ROLE OF THE IMMUNE SYSTEM IN TUMOR PROGRESSION 1

ROLE OF THE IMMUNE SYSTEM IN TUMOR PROGRESSION TOH PANG KIAT, BENJAMIN (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements I would like to acknowledge my supervisor, Jean-Pierre Abastado, for his continued help and support throughout the course of my PhD He has been a great mentor and guide from whom I have learnt a great deal I would also like to thank my thesis advisory committee members, Ren Ee Chee and Laurent Renia, for their guidance and help I would like to show my appreciation to all members of the Tumor Immunology Laboratory in the Singapore Immunology Network (SIgN) Special thanks go to Karen Khoo, Jeremy Wang and Jo Keeble for their help and discussions about the work presented in this thesis Next, I would like to thank my collaborators for their help in this project Laurent Renia (SIgN) for the NIMP-R14 antibody, Ng Lai Guan (SIgN) for providing me with the IL8R-KO and tdTomato mice, Esther Koh for help in the cell tracking software, Poon Lai Fong (SIgN) for help with cell sorting, Josephine Lum (SIgN) for the microarray work and Wong Wing Cheong (BII) for the analysis of the microarray data Special thanks to Masashi Kato (Chubu University, Aichi, Japan) and Armelle Prevost-Blondel (Institut Cochin, Paris, France) for providing the RET mice I would also like to thank Jean-Paul Thiery (IMCB) and Sim Wen Jing (IMCB) for their help and discussion regarding the EMT assays Last but not least, I would like to thank my family for their support, especially my wife, who painstakingly helped with the editing of this thesis i Table of Contents Acknowledgements i Summary v List of Tables vi List of Figures vii List of Appendices ix List of Videos ix List of Publications .x List of Abbreviations xi Introduction Inflammation, immunity and cancer Immunosurveillance theory Immunoediting theory Roles of immune cells Melanoma Diagnosis and treatment .11 Melanoma and the immune system 15 Objectives 16 Experimental Procedures 18 Mice 18 In vivo PMN-MDSCs depletion 18 Flow cytometry analysis 19 Isolation of PMN-MDSCs and macrophages 20 Microarray analysis 21 Cytospin and May-Grunwald/Giemsa stain 22 Tumor cell detection by qRT-PCR 23 Low density microarray 23 Immunohistochemistry and calculations 24 Migration assay 25 Tumor proliferation assay 26 ii OVA-specific T cell proliferation assay 27 E-Cadherin assays 27 MT assay 28 Statistics 29 Chapter 1: Not All Tumors Are Made Equal 30 Introduction 30 RETAAD – Spontaneous mouse model of melanoma 31 Results 35 Different tumors from the same mouse have different immune infiltrates 35 Granulocytes are increased in the periphery during tumor progression 39 CXCL1, and and CCL19 are up-regulated in primary tumors .41 CXCR2 ligands are able to attract PMN-MDSCs 42 CXCR2 is necessary for the attraction of PMN-MDSCs to the tumor in vivo 43 CXCL1 and are expressed by the PMN-MDSCs while CXCL5 is expressed in tumor cells .44 Discussion 46 Chapter 2: Role of PMN-MDSCs in Tumor Progression 53 Introduction 53 Myeloid-derived suppressor cells 53 Metastasis and epithelial-mesenchymal transition .62 Results 65 Depletion of PMN-MDSCs reduces tumor growth in vivo 65 PMN-MDSCs promote cancer cell proliferation in the primary tumor .67 PMN-MDSCs secrete soluble factors that promote cancer cell proliferation in vitro .69 PMN-MDSCs favor multinodular development of primary tumors 70 PMN-MDSCs induce cancer cell dissemination to regional and distant sites 71 PMN-MDSCs favor metastatic outgrowth 73 PMN-MDSCs induce cancer cell MT in vitro .74 iii PMN-MDSCs induce down-regulation of epithelial marker, ECadherin, in melanoma cells 77 PMN-MDSCs induce melanoma cell MT in vivo .78 Tumor cells express EGF, while PMN-MDSCs express HGF and TGF-β1 81 PMN-MDSCs induce MT through multiple pathways 82 Discussion 84 Induced proliferation of cancers cells by PMN-MDSCs .84 PMN-MDSCs induce MT in cancer cells 86 Chapter 3: Significance and Implications 92 MT, metastasis and phenotype-switching 92 Multiple roles of PMN-MDSCs 101 Tumor dormancy 105 Therapeutic implications 109 Lymph node excisions .109 Inflammation 112 PMN-MDSCs 114 Chemotherapy and immunotherapy 118 Conclusion 121 Bibliography 124 Appendices……………………………………………………………… A iv Summary In order to metastasize, cancer cells need to acquire a motile phenotype Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus occur late in cancer progression However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype Using a spontaneous murine model of melanoma, I show that a subset of bone marrow-derived immune cells (myeloidderived suppressor cells or MDSCs) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing mesenchymal transition (MT) In vitro and in vivo assays using purified MDSCs showed attraction of MDSCs to the primary tumor is CXCR2-dependent and that TGF-β, EGF and HGF signaling pathways are all used by MDSCs to induce MT in cancer cells These findings explain how cancer cells acquire a motile phenotype early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression v List of Tables Table 1: Roles of different immune cell subsets in cancer Table 2: The ‘ABCD’ method of identifying early melanoma lesions 12 Table 3: List of antibodies used 20 Table 4: Summary of the characteristics and differences of MDSCs 61 Table 5: Summary of the characteristics and differences of MDSCs including the present research 91 vi List of Figures Figure 1: Illustration of immunoediting theory Figure 2: Overall analysis of tumors 35 Figure 3: Differential accumulation and morphology of macrophages and granulocytes 37 Figure 4: Comparison of tumors by immune subset .38 Figure 5: Accumulation of granulocytes in tumor bearing mice 40 Figure 6: Differential expression of chemokines and cytokines between primary tumor and cutaneous metastases .41 Figure 7: Migration of PMN-MDSCs to CXCR2 ligands 43 Figure 8: CXCR2 ligands are important and necessary for PMN-MDSCs migration to the primary tumor .44 Figure 9: Expression of CXCR2 and its ligands in PMN-MDSCs and tumor cells 45 Figure 10: Depletion of intra-tumoral PMN-MDSCs .66 Figure 11: Depletion of PMN-MDSCs reduces tumor growth but not tumor vasculature 67 Figure 12: Depletion of PMN-MDSCs reduces proliferation in young eye tumors .68 Figure 13: PMN-MDSCs induce proliferation of tumor cells in vitro through soluble factors 70 Figure 14: Depletion of PMN-MDSCs reduces nodular structure of the primary tumor 71 vii Figure 15: Depletion of PMN-MDSCs reduce metastasis to lung and lymph nodes 72 Figure 16: Depletion of PMN-MDSCs reduces number of cutaneous metastases but not their size .73 Figure 17: PMN-MDSCs induce MT in vitro 75 Figure 18: PMN-MDSCs down-regulate E-Cadherin expression 77 Figure 19: S100A4 expression in primary tumors 80 Figure 20: Differential expression of TGF-β1, HGF and EGF in PMNMDSCs and tumor cells 81 Figure 21: Inhibition of PMN-MDSC induced mesenchymal transition 82 Figure 22: Diagram illustrating the differences between the two models of tumor progression 96 Figure 23: Diagram illustrating the progression of tumors in RETAAD mice 99 Figure 24: In silico modeling of tumor growth in non-vascularised tumors 103 Figure 25: Graph illustrating that micrometastases occur before detection of the primary tumor .106 Figure 26: Illustration of interactions between PMN-MDSCs and tumor cells .122 viii List of Appendices Appendix 1: Calculations of proliferation and estimate of size ………………… B Appendix 2: Immune profiling gating strategy ………………………………… … E Appendix 3: Table of chemokine/cytokine expression …………………………… F Appendix 4: Mitf expression 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tumor. .. and/or (2) allow metastasis of tumor cells These functions vary depending on the type of tumor and the location of the TAMs in the tumor e.g TAMs in hypoxic areas of the tumor express more angiogenic