The Role and Mechanism of Action of CD200:CD200R1 Interaction in Breast Cancer by Anna Podnos A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Anna Podnos 2015 The Role and Mechanism of Action of CD200:CD200R Interaction in Breast Cancer Anna Podnos PhD Institute of Medical Science University of Toronto 2015 Abstract Cancer cells can use immune inhibitory receptors to evade the host’s anti-tumour responses and establish immunosuppressive networks in the tumour microenvironment. In this thesis, we investigated the interaction between the immunosuppressive molecule CD200 with its receptor, CD200R1, in breast cancer. We found that CD200 is expressed in the local tumour microenvironment in human breast cancer patients and developed a mouse model to study the effect of CD200 on tumour growth and metastasis. Using mouse-derived EMT6 breast cancer cells and BALB/c female hosts, we explored the effects of overexpressing and silencing the expression of CD200 and CD200R1 in hosts and tumour cells. CD200 expression by host and tumour cells enhanced tumour growth and metastasis to draining lymph nodes (DLN). Silencing CD200 expression in EMT6 tumour cells led to a reduction in primary tumour size and metastasis, as well as an increase in anti-tumour cytotoxic responses in the host. Lack of CD200R1 expression in the host resulted in a marked decrease in breast cancer development and CD200R1-/- mice were able to mount specific anti-EMT6 immune response that could be adoptively transferred to wild type naïve hosts. In addition, we extended our findings to a model in which anti-tumour immunity was explored in EMT6 tumour-bearing ii hosts lacking CD200 expression and treated with a combination of immunotherapy with the non- conventional chemotherapeutic agent, metformin. The findings suggest that CD200 may be an important prognostic marker and a target for breast cancer treatment that could synergize with other therapies and improve outcomes in patients. iii Acknowledgments First and foremost, I would like to express my gratitude to my supervisor and mentor, Reg Gorczynski, for his unconditional support, patience, generosity, and guidance. His passion for science has fueled my interest in biomedical research, and his vivacious energy and sense of humour have motivated me and made my time in his laboratory an enjoyable experience that I will never forget. I would also like to thank my colleagues and lab mates, who have supported me through the ups and downs of research over the last five years. I would especially like to thank our lab manager and a great teacher, Ismat Khatri, who taught me everything I know in the lab and has become a close personal friend and mentor. I am grateful for the encouragement and help from lab members Olha Kos and Fang Zhu, who have been reliable and supportive throughout this journey. Also, I thank Hassan Sadozai and Ramzi Khattar for their help with planning experiments and editing my thesis. I thank my program advisory committee members, Shannon Dunn and David Spaner, for their helpful comments and suggestions that have made this work better and for their guidance in resolving lab related problems. I also thank the Institute of Medical Science, particularly Mingyao Li, Cindy Morshead, and the administrative staff, for helping me navigate complicated situations and complete my thesis. Finally, I am thankful to Gillian Einstein, who has been my mentor for the Collaborative Graduate Program in Women’s Health and has expanded my research interests during my PhD. I am eternally grateful to my fiancé, Tom, for believing in me, supporting me through the hard times, celebrating with me in good times, and editing countless copies of my thesis. I also want to thank my parents, who have taught me to work hard and persevere, and my little brother David, who can always put a smile on my face. Last but not least, I would like to give a shout- out to my dogs, Chuba and Xena, for their unconditional love and slobbery kisses. iv Contributions Dr. Reg Gorczynski1,2 assisted with limiting dilution assays, chromium release assays, and mouse injections. Dr. Ismat Khatri2 assisted with human and mouse sCD200 ELISAs and data analysis. Dr. Kai Yu2 helped with genotyping and maintaining animal colonies. Dr. Nuray Erin3 assisted with the mixed lymphocyte culture assays. The Ontario Tumour Bank provided the human breast cancer plasma and serum samples as well as histology sections. 1 Institute of Medical Science, University of Toronto, Toronto, ON, Canada 2 University Health Network, Toronto General Hospital, Toronto, ON, Canada 3Department of Medical Pharmacology, School of Medicine, Akdeniz University, Antalya City, Antalya, Turkey v Table of Contents Acknowledgments .......................................................................................................................... iv Contributions ................................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Abbreviations ................................................................................................................... xiv List of Appendices ....................................................................................................................... xix Chapter 1 Introduction and literature overview .............................................................................. 1 1 Introduction and literature overview .......................................................................................... 1 1.1 Cancer and the immune system .......................................................................................... 1 1.1.1 Immunity and cancer ............................................................................................... 1 1.1.2 Tumour microenvironment ..................................................................................... 2 1.1.3 Non-immune cells in the tumour microenvironment .............................................. 5 1.1.4 Innate immunity in cancer ...................................................................................... 7 1.1.5 Adaptive immunity in cancer ................................................................................ 11 1.1.6 Immune inhibitory receptors in the tumour microenvironment ............................ 19 1.2 Metastasis and the immune system ................................................................................... 20 1.2.1 Metastasis is a hallmark of cancer ........................................................................ 20 1.2.2 Immunity and metastasis ....................................................................................... 22 1.3 Breast cancer ..................................................................................................................... 23 1.3.1 Clinical features of breast cancer .......................................................................... 23 1.3.2 Tumour microenvironment in breast cancer ......................................................... 28 1.3.3 Animal models of breast cancer ............................................................................ 37 vi 1.4 CD200 and CD200R ......................................................................................................... 42 1.4.1 CD200:CD200R overview .................................................................................... 42 1.4.2 CD200:CD200R in cancer .................................................................................... 43 1.5 EMT6 breast cancer model ............................................................................................... 47 1.6 Objectives and hypotheses ................................................................................................ 50 Chapter 2 Evidence for a role of tumour CD200 expression in breast cancer metastasis: decreased metastasis in CD200R1-/- mice or using EMT6siCD200 breast cancer cells ............... 52 2 Studies in WT and CD200R1-/- mice with EMT6, EMT6CD200tg, and EMT6shCD200 tumour cells .......................................................................................................................................... 52 2.1 Abstract ............................................................................................................................. 52 2.2 Introduction ....................................................................................................................... 53 2.3 Materials and methods ...................................................................................................... 55 2.3.1 Mice ...................................................................................................................... 55 2.3.2 Monoclonal antibodies .......................................................................................... 55 2.3.3 EMT6 breast tumour cells, induction of tumour growth in BALB/c mice, and limiting dilution assays ........................................................................................