin for cancer. Progenitors have limited self-renewal capacity and are generated from a symmetric (but depleting) or

asymmetric stem cell division [261, 262]. Expression of the leukemia associated MLL-ENL fusion protein equally transforms murine HSCs, common myeloid progenitors, and granulocytic/monocytic-restricted progenitors [263]. When purified populations of these fusion- protein expressing cells were injected into mice, all cell types generated leukemias of a similar phenotype, and expression profiling demonstrating that the same leukemia developed regardless of the cell type targeted. Similarly, expression of the oncogenic breakpoint cluster region (Bcr) c-abl oncogene 1 (Abl) fusion protein (Bcr-Abl) in committed myelomonocytic cells is enough to produce chronic myelogenous leukemia in mice [264]. Finally, the cell-of-origin in mouse brain tumours has been actively investigated in the past few years, and is discussed in context of our findings in Chapter 5. However, results suggest that stem cell transformation is not a requirement of cancer, and progenitor and differentiated cells have the capacity to produce tumours in various tissues, including the brain.

1.6 Hypothesis, Potential Significance and Specific Aims of this Ph.D. Thesis

Given the identification of CSCs in human glioma and medulloblastoma, we hypothesized that brain tumour stem cells are responsible for the propagation of mouse brain tumours. The significance of finding this result is clear: firstly it would validate the existence of CSCs in human brain tumours, arguing against their identification as being a technical artefact; secondly, it would validate the clinical representation of the mouse models investigated; thirdly, it would provide an important resource for the in vitro and in vivo interrogation of the mechanisms driving CSC induction, self-renewal, maintenance and survival and; lastly, it would provide an in vitro and in vivo platform on which preclinical drug-discovery experiments could be performed.

1.6.1 Specific Aims

1 - To identify brain tumour initiating cells from Ptc1+/- MBs by in vitro and in vivo analysis.

In vitro analysis will include the determination of the neurosphere or NS culture capacity of primary tumours, the phenotype and behaviour of the resulting cell lines, and the molecular characterization of the signalling pathways known to be involved with the disease. In vivo analysis will include a limiting dilution assay of freshly dissociated tumour cells, a comparison of the tumourigenic potential of prospectively isolated populations and a phenotypic analysis of identified populations.

2 – To identify brain tumour initiating cells in a chemical-genetic mouse model of glioma.

Mouse gliomas will be generated by transplacental ENU treatment of p53-/- mice. An in vitro analysis of glioma growth capacity in neurosphere or NS culture conditions will be performed, as will a determination of the physical, functional, chemical and molecular properties of neurosphere/NS cell lines. In vivo, an analysis of the phenotypic and functional characteristics of freshly dissociated tumours and cell lines will be performed, as will a determination of the tumourigenic potential of prospectively isolated populations.

Chapter 2

2 Multipotent CD15+ Cancer Stem Cells in Patched-1

Deficient Mouse Medulloblastoma

2.1 Abstract

Subpopulations of tumourigenic cells have been identified in many human tumours, though these cells may not be very rare in some types of cancer. Here we report that medulloblastomas arising from Patched-1 deficient mice contain a subpopulation of cells that demonstrate a neural precursor phenotype, show clonogenic and multilineage differentiation capacity, activated

Hedgehog signalling, wild-type Patched-1 expression, and the ability to initiate tumours following allogeneic orthotopic transplantation. The normal neural stem cell surface antigen

CD15 enriches for the in vitro proliferative and in vivo tumourigenic potential from uncultured medulloblastomas supporting the existence of a CSC hierarchy in this clinically relevant mouse model of cancer.

2.2 Introduction

Medulloblastoma is a cancer of neuronal phenotype and is the most frequent malignant brain

tumour found in children. Disruptions of the Hedgehog signalling pathway occur in roughly

30% of human MBs analyzed, and Gorlin’s syndrome patients, who have germline mutations in

the Hh receptor Patched-1, are at an increased risk of developing MB and cancers of other types

[265]. This clinical observation is recapitulated in mice heterozygous for the Ptc1 allele [48].

Ptc1+/- mice spontaneously develop MB at a reproducible frequency of 5-30% [48]. Ptc1+/- combined with postnatal irradiation or homozygous deletion of the tumour protein 53 augments the severity and frequency (to ~100% at 8-12 weeks) of MB occurring in these mice [50, 51,

266].

It remains unclear if representative Ptc1+/- MB cells can be propagated in serum-free neural stem

cell conditions, as bFGF is reported to differentiate Ptc1+/- MB cells when analyzed up to 72hrs post dissociation [267, 268]. Similar results were observed with bone morphogenic protein treatment [269] but importantly, there are no reports describing the generation of long term, tumourigenic cell lines with activated Hedgehog signalling from Ptc1+/- MB in stem cell media.

Cancer stem cells, tumour-initiating cells (TIC), or tumour-propagating cells (TPC), are defined

functionally as an enriched population of cells within a primary tumour capable of transplanting

a representative copy of that disease in vivo [270, 271]. Subpopulations of cells meeting this

functional criteria have now been identified in many human cancers [270]. Human brain TICs

were isolated based on the expression of CD133 [220, 229] and are reported to be relatively

resistant to both radiation- and chemo-therapy [245, 246]. In addition, the application of stem

cell methods to the study of brain cancer revealed that cells propagated in NSC media more

30 closely represent the tumour from which they were derived than do those established and propagated in serum-containing media [272].

Here we identify a rare, phenotypically primitive, multipotent, and tumourigenic population of

Ptc1+/- MB cells that can be propagated without limit and studied in vitro in stem cell conditions.

These cells retain activated Hh and Notch signalling, and do not necessarily display Ptc1 loss of heterozygosity or loss of WT Ptc1 gene expression. Finally, the NSC marker Lewis X/Stage

Specific Embryonic Antigen 1 (SSEA-1)/CD15 [214] prospectively enriches for proliferative cells in vitro and tumourigenic cells in vivo, identifying a subpopulation of CSCs in this mouse model of MB.

2.3 Materials & Methods

2.3.1 Mouse Husbandry and Tumour Processing

All mouse procedures were approved by the Hospital for Sick Children’s Animal Care

Committee. C57/B6 Trp53+/-, GFAP-TK mice (Jackson Laboratory, Maine, USA) and Ptc1+/- mice were mated to generate a breeding colony. Medulloblastomas were microdissected, dissociated by gentle pipetting in PBS followed by a 10-15min Accutase (Sigma-Aldrich) digestion and filtered sequentially through 70μm and 40μm nylon filters.

2.3.2 Flow Cytometry, Cell Sorting and In vivo Injections

Cells were stained with 1μl anti-CD15-FITC (BD Biosciences) and 1μl anti-Ter-119-APC per

100μl ice cold PBS for at least 30 min, washed with PBS and filtered (40 μm) at least once post

staining, suspended in 2μl propidium iodide (PI)/ml ice cold PBS, and processed via MoFlo

(Dako Cytomation) FACS. Cells were sorted into growth factor free DMEM/F12 and the purity

of each population was assessed at the end of each sort. Freshly sorted uncultured cells, or cells

from established cell lines were suspended in approximately 2-5μl cold PBS and injected into

the cerebellum of NOD/SCID recipients using a rodent stereotaxic headframe as previously

described [220]. For intracellular Nesting flow cytometry, cells were fixed in 4% PFA for 10min

at room temperature, and stained for 30min at room temperature in PBS/0.5% Tween20 with 1μl anti-Nestin followed by 1:16000 anti-mouse-488.

Embryonic day 14.5 hindbrains were processed as described above. Cells were plated in mouse

NSC media [150] overnight before FACS. Cells were collected, dissociated by 5-10min

Accutase digestion and stained with 2μl anti-CD15-FITC and 1μl anti-Ter119-APC per 100μl

PBS for a minimum of 30min, on ice. Thereafter, cells were processed as described above.

Cells were centrifuged and plated at a density of 1x104 cells/well in a 96 well plate. 7days post

sort, the number of neuro-spheres (minimum size > 50μm) in each population was counted.

2.3.3 Intracerebellar Ganciclovir Infusion

Ptc1+/- mice were mated with GFAP-TK+ mice (Jackson Laboratory, Maine, USA) and pups

were irradiated (3Gy) on post-natal day 0. On p21 Ptc1+/-GFAP-TK+ mice were anesthetised,

mounted on a stereotaxic headframe and a small incision was made to expose the base of the skull. A small hole was drilled at stereotaxic coordinates -6mm A/P posterior to bregma, 0mm

M/L, and an Alzet Brain Infusion Kit #3 cannula was installed through this hole and secured to

the surface of the skull. A primed miniosmotic pump containing 200μM GCV with a constant

delivery flow rate of 0.5μl/hr was attached to the cannula via a short (1cm) catheter. Both pump

and catheter introduced into a sub-cutaneous pocket opened between the shoulders, and the

incision was closed by interrupted sutures. One week later mice were anesthetised, the cannula

catheter and minipump were removed, and the incision closed by interrupted sutures.

2.3.4 Tissue Culture, DNA, RNA and Protein Analysis

In vitro cells were grown in mouse NSC media (20ng/ml epidermal growth factor (EGF) and

20ng/ml basic fibroblast growth factor (FGF)) [150] on Primaria culture plates (BD

Biosciences). Time lapse photo-microscopy and calculations of well confluency were performed

using the IncuCyte live-cell imaging system (Essen Instruments, Michigan, USA).

Hh and Notch target genes were analyzed by PCR (Supplementary Methods & Methods) and/or Western by standard procedures. Cells and mouse ear punches were genotyped as

33 previously described [273], as was analysis of WT Ptc1 RNA expression [60] (Supplemental

Materials & Methods).

In vitro limiting dilution analysis was performed in 96 well plates as previously described [181] and analyzed for the presence of adherent, proliferating colonies at least two weeks post culture.

For dose response analysis, established cell lines were seeded at a density of 2500 cells per well in 96 well format, and analyzed by MTT assay after 7days growth in the absence or presence of indicated concentrations of Cyclopamine (Sigma Aldrich) or DAPT (Sigma Aldrich).

Established cell lines or freshly dissociated tumours were plated in indicated concentrations of

Ganciclovir (Sigma Aldrich) and analyzed as described for MTT assay or limiting dilution assay.

For differentiation assays, cells were subjected to a two step, sequential EGF/FGF withdrawal differentiation protocol [204] performed over two weeks with or without the addition of 10% foetal bovine serum (FBS).

2.3.5 Immunocytochemistry & Immunohistochemistry

Freshly dissociated tumour cells were cytospun onto glass slides (105 cells/slide) and established cell lines were grown on Poly-L ornithine/Laminin coated glass coverslips. Cells were fixed in

4% paraformaledhyde for 30 min at room temperature, washed with PBS and permeablized in

0.3% Triton X for 30min. 4% paraformaldehyde fixed tissue was paraffin embedded and sectioned to generate 6μm tissue slices. Tissue sections were processed by standard protocol and stained with indicated primary antibodies (Supplemental Materials & Methods).

2.4 Results

2.4.1 Rare, Phenotypically Primitive and Multipotent Ptc1+/- MB Cells

can be Propagated In Vitro.

We sought to determine if a population of Ptc1+/- MB cells could be propagated in NSC media if

analyzed beyond a 72hr time point [267]. Primary tumours were plated in serum-free media

containing 20ng/ml EGF and 20ng/ml bFGF. Twenty-four hours later, cells from Ptc1+/-p53+/+,

Ptc1+/-p53+/-, Ptc1+/-p53-/- and IR Ptc1+/- MB aggregated into clusters, the majority of which did

not go on to proliferate (Figure 2-1A). One to three weeks later, adherent proliferating colonies

were observed from every MB tested. Ptc1+/- MB cells primarily grew adherent to plastic, but

occasionally as semi-attached or free-floating spheres (Supplemental Figure 2-1), and could be

made adherent by coating plates with 0.1% gelatin [203]. In X-gal substrate, cells stained

positive for β−galactosidase expression suggesting activated Hh signalling (Supplemental

Figure 2-1), and could be propagated long term (>50 passages) in vitro without reduction of

proliferative potential. To ensure that expanded cells were of MB origin, we transplanted

uncultured tumour cells into the brains of NOD/SCID mice and expanded individual colonies

from the resulting allografts (Supplemental Figure 2-2). We observed that that all cultures were

Ptc1-/- and LacZ/Neo+, discounting the possibility that cultures contained contaminating normal

cells.

Figure 2-1 (A) Primary Ptc1+/- MB were dissected and dissociated into single cells and cultured in serum-free media. Within 12-24h, cells amalgamated into floating clusters, the majority which did not go on to proliferate (red arrows). 1-3 weeks later, adherent proliferating colonies (green arrows) could be observed from every MB tested and expanded as cell lines, irrespective of p53 genotype or early postnatal irradiation. B) Ptc1+/- MB cell lines demonstrated a precursor phenotype, expressing Nestin, Sox2, and Math1. EGF and FGF withdrawal induced markers of mature glial (S100β, CNPase) and neuronal (βIII-tubulin) cell types. (C) Primary Ptc1+/- MB cells were cytospun and stained for Nestin and Math1, GFAP, Sox2, or Map2 to identify cells of neural precursor phenotype. (D) Established Ptc1+/- MB cell lines or primary tumours, derived from GFAP-TK+ or GFAP-TK- mice, were plated in the absence or presence Ganciclovir in vitro. GFAP-TK+ cells were significantly inhibited in their proliferative capacity, or ability to establish proliferative colonies in vitro. (t test, *p<0.05, **p<0.01) (E) Irradiated Ptc1+/- mice were untreated or treated with 200mM Ganciclovir for 7 days by intracerebellar cannulation and osmotic minipump delivery. Untreated or treated mice were followed for 120 days past birth (logrank survival curve comparison, *p=0.0534).

We performed an in vitro limiting dilution growth analysis of primary MB cells to determine their clonogenic capacity. All tumours studied contained a rare (<1%) population of clonogenic cells. Tumours derived from Ptc+/-p53-/- and IR Ptc+/- demonstrated the highest clonogenic capacity (1:4000 cells (n=8 tumours), 1:8000 cells (n=3 tumours), respectively) compared to

Ptc+/-p53+/- and Ptc+/-p53+/+ tumours (both >1:50000 cells (n>6 tumours)). Cell lines demonstrated a neural precursor phenotype expressing Nestin, Sox2, Musashi, GFAP, and Math1 but not neuronal (βIII tubulin, MAP2) or glial (S100β, CNPase) cell markers (Figure 2-1B,

Supplemental Figure 2-3 and data not shown). Growth factor withdrawal or serum treatment, induced expression of mature astrocytic, oligodendral and neuronal cell markers (S100β,

CNPase, βIII tubulin respectively) indicating a capacity of these cell lines to undergo multilineage differentiation (Figure 2-1B, Supplemental 2-3). Differentiated single cell derived cultures also expressed all lineage markers, including CNPase, further demonstrating the multilineage differentiation capacity of MB cells (data not shown). Conversion of cells to serum-containing media dramatically altered cell morphology, inhibited proliferation, decreased

Nestin expression from >90% of the cells to undetectable levels, but did not reduce Math1 expression suggesting that this transcription factor is expressed by both proliferating and non- proliferating MB cells (Supplemental Figure 2-4).

To determine if cells of precursor phenotype could be identified in primary tumours, we cytospun freshly dissociated Ptc1+/- MB cells onto glass slides and analyzed their phenotypes.

Greater than 97% of all cells stained positive for Math1, and a relatively rare (<5%) population of cells co-expressing the neural precursor markers Nestin, Sox2 and GFAP, a phenotype similar to that of forebrain and cerebellar mouse NSCs [60, 142] (Figure 2-1C).

To test the possibility that serum-free culture conditions expanded rare Nestin+Sox2+GFAP+ cells within the primary tumour, we crossed our Ptc1+/- mice with mice expressing the viral gene

thymidine kinase (TK) from the GFAP promoter (GFAP-TK) which marks NSCs in both the

cerebellum and the forebrain. Ganciclovir (GCV) infusion into the brains of GFAP-TK mice

ablates the NSC population in vivo [173]. The proliferation of established cell lines and freshly

dissociated tumours derived from Ptc1+/-GFAP-TK+ MBs was significantly inhibited upon the

addition of 0.25-2μm GCV, concentrations that did not demonstrate any non-specific toxicity or

bystander effect in controls (Figure 2-1D & Supplemental Figure 2-5). Additionally,

continuous delivery (0.5μl/hr) of 200μM GCV for 7 days into the cerebellum of 21 day old

irradiated Ptc1+/-GFAP-TK+ mice extended the percentage of mice surviving to 120days, and

decreased the frequency of MB compared to untreated Ptc1+/- littermate controls (6/10 MB in

GCV treated mice, versus 10/11 MB in untreated Ptc1+/- mice (Figure 2-1E).

2.4.2 Ptc1+/-p53-/- MB Cell Lines Initiate the Growth of Phenotypically

Representative Tumours In Vivo.

We injected 105 cells from established Ptc1+/-p53-/- cell lines (n=3) into the cerebella of

NOD/SCID mice. Eight to twelve weeks after injection, MBs were clearly apparent and

mirrored the primary tumour histology, demonstrating expression of neuronal (Map2), astrocytic

(GFAP and S100β) and oligodendral (CNPase) cell lineages (Figure 2-2). Despite >90% Nestin expression in Ptc1+/- MB cells in vitro (Supplemental Figure 2-4), only rare Nestin+ cells could be identified in the tumours arising from orthotopically injected cell lines. Nestin+ and

S100β+ cells could also be observed when freshly dissociated Ptc1+/-p53-/- tumours or established

cell lines were injected subcutaneously into the flanks of NOD/SCID mice, suggesting that cells

of glial and stem cell phenotype are tumour derived, versus normal brain (Supplemental Figure

2-6).

Interestingly, despite the proliferative and phenotypic similarities between p53+/+ and p53-/-

Ptc1+/- MB cells in vitro, we observe a difference in their tumourigenic capacity based on their

respective p53 genotype. Thus far, we have not generated tumours in NOD/SCID recipients

when injecting up to 106 cells from Ptc1+/-p53+/+ cell lines.

Figure 2-2 Primary Ptc1+/- MB (Left) and tumours arising from injections of 1x105 MB cell lines (Right) were stained with (A) hematoxylin and eosin, or (B) DAPI, and immunostained for the expression of Nestin, Map2, GFAP, S100β, and CNPase.

2.4.3 Ptc1+/- MB Cell Lines Maintain Activated Hedgehog and Notch Signalling Pathways.

Ptc1+/- MB cell lines of differing genotypes (n=6) all demonstrated constitutively activated Hh

and Notch signalling, expressing all Gli transcripts, Ptc2, Hes1 and Hes5 mRNA (Figure 2-3A).

Expression of Gli proteins was observed, for Gli3 in full length (FL)/activator and repressor

(Rep) forms (Figure 2-3B). Growth of all cell lines tested was inhibited after treatment with the

Hh signalling inhibitor cyclopamine at 50% inhibitory concentrations (IC50) between 1-3μM,

correlating with downregulation of expression of Hh signalling components (Figure 2-3B).

Expression of all three Gli proteins was greatly reduced in serum-treated cells consistent with lack of activated Hh signalling in serum cultured Ptc1+/- MB cell lines [268] (Figure 3C).

Similarly, γ-Secretase inhibition by DAPT treatment resulted in proliferative inhibition (IC50

10μM) and downregulation of Notch target genes Hes1 and Hes5 indicating an active Notch signalling pathway in vitro (Figure 3C).

Figure 2-3 (A) Ptc1+/- MB cell lines express Hedgehog (Gli1, Gli2, Ptc2) and Notch (Hes1, Hes2) target genes as determined by RT-PCR (e14.5 HB, embryonic day 14.5 hindbrain tissue, GAPDH, control). (B) Proliferation (IC50 = 1-3μM) and expression of Hh target genes is inhibited by 5μM cyclopamine treatment. Serum treatment abolished protein expression of Hh targets (Gli1, Gli2, Gli3-Full Length (FL), Gli3-Repressor (Rep) & Actin protein analysis by Western; Ptc2 and GAPDH expression analysis by RT-PCR). (C) Proliferation (IC50 = 10μM) and expression of Notch target genes is inhibited by treatment with 10μM of the γ-secretase inhibitor DAPT (Hes1 and Hes5 expression analysis by RT-PCR, and confirmation of Hes1 expression by Real Time PCR).

2.4.4 Loss of Ptc1 Heterozygosity, or WT RNA Expression, is not

required for Ptc1+/- MB Development.

We genotyped our MB cell lines and in some cases we could detect a WT Ptc1 allele, and in others LOH for Ptc1 in multiple established cell lines, with or without loss of p53 (n=21 cell lines analyzed, Ptc1-/- n=11, Ptc1+/- n=10) (Figure 2-4A). MB cultures showed the same Ptc1 genotype when compared to the primary tumour from which they were derived (Figure 2-4B), and cell lines that retained the WT Ptc1 allele expressed WT Ptc1 mRNA when analyzed by RT-

PCR using exon2 specific primers (Figure 2-4C). Single cell sorting and clonal expansion of two lines, revealed that all subsequent clones (>10 analyzed) had the same genotype as the parental cell line, discounting the possibility of Ptc1 allelic heterogeneity within the bulk culture

(Figure 2-4D). Importantly, tumours developed when Ptc1 mRNA expressing cell lines Ptc+/- p53-/- 266 and 302 were injected orthotopically into NOD/SCID recipients demonstrating that tumourigenicity is not dependent on Ptc1 LOH (Supplemental Figure 6).

Figure 2-4 (A&B) Ptc1+/- MB cell lines were genotyped by PCR reaction of genomic DNA. The Ptc1 WT allele could be detected in some cell lines (+), and (B) in some primary tumours from which they were derived. (C) WT Ptc1 RNA could be detected in the cell lines that retained the WT Ptc1 allele as determined by RT-PCR reaction with WT specific primers. e14.5 HB, WT embryonic day 14.5 hindbrain tissue, negative control for LacZ/Neo cassette. Below, schematic showing the disruption of the WT Ptc1 allele by LacZ/Neo cassette. (D) Heterogeneity for the Ptc1 allele does not exist within established cell lines. Single cells were deposited by FACS, expanded clonally, and genotyped. All subclones demonstrated the same genotype as the parental cell lines from which they were derived.

2.4.5 CD15 Enriches for Proliferative Cells In Vitro and Tumourigenic

Cells In Vivo

Both normal human NSCs and human medulloblastoma CSCs can be enriched by cell sorting for

CD133 [161, 220] Human brain cancer cells are known to share other similarities, in

phenotype and behaviour, to that of normal NSCs [220, 239, 240, 247, 248] We sought to

determine if Ptc1+/- MBs contained a population of cells enriched for tumour initiating capacity,

and reasoned that the same cell surface markers that enrich for normal mouse NSCs may also

enrich for Ptc1+/- MB TICs. Multipotent mouse NSCs can be enriched from embryonic brains

using the carbohydrate cell surface antigen CD15 [214]. We reproduced this observation using

mouse embryonic hindbrain tissue (Figure 2-5A) and observed that CD15 is a more reliable

marker compared to Prominin-1 when cell sorting mouse NSCs (data not shown). Further, when

we analyzed freshly dissociated Ptc1+/- MB cells for the expression of Prominin-1 we were unable to appreciate a convincing Prominin-1+ population (Supplemental Figure 2-7).

Therefore, we selected CD15 as a candidate marker to enrich for Ptc1+/- MB TICs.

Freshly dissected, uncultured Ptc1+/- MB cells (n=11 independent tumours analyzed), were

stained with anti-CD15-FITC, propidium iodide and the erythroid lineage marker Ter119-APC.

Live, Ter119- and CD15+ or CD15- cells were sorted by fluorescence activated cell sorting. The

purity of sorted populations was verified post-FACS, and all CD15- populations were >95%

(mean 97%, median 98%) pure while CD15+ populations ranged in purity (mean 74%, median

75%) (Figure 2-5B). Cells were immediately cultured in NSC media or injected orthotopically into the cerebella of NOD/SCID mice. Unsorted, bulk Ptc1+/- cells from eight MBs were also

injected to determine the tumourigenic capacity of freshly dissociated tumours.

In vitro, freshly isolated tumour cells capable of growing in NSC media were enriched in the

CD15+ population, however after cultures were established both CD15+ and CD15- populations give rise to CD15 heterogeneity (Supplementary Figure 2-8). Similarly, the CD15+ population

transplanted the disease to NOD/SCID recipients in three of four orthotopic transplants at 100

000 cells injected, and in five of six transplants at 10 000 cells injected (Table 2-1). Injections

of CD15- cells only resulted in three tumours from eight injections at 100 000 cells and no

tumour formation in five attempts, comprising independent tumours, at 10 000 cells injected

(Table 2-1). Consistent with previous findings, unsorted bulk tumour cells demonstrated very

little capacity to transplant the disease at injections of 100 000 cells or more [266, 274], and no

tumours developed from injections of 10 000 cells (Table 2-1). No tumour formation was

observed for any population of cells when fewer than 10 000 cells were injected suggesting that

greater purification of the CD15+ population is required to further enrich for Ptc1+/- MB TICs.

Figure 2-5 (A) Representative FACS profiles of embryonic day 14.5 (e14.5) WT hindbrain (HB) cells stained for CD15-FITC and the erythroid lineage marker Ter119-APC. APC-negative (Ter119-APC-), CD15-positive (CD15+) or -negative (CD15-) populations were sorted by FACS. The purity of each population was determined immediately post sort, and both populations were plated in vitro. Seven days post sort, the number of neuro-spheres in each population was counted. Sphere assay data from 3 independent time-mated litters and sorts. * t test p<0.05. (B) Representative data from Ter119-APC-, CD15+ or CD15- sorted cells derived from a freshly dissociated, uncultured Ptc1+/- MB. The purity of each population was determined immediately post sort, cells were plated in mouse NSC media, and imaged 14 days post sort. CD15+ cells quickly grew to confluence.

Table 2-1 Bulk, unsorted Ptc1+/- MB cells, and freshly sorted, uncultured CD15+ or CD15- cells were injected orthotopically in the cerebella of NOD/SCID recipients at the indicated cell densities. Data indicates the number of tumours formed/number of injections at each density. Data set comprises injections from primary Ptc1+/- MBs, n=8 independent MBs for unsorted injections, n=11 independent MBs for CD15+ and CD15- injections. ND, not determined. *Statistically significant versus bulk, unsorted. χ 2= 7.639, p = 0.0057.

2.5 Discussion

Ptc1+/- mice closely recapitulate clinical observations made from Gorlin’s patients, both which

have been reported to display desmoplastic forms of the disease [47, 57]. Irradiated and p53-

deficient Ptc1+/- mice are thought to represent the aggressive, classical version of human MB

[58]. Approximately 20% of human MBs of the classic variant display aberrations within the

p53-ARF signalling pathway [275] and 10% of Li-Fraumeni patients develop MB within their

first decade of life [100]. Although MB is now being modeled in mice via biallelic deletion of

the Ptc1 gene [53], an analysis of clinical specimens suggests that this strategy models a limited

spectrum of the disease: Ptc1 mutations, or loss of heterozygosity for the Ptc1 allele, only occur

in a minority (~10%) of human MB [44, 47, 276]. Pietsch et al. detected these aberrations

exclusively within the desmoplastic MB variant, and not in any of 57 samples of classic

histopathology [47]. Modeling MB with p53-deficiency may allow a more representative

spectrum of the mutations observed in the aggressive and clinically important MB of classic

histopathology.

A stem cell based methodology revealed that a rare population of Ptc1+/- MB cells does have the

capacity to grow in vitro in serum-free conditions containing EGF and FGF, as is observed with

human MB cells. Paradoxically, Fogarty et al. reported that FGF, a principle component of

serum-free media that supports the growth of human MB cells and normal NSCs in vitro,

differentiates isolated Ptc1+/- MB cells and suggested that FGF may serve as an effective

treatment for human MB [267]. Read et al. recently reported that the inability to propagate

Ptc1+/- MB cells in stem cell conditions, and the failure of Prominin1 to identify TICs, suggested that MB propagating cells display a progenitor, and not stem cell phenotype [274]. The fact that

tumourigenic human MB cells with stem cell phenotype can be propagated in FGF-containing media [229, 239], and the results presented in our study, challenge these conclusions.

We observed that all Ptc1+/- MB cell lines growing in NSC media display a neural precursor

phenotype [142] and demonstrate the capacity for multilineage differentiation, including cultures

derived from single cells. Cells capable of establishing cultures from fresh tumours demonstrate

a stem cell phenotype, pointing to a more primitive population of cells which drive tumour

growth in vivo than unipotent GCPs. Consistent with this idea is the recent observation that

perivascular Nestin+ Ptc1+/- MB cells re-enter the cell cycle post irradiation to repopulate the

tumour in vivo [277], results that closely mirror the behaviour of human CSCs in vivo [245, 278].

Doubt regarding the utility of serum derived Ptc1+/- MB cell lines was first reported by Sasai et

al. from the observation that these cells do not retain activated Hh signalling in vitro, nor could pathway activity be restored when cell lines were injected in vivo [268]. Similar results have been observed with traditional serum grown human brain tumour cell lines compared to primary patient samples, providing further evidence that serum established and propagated cell lines

sometimes bear little resemblance to the disease from which they were derived [272]. In serum- free conditions normal NSCs demonstrate activation of numerous developmental pathways, including the Hh and Notch signalling [186, 187, 279]. It is perhaps not surprising then, that

activated Hh and Notch signalling is observed in our Ptc1+/- MB cell lines. In agreement with the

differentiating role of serum, expression of Hh pathway components was abolished when our

Ptc1+/- MB cell lines were treated with 10% serum. Unlike Ptc1+/- MB cells grown in 10-20%

serum, cell lines established and propagated in NSC media retain the activated Hh and Notch

signalling observed the in vivo disease [55].

We observed that some, but not all Ptc1+/- MBs show Ptc1 LOH. Importantly, cell lines

demonstrate the same genotype as the tumours from which they were derived, express WT Ptc1

RNA if they retain the WT allele, and initiate the growth of tumours in the absence of Ptc1 LOH.

These observations reconcile some of the debate in the past literature, and support both positions in that Ptc1 LOH does occur in the tumourigenic process, but is not absolutely required in every instance. This result suggests that these tumours are more genetically complex than widely believed, and that studies that only model the tumourigenic process in the context of Ptc1 LOH may not fully consider the repertoire of mutations or tumour initiating events arising in these mice. What are the additional contributing events which lead to MB in tumours that retain expression from the WT Ptc1 gene? Though not addressed in this study, possibilities include point mutations within the WT Ptc1 sequence [280] changes in post-transcriptional and/or post-

translational modifications, or aberrations in other components of the Hh signalling pathway as is

observed in the human disease [46].

Finally, the CSC hypothesis continues to attract debate [281], and the identification of CSCs

requires an enrichment in tumour initiating capacity compared to the bulk tumour population.

Recently, Read et al. reported that 3x105 unsorted and 3x105 CD15+ Ptc1+/- MB cells could

transplant the disease in vivo, while 3x105 CD15- cells did not [274]. Here we report that TICs from Ptc1+/- MBs are enriched within the CD15+ population. CD15+ cells reliably transplanted

the disease when 1x104 cells were injected, whereas CD15- and unsorted cells did not. As is consistent with TICs from human brain cancer, this same cell surface marker also prospectively identifies multipotent mouse NSCs from the forebrain and hindbrain [214]. These results recapitulate those made from CD133+ human brain TICs, although with a different marker, and

suggest that the identification of human CSCs is not simply a consequence of

xenotransplantation, or a failure of human cells to properly engage the mouse microenvironment.

We are the first to report that cells from Ptc1+/- tumours can be propagated long term in vitro in

serum-free conditions containing EGF and FGF, demonstrating stem cell properties, tumour

initiating capacity, and activated developmental signalling pathways. These results are in close

agreement with those obtained when stem cell based methodologies were applied to the study of

human cancer, and suggest that the Ptc1+/- MB tumour initiating cell demonstrates a stem cell,

and not lineage restricted phenotype. Our genetic analysis of tumours from Ptc1+/- mice indicates a molecular heterogeneity for Ptc1 LOH, and suggests distinct molecular mechanisms of MB initiation in this model. In summary, our stem cell-based interrogation of this clinically representative mouse model of cancer demonstrates that they recapitulate the functional hierarchy observed within human MB, with a tumour initiating population identified by a cell surface marker: a critical finding for this intensively studied preclinical therapeutic model.

Supplemental Figure 2-1 (A&B) Ptc1+/- MB cell lines grew adherent to plastic (A), but occasionally as free-floating spheres (B). (C) Addition of X-gal substrate indicated expression of β-galactosidase from the mutant Ptc1 allele suggestive of an activated Hedgehog signalling pathway.

Supplemental Figure 2-2 Ptc1+/- MB cells (LacZ/Neo+) were directly transplanted into the cerebellum of a NOD/SCID recipient (LacZ/Neo-). The resulting tumour was plated in vitro at low density. Distinct colonies were isolated, expanded separately and genotyped. PCR reaction demonstrated that all clones contain the Ptc1+/- derived LacZ/Neo cassette and demonstrated Ptc1 loss of heterozygosity (LOH) confirming their MB origin.

Supplemental Figure 2-3 Ptc1+/- MB cells grown in NSC media stained for (A) Nestin, Sox2, Math1, Musashi, GFAP and DAPI. (B) After EGF and FGF withdrawal, cells expressed markers of mature glial (s100β, CNPase) and neuronal (βIII-tubulin) cell lineages.

Supplemental Figure 2-4 (A) Intracellular flow cytometry demonstrating >90% Nestin expression in cell lines when grown in stem cell media (mNSC), and no expression after 7days in 10% serum. Left: non- specific fluorescent signal from secondary antibody alone; Centre and Right: specific staining for Nestin. (B) In 10% foetal bovine serum, the proliferation of Ptc1+/- MB cell lines was significantly inhibited (n=3 independent MTT assays), and (C) cells adopted a large, flattened morphology. (C) Cells lost detectable expression of Nestin, but expressed Math1, and the neuronal lineage marker Map2. (*t test p<0.05).

Supplemental Figure 2-5 Media from GFAP-TK+ Ptc1 MB cells with or without GCV was collected two weeks after the primary tumours were initially treated. Conditioned media was mixed 1:1 with new media and added to a freshly dissociated GFAP-TK- Ptc1 MB. Conditioned media had no effect on the clonogenic capacity of GFAP-TK- cells.

Supplemental Figure 2-6 (A) 5x104 cells from fresh tumours or cell lines were injected subcutaneously. Resulting tumours were analyzed for Nestin and S100β expression. (B) Cell lines that retain expression from the WT Ptc1 allele initiate tumours when injected into the cerebellum of recipients. Right: scale in cm. Right and left: * indicates location of MB.

Supplemental Figure 2-7 Representative anti-Prominin1-PE staining of primary Ptc1+/- MB. Data from three independent tumours are shown, including unstained and isotype (Rat IgG1-PE) controls.

Supplemental Figure 2-8 (A) Freshly dissociated CD15+ or CD15- cells were seeded in 96 well plates at 5x104 cells/well monitored in culture by time lapse photo microscopy. Cells capable of proliferating in serum-free conditions are enriched in the CD15+ population. (B) Both the CD15+ and CD15- populations regenerate heterogeneous CD15 expression after limited expansion in vitro (<4 passages).

Chapter 3 3 Percoll Density Centrifugation Separates Functionally Distinct CD15+ Patched-1 Mouse Medulloblastoma Cells.

3.1 Abstract

Human and mouse brain CSCs are reported to display a neural stem cell phenotype and behaviour. Interestingly, the phenotype and behaviour of CSCs identified from Patched-1 heterozygous mouse medulloblastomas remains debated, as being that of either a unipotent neuronal precursor or a more primitive multipotent stem cell. Here we demonstrate that CD15+ tumour initiating cells exist in both interfaces of a two-step Percoll centrifugation gradient which separates small, dense cells of granule neuron phenotype in the bottom interface, from larger, more complex cells of astroglial phenotype in the top interface. However, cells isolated from the top interface are tumourigenic and enriched for CD15+ cells and clonogenic capacity in vitro. These studies suggest that the CD15+ population is heterogeneous, and that cells of stem cell phenotype are enriched with the capacity to drive Patched-1 medulloblastoma growth.

3.2 Introduction

Medulloblastoma is a tumour of neuronal phenotype most often occurring in the cerebellum of children. In the early postnatal mammalian hindbrain, Patched-1 is a receptor for the Sonic Hedgehog ligand which mediates a proliferative signal upon unipotent granule cell precursors of the external granule layer [141]. Patched-1-deficient mice develop medulloblastoma that recapitulate many features of the human disease [48]. These tumours have been shown to arise from either unipotent Math1+ CGCPs or from multipotent GFAP+ or Olig2+ stem cells which reside within the rhombic lip [53, 54], and may or may not depend on loss of Ptc1 heterozygosity [59, 60, 62, 215].

Cancer stem cells, tumour initiating cells, or tumour propagating cells comprise a tumourigenic population enriched with the capacity to transplant the disease in vivo [224]. Human brain CSCs were prospectively identified from MBs using the cell surface marker CD133 [220]. These MB CSCs could be propagated in vitro in the same conditions that support the growth of normal human neural stem cells (in serum-free media containing EGF and bFGF) [239] and demonstrated multilineage differentiation capacity in vitro [229, 239] and in vivo [220].

CSCs were recently prospectively identified from Ptc1+/- MBs using the stem cell marker CD15 [215, 274]. Interestingly, a significant difference regarding the phenotype and behaviour of Ptc1+/- MB CSCs is suggested in these two publications: Read and colleagues suggest that CD15+ CSCs are of unipotent cerebellar granule cell precursor phenotype while our analysis suggested that they demonstrate a more primitive stem cell phenotype and behaviour.

When studying the postnatal cerebellum, a two-step (35%/65%) Percoll gradient can be used to physically separate small dense granule neurons and their precursors (CGCPs), which are isolated from the bottom interface of the gradient, from larger more complex astroglia, which are isolated from the top interface of the gradient (Figure 3-1) [282]. The conclusion that the Ptc1+/- MB CSC is of CGCP phenotype may relate to the isolation and study of cells from only the bottom fraction of a Percoll gradient [274]. Here we demonstrate that cells isolated from the top interface of the gradient are tumourigenic, enriched for CD15+ TICs and in vitro clonogenicity when compared to cell isolated from the bottom interface of the gradient. These results demonstrate that physical and functional heterogeneity exits within the CD15+ TIC population of

Ptc1+/- MB and suggest that cells of stem cell phenotype are enriched with the ability to drive MB growth.

3.3 Materials and Methods

MBs from Ptc1+/- mice were processed as previously described [215]. Briefly, tumours were microdissected, mechanically dissociated, and sequentially filtered (70μm then 40μm mesh filters (BD Biosciences)) to derive a single cell suspension. 1x107 cells were under layered with a two step 35%/65% Percoll density gradient, in 50ml conical tubes. Cells were spun for at least 15min at 1000RCF and cells from both interfaces were collected, washed twice in PBS and counted by haemocytometer. Cells were 1) plated in limiting dilution analysis assays in vitro in serum-free stem cell media containing EGF and bFGF as previously described [215]; 2) harvested for standard protein analysis by Western blot; 3) stained with anti-CD15-FITC and propidium iodide and analyzed by flow cytometry using a BD LSRII cytometer as previously described [215] and; 4) suspended in 200μl PBS and injected into the subcutaneous flanks of NOD/SCID recipients. At six week post injection tumours were harvested, fixed in 4% PFA and processed by standard immunohistochemical protocols.

Figure 3-1 Cartoon representation of a two-step (35%/65%) density-centrifugation Percoll gradient. Small dense cells can be isolated from the bottom (35%/65%) interface of the gradient, larger filamentous cells can be found at the top (PBS or Media/35%) interface and debris and red blood cells spin through to the bottom of the tube [282].

3.4 Results

A two-step Percoll gradient has been used to physically separate small, dense granule neurons and their precursors (CGCPs) and larger, more complex astroglial cells, from the normal cerebellum and mouse MBs [60, 282]. To determine if a Percoll gradient separates functionally and phenotypically distinct tumour cell populations, we dissected, mechanically dissociated and sequentially filtered (70μm then 40μm) primary Ptc1+/- mouse medulloblastomas (n=5) to derive single cell suspensions. 1x107 cells were then under layered with a two-step 35%/65% Percoll gradient in a 50ml conical tube, and spun at 1000RCF for at least 15min, as previously described [60, 267]. Cells from both interfaces were harvested, washed in PBS and counted. We observed that approximately 25% of all cells recovered from the gradient were found at the top interface. Cells were harvested for protein analysis, or directly plated in vitro in primary limiting dilution analysis assays in EGF- and bFGF- containing serum-free media [150]. As previously reported, filamentous GFAP+ cells are separated and enriched at the top interface of the Percoll gradient (Figure 3-2A). Two weeks after plating cells in vitro, wells were scored as containing at least one proliferating adherent colony, or not, and the average percentage of wells with colonies at each cellular density was calculated. We observed that in vitro clonogenic cells were significantly enriched within the top interface of the Percoll gradient (Figure 3-2B&C), and that in three of the five tumours tested the small, dense cell population isolated from the bottom interface of the Percoll gradient had little capacity to generate cultures in vitro, and aggregated into clusters that did not go on to proliferate even at relatively high cell densities (1x105 and 5x104 cells/well) (Figure 3-2B&C).

To exclude the possibility that cells derived from the bottom layer of the interface were less viable compared the cells found at the top interface we stained cells with the cell viability marker propidium iodide. Flow cytometry analysis revealed that cells harvested from both interfaces contained a similar percentage of PI-negative/live, single cells immediately after centrifugation (Figure 3-3A). Additionally, staining live cells with anti-CD15-FITC demonstrated that CD15+ cells could be found in both fractions of the gradient (Figure 3-3B) but were enriched within the top fraction (Figure 3-3B&C, average fold enrichment 1.43, n=4, t-test p=0.0176) of the Percoll gradient.

Freshly dissociated Ptc1+/- MB cells can initiate the growth of representative tumours in recipient mice at both orthotopic and subcutaneous sites of injection [215, 266, 274]. We next tested the tumourigenic ability of cells from each fraction and injected 2x105 cells from each layer subcutaneously into the right flank of NOD/SCID mice, and six weeks later mice were sacrificed due to the presence of obvious tumours. In 4 of 4 injections of cells isolated from the top layer of the Percoll gradient large subcutaneous tumours were apparent, and in 3 of 4 injections of cells isolated from the bottom layer tumours could be found, though one was exceptionally small (Figure 3-4).

Figure 3-2 Astrocytic cells derived from the top interface are enriched with in vitro clonogenic capacity compared to cells isolated from the bottom interface. (A) Western blot for the astrocytic marker GFAP or βactin in protein lysates of Ptc1+/- MB cells isolated from the top or bottom interface of a Percoll gradient. (B) 1x107 freshly dissected and dissociated Ptc1+/- MB cells (n=5 independent tumours) were loaded onto a two-step Percoll gradient (5ml 35% Percoll, 5ml 65% Percoll, in a 50ml conical) and spun at 1000RCF for 15 min. Cells were collected from each interface, washed twice in 40ml PBS and plated in a limiting dilution analysis assay, in neural stem cell conditions (serum-free media with 20ng/ml EGF and 20ng/ml bFGF) as previously described [215]. Two weeks thereafter, wells were scored as either containing a proliferating colony or not, and the average number of wells with colonies calculated. *t-test, top interface vs. bottom interface, p-value <0.05. (C) Representative images of wells two weeks post initial in vitro plating at 5x104 cells/well. Proliferating colonies are clearly apparent from cells derived from the top interface of the Percoll gradient, whereas cells isolated from the bottom interface show little in vitro clonogenic capacity.

Figure 3-3 Gating strategy to identify live, CD15+ medulloblastoma cells. Cells were spun in a Percoll gradient as described above, harvested from either interface, washed with PBS, stained with (A) 2μl/ml Propidium Iodide (PI) and (B) PI and anti-CD15-FITC antibody as previously described [215]. A) Single cells were selected (hierarchical Gates 1 and 2), and their viability was analyzed by propidium iodide staining (hierarchical Gate 3). Equivalent percentages of live single cells can be isolated from both top and bottom fractions of the Percoll gradient. B) Single, live cells were analyzed for the expression of CD15. CD15+ medulloblastoma cells can be found in both fractions of a Percoll gradient, and are enriched in cells isolated from the top fraction of the gradient (B&C).

Figure 3-4 H&E staining of individual tumours arising after injection of cells isolated from either top or bottom interface of a Percoll gradient. 2x105 cells from either interface were injected into the subcutaneous flank of NOD/SCID mice. Six weeks later, mice were analyzed for the presence of tumours. Large tumours were easily apparent in four of four mice injected with cells from the top interface, however tumours were observed in only three of four mice injected with cells from the bottom interface, one of which was exceptionally small (top right). Scale bar, 0.5cm.

3.5 Discussion

Physical purification of brain cells using a two-step Percoll density centrifugation gradient was first described for the purpose of separating neurons from glia after dissection of the post-natal cerebellum [282]. This methodology was reported to rapidly isolate distinct populations of cells, as larger astroglia could be harvested from the top interface and denser cerebellar granule neurons, and their precursors (CGCPs) could be isolated from the bottom interface. The application of the Percoll gradient to the study of medulloblastoma was first published by Oliver and colleagues with their report that 50-600 million CGCP-like MB cells could be isolated from the bottom fraction of the gradient [60]. These cells were later reported to differentiate in the presence of bFGF, when studied up to 72hrs after initial dissection, and it wasn’t thought possible to propagate Ptc1+/- MB cells in vitro in the presence of this growth factor [267]. Recently, Read and colleagues demonstrated that CD15 could be used to identify a population of TPCs, but they again studied cells isolated from the bottom fraction of the gradient [274]. Consistent with Fogarty and colleagues, they reported that CD15+ TPCs could not be grown in vitro in serum-free media containing bFGF, and did not have any capacity for multilineage differentiation. Accordingly they concluded that Ptc1+/- MB TPCs were of precursor phenotype, resembling the Math1+ CGCP.

In a similar set of experiments, we also observed that the CD15+ subpopulation of freshly dissociated Ptc1+/- MB cells was enriched with tumour initiating capacity [215]. In our analysis, which did not included a Percoll purification step, we identified multipotent and phenotypically primitive Nestin+Sox2+ cells in primary Ptc1+/- MBs, freshly dissociated cell suspensions, in established cell lines propagated in serum-free conditions containing EGF and bFGF, and in secondary tumours derived from the injection of both cultured and uncultured MB cells. Though these cells also expressed the precursor marker Math1+, their behaviour and phenotype was consistent with that a stem cell, accordingly we suggested that the Ptc1+/- MB CSC displayed physical and functional properties more similar to multipotent cerebellar stem cells [215].

One explanation for these different observations may be the different methods of processing freshly dissociated cells. In their study, Read and colleagues were unable to generate neurosphere cultures when cells were plated at low density (10 cells/μl) and analyzed after 10 days in vitro. In contrast, we observed that cells grew readily, but adherent to plastic and not as

spheres, and calculated that the in vitro clonogenic frequency of multipotent cells within the tumours to be only one in many thousand cells [215]. Therefore, our ability to generate cell lines may relate to the fact that adherent cultures are preferentially established from these tumours at a low frequency. Another explanation may relate to the functional consequence of purifying and studying MB cells harvested from the bottom interface of a Percoll gradient. As far as we are aware the functional properties of Ptc1+/- MB cells isolated from the top interface of the Percoll gradient have not been previously reported.

Here we demonstrate that the population of cells isolated from the top fraction of a Percoll gradient are clonogenic in vitro in serum-free conditions containing EGF and bFGF, contain an equivalent number of viable, single cells as the population of cells isolated from the bottom fraction, contain CD15+ cells, and are tumourigenic when injected into NOD/SCID recipients. Interestingly, cells isolated from the bottom fraction of the Percoll gradient also contained CD15+ cells, but demonstrated a significantly reduced capacity to establish colonies in vitro, and only generated tumours in three of four recipients when injected in vivo. Though not thoroughly analyzed with a full set of in vivo limiting dilution analysis experiments, our data may indicate that tumour initiating capacity also exists in the population of MB cells isolated from the top interface of a Percoll gradient.

A question remains regarding the nature of cells isolated at either interface of the Percoll gradient. The in vitro clonogenic capacity of isolated cells is distinct, yet in vivo both populations can give rise to tumours. One possible explanation for this observation is that there exist two distinct populations of TICs, perhaps with differing tumourigenic potencies: one with a stem cell phenotype and behaviour, the other with a precursor phenotype and properties. Support for this possibility comes from observations describing the development of Ptc1+/- MB from both GFAP+ or Olig2+ stem cells, or Math1+ precursors [53, 54]. Thus it is conceivable that both populations of TICs could exist within a tumour, representing a hierarchical organization in the tumour initiating population.

Our results suggest that studying the population of cells found at the bottom interface of the Percoll gradient neglects an important tumourigenic and clonogenic fraction of cells. MB cells of stem cell phenotype, and multilineage differentiation capacity have also been suggested by

others [54, 277, 283], therefore all cell fractions should be evaluated when characterizing the TIC population.

Our data has clear implications for the study of human MB. Ptc1+/- MBs are thought a representative model of the disease, yet results derived from experiments excluding the population of cells isolated from the top interface of the Percoll gradient seem at odds with observations made from human samples; for example that these cells differentiate in the presence of bFGF [267] and contain unipotent CSCs that demonstrate a precursor phenotype and behaviour [274]. Human MB CSCs are phenotypically primitive, multipotent in vitro and in vivo, and can be propagated as tumourigenic spheres in bFGF-containing media [220, 229, 239, 242]. Accordingly, an unbiased analysis of all the phenotypically diverse Ptc1+/- MBs is a powerful tool for understanding the functional hierarchy existent within tumours of this phenotype.

Chapter 4 4 Cellular, Molecular and Chemical Profile of Clinically Representative Cancer Stem Cells from a Chemical- Genetic Mouse Model of Glioma.

4.1 Abstract

High grade glioma is the most frequent and aggressive form of brain cancer occurring in adults. We are interested in studying brain CSCs and identifying novel therapeutic approaches in representative preclinical models of the disease. We treated neural-specific p53-deficient mice in utero with the chemical carcinogen N-ethyl-N-nitrosourea and all exposed mice developed spontaneous brain tumours by 6 months of age. Like human tumours, primary mouse gliomas were phenotypically and functionally heterogeneous and contained clonogenic glioma stem cells enriched in the CD15+ fraction of freshly dissociated tumours. When expanded as adherent stem cell cultures in vitro, cells displayed a heterogeneous neural precursor phenotype and could initiate the growth of representative tumours when as few as 1000 cells were injected orthotopically. Microarray expression analysis identified the novel, p53-independent Shox2/Arx/Irx gene regulatory network as commonly dysregulated in both mouse and human glioma stem cells. Finally, we identify numerous compounds capable of selectively inhibiting mouse and human glioma stem cell growth, but not fibroblast controls. Together our results identify the phenotypic, functional, molecular and chemical profiles common between mouse and human glioma stem cells, identifying novel molecular mediators of glioma and compounds that may prove effective in the treatment of the disease.

4.2 Introduction

Gliomas are primary human brain tumours containing cells of glial phenotype [1]. Grade four gliomas, called glioblastoma multiforme, are one of the most aggressive and therapeutically refractory types of cancer occurring in adults, with a median survival of ~15 months even after intensive therapy [63]. Large scale genomic profiling of GBM samples revealed that genes within three main signalling pathways were commonly deleted/mutated in a high percentage (>75%) of all tumours tested: the EGFR/PI3K, p53 and RB signalling pathways [74]. Individual genes that were commonly deleted/mutated were also identified, one of the most prevalent being that of the tumour-suppressor protein p53, demonstrating alterations in 35%-40% of all samples tested [73, 74].

Cancer stem cells, tumour-initiating cells or tumour propagating cells are defined as the tumourigenic population of cancer cells that have the capacity to regenerate a phenocopy of the original tumour in vivo [224, 271]. Human brain CSCs were first prospectively identified by isolating the CD133+ cell population within freshly dissociated tumours, and were enriched with tumour initiating capacity in vivo, and clonogenic capacity in vitro [220, 229]. CD133+ CSCs are thought to be relatively resistant to irradiation, chemotherapy and the likely source of disease recurrence [245, 246, 284]. More recently, human GBM and mouse medulloblastoma CSCs were prospectively identified using the cell surface marker CD15, an epitope that enriches for a number of stem cell types including both mouse and human neural stem cells [214, 215, 274, 285]. When grown on laminin-coated plates in serum-free media containing epidermal growth factor and basic fibroblast growth factor, human brain CSCs can be propagated long-term as a relatively homogeneous population of tumourigenic ‘NS’ cells, and utilized in chemical and genetic screens for biological and drug discovery purposes [201].

To study high grade glioma, and to identify novel therapeutics in preclinical models of the disease, a number of mouse models of glioma have been developed [102, 117, 119, 286]. Older strategies to induce high grade glioma in rodents involved the administration of carcinogen directly into the brains of animals [287]. Transplacental administration of the N-nitroso carcinogen N-ethyl-N-nitrosourea to pregnant rats was proven an effective method to generate gliomas in wild-type pups, however brain tumours were only observed in p53-/- mice when following this protocol [99, 288]. More recently, genetic strategies to delete tumour-suppressor

genes and/or overexpress oncogenes in targeted cellular compartments have been utilized to generate aggressive brain tumours in mice. However, in some cases these mouse models exploit genetic aberrations that are observed in the human disease only very infrequently [117, 118].

We were interested in generating a diverse and representative mouse model of high grade glioma. We administered ENU to p53-/- mice in utero to generate high incidence, and functionally and phenotypically heterogeneous gliomas. Clonogenic GM cells were enriched in the CD15+ population of freshly dissociated tumours and adherent, tumourigenic NS cell cultures were established and studied. Mouse glioma NS cell lines closely recapitulate the patient- specific phenotypic and functional diversity observed in human glioma NS cell lines [201]. Comparative microarray analysis of mouse GM NS cells, human GM NS cells, and embryonic NS cell controls separated a p53-deficient gene signature from a glioma-gene signature and elucidated a dysregulated Shox2/Arx/Irx gene regulatory network, known to be critically important in developmental regionalization of the brain. Finally, we screened approximately 700 neuroactive compounds and identified numerous chemicals that selectively inhibit the proliferation of mouse and human glioma NS cells, but not fibroblast controls. Our study harnesses the utility of modeling glioma in mice, uncovers novel molecular mediators of human and mouse glioma, and identifies promising chemical compounds that can be rapidly evaluated in vivo in this representative preclinical model of the disease.

4.3 Materials & Methods

4.3.1 Mouse Husbandry, Mating Strategies & Carcinogen Administration

p53+/- mice and Nestin-Cre+/- mice were obtained from the Jackson Laboratory. p53floxed mice were obtained from the National Cancer Institute at Frederick Animal Health Diagnostic Laboratory. Ptc+/- were provided by C.c. Hui at the Hospital for Sick Children, Toronto, Canada as previously described [215]. All animals were housed and utilized in accordance with the Hospital for Sick Children’s Animal Care Committee guidelines. Timed-pregnant females were injected intra-perotoneally with freshly prepared 25mg/kg ENU (Sigma-Aldrich) (in PBS) at indicated embryonic time points as previously described [99].

4.3.2 Orthotopic Injections of Freshly Dissociated Tumours and Established Cell Lines

Mice demonstrating neurologic symptoms were sacrificed by CO2 asphyxiation and whole brains were immediately dissected into PBS. Tumours were microdissected under magnification, dissociated by mechanical and Accutase (Sigma-Aldrich) enzymatic tissue disruption, and passed through 70μm and 40μm nylon filters (BD Biosciences) to generate a single cell suspension. Established cell lines were suspended in PBS following a brief Accutase digestion. Cells were counted by hemocytometer and kept on ice until injected. NOD/SCID mice were obtained from the Centre for Phenogenomics (Toronto, Canada) and were prepared for stereotaxic surgery as previously described [215, 220]. Glioma cells were injected in a maximum volume of 2μl into the cortex of NOD/SCID recipients at stereotaxic co-ordinates 2mm anterior to the coronal suture, 3mm right of midline and 3mm deep relative to the surface of the brain.

4.3.3 Fluorescent Activated Cell Sorting of Mouse Gliomas, Establishment of Glioma Stem Cell Lines In Vitro and Differentiation Assays

Primary mouse brain tumours were dissociated to a single cell suspension as described above. For FACS, cells were stained with CD15-FITC (BD Biosciences, Clone MMA), Ter119-APC (BD Biosciences) at ratios of 1:500 and 1:100 in cold PBS, respectively, for at least 30 minutes on ice. Cells were washed and suspended in cold PBS containing 2μl propidium iodide/ml and

sorted with a Becton Dickinson FACS Aria into growth factor-free DMEM/F12 (Wisent). Sorted and unsorted cells were plated on 0.1% gelatin coated Primaria (BD Biosciences) culture dishes in mouse neural stem cell media (including 20ng/ml EGF and 20ng/ml bFGF). Cell lines were propagated on gelatin coated Primaria dishes in mouse neural stem cell media and routinely subcultured post brief Accutase digestion and suspension. Differentiation was induced by growth factor withdrawal with or without the addition of 10% serum as previously described [201, 203, 215].

4.3.4 Histology, Immunocytochemistry and Intracellular Flow Cytometry

Histological tissue samples were generated via standard protocols for paraffin embedded or frozen sections. H&E staining was performed by standard methods. Immunohistochemistry was performed with indicated primary and secondary antibodies (Supplementary Methods). Cells for immunocytochemistry were grown on poly-L-ornithine/laminin coated plates. Cells for immunocytochemistry and intracellular flow cytometry were fixed briefly with 4% paraformaldehyde at room temperature and stained with indicated primary and secondary antibodies in 10% normal goat serum and 0.5% Tween (Supplementary Methods).

4.3.5 Microarray Analysis

Three independent e14.5 p53+/+ NS (A1, A2, A3) and e14.5 p53-/- NS (223-3, 225-1, 225-6) cell lines were generated as previously described [203, 204]. RNA from early passage (p<5) e14.5 p53+/+ NS, e14.5 p53-/- NS cells and mouse glioma NS cells (NC-156-G, NC-159-G, NC-166-G) was harvested and gene expression was analyzed at the Hospital for Sick Children’s The Center for Applied Genomics (TCAG, http://www.tcag.ca) with Affymetrix GeneChip Mouse Genome 430 2.0 microarray chips (Santa Clara, California). Human microarray data was previously generated [201]. Data and ANOVA statistical analysis was performed using Partek Genomics Suite (St. Louis, Missouri) and Microsoft Excel (Redmund, Washington). Microarray expression data was validated by Real-Time PCR using the SA Biosciences Mouse Cancer PathwayFinder RT² Profiler (Frederick, Maryland). Average-linkage unsupervised and supervised clustering of microarray data was performed using Cluster 3.0 (http://rana.lbl.gov/EisenSoftware.htm) and visualized with JavaTreeview (version 1.1.4r3, http://jtreeview.sourceforge.net/). Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO)

analysis was performed using the DAVID Bioinformatics Database (http://david.abcc.ncifcrf.gov/home.jsp) [289, 290].

4.3.6 BIOMOL Chemical Library Screen of Mouse and Human Glioma Stem Cell Lines

Human glioma NS cell lines were grown as previously described [201], and human and mouse fibroblasts were grown in DMEM media supplemented with 10% foetal bovine serum (Wisent). Cells were plated at a density of 1000 cells/well in 384 well plates and screened with the BIOMOL International Neurotransmitter Chemical Library (Enzo Life Sciences, Farmingdale, New York) at a final chemical concentration of 3μM/well. On day five, a standard Alamar Blue (Invitrogen) assay was performed to identify chemicals that inhibited cell growth, and individual wells were checked visually to confirm hits.

4.4 Results

4.4.1 Chemical mutagenesis of p53-deficient embryonic mice generates glioma, as well as other tumour types.

We were interested in generating a diverse array of clinically representative high grade gliomas in mice. Oda and colleagues reported that the administration of 25mg/kg ENU to p53-/- pups in utero generated high incidence (~70%) glioma, but did not report any association regarding the developmental timing of carcinogen administration and tumour incidence although their sample size was limited (n=17) [99]. We repeated these experiments, but included a more thorough analysis of the incidence of glioma and the timing of ENU administration. We mated p53- heterozygous (p53+/-) mice, injected timed-pregnant females with 25mg/kg ENU at e12.5 to e18.5 and followed the exposed pups for a minimum of twenty weeks after birth or until signs of illness were apparent, at which point mice were sacrificed and subjected to general necropsy and H&E analysis of serial brain sections.

We observed that all but one p53-/- mouse, irrespective of when ENU was administered, were dead by 20 weeks of age succumbing to gliomas, thymic lymphomas, lung tumours and sarcomas as previously described (Table 4-1, Figure 4-1, Supplemental Figure 4-1) [99]. Histological analysis of ENU-treated p53-/- brains revealed the presence of tumours associated with the neurogenic regions of the brain: the lateral ventricles of the forebrain or the dentate gyrus of the hippocampus (Figure 3-1) [149]. Tumours were predominantly observed in animals receiving ENU at e12.5, e13.5 and 14.5, but also in 2 of 4 mice treated at e18.5 (Table 4-1, Figure 4-1). In contrast, 26 of 28 ENU-treated p53+/- littermate controls survived without symptoms to 20 weeks, and none demonstrated any abnormalities indicative of brain cancer (Table 4-1).

Table 4-1 Survival and brain tumour incidence of p53-/- and p53+/- mice treated with ENU in utero at different embryonic (e) days. Timed pregnant females were injected with 25mg/kg ENU at the indicated times and the resulting pups were followed for signs of illness. Brain tumours were predominantly observed in pups exposed to ENU at earlier developmental stages. Conversely, no brain tumours were observed in ENU exposed p53-heterozygous littermate controls.

Figure 4-1 H&E staining of p53-deficient adult mouse brains after exposure to ENU at indicated embryonic (e) days. Hypercellular regions (circled) occurred in close proximity to neurogenic regions of the brain: the lateral ventricles of the forebrain or the dentate gyrus of the hippocampus.

4.4.2 ENU Administration to Ptc1+/- Mice Generates High Incidence Medulloblastoma.

Hedgehog pathway activity is reported to regulate the growth and survival of human GBM cells [248], therefore we questioned if administration of ENU to mice with aberrant Hh signalling might also induce glioma formation. Ptc1+/- mice spontaneously develop medulloblastoma and other tumour types [48], but there are no reports describing gliomagenesis in this mouse model. However, brain tumours in these mice demonstrate activated Hh signalling [48, 291]; therefore we treated timed-pregnant Ptc1+/- mice with 25mg/kg ENU at e10.4, e14.5 and e17.5 and monitored Ptc1+/- pups for signs of illness. Untreated Ptc1+/- mice spontaneously developed MB at a frequency of 38% by 28 weeks, and in utero ENU exposure did not generate glioma at any treatment time point (0/40). Interestingly, administration of ENU at e17.5 did not alter the frequency of MB arising in these mice however administration of ENU at either e10.5 or e14.5 increased MB frequency to 90% in both groups, similar to the observation that early post-natal irradiation can augment the frequency of MB in Ptc1+/- mice [52] (Supplemental Figure 4-1).

4.4.3 Tissue Specific p53-Deletion and Chemical Mutagenesis Generates High Incidence Glioma in the Absence of Other Tumour Types.

We were interested in generating high incidence glioma in mice in the absence of other tumour types, therefore we mated Nestin-Cre+;p53+/- mice with p53flox/flox mice to specifically delete p53 in the nervous system of pups. As glioma frequency in p53-/- ENU-exposed pups was greatest between e12.5 and e14.5 (Table 4-1), we administered 25mg/kg ENU to timed-pregnant females at e13.5 and followed pups for signs of illness. Beginning at 10 weeks of age, ENU-treated Nestin-Cre+;p53flox/- (ENU+NC+p53f/-) mice demonstrated neurological symptoms (ataxia/paralysis) and domed heads necessitating their sacrifice (Figure 4-2A). Histological analysis of ENU+NC+p53f/- brains revealed that all mice harboured brain tumours (10 GM and 1 MB) located in close proximity to neurogenic regions of the brain, with a median survival of approximately 20 weeks and in the absence of tumours of other types (Figure 4-2B, Supplemental Figure 4-3). Each tumour was given a unique identification, based on the genotype of the mouse from which it was derived (NC+p53f/-. NC), and the histological appearance of the primary tumour (Glioma: G, Medulloblastoma: MB). Three ENU+NC+p53f/- tumours (NC-148-G, NC-169-G and NC-171-MB) were stained for the expression of precursor

(Nestin), astrocytic (GFAP, S100β), oligodendroglial (CNPase) and neuronal (βIII-tubulin) cell markers (Figure 4-2C). Gliomas demonstrated robust but heterogeneous staining for glial markers GFAP, CNPase and S100β, and limited expression of Nestin. NC-171-MB expressed high levels of βIII-tubulin, but also Nestin and GFAP consistent with heterogeneous neuronal and glial/precursor marker expression in mouse models of MB [215].

Figure 4-2 Brain tumours occurring in ENU exposed Nestin-Cre;p53flox/- (ENU+NC+p53f/-) mice. (A) ENU+NC+p53f/- mice all succumbed to brain tumours, in the absence of other tumour types, with a median survival of 19.3 weeks. (B) H&E staining of ENU+NC+p53f/- gliomas (G) and medulloblastoma (MB). NC-148-G, NC-156-G, NC-163-G and NC-169-G are shown at lower magnification and tumours are circled with dashed lines. NC-159-G and NC-171- MB are shown at higher magnification. (C) Phenotypic analysis of primary ENU+NC+p53f/- brain tumours in situ. Cells were stained with stem (Nestin), astrocytic (GFAP & S100β) oligodendroglial (CNPase) or neuronal (βIIITubulin) cell lineage markers. DAPI, nuclear marker.

4.4.4 ENU+NC+p53f/- Gliomas are Tumourigenic and Demonstrate Functional Heterogeneity In Vivo.

Human brain tumours can be propagated long term in vivo when injected into the brains of immune-compromised mice [220, 239, 240]. We were interested in determining if gliomas arising in ENU+NC+p53f/- mice were similarly tumourigenic and injected 1x105 freshly dissociated cells into the forebrains of NOD/SCID recipients. Two independent tumours were tested, both generating tumours within 12 weeks of injection in primary, secondary and tertiary NOD/SCID recipients when 1x105 cells freshly dissociated cells were injected (Data not shown).

Human brain tumours demonstrate a cell-dose dependent capacity to transplant the disease in vivo [243], indicating functional heterogeneity within the bulk tumour population. To determine the tumourigenic capacity of freshly dissected and dissociated glioma cells, we performed an in vivo limiting dilution analysis of ENU+NC+p53f/- gliomas and injected 5x105 to 1x103 cells orthotopically into NOD/SCID recipients (Table 4-2). Similar to human gliomas [220, 243], we observed a cell-dose dependent correlation with tumour formation in vivo, with high cell densities (5x105 cells/mouse) generating tumours in all recipients (3/3) while low cell densities (1x103 cells/mouse injected) showing more limited capacity to transplant the disease (1/3).

Table 4-2 In vivo limiting dilution analysis of freshly dissected and dissociated ENU+NC+p53f/- mouse glioma cells. Mouse gliomas were dissected and dissociated to generate a single cell suspension and directly injected orthotopically into NOD/SCID recipients. Mouse glioma cells demonstrated a cell-dose dependent capacity to initiate tumours in recipients, suggesting functional heterogeneity within the primary tumour cell population.

4.4.5 CD15 Enriches for Clonogenic Mouse Glioma Cells In Vitro

Cancer stem cells from human brain tumours were first prospectively identified by isolating the CD133+ population within freshly dissociated patient samples [220]. More recently, CD15 was reported to functionally enrich human glioma CSCs and mouse medulloblastoma CSCs both in vitro and in vivo [215, 243, 274]. We were interested in determining if a functional hierarchy existed within ENU+NC+p53f/- mouse brain tumours and FACSed freshly dissociated tumours to isolate CD15+ and CD15- cell populations. Each population was cultured in a limiting dilution assay in 96-well plates and grown in serum-free media containing EGF and bFGF. Two weeks thereafter, each well was evaluated as containing at least one proliferating colony/sphere, or not, and the average number of wells without colonies/spheres at particular cell densities was calculated, for both CD15+ and CD15- populations. We observed a varying degree of CD15- FITC staining in the ENU+NC+p53f/- gliomas tested, ranging from 2% to 65% of live tumour cells (Figure 4-3A). However, in all cases (n=5 freshly dissociated tumours) CD15+ cells were enriched for in vitro clonogenic capacity compared to CD15- cells when plated at the same cell densities (Figure 4-3B). Moreover, when CD15+ derived cultures were expanded (<3 passages) and re-analyzed for the expression of CD15, we observed that the CD15+ population regenerated CD15- cells indicating the capacity of CD15+ cells to repopulate the marker heterogeneity observed in the primary tumour (Figure 4-3C).

We were interested in determining if the CD15+ population was also enriched for tumour initiating capacity in vivo, and injected 1x104 freshly sorted CD15+, CD15-, and live-gated cells from three independent tumours, orthotopically into the brains of NOD/SCID recipients. CD15+ population yielded tumours in two of eleven injections, while no tumours were observed in mice injected with the CD15- (0/13) or live-gated (0/4) populations, up to 24 weeks post injection (Data not shown).

Figure 4-3 CD15 enriches for clonogenic ENU+NC+p53f/- mouse glioma cells in vitro. Tumours were freshly dissected and dissociated, and stained and sorted for the cell surface marker CD15. (A) A varying percentage of CD15+ cells were observed for individual tumours (n=6), however on average CD15+ cells were in the minority population (mean and median indicated). (B) CD15+ and CD15- were sorted and immediately cultured in limiting dilution analysis assays. In vitro, clonogenic ENU+NC+p53f/- mouse glioma cells are enriched in the CD15+ population. (C) After limited expansion in vitro (p<3), the CD15+ population regenerated CD15- cells and the marker heterogeneity observed within the primary tumour.

4.4.6 Mouse Tumour Stem Cell Lines Are Readily Established and Demonstrate Unique Phenotypes and Properties In Vitro.

Our lab recently described the generation of adherent glioma ‘NS’ cell lines from human brain tumours and demonstrated that these cells could be propagated long-term in vitro, retain tumourigenic potential when low cell numbers are injected orthotopically in vivo, and can be utilized in high-throughput (HTP) chemical and genetic screens [198, 201]. These human glioma NS cell lines were derived and propagated on laminin-coated plastic and grown in the stem cell conditions that support the growth of human foetal NS cells [203]. We were interested in generating similar cell lines from ENU+NC+p53f/- gliomas and plated freshly dissociated tumour cells on gelatin-coated Primaria tissue-culture plates, as described for the culture of adherent embryonic mouse NS cell lines [203]. Adherent mouse glioma NS cultures were quickly established (n=3 cell lines from independent tumours), all were genetically deficient for p53 and could be propagated long-term (>25 passages) without reduction in proliferative potential (Supplemental Figure 4-4 and Data Not Shown). Interestingly, NS cultures derived from independent tumours demonstrated unique morphologies and behaviours. NC-156-G grew as loosely adherent clusters, NC-159-G grew as complex, adherent single cells, and NC-166-G grew as adherent spheres (Figure 4-4, Brightfield).

To determine the clonogenic potential of cells within each ENU+NC+p53f/- NS cell line, cells were dissociated to a single cell suspension and one live cell was FACSed into each well of a 96 well plate (n≥4 independent single-cell sorts per cell line). At least two weeks later, the number of wells containing a proliferating colony was evaluated and the clonogenic frequency of each cell line was determined. We calculated that NC-156-G showed the lowest clonogenic capacity at 4% (±2%), while NC-159-G and NC166-G displayed similar clonogenic frequencies of 8% (±6%) and 10% (±2%) respectively.

Figure 4-4 Morphological (Brightfield) and phenotypic analysis of three established ENU+NC+p53f/- mouse glioma cell lines for the expression of stem (Sox2, Nestin), astrocytic (GFAP, S100β), oligodendroglial (CNPase) and neuronal (βIIITubulin, Map2) cell lineages when grown in stem cell conditions. DAPI, nuclear marker.

In vitro human glioma NS cells represent a relatively homogeneous population, expressing Nestin, Vimentin, and Sox2 [201], however unlike normal neural stem cells, glioma stem cell lines can also display patient-specific expression of mature lineage markers when grown in stem cell conditions (Supplemental Figure 4-5) [201]. To determine the in vitro phenotype of our mouse glioma NS cultures cells were grown on poly-L-ornithine/laminin-coated glass coverslips and immunostained for markers of stem/precursor (Nestin, Sox2), astrocytic (GFAP, S100β), oligodendroglial (CNPase), or neuronal (Map2 and βIIITubulin) cell types (Figure 4-4). All cultures displayed a neural precursor phenotype, with the majority of cells in all NS cell lines expressing Nestin (96%-100%) and Sox2 (70%-90%). We also observed considerable heterogeneity between cell lines regarding lineage marker expression, even when cells were grown in NS conditions. Many βIIITubulin+ cells and rare, complex, GFAP-bright cells were observed in all three mouse glioma cell lines, while s100β+ and rare CNPase+ cells were present in NC-156-G and NC159-G cultures and NC-166-G cells demonstrated expression of Map2 (Figure 4-4).

In vitro, normal neural stem cells stop proliferating and upregulate the expression of differentiated cell markers when growth factors are withdrawn from the culture media, with or without the addition of serum [203, 204]. Similarly, brain tumour stem cells express mature cell markers consistent with their tumour phenotype in differentiating conditions, but do not necessarily stop proliferating suggesting a blockage in their terminal differentiation capacity [201, 203, 215]. We were interested in determining the differentiation capacity of our mouse glioma stem cell lines and sequentially withdrew EGF then bFGF over a three week period [203]. Cells were then stained for the expression of mature cell markers qualitatively by immunocytochemistry (Figure 4-5) or quantitatively by intracellular flow cytometry (Supplemental Figure 4-6). All three glioma NS cell lines downregulated the expression of Nestin, and NC-156 and NC-159-G increased the expression of astrocytic markers GFAP and s100β. Rare CNPase positive cells were observed in differentiated cultures of NC-159-G and NC-166-G, but not NC-156-G. Interestingly, NC-166-G demonstrated continued and strong expression of Map2, but little expression of glial cell markers (GFAP, s100β) (Figure 4-5A). To further characterize the differentiation capacity of NC-166-G, we withdrew growth factors and supplemented media with 10% FBS for seven days. Cells were stained by immunocytochemistry and we observed s100β+ cells and robust staining for GFAP, again in the presence of many

Map2+ cells (Figure 4-5B). Interestingly, the growth of NC-166-G slowed in the presence of serum, versus NC-156-G and NC-159-G NS cell lines which continued to proliferate at a similar rate, while normal neural stem cells (p53-WT: A2, p53-/-: 225-1) stopped proliferating altogether (Supplemental Figure 4-6).

Figure 4-5 Phenotypic analysis of three established ENU+NC+p53f/- mouse glioma cell lines after differentiation by (A) sequential EGF and bFGF withdrawal or (B) in the presence of 10% FBS. Cells were stained for the expression of stem (Nestin), astrocytic (GFAP, S100β), oligodendroglial (CNPase) and neuronal (βIIITubulin, Map2) cell lineages. NC-156-G and NC-159-G upregulated the expression of glial lineage markers in the absence of growth factors, conversely NC-166-G upregulated the expression of neuronal lineage markers after EGF and bFGF withdrawal and glial lineage markers when treated with 10% serum. DAPI, nuclear marker.

4.4.7 Mouse Glioma NS Cell Lines are Tumourigenic when Low Cell Densities are Injected In Vivo.

Human glioma NS cell lines initiate the growth of representative tumours in the brains of immune-compromised mice when as few as 100 cells are injected [201]. We tested the tumourigenic capacity of mouse glioma NS cell lines, expanded in vitro for 10 passages, by injecting 1x105, 1x104, or 1x103 cells into the forebrains of 8-12 week old NOD/SCID recipients (Table 4-3). With injections of 1x105 and 1x104cells, all cell lines readily generated large tumours within 8-12 weeks of transplant. Additionally, NC-159-G and NC-166-G were capable of generating tumours in two of three recipients when as few as 1000 cells were injected. To determine the phenotype of these tumours we stained tumour sections by immunohistochemistry (Figure 4-6). Interestingly, all cell lines generated tumours phenotypically consistent with glioma, expressing the astrocytic markers s100β, and GFAP. Despite near 100% Nestin staining in vitro, a more limited expression of Nestin was observed in tumours in vivo. βIIITubulin expression could be observed in tumours derived from NC-156-G and NC-159-G. CNPase expression was observed in all three cell lines in vitro, however its expression could only be appreciated in tumours arising from NC-159-G in vivo. Despite strong expression of Map2 in vitro, NC-166-G tumours demonstrated little expression of neuronal markers (βIIITubulin, Map2) in vivo, but did demonstrate strong expression for s100β consistent with the glioma phenotype of the original tumour.

Table 4-3 ENU+NC+p53f/- mouse glioma cell lines are tumourigenic and NC-159-G and NC-166-G initiate the growth of tumours when as few as 1000 cells are injected orthotopically into NOD/SCID recipients.

Figure 4-6 ENU+NC+p53f/- mouse glioma cell lines initiate the growth of gliomas in vivo. Tumours were stained with H&E (top) or by immunohistochemistry for the expression of stem (Nestin), astrocytic (GFAP, S100β), oligodendroglial (CNPase) and neuronal (βIIITubulin, Map2) cell lineages. DAPI, nuclear marker.

4.4.8 Microarray Expression Analysis of Mouse Glioma NS Cells Reveals a p53-Deficient- and Glioma-Associated Expression Signature.

To investigate the molecular mechanisms that may govern the tumourigenic phenotype of glioma NS cells we performed a microarray expression analysis of early passage (<10) mouse glioma NS cell lines (NC-156-G, NC-159-G and NC-166-G), and three independent e14.5 p53+/+ (A1, A2, A3) and e14.5 p53-/- (223-2, 225-1, 225-6) mouse NS cell lines. Principle component analysis of global gene expression revealed considerable heterogeneity in expression between mouse glioma NS cell lines, but relative homogeneity regarding the expression profile of embryonic NS cell controls, both p53+/+ and p53-/- (Supplemental Figure 4-7).

We compared the expression of mouse glioma NS cells and e14.5 p53-/- NS cells to e14.5 p53+/+ NS cell lines and validated a set of results by real-time PCR (Supplemental Figure 4-8). Unsupervised average clustering of statistically significant differentially expressed genes (glioma NS vs. e14.5 p53+/+, ANOVA p<0.05, > 2-fold change in expression) revealed two distinct patterns of expression: one associated with p53-deficiency, and one associated with glioma (Figure 4-7). The p53-/- associated gene signature contained transcripts similarly expressed by mouse glioma NS cells and p53-/- NS cells (Figure 4-7, p53-Deficient Associated Genes). Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of probsets within this signature revealed enrichment of genes involved in the p53-, apoptosis-, and mitogen-activated protein kinase- (MAPK) signalling pathways (Supplemental Table 4-1). As expected, downregulated genes within the p53-signalling pathway included p53 and its transcriptional targets Apoptotic Peptidase Activating Factor 1 (Apaf-1), Bcl2-associated X protein (Bax), p53 apoptosis effector related to PMP-22 (Perp) and Tumour Necrosis Factor Receptor Superfamily, Member 10b (Tnfrs10b)) (Supplemental Table 4-1).

Figure 4-7 Unsupervised average clustering of probsets (p<0.05) demonstrating > 2-fold change in expression when ENU+NC+p53f/- NS cells were compared to p53WT NS cells. Similar comparison of p53-deficient NS cells revealed two patterns of expression in glioma: transcripts associated with p53-deficiency and transcripts associated with glioma NS cells.

Additionally, we identified approximately 7000 differentially expressed probsets between mouse glioma NS cell lines and embryonic NS cell lines (Figure 4-7, Glioma Associated Genes). KEGG pathway analysis of glioma-associated probsets demonstrated enrichment of transcripts involved in multiple cancer types (glioma, colorectal, prostate and non-small cell lung cancer), metabolic pathways (valine, leucine and isoleucine degradation, fatty acid metabolism, oxidative phosporylation, pyrimidine metabolism and the citrate cycle), signalling pathways (mechanistic target of rapamycin (mTOR) signalling, insulin signalling, long term potentiation) and cellular processes (ubiquitin mediated proteolysis, DNA polymerase, focal adhesion and regulation of the actin cytoskeleton) (Supplemental Table 4-2), and many of these same pathways were also represented when a similar analysis was performed with microarray results comparing human glioma NS cell lines (n=6) with normal human foetal NS cell lines (n=3) (Supplemental Table 4-3) [201].

4.4.9 Identification of a Misregulated Homeobox Gene Regulatory Network in both Mouse and Human Glioma Stem Cells.

Gene ontology (GO) analysis of glioma-associated transcripts (demonstrating > 2-fold change in expression compared to p53-WT NS cells) revealed enrichment for homeodomain-containing proteins (Supplemental Table 4-4). Of all homeodomain-containing protein transcripts, the Arx and Otx2 homeobox transcripts demonstrated the greatest decrease in expression, while Short stature homeobox 2 (Shox2) and Irx protein transcripts demonstrated some of the greatest increases in expression when glioma NS cells were compared to foetal NS controls (Figure 4-8). These homeodomain proteins are highly conserved and serve to establish anterior-posterior regionalization and patterning of the developing brain. Arx is a transcription factor known to directly repress the expression of Shox2 and multiple Irx-family transcription factors [148, 292]. Shox2 is transcriptional regulator and positively regulates the expression of Runt-related transcription factor 2 (Runx2), which has known roles in the pathology of multiple cancers [293- 297]. Finally, the Irx family of homeodomain-containing proteins negatively regulate the expression of Arx and Otx2, induce the expression of Gbx2, and are regulated by a positive feedback loop involving Fgf and MAPK signalling to establish the position of the cerebellum (Figure 4-8A) [137]. Real time PCR analysis of Irx1/2/3/5, Gbx2, Shox2, Runx2, Arx and Otx2 confirmed our microarray results. Intriguingly, in both mouse and human glioma NS cell lines this gene regulatory network appears dysregulated (Figure 4-8), demonstrating decreased Arx

100 and Otx2 expression, and increased expression of Shox2, Runx2, Gbx2 and Irx family transcripts, suggesting that overexpression of these transcription factors and loss of Arx expression and/or Otx2 expression may be functionally important for the development of glioma.

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Figure 4-8 A Shox2/Arx/Irx gene regulatory network is dysregulated in mouse and human glioma stem cells. (A) Relative expression of homeodomain-containing transcription factors in human (right) and mouse (left) glioma stem cells relative to foetal NS controls and (B) confirmation of results by real-time PCR reaction. C) Arx is a transcriptional repressor that inhibits the expression of Shox2 and Irx to establish regional specification during embryonic forebrain development. Cerebellar specification is established by FGF8/MAPK signalling leading to phosphorylation of Irx2, expression of GBX2, and repression of Otx2 and BMP4. This gene regulatory network is dysregulated in (A) mouse and human glioma stem cells, with downregulated Arx and Otx expression, and overexpression of Shox2, and Irx family transcripts (*) as determined by (A) microarray and (B) real-time PCR expression analysis when compared to foetal controls (Human glioma cell lines & microarray data from Pollard et al, 2009; HF, human foetal).

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4.4.10 Chemical Screening of Human and Mouse Glioma Stem Cell Lines Identifies Selective Pharmacological Inhibitors.

Ideally mouse models of cancer should recapitulate similar pharmacological profiles towards chemical compounds as human cancers such that they may serve as predictive in the preclinical development of anti-neoplastic therapeutics. Previously we reported that compounds targeting neurotransmitter pathways inhibit the growth of human glioma NS cells [198]. Therefore we were interested in determining the effect of neurotransmission modulating agents on our mouse glioma NS cell lines versus human glioma NS cell lines. We screened approximately 700 compounds of the BIOMOL International Neurotransmitter Chemical Library against two human glioma NS cell lines (G144, GliNS1 [201]), three mouse glioma NS cell lines (NC-156-G, NC159-G NC-166-G), and human and mouse fibroblasts (BJ fibroblasts and NIH 3T3 fibroblasts, respectively) and assessed cell viability by standard Alamar Blue assay 5 days after treating cells. Generally cytotoxic agents inhibited the growth of all cell types (n=12), and mouse glioma cells demonstrated sensitivity to more compounds compared to human glioma cells (n=148 versus n=24, respectively) (Figure 4-9). All but two chemicals selective for human glioma stem cells also inhibited mouse glioma stem cells (n=13) (Figure 4-9). These compounds are known to modulate a number of neurotransmitter pathways, including Cholinergic, Dopaminergic, Adrenergic, Histaminergic, Serotonergic and GABAergic signalling pathways, as previously described [198]. Together, these results demonstrate that mouse glioma NS cells and human glioma NS cells are sensitive to a common set of pharmacological compounds, validating the use of our novel mouse glioma NS cells in preclinical drug development assays.

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Figure 4-9 Mouse glioma stem cells, human glioma stem cells (from Pollard et al, 2009), mouse NIH 3T3 fibroblasts and human BJ fibroblasts were treated with the BIOMOL neurotransmitter library ([compound] = 3μM) and cell viability was determined 5 days post treatment. (A) Venn diagram depicting the number of compounds capable of inhibiting each cell type and combination. All but two compounds selective for human glioma stem cells also effectively inhibited the proliferation of mouse glioma stem cells. (B) The 13 compounds that selectively inhibit the proliferation of glioma stem cells, but not fibroblast controls, target a number of different neurotransmitter pathways (Fold Selectivity, %Inhibition Glioma NS cells/%Inhibition Fibroblast; Rank, order of inhibition, greatest to smallest).

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4.5 Discussion

The utility of mouse models of cancer comes from interrogating their biology and by their use in preclinical drug discovery assays. To generate clinically relevant information, representative mouse models should recapitulate the genetic, phenotypic and functional characteristics that distinguish the human disease. In this regard, certain mouse models of glioma are thought to better represent the human disease than others, as their targeted genetic aberrations are also observed in a large percentage of patient samples [102, 286, 298]. Conversely, some models likely represent a limited spectrum of human glioma as the aberrations driving their tumourigenic process are rarely implicated in clinical samples [117, 118].

Here we report the generation and characterization of high incidence, diverse and aggressive mouse glioma. We have modeled brain tumours using a chemical-genetic approach, with random mutagenesis during embryonic development in a p53-/- genetic background. Large scale genetic analysis of human GBM samples revealed that alteration of p53 function or expression is one of the most common aberrations to occur in GBM [73, 74]. Exposure to the carcinogen vinyl chloride has long been associated with glioma [69] and more recently, maternal consumption of N-nitroso containing foods during pregnancy is associated with an increased risk of brain tumours in children, particularly of astroglial phenotype [7]. While the causes of sporadic glioblastoma are largely unknown, our chemical-genetic approach to generate gliomas in mice recapitulates known genetic and environmental influences thought to be involved in the human disease. Additionally, the power of our strategy is appreciated when considering the diversity of phenotypes observed in primary tumours and established cell lines, similar to the patient-specific phenotypes observed when human gliomas were analyzed [201].

Primary mouse gliomas were phenotypically heterogeneous, demonstrating expression of stem-, glial-, and oligodendroglial-cell lineages. Additionally, primary mouse gliomas self-renewed in vivo as tumours could be propagated long term (>3 passages) in the brains of recipients. Like the human disease, mouse gliomas demonstrated a cell-dose dependent capacity to transplant tumours in vivo, suggesting functional heterogeneity within the tumour cell population and the existence of a CSC hierarchy. CD15 has been shown to enrich for human glioma- and mouse medulloblastoma CSCs [215, 243, 274]. In vitro, clonogenic mouse glioma cells were enriched in the CD15+ population of freshly dissociated tumours. In vivo, only the CD15+ population

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generated tumours when cells were injected orthotopically in NOD/SCID recipients, while the CD15- and live-gated populations did not suggesting that, like human glioma stem cells, mouse glioma stem cells are enriched when FACSing with this cell surface marker. However, the low frequency of engraftment in our in vivo experiments precludes a definitive conclusion regarding this point, likely due to reduced cell viability after live-cell FACSing, as unsorted cells were able to initiate the growth of tumours (in five of seven injections) when the same number of cells were injected.

We recently reported that adherent, tumourigenic and long-term cell lines could be readily generated when human gliomas were cultured in serum-free conditions containing EGF and bFGF and grown on laminin-coated dishes [201]. Here we demonstrate that adherent mouse glioma cell lines are readily established in serum-free media containing EGF and bFGF. These cells were tumourigenic when as few as 1000 cells were injected orthotopically in NOD/SCID recipients and regenerated tumours of glioma phenotype in vivo. Despite uniform p53- deficiency, we observed considerable heterogeneity regarding the phenotypic and behavioural characteristics of mouse glioma cell lines, possibly reflecting the unique mutations induced by carcinogen exposure during tumour initiation. In NS cell conditions, all cell lines demonstrated a neural stem cell phenotype with the majority of cells expressing Nestin and Sox2. Interestingly, mouse and human glioma NS cell lines also expressed mature cell lineage markers, to a varying degree, when grown in stem cell conditions. Expression of the neuronal precursor marker βIII- tubulin was observed in all glioma NS cell lines, human and mouse, consistent with the observation that elevated βIII-tubulin expression correlates with high grade astrocytoma and poor clinical outcome [299, 300]. Bright GFAP+, S100β+, CNPase+ or Map2+ cells were also observed, though at low frequency in some lines, in mouse and human glioma NS cell lines propagated in stem cell media. One explanation for this observation is that some cells within the culture are lineage primed [301] and express mature cell markers while retaining stem cell properties. Alternatively, cells expressing mature cell markers may have differentiated spontaneously and may no longer contribute to the propagation of the culture. While we are not certain, evidence for spontaneous differentiation and functionally heterogeneous NS cultures is derived from the observation that <10% of single sorted mouse glioma NS cells are clonogenic.

Upon sequential withdrawal of EGF and bFGF, NC-156-G and NC-159-G cell lines upregulated the expression of the mature glial cell markers GFAP and s100β. NC-166-G demonstrated a

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dramatic increase in the expression of neuronal cell markers, βIII-tubulin and Map2, and limited expression of glial lineage markers. In the presence of 10% FBS, a differentiating agent known to promote glial lineage specification [189], cells within the NC-166-G culture demonstrated robust expression of GFAP and s100β. However, all three cell lines produced tumours of glial phenotype in vivo, particularly those derived from NC-166-G which showed little, if any, Map2 expression in established tumours. This observation differs from tumours initiated by NC-159- G, a cell lines that demonstrated expression of CNPase in vivo and in both NS and differentiating conditions in vitro. This may suggest a difference in differentiation programming between mouse glioma NS cell lines, with NC-166-G cells responding in a non-cell autonomous manner to differentiation stimuli, versus NC-156-G and NC-159-G cells which may differentiate down fixed glial lineages. This interpretation could have implications for putative differentiation therapies which promote CSC lineage commitment [247].

The Gli Hh signalling pathway targets were first identified as amplified sequences in human glioma samples [35], and functional activation of the Hedgehog pathway is reported in GBM CSCs [248]. Activation of the pathway also generates medulloblastoma in humans and mice, as well as other cancer types including basal cell carcinoma and rhabdomyosarcoma [42, 44, 48, 49]. We questioned if Patched-1 haploinsufficiency in combination with in utero ENU exposure might induce gliomas in pups, however we observed medulloblastoma, exclusively, in ENU treated Ptc1+/- mice. Of particular interest is the observation that ENU exposure of pups at e10.5 and e14.5 generated medulloblastoma at high frequency (~90%), whereas exposure of pups at e17.5 did not alter tumour frequency above the background incidence rate (~40%). Similarly, postnatal gamma-irradiation of Ptc1+/- mice revealed an age-dependent correlation with medulloblastoma induction, as exposure augmented medulloblastoma frequency to ~100% at p0/1, 50% at p4, but had no effect at p10 [61]. Together these observations suggest a role for stem cells, versus unipotent CGCPs, as the cells initiating the disease. CGCPs are first born at e12.5 [140], expand in numbers until post-natal day 8 [302, 303], and differentiate to depletion by p21 [57, 60]. Conversely, Nestin+ CNSCs are proliferating at e10.5, peak in numbers at e14.5 and are restricted to a limited population shortly after birth [210]. Interestingly, ENU and irradiation both augment medulloblastoma frequency proportional to the size of the CNSC pool, and inversely to the size of the CGCP population. However, CNSCs and CGCPs can both generate medulloblastoma with an incidence of 100% by targeted homozygous deletion of Ptc1,

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but in the case of CGCPs as long as genetic excision occurs by p8 after which point medulloblastomas are observed infrequently [53]. This data would suggest that specifically targeting both CNSCs and CGCPs can generate medulloblastoma, but induction by random mutagenesis (carcinogen or irradiation exposure) may initiate tumours from within the stem cell, versus CGCP compartment.

Similarly, we noticed that ENU-induced mouse gliomas occurred in close proximity to the neurogenic regions of the brain, as was previously reported for ENU-induced gliomas in rats [89, 90]. ENU exposure of mouse pups at e12.5-e13.5 is reported to generate apoptotic and cytogenetically aberrant neural stem cells in vitro and in vivo [304]. Recent reports suggest that gliomas arising in p53-mutant, p53-mutant/Nf1-/-, p53-/-/Nf1-/- or p53-/-/Nf1-/-/Pten-/- mice are derived from a stem cell cell-of-origin [102, 286]. While a thorough analysis was not performed in this study, our observations regarding the location of early lesions, the ability of the stem cell marker CD15 [214] to enrich for clonogenic cells in vitro and likely in vivo, and our ability to propagate tumourigenic cells with stem cell phenotype and multipotent differentiation capacity is consistent with a stem cell cell-of-origin for ENU-induced gliomas.

Microarray analysis of mouse gliomas, e14.5 p53-/- and e14.5 p53+/+ mouse NS cell lines allowed us to elucidate distinct gene expression patterns associated with p53-deficiency and glioma. Principal component analysis revealed highly similar global expression patterns amongst embryonic controls of the same p53-status, but heterogeneity between mouse glioma NS cell lines likely reflecting the random mutagenic effect of ENU administration, and the unique natural histories of each tumour. Cultured embryonic mouse NS cells do not form tumours in vivo [203]. p53-/- mouse neural stem and astrocyte cell lines are rarely, if ever, gliomagenic [113, 305, 306], and paradoxically, potentially more genetically stable when compared to p53- heterozygous controls [307]. In vivo, neural-specific p53-/- mice only develop brain tumours after a prolonged latency (50% survival, 43 weeks) and the accumulation of co-operating transforming mutations [102] signifying that p53-deficiency and the accompanying changes in gene expression are not transforming. KEGG pathway analysis of p53-associated probsets revealed enrichment of transcripts involved in cancer development (p53-, MAPK-, apoptosis- and Wnt-signalling). However, the fact that p53-/- neural stem cells require additional genetic hits to be fully transformed suggests that these changes provide a permissive background in which additional aberrations may generate glioma. Comparison of mouse glioma NS cell lines

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to embryonic controls allowed us to appreciate the unique gene expression changes, independent of p53-deficiency, common amongst glioma cell lines. We identified approximately 1500 probsets, representing roughly 1150 genes, as being up- or down-regulated by at least two-fold. KEGG signalling analysis revealed enrichment of transcripts involved in numerous signalling pathways including: metabolic pathways, consistent with the expression of aberrant metabolic enzymes in glioma, though no IDH1 R132 mutations were identified in mouse glioma NS lines (data not shown) [308]; mTOR signalling, consistent with its role in glioma cell survival and the apoptotic effect of rapamycin on mouse gliomas in vitro and in vivo [309]; and long term potentiation, consistent with the antiproliferative effect of neurotransmission modulating agents on neural stem cells and glioma stem cells [198, 201, 310, 311]. Importantly, these same pathways are enriched with transcripts when microarray data from human glioma NS cells is compared to human foetal NS controls, suggesting that aberrations in these signalling pathways may be of great functional consequence for tumour development and perhaps a defining feature of glioma.

GO analysis identified homeodomain-containing proteins as enriched in our glioma-associated probset list. Within this gene set we identified the Shox2/Arx/Irx gene regulatory network as being dysregulated in glioma NS cells compared to embryonic controls. This gene regulatory network is critical for anterior-posterior patterning of the developing brain, and Arx acts as direct transcriptional repressor of Shox2 [292] and serves to repress the anterior expression of Irx proteins [148]. Little is known regarding the roles of Arx, Shox2, Gbx2, and Irx family proteins in cancer, particularly in glioma. Irx2 is expressed in human mammary tumours [312], and knockdown of Irx5 was shown to decrease viability and increase apoptosis in LNCaP prostate-, HCT 116 colon-, and MCF-7 breast-cancer cell lines [313]. In contrast, Irx1 is methylated and down-regulated in head and neck squamous cell carcinoma, and was characterized as a putative tumour-suppressor protein [314]. Otx1 and Otx2 are reported to be highly expressed in medulloblastoma and retinoblastoma, and in the case of medulloblastoma, knockdown of Otx2 inhibited cell growth in vitro, potentially suggesting a divergent function in glioma [315-317].

Some insights regarding the roles of these proteins in glioma can be gained from the functional consequences of perturbing their expression. In both mice and humans, germline mutations within Arx cause mental retardation and epileptic seizures, and in Arx knockout mice an accumulation of neuronal precursors within the periventricular ganglionic emini due to reduced

109 cell migration [318, 319]. Further, Arx is a marker of embryonic and adult mouse neural stem cells, its expression is dramatically upregulated upon differentiation, and benign brain cysts were observed in one patient with a 24 bp duplication mutation in exon 2 of the gene [320, 321]. Runx2 overexpression promotes cell survival, proliferation and transformation, and is associated with poor clinical prognosis in a variety of human cancers [295-297]. In the developing limb bud Shox2 knockout leads to reduced chondrocyte proliferation and differentiation owing to reduced Runx2 expression [293, 294]. Taken together, it is conceivable that tumour initiation or progression may occur by loss of Arx expression with concurrent overexpression of Shox2, Runx2 and Irx proteins, with the cellular consequence of enhanced proliferation and survival, and decreased migration and differentiation. In vitro and in vivo functional analysis of Arx overexpression, and knock down of Shox2 and Irx proteins will determine the consequence of perturbing this gene regulatory network in glioma cells.

Cancer stem cells are thought a likely source of disease recurrence and are reported to be relatively resistant to traditional therapies [245, 246, 284, 322]. Novel, selective and effective therapies for the treatment of glioma are required, necessitating the development of representative preclinical systems in which large chemical libraries can be screened. As a proof- of-principle experiment we screened human and mouse glioma NS cells, and human and mouse fibroblasts with the BIOMOL neurotransmitter chemical library. Mouse glioma NS cells were sensitive to four-times as many compounds as compared to the human glioma cells, possibly indicating that mouse cells are generally more sensitive to compounds than human NS cells. Nonetheless, all but two compounds that selectively inhibited the proliferation of human glioma NS cells also inhibited the growth of mouse glioma NS cells with equal or greater efficacy, promoting the use of this model in preclinical drug discovery efforts.

We have modeled glioma is mice by random mutagenesis in a p53-deficient genetic background. This strategy allowed us to generate phenotypically and functionally heterogeneous tumours with unique natural histories and initiating mutations, like in the human disease. Mouse glioma stem cells were enriched using the cell surface marker CD15, and individual tumours demonstrated unique properties and behaviours like the patient-specific observations made with human cells. Microarray analysis of mouse glioma NS cells allowed us to appreciate the common p53- independent expressional changes associated with glioma and identify a novel gene regulatory network as being dysregulated in both human and mouse tumours. Finally our chemical library

110 screen identified compounds selective in inhibiting glioma NS cells, affording the possibility of high-throughput in vitro and in vivo preclinical drug discovery assays for development of anti- glioma stem cell therapies.

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4.6 Supplemental Figures

Supplemental Figure 4-1 Examples of non-brain tumours arising in ENU-treated p53-/- mice. (A) Thymic lymphoma/leukemia in ENU-treated p53-/- in comparison to age-matched ENU-treated p53+/- littermate control. (B) A sarcoma occurring in ENU-treated p53-/- mouse, outlined with white-dashed line.

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Supplemental Figure 4-2 (Top) Survival curves and medulloblastoma incidence of Ptc1+/- mice with or without exposure to 25mg/kg ENU at embryonic day (e) 10.5, 14.5, or 17.5. *Logrank statistic indicating significance between survival curves. (Bottom) H&E staining of medulloblastoma arising in Ptc1+/- mice administered ENU at e10.5, e14.5 or e17.5.

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Supplemental Figure 4-3 Macroscopic image of medulloblastoma NC-171-MB (circled), derived from an ENU+NC+p53f/- mouse. MB, medulloblastoma; Cb, normal cerebellum; Ctx, cortex.

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Supplemental Figure 4-4 PCR analysis of genomic DNA for the presence of wild-type p53 alleles. ENU+NC+p53f/- gliomas are all genetically deficient for p53.

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Supplemental Figure 4-5 Phenotypic analysis of human glioma stem cell lines G374NS and G377NS. Cells were grown in stem cell conditions and stained with stem (Nestin, Sox2) astrocytic (GFAP, S100β) neuronal (Map2, βIIITubulin) or oligodendroglial (CNPase) lineage markers. DAPI, nuclear marker.

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Supplemental Figure 4-6 (A) Intracellular flow cytometry of undifferentiated and differentiated (2-step, sequential EGF and bFGF withdrawal) mouse glioma NS cells for the expression of Nestin (top), GFAP (middle) or S100β (bottom). (B) Effect of serum treatment on proliferation of mouse glioma stem cells, e14.5 p53WT NS cells (A2) and e14.5 p53-/- NS cells (225-1). Cells were grown in stem cell conditions (serum-free media) or in DMEM/F12 plus 10% FBS for 7 days and proliferation was measured by standard MTT assay. *One-sample t-test p<0.01.

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Supplemental Figure 4-7 Principal component analysis of global gene expression of e14.5 p53+/+ NS, e14.5 p53-/- NS and mouse glioma NS cell lines.

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Supplemental Figure 4-8 Real time PCR validation of microarray expression results comparing mouse glioma NS cells to e14.5 p53+/+ NC cells. (A) Fold change in expression of indicated genes as determined by microarray analysis and (B) by real time PCR, in comparison to e14.5 p53+/+ NS cells.

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Supplemental Table 4-1 KEGG signalling pathway analysis of p53-associated probsets, determined as previously described [289, 290]

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Supplemental Table 4-2 KEGG signalling pathway analysis of glioma-associated probsets, determined as previously described [289, 290]

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Supplemental Table 4-3 KEGG signalling pathway analysis of >2 fold expressed probsets (p<0.05) from comparison of human glioma NS cell lines (n=6) versus human foetal cell lines (n=3), determined as previously described [289, 290]. Highlighted pathways are represented in mouse glioma NS cells, and are categorized as associated with p53-deficiency or glioma.

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Supplemental Table 4-4 Gene ontology (GO) analysis of >2-fold expressed glioma-associated probsets revealed enrichment for homeodomain containing proteins within this gene signature. Analysis performed as previously described [289, 290]

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Chapter 5

5 General Discussion

5.1 The Cancer Stem Cell Hypothesis: a Lingering Debate.

The observation that unselected single-sorted human melanoma cells can engraft and initiate the growth of tumours in a high frequency of recipient mice has fuelled much discussion regarding the validity of the CSC hypothesis, and the experimental evidence supporting its existence [256, 323, 324]. Fundamentally these findings do not call into question the validity of the CSC hypothesis, rather the estimated frequency of tumour initiating cells in certain patient samples. In fact, despite the propagation and expansion of melanomas in mice before the patient samples were studied, the best calculated frequency of melanoma tumour initiating cells was 33%, suggesting that the CSCs still represented the minority population [256]. Further, the assertion that the identification of CSCs is a consequence of injecting isolated human cells into rodents is undermined by the identification of CSCs from an ever increasing number of mouse tumour models, including leukemia/lymphoma [325-329]. With respect to mouse brain tumours, we and the Wechsler-Reya lab identified CSCs in the CD15+ population of Ptc1+/- MB [215, 274]. Mouse glioma stem cells have been identified from genetic models of the disease by functional characteristics (side population analysis) or promoter driven GFP expression [330-332]. In Chapter 4 we describe the identification of mouse glioma CSCs by phenotypic characterization (CD15 expression), and from a chemical-genetic model of the disease [185]. Together these results strengthen the argument favouring the cancer stem cell hypothesis and question the validity of mouse models in which CSCs cannot be identified [255]. Clinically, the identification of CSCs suggests that highly effective cancer therapies may require some anti-CSC component. In the chemical screen presented here (Chapter 4), and in previous reports from our lab, it appears that neurotransmission modulating drugs may serve as protective against brain tumour development and/or useful in their treatment, with the added benefit that many are already in clinical use [198, 333]. Further in vivo testing of these compounds is obviously required, however, the need for novel anti-brain tumour therapies is enormous and any amelioration of

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patient survival will be of great consequence, especially for individuals with a uniformly fatal GBM.

5.2 The Medulloblastoma Cancer Stem Cell Phenotype: Differences in Perspective, Methodology or Both?

Our analysis of Ptc1+/- MB CSCs led to the conclusion that these cells are rare, multipotent, and of cerebellar stem cell phenotype and behaviour (Chapter 2 and 3). In contrast, the Wechsler- Reya lab concluded that they more closely resembled a unipotent CGCP and did not display stem cell characteristics. This conclusion is consistent with their previous report suggesting that bFGF differentiates MB cells in vitro and in vivo [267]. These differences may relate to the use of a Percoll density centrifugation gradient during the preparation of their single cell suspension (Chapter 3). It may also relate to differing perspectives regarding the nature of Ptc1+/- MBs, with our hypothesis driven by the identification of CSCs from human MBs, and theirs based on the idea that CGCPs are the source of the disease [60, 141]. On one point we can certainly agree, that Ptc1+/- MBs recapitulate the human disease [215, 265, 334]. It is therefore paradoxical that their results suggest that Ptc1+/- CSC are of precursor phenotype and differentiate in the presence of bFGF when human MB CSCs demonstrate a stem cell phenotype and behaviour, and robust capacity to proliferate in vitro in the presence of bFGF [220, 229, 239, 242, 335]. Undoubtedly confirmation of both findings by independent labs will resolve these discrepancies, however tumourigenic Ptc1+/- neurospheres are now being reported by the Galli lab and are said to demonstrate an expression profile similar to that of the normal cerebellar NSC [336].

A remaining issue is a definitive lineage relationship between freshly dissociated Nestin+Sox2+ Ptc1+/- MB cells and the subsequent cell lines of the same phenotype. To address this concern we mated Nestin-GFP mice with Ptc1+/- mice in hopes that a rare, distinct Nestin-GFP+ population could be identified from Ptc1+/- MBs and lineage tracked in vitro. Unfortunately Nestin-GFP+Ptc1+/- MBs did not harbour distinct GFP-/low or GFP+/high populations, but rather a relatively homogeneous GFP+ cluster of cells, as analyzed by flow cytometry (data not shown). Therefore, we cannot say definitively that freshly dissociated Nestin+Sox2+ Ptc1+/- MB cells are uniquely responsible for establishing adherent MB NS cell lines, but their rarity and the low in

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vitro clonogenic frequency of primary tumours suggests a relationship. We are not unaware of the possibility that the saturating concentrations of EGF and/or bFGF in our media might induce a stem cell phenotype in vitro [337], however the observation that non-proliferative CD133- human glioma cells retained the expression of mature cell markers when plated in EGF and bFGF media argues against this possibility [229]. Further, the fact that the Wechsler-Reya lab observes an anti-proliferative effect of bFGF on purified CGCPs suggests that the induction of a stem cell phenotype is not the principle effect of bFGF on these cells. Therefore, while we acknowledge this possible effect of EGF and bFGF we think it is more likely that a rare, phenotypically primitive population within the primary tumour is expanded and propagated in serum-free culture in vitro.

5.3 The Cell-of-Origin of Brain Tumours: Elucidated, but Completely Unresolved.

What are the cells-of-origin for human brain tumours? This question has been the focus of intensive study in the past few years. For Ptc1-/- MB, it is now reported that either CNSCs or CGPCs can serve as the cells-of-origin for the disease. As GFAP+ or Olig2+ CNSCs gave rise to MBs but not tumours of other rhombic lip derived structures, the acquisition of a CGCP fate in which Ptc1-deletion can exert an effect is thought the most important determinant of MB development [54]. Interestingly ENU administration, IR and targeted Ptc1-/- all demonstrate an age-related capacity to augment MB frequency, despite the persistence of proliferating CGCPs at ages when MBs can no longer be induced. This may suggest a relationship between MB and the CNSC pool (as discussed in Chapters 2 and 4 ), or a molecular switch between a permissive and resistant state of transformation in proliferating CGCPs; for example the upregulation of senescence or differentiation signalling in p8-p21 CGCPs may prevent tumourigenesis. Another outstanding question is whether targeted Ptc1-deletion in postnatal CNSCs gives rise to MB, and if the same window for tumourigenesis exists. If MBs can be initiated by postnatal CNSCs at >p10, there might be divergent temporal, as well as cellular, origins for the disease.

Similarly, aggressive gliomas in mice can be initiated by physically targeting SVZ NSCs or differentiated glial cells in the cortex [118, 338]. Transformation of a limited number of differentiated astrocytes required forced expression of Ras and Akt oncoproteins in a p53+/-

126 genotype, demonstrating that non-proliferating brain cells can generate the disease [118]; however, the clinical relevance of this strategy is likely limited, as discussed previously (Chapters 1 and 4). Nonetheless, experimental evidence now exists to demonstrate that aggressive gliomas and medulloblastomas can be initiated from multiple cellular origins and neural stem cells, their precursors and their differentiated progeny are all validated candidates for transformation. In some respects these results make the original question moot, with all cell types capable of generating brain tumours. However, the most fundamental question remains unanswered: what are the cells-of-origin for human brain tumours? Further, is it fair to assume that all experimental brain tumours accurately depict their respective disease? A strong argument can be made against the validity of using activated Ras and Akt proteins for generating mouse brain tumours, as mutations in these genes are very rarely observed clinically. Deletion of Nf1, p53 and/or PTEN by physically targeting adult neural stem cells in the SVZ induced gliomas, however no tumour formation was observed when cortical astrocytes were targeted with the same deletions [338]. The earliest microtumours observed after transplacental ENU treatment were composed of proliferating neural stem cells adjacent to the SVZ of the lateral ventricles [89, 90](and as described in Chapter 4). Together these results suggest that clinically relevant induction methods generate experimental gliomas from within the stem cell pool, and not from differentiated astrocytes, supporting the role of NSCs as the cells-of-origin for human brain tumours. However, a definitive conclusion regarding this point remains elusive if not impossible to obtain; accordingly it’s fair to summarize the brain tumour cell-of-origin debate as being completely elucidated, and at the same time completely unresolved.

5.4 Brain Cancer: A Disease of Misplaced Identity?

It is particularly interesting that our microarray analysis (Chapter 4) identified homeodomain- containing transcription factors as some of the most differentially expressed genes when human and mouse glioma NS cell lines were compared to normal foetal NS controls. Further, these changes relate to the expression of posterior prepattern genes in forebrain gliomas, for example the overexpression of posterior Hox and Irx genes, and underexpression of forebrain Arx and Otx genes. During development Arx and Otx genes are expressed anterior to the ZLI and MHB, respectively [148, 339]. Arx expression is related to, but not necessarily dependent on Dlx and

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Fezf expression to establish the prethalamus/thalamus boundary, while Otx1 and 2 cooperate with Emx2 and Pax6 to pattern the caudal forebrain [148, 292, 339]. In the hindbrain, Hox genes are expressed posterior to the MHB and in rhombomere specific patterns while the cerebellum is localized by the induction of Gbx2 and inhibition of Otx2 by Irx2 and Fgf8 [137, 138] . Interestingly, posterior Hox, Gbx and Irx genes are all overexpressed in human and mouse forebrain gliomas, while Otx and Arx genes are highly downregulated. Paradoxically, the forebrain Otx2 gene is amplified and overexpressed in MBs, and knocking down its expression prolongs the survival of tumour bearing mice [340, 341]. Together these results suggest that brain tumours may be a disease of mislocalized tissue when analyzed at the developmental patterning level, and that gliomas are posteriorized tissue existing within the forebrain and MBs are anteriorized tissue existing in the hindbrain. It is easy to hypothesize that cells with mislocalized expression patterns may not respond to the environmental and molecular controls that restrain growth or promote differentiation. For example, the known targets of Arx repression are Shox2, early B-cell factor 3 (Ebf3), LIM domain only 1 (Lmo1). Shox2 was identified as one of the most upregulated genes in glioma NS cells compared to normal controls, and an induces the expression of Runx2, a known mediator of the cancer phenotype [292, 294, 295]. Accordingly, re-establishing the appropriate regional gene signature within brain tumour cells may attenuate the disease by reintegrating cancer cells with their environment and allowing their proper response to endogenous signals. Validation of this hypothesis is required, however if true it might suggest that brain tumours are a disease of mistaken anterior-posterior identity.

5.5 Future Experimental Directions

There are some obvious and immediate experiments that derive from the results presented herein. The first is to understand the molecular control of Ptc1+/- MB CSCs. Regardless of their phenotype, we have now generated an experimental system in which freshly dissociated CD15+ MB CSCs and the resulting NS cell lines can be interrogated. We have begun this process by microarray analysis of Ptc1+/- MB NS cell lines derived from p53+/+, p53-/-, IR- and ENU-treated mice. What are the molecular events common to MBs regardless of the method of induction? What common pathways drive tumour growth and can they be exploited therapeutically to

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slow/stop the disease in vivo? Are these pathways shared by human MB cells, and are they of therapeutic significance?

Secondly, we have identified functionally and physically distinct populations of Ptc1+/- MB cells isolated in the top and bottom fraction of the Percoll gradient. Both cellular populations were tumourigenic, but a full in vivo limiting dilution analysis is required (and ongoing) to determine if a differential tumour initiating capacity exists between physically different populations. Very recently, human GBM CSC were identified based on a larger forward scatter/side scatter profile and increased autofluorescence when analyzed by flow cytometry/FACS [244]. It is an intriguing possibility that brain CSCs are larger, more complex and autofluorescent compared to non-CSCs in general, reminiscent of the filamentous type ‘B’ SVZ cells giving rise to the smaller migrating type ‘A’ cells in the forebrain, or GFAP+ CNSCs giving rise to small migrating CGCPs in the rhombic lip [53, 168]. Though we have previously investigated this possibility, it is now important to revisit this hypothesis with more determination.

Thirdly, we are actively pursuing the functional consequences Arx, Otx1 and/or Otx2 expression and knockdown of Shox2 and Irx genes in mouse and human glioma NS cell lines. We are currently validating our microarray data by real-time PCR and Western analysis, and will begin by overexpressing Arx in glioma cells in vitro and analyzing the proliferation, apoptosis and differentiation potential of transfected cells. We hypothesize that Arx overexpression will reset the developmental signalling profile of glioma NS cells, reducing Shox2 and Runx2 expression and attenuating their tumourigenic potential. Similar experiments will be performed with Otx1 and Otx2, as will experiments involving the knockdown of Shox2 and Irx genes. Ultimately it may be important to model glioma in mice deficient for Arx, Otx and Irx genes. This could be approached in at least two ways, the first is to use Irx-deficient mice in combination with p53-/- and ENU treatment to determine if glioma frequency is reduced or survival prolonged. The second is to ENU-treat Arx-deficient mice (with or without the presence of p53-/-) to determine if random mutagenesis in an Arx-deficient genotype generates glioma.

Lastly, we have validated the CSC hypothesis in two experimental models of the human disease and are now performing high-throughput chemical library screening on human and mouse brain tumour NS cell lines, normal foetal NS controls and irrelevant cell lines to identify selective anti- CSC compounds. These compounds can then be characterized in vitro and their therapeutic

129 potential can be determined in vivo in these preclinical mouse models of glioma and medulloblastoma.

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6 Summary and Conclusion

We have identified brain CSCs from two clinically representative mouse models of brain cancer. Mouse glioma and medulloblastoma CSCs are prospectively identified using the cell surface marker SSEA-1/LewisX/CD15. Mouse brain CSCs can be propagated as adherent cultures in serum-free conditions containing EGF and bFGF, demonstrate a stem cell phenotype and behaviour, and initiate the growth of phenotypically representative cancers in the brains of recipient mice. Phenotypic and physical characterization of MB CSCs suggests that they share many similarities to the normal cerebellar stem cell, and not the unipotent cerebellar granule cell precursor. Molecular characterization of mouse glioma CSCs identifies numerous homeodomain-containing transcription factors involved in hindbrain regionalization as differentially expressed suggesting that brain cancer may derive from expressionally mislocalized NSCs. Together these results have profound implications, validating the CSC hypothesis in the human disease, and identifying key cellular and molecular mediators driving glioma and medulloblastoma. Finally, we identify many neurotransmission modulating compounds as effective at inhibiting the proliferation of mouse and human glioma CSCs, drugs that can be evaluated in vivo in these representative preclinical models of human medulloblastoma and glioma.

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Supplemental Methods

Primer Name Primer Sequence Purpose p53 Ex6 CCC GAG TAT CTG GAA GAC AG p53 genotyping p53 Ex7 W3 TAT ACT CAG AGC CGG CCT p53 genotyping p53 Neo M5 CTA TCA GGA CAT AGC GTT GG p53 genotyping Ptc1 WT3 TTG CGG CAA GTT TTT GGT TG Ptc1 genotyping Ptc1 WT4 AGG GCT TCT GGT TGG CTA CAA G Ptc1 genotyping Ptc1Neo3 TGT CTG TGT GTG CTC CTG AAT CAC Ptc1 genotyping Ptc1 PT3 TGG GGT GGG ATT AGA TAA ATG CC Ptc1 genotyping p53-Flox F CAC AAA AAC AGG TTA AAC CCA G Flox genotyping p53-Flox R AGC ACA TAG GAG GCA GAG AC Flox genotyping Cre-F GCG GTC TGG CAG TAA AAA CTA TC Nestin-Cre genotyping Cre-R GTG AAA CAG CAT TGC TGT CAC TT Nestin-Cre genotyping Ptc1-Ex2-259 TTT TGG TTG TGG GTC TCC TC Ptc1 exon2 RT PCR Ptc1-752 TAG GAA TTC CAA GGG GTC AA Ptc1 exon2 RT PCR Ptc1-Ex4 F AAC AAA AAT TCA ACC AAA CCT C Ptc1 exon4 RT PCR Ptc1-Ex4 R TGT CTT CAT TCC AGT TGA TGT G Ptc1 exon4 RT PCR Hes1-F AGG CTGG AGA GGC TGC CAA GGT TT Hes1 RT PCR Hes1-R ACA TGG AGT CCG AAG TGA GCG AG Hes1 RT PCR Hes1-F CAC AGA CCC GAG CGT GTT G Hes1 Real Time PCR Hes1-R GAC AGG AAG CGG GTC ACC TC Hes1 Real Time PCR Hes5-F TTC AGC AAG TGA CTT CTG CGA AGT TC Hes5 RT PCR Hes5-R GGC CAT GTG GAC CTT GAG GTG AG Hes5 RT PCR Ptc2 F TGC CTC TCT GGA GGG CTT CC Ptc2 RT PCR Ptc2 R CAG TTC CTC CTG CCA GTG CA Ptc2 RT PCR Gli1 F TTC GTG TGC CAT TGG GGA GG Gli1 RT PCR Gli1 R CTT GGG CTC CAC TGT GGA GA Gli1 RT PCR Gli2 F TTC GTG TGC CGC TGG CAG GC Gli2 RT PCR Gli2 R TTG AGC AGT GGA GCA CGG AC Gli2 RT PCR Irx1 F CGC ACC CAA CTA CAG CGC CT Irx1 Real Time PCR Irx1 R CAG CGT GCT GGT GCT CTC CC Irx1 Real Time PCR Irx2 F CTC CGC GTT CAG CCC GTA CC Irx2 Real Time PCR Irx2 R CGT GGC TAT CTC GGC CAG CG Irx2 Real Time PCR Irx3 F GGC CGC CTC TGG GTC CCT AT Irx3 Real Time PCR Irx3 R GAG CGC CCA GCT GTG GGA AG Irx3 Real Time PCR Irx5 F CCC GGT GCA AAG AGC GAG GG Irx5 Real Time PCR Irx5 R GCG CGT TGG CAA ACC AGG TG Irx5 Real Time PCR Gbx2 F CTG CCC GCA AGT TCG CTC CA Gbx2 Real Time PCR Gbx2 R AGC GCT CGG TCA GGG AGA GG Gbx2 Real Time PCR Runx2 F ACG CCG CTG TCT TCC ACA CG Runx2 Real Time PCR Runx2 R TGC TGT GGC TTC CGT CAG CG Runx2 Real Time PCR Shox2 F AGC GGC CGC GAT GGA AGA AC Shox2 Real Time PCR Shox2 R GCA GCT GCG CCT GAA CCT GA Shox2 Real Time PCR Arx F CGC CGC AGG TGA GCA TCA GT Arx Real Time PCR Arx R TCT CCC GCT TGC GCC ACT TG Arx Real Time PCR Otx2 F TTT GGG CCG ACT TTG CGC CT Otx2 Real Time PCR Otx2 R GGG GAG ATG GAC GCT GGG CT Otx2 Real Time PCR

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Antibody Supplier Purpose Dilution & Condition Immunocytochemistry 1:500 1hr RT BD Biosciences, mouse anti-Nestin Canada Immunohistochemistry 1:125 O/N 4'C Immunocytochemistry 1:125 1hr RT mouse anti-MAP2 Chemion, USA Immunohistochemistry 1:100 O/N 4'C

rabbit anti-Math1 Biovision, USA Immunohistochemistry 1:100 O/N 4'C rabbit anti-Math1 Abcam, USA Immunocytochemistry 1:125 1hr RT Immunocytochemistry 1:1000 1hr RT DakoCytomation, rabbit anti-GFAP Denmark Immunohistochemistry 1:500 O/N 4'C mouse anti-Tubulin, beta Chemion, USA Immunohistochemistry 1:100 O/N 4'C III Immunocytochemistry 1:500 1hr RT rabbit anti-Sox2 Chemion, USA Immunohistochemistry 1:500 O/N 4'C Immunocytochemistry 1:500 1hr RT mouse anti-CNPase Sigma, USA Clone 11-5B Immunohistochemistry 1:500 O/N 4'C

rat anti-Musashi, 14H1 Dr. Okano, Japan Immunocytochemistry 1:250 1hr RT

anti-CD15-FITC clone BD Biosciences, FACS 1ul/100ul PBS MMA Canada anti-Prominin1-PE clone eBioscience, FACS 2ul/100ul PBS 13A4 Canada BD Biosciences, anti-Ter119-APC FACS 1ul/100ul PBS Canada

Overnight: O/N Room RT: Temperature

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