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The Role of Lymph Node-Derived Lymphatic Endothelial Cells in Immune Modulation in the Tumour Microenvironment

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The Role of Lymph Node-Derived Lymphatic Endothelial Cells in Immune Modulation in the Tumour Microenvironment The role of lymph node-derived lymphatic endothelial cells in immune modulation in the tumour microenvironment Jennifer Nicole Harris Darwin College Student Name (CRSid): Jennifer Nicole Harris (jnh42) Dissertation Title: The role of lymph node-derived lymphatic endothelial cells in immune modulation in the tumour microenvironment Abstract: The lymphatic vasculature is a key player in progression of many cancers, with lymphangiogenesis at the primary tumour and tumour-draining lymph nodes (TDLNs) associated with poor patient prognosis. As well as providing a highway for metastatic tumour cells, recent reports propose lymphatics as modulators of immunity, highlighting a need for greater understanding of immune regulation by lymphatics. The specific role of lymphatic endothelial cells (LECs) in this context, particularly in TDLNs, is unknown. As TDLNs are immune hubs, yet anti-tumour immune responses are often ineffective, this thesis aimed to investigate functional changes to lymphatics in TDLNs and the role of TDLN-derived LECs in anti-tumour immunity. I hypothesised that factors from the tumour microenvironment alter functionality of TDLN-LECs from early stages of tumour development. I further hypothesised that these changes would promote immune tolerance, with this thesis exploring specific impact on dendritic cell (DC) mediated immunity. Using the B16-F10 melanoma model, this work confirmed expansion of TDLN- LECs prior to metastasis and demonstrated transcriptional reprogramming of immune- associated pathways in LECs isolated from early TDLNs. This was accompanied by differentially localized migratory DCs, clustered at lymphatic subcapsular sinuses. In vitro using co-culture assays revealed mature DCs undergo prolonged interactions with LECs conditioned with B16-F10 tumour-conditioned media, suggesting a change in the physical interactions occurring in vivo in early TDLNs. Additionally, we investigated possible mechanistic contributors, demonstrating using in vitro and in vivo blockade and knockout models, a role for lymphatic expressed Podoplanin in DC interactions and migration. Prolonged physical interactions were further found to facilitate antigen transfer from ovalbumin-loaded LECs to DCs yet inhibit DC priming of T-cells, with DCs found capable of acquiring TDLN-LEC archived antigen in vivo. These results show that in lymph nodes conditioned by factors derived from the tumour microenvironment, prolonged physical interactions between LECs and DCs impact DC migration and T-cell priming. As immune tolerance is a key feature of the tumour microenvironment, this work has highlighted lymphatics as key modulators of the anti-tumour immune response. Furthermore, this work provides new insight into lymphatic involvement during tumour development, identifying lymphatics as a potential target for early intervention therapies. 2 i. Declaration This dissertation is the result of my own work and includes nothing, which is the outcome of work done in collaboration except as declared in the Preface and specific in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma of other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma, or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed work limit of 60,000 words for the Clinical Medicine and Clinical Veterinary Medicine Degree Committee. Miss. Jennifer Nicole Harris 3 ii. Acknowledgements First and foremost, I would like to thank my PhD supervisor Dr Jacqui Shields, who not only has given me the opportunity to work in her lab for the past few years but has also been incredibly supportive and helpful. From experimental design to conversations about cycling and running – you have genuinely been a friend as well as a mentor. Secondly, I would like to thank every single Agent of S.H.I.E.L.D who have been best friends as well as colleagues, thank you so much for always being there – Luisa Pedro, Matthew Lakins, Angela Riedel, Hafsa Munir, Ehsan Ghorani, James Jones, Sarah (Dave) Davidson, Jake Cridge, Carlo Zimarino, Rebecca White, Bastian Schmied, Lisa Haas, Jonathan Swietlik, Garrett Beeghly and Will Moody. I would also like to thank Emma Kerr, Frances Turrell, Eleanor Heaton, Annalise Katz-Summercorn and Jason Crawte for always being there as a friend to have a laugh with and to turn to in tough times. Finally, Nadeera de Silva – I wish you had been here to share this PhD rollercoaster with us; you are forever missed. Thirdly, I would like to thank Thilo Richter who has been my best friend and partner throughout, supporting me and helping me through all the late nights, long weekends and celebratory wins along the way - I love you so very much. Alongside all those at Darwin College and beyond, especially BarComm, Alki, Gaby and Joana, you have been my Cambridge bedrock through the happiest and toughest moments of this PhD, and without you all, I’m not sure how I would have coped – many many thanks. Last but not least, I would like to thank my beautiful family. Coming to the University of Cambridge was always an ambition of mine and you have all sacrificed a lot to allow me to fulfil that goal. From that life-changing moment in 2004, right up to current day, you have all been nothing other than a gift. My support network and my constant – I thank you from the bottom of my heart for being the best family a woman could ask for. So, Josephine Harris-Cook, Rachel Harris, Bex-Harris Cook, Arwyn Harris-Cook, Maisie Double-Cook and Leo Double-Cook – I love you all and I am forever grateful and indebted. 4 iii. List of presentations and prizes awarded at scientific meetings Poster presentations • Cambridge Immunology Network PhD & Post-Doc Day, Cambridge, June 2016 • Hutchison / MRC Research Centre Annual Retreat, Cambridge, November 2015 and November 2017 • BMS-UKCAS Conference, Birmingham, April 2017 • 1st Crick International Cancer Conference, London, September 2017 Oral presentations • Hutchison / MRC Research Centre Lunchtime Seminar, Cambridge, May 2016 • Hutchison / MRC Research Centre Annual Retreat, Cambridge, December 2016 Prizes • Best Oral Presentation at the Hutchison / MRC Research Centre Annual Retreat, Cambridge, December 2016 • Poster Prize at the joint British Microcirculation Society (BMS) and UK Cell Adhesion Society (UKCAS) conference, Birmingham, April 2017 Other Attended Conferences • Cambridge Cancer Centre Annual Symposia, Cambridge, June 2014, 2015, 2016, 2017 • Cancer Research UK Cambridge Institute Annual International Symposium, Cambridge, March 2015 • 2nd AstraZeneca-MedImmune Cambridge Cancer Science Symposium, Cambridge, September 2015 5 Table of Contents Abstract 2 i. Declaration 3 ii. Acknowledgements 4 iii. List of presentations and prizes awarded at scientific meetings 5 Table of Contents 6 List of Figures 8 List of Tables 11 Abbreviations 13 CHAPTER 1 15 INTRODUCTION 15 1. General Introduction 16 1.1. Overview of lymphatics in cancer 16 1.2. Structure and function of the lymphatic system 17 1.3. The role of lymphatics in inflammation 31 1.4. The role of lymphatics in cancer 36 1.5. Summary 43 1.6. Hypothesis and Aims 44 CHAPTER 2 46 METHODS 46 2. Methods 47 2.1. Cell Culture 47 2.2. Isolation of primary murine cell populations 48 2.3. In vivo mouse studies 50 2.4. RNA Isolation, Amplification and Quantification 58 2.5. Microarray Hybridisation and Data Normalisation 61 2.6. Quantitative real-time polymerase chain reaction (qRT-PCR) 62 2.7. Dendritic Cell and Antigen Presenting Cell PCR Array 63 2.8. Flow Cytometry analysis of primary and cell line populations 67 2.9. Immunofluorescent Imaging (IF) 68 2.10. Functional Assays 72 2.11. Statistical Analysis 78 CHAPTER 3 82 RESULTS 82 3. Functional characterisation of lymphatics from melanoma TDLNs 83 3.1. Introduction 83 3.2. Methods 84 3.3. Results 89 3.4. Discussion 114 6 CHAPTER 4 118 RESULTS 118 4. Determining the interactions between DCs and LECs in TDLNs 119 4.1. Introduction 119 4.2. Methods 121 4.3. Results 126 4.4. Discussion 164 CHAPTER 5 171 RESULTS 171 5. Determining the role of lymphatic-DC cross-talk in T-cell priming in TDLNs 172 5.1. Introduction 172 5.2. Methods 174 5.3. Results 179 5.4. Discussion 216 CHAPTER 6 220 DISCUSSION 220 6. General Discussion 221 6.1. Project rationale and overview 221 6.2. Lymphangiogenesis in the tumour microenvironment 222 6.3. Modulation of DC migration in TDLNs mediated by Podoplanin positive lymphatics 225 6.4. Lymphatics as modulators of antigen-mediated immune priming 230 6.5. Driving factors involved in altered lymphatic and DC behaviour in the tumour microenvironment 232 6.6. Summary and Future Perspectives 234 Bibliography 238 APPENDICES 262 Appendix 1 – Altered gene targets in TDLN-derived LECs 263 Appendix 2 – Gene targets altered in Day 4 and Day 11 TDLN-derived LECs 264 Appendix 3 – Gene ontology pathways for altered genes 265 Appendix 4 – Altered canonical pathways and associated gene targets 267 Appendix 5 – Manual categorisation of canonical pathways 269 Appendix 6 – Altered Immunity associated gene targets 272 Appendix 7 – Flow cytometry gating for profiling dendritic cells 273 Appendix
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