TUMOUR-CONDITIONED MACROPHAGES UP-REGULATE ENDOTHELIAL-DERIVED FIBRONECTIN TO FACILITATE LUNG METASTASIS IN BREAST CANCER LOW PIN YAN (B.Sc (Hons), NUS) THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2015 i ii ACKNOWLEDGEMENTS First and foremost, I would like to express immense gratitude to my supervisor, Dr Lim Yaw Chyn Without her, this project would not have been possible I am extremely grateful for her continuous supervision and strong support throughout my graduate studies You have imparted valuable scientific research knowledge which is very helpful to my future career Your critical scientific input has contributed to what I have achieved today I sincerely thank you for the countless opportunities that you have given me Other than a supervisor, you have been a great companion both in the lab and outside of the lab I will not forget our delightful conference/travel experience in San Diego Thank you for being a wonderful mentor and a listening ear all these years I would also wish to extend my sincere thanks to Assoc Prof Chong Siew Meng and Assoc Prof Celestial Yap for the invaluable guidance and the critical inputs throughout this project To A/Prof Chong, every discussion with you has been educational and brain teasing I greatly appreciate you for taking time off from your busy schedule to attend my presentation rehearsals and helping me perfect every presentation I also greatly appreciate A/Prof Celestial Yap and her lab members for their help throughout my graduate studies Thank you for the reagents and experimental tools that you have generously shared with me To my thesis advisory committee (TAC) members Prof Peter Lobie and Dr Seet Ju Ee, thank you for the assistance and advice during the last two years of my studies To my fellow lab mates, Joe Thuan, Chi Kuen, Chee Wai, Kim Yee and Huey Jin, thank you for making my lab experience so memorable and enjoyable I truly enjoy working with them and they have made this journey a lot easier Outside of lab, you guys have been such dear friends I treasure our friendship and thank you for helping me survive all the stress i To my boyfriend, Stephen, thank you for being so understanding, supportive and caring during the last year of my study Thanks for sharing my joy, sorrow and hardship Thank you for being there and looking out for me all these while Last but not least, I would like to express deep gratitude to my family: my parents and my brother, for their support and patience throughout this entire period Thank you for taking care of me and allowing me to fully focus on my research ii Contents ACKNOWLEDGEMENTS i SUMMARY .vii LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiv CHAPTER 1: GENERAL INTRODUCTION 1.1 Metastasis 1.1.1 Organ specific metastasis 1.2 Tumour adhesive interactions with endothelium 1.2.1 E-selectins and Immunoglobulin superfamily 1.2.2 Integrins 1.2.3 E-selectins and Immunoglobulin superfamily in cancer 1.2.4 Integrins in cancer 10 1.3 Extravasation of tumour cells 11 1.3.1 Endothelium disruption by tumour cells 12 1.3.2 Matrix metalloproteinases 14 1.3.3 Matrix metalloproteinases in cancer 16 1.4 Tumour microenvironment 17 1.4.1 Inflammation and cancer 18 1.4.2 Tumour Necrosis Factor-alpha (TNF-α) and its role in cancer 21 1.4.3 Interleukin-6 (IL-6) and its role in cancer 24 1.4.4 Pre-metastatic niche 28 1.4.5 Fibronectin and its role in cancer 33 1.5 Signalling pathways in cancer 36 1.5.1 JNK/cJUN signalling and its role in cancer 37 1.5.2 STAT3 signalling and its role in cancer 39 1.6 Breast cancer 42 1.6.1 Tumour microenvironment and breast cancer progression 43 1.7 Aims and objectives 45 CHAPTER 2: MATERIALS AND METHODS 49 2.1 Breast Cells Culture 49 iii 2.1.1 Preparation of tumour-conditioned media 49 2.2 Endothelial Cells Culture 50 2.2.1 Preparation of gelatin coated dishes 50 2.2.2 Isolation of Human Umbilical Cord Vein Endothelial cells 50 2.2.3 Isolation of Saphenous Vein Endothelial Cells (SVEC) 52 2.2.4 Isolation of Fetal Lung Endothelial Cells (FLEC) and Adult Lung Endothelial Cells (ALEC) 53 2.3 Monocytes, Macrophages, Dendritic Cells and Tumour-conditioned Macrophages culture 55 2.3.1 Monocytes isolation 55 2.3.2 Macrophages and Tumour- conditioned Macrophages differentiation 56 2.3.3 Dendritic cells differentiation 57 2.4 Flow cytometry 58 2.4.1 Cell surface markers and adhesion molecules expression 59 2.4.2 MCSF-mϕ, MDA-mϕ, M1-mϕ, M2-mϕ and DC characterisation 62 2.5 Tumour cell-endothelial cell interaction study 65 2.5.1 Tumour cell binding assay 65 2.5.2 Trans-endothelial migration assay 67 2.6 Immunofluorescence staining 70 2.6.1 Tumour endothelial interactions 70 2.6.2 Fibronectin staining 73 2.7 Western blot 74 2.8 Enzyme-linked immunosorbent assay (ELISA) 76 2.9 PCR array 77 2.10 SiRNA Transfection 78 2.11 Statistical analysis 80 CHAPTER 3: Interactions of breast cancer cells with endothelial cells from metastatic sites and non-metastatic sites 81 3.1 Introduction 81 3.2 Results 84 3.2.1 Endothelial cells from various organ sites were able to support the adhesion of breast cells 84 3.2.2 Invasive MDA-MB-231 most readily transmigrated across lung endothelial monolayer 87 iv 3.2.3 FLEC is most susceptible to invasion by invasive breast tumour cells, MDA-MB-231 92 3.2.4 Matrix metalloproteinases (MMPs) 2, MMP3 and MMP8 are involved in facilitating trans-endothelial migration of MDA-MB-231 99 3.3 Discussion 105 CHAPTER 4: Primary tumour-derived factors could transform microenvironment of distant organs into potential sites of metastasis 114 4.1 Introduction 114 4.2 Results 118 4.2.1 Enhancement of tumour interactions by TNF-α is specific to the lung endothelial cells 118 4.2.2 Tumour secreted factors in MDA-MB-231 conditioned medium did not enhance tumour-endothelial interactions 122 4.2.3 Tumour-conditioned macrophages play a key role in enhancing tumour cell interactions specifically to lung endothelium 125 4.2.4 TNF-α and MDA-mϕ CM stimulation of FLEC up-regulated expression of MMP2, MMP3, collagen, fibronectin, veriscan, TGFBI and integrins α2, 135 4.2.5 Integrins α5β1, α6β1 and α6β4 on tumour cells are involved in the adhesion of MDA-MB-231 and MCF-7 to TNF-α- and MDA-mϕ CMstimulated FLEC 138 4.2.6 Breast tumour-lung endothelium adhesion is mediated by the binding of α5β1 integrin on breast tumour cells to fibronectin on the lung endothelium 147 4.3 Discussion 156 CHAPTER 5: MDA-mϕ derived IL6 up-regulates endothelial fibronectin expression via activation of the STAT3 signalling pathway 167 5.1 Introduction 167 5.2 Results 171 5.2.1 MDA-mϕ CM stimulation activates the JNK/cJUN and STAT3 signalling pathways in FLEC 171 5.2.2 JNK/cJUN and STAT3 signalling pathways are involved in regulating FLEC fibronectin surface expression 174 5.2.3 MDA-mϕ secretes higher concentration of IL-6 compared to MCSF-mϕ 191 5.2.4 MDA-mϕ-derived IL-6 plays a role in the up-regulation of endothelial surface fibronectin and tumour adhesion 195 v 5.3 Discussion 203 5.3.1 MDA-mϕ CM activates JNK/cJUN and STAT3 signalling pathways in lung endothelium 203 5.3.2 JNK/cJUN and STAT3 pathways regulate fibronectin expression on MDA-mϕ CM stimulated lung endothelium 205 5.3.3 MDA-mϕ-derived IL-6 is the stimulating factor that up-regulates fibronectin expression in lung endothelium 207 CHAPTER 6: GENERAL DISCUSSION 212 6.1 Activated endothelium is the gateway to successful colonisation of the distant target organ 213 6.2 Tumour-conditioned macrophages promote pre-metastatic niche formation 217 6.3 Conclusion 220 6.4 Future work 223 REFERENCES 227 APPENDIX I 261 vi SUMMARY Organ-specific metastasis is dependent on both the intrinsic properties of the tumour cells and the receptiveness of the host organ Primary tumour cellderived factors can modulate distant microenvironment to become more receptive to colonisation by circulating tumour cells Such sites are known as the pre-metastatic niche The endothelium is a physical barrier that the disseminated tumour cells encounter during extravasation into the secondary site However, it is not known whether tumour secreted factors could alter endothelial function during pre-metastatic niche formation to augment tumourendothelial interactions The aim of this study is to develop a robust co-culture model using breast tumour cells, endothelial cells derived from lung (FLEC), and in-vitro differentiated macrophages to systematically examine the cell-cell interactions between these three cell types Data from this study will help elucidate the mechanisms underlying the homing and extravasation of breast cancer cells to the lung Using breast tumour cells (MDA-MB-231 and MCF-7) and endothelial cells isolated from various organs, tumour-endothelial interactions including stable binding and trans-endothelial migration were examined in-vitro In order to simulate the systemic effect of tumour-secreted factors on extravasation, the different types of endothelium were subjected to stimulation with various factors (TNF-α, conditioned media from MDA-MB-231 (MDA-MB-231 CM) and from tumour-conditioned macrophages (MDA-mϕ CM)) prior to use in the tumour-endothelial interaction assays The adhesion molecules involved in these interactions and the signalling pathways that regulate the endothelial responses were also studied vii My data revealed that FLEC was more susceptible to invasion by breast cancer cells, MDA-MB-231, compared to endothelium isolated from the umbilical cord vein and saphenous vein, which represent the vasculature at nonmetastatic sites In addition, FLEC-derived MMP2 and a yet unidentified MMP(s) secreted by MDA-MB-231 acted in concert to disrupt FLEC monolayer integrity This resulted in the formation of “gaps” in the FLEC monolayer, thus allowing the tumour cells to transmigrate across endothelial monolayer more readily FLEC subjected to stimulation with TNF-α and MDA-mϕ CM exhibited significantly more interactions with MDA-MB-231 and MCF-7 cells than untreated FLEC or FLEC treated with MDA-MB-231 CM Such enhanced interactions were not observed with endothelium from non-metastatic sites under similar conditions The observed tumour cell-FLEC interactions were mediated by the binding of tumour-α5β1 integrin to fibronectin expressed on the MDA-mϕ-primed FLEC surface The enhanced expression of fibronectin on MDA-mϕ primed FLEC was regulated by the activation of the JNK/cJUN and STAT3 signalling pathways My data also identified MDA-mϕ-derived interleukin (IL-6) as the dominant mediator that primed FLEC in this model system Data from this study suggest that factors secreted by breast cancer cells can transform resident macrophages in target organs to become M2-like with protumour properties; and these conditioned-macrophages in turn prime the organ microenvironment Macrophage-derived IL-6 enhances fibronectin expression in the endothelial cells via the 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fragment circulating level increases in patients with metastatic pancreatic ductal cell adenocarcinomas Cancer Lett 225, 167-76 (2005) 260 APPENDIX I Poster Presentation P Y LOW, C YAP, Y C LIM, “Redistribution of endothelial junction protein PECAM-1 and MMP activity are important for transmigration of breast tumour cells across lung endothelial monolayer in-vitro” (Second AACR International Conference on Frontiers in Basic Cancer Research, Sep 14 - 18 2011, InterContinental San Francisco, San Francisco, United States) P Y LOW, Y C LIM, “Upregulation of fibronectin expression on endothelial cells by tumour-conditioned macrophages facilitates lung metastasis in breast cancer” (Cellular Heterogeneity in the Tumour Microenvironment, AACR Special Conference, Feb 26 –March 2014, Westin Gaslamp Quarter, San Diego, CA, United States) Oral Presentation Y C LIM, P Y LOW, “Breast cancer cells modulate the tissue microenvironment of distant sites to facilitate metastasis” (CSH Asia/ International Cancer Microenvironment Society ICMS Joint Conference on Tumour Microenvironment, Nov 13-17 2012, Suzhou, China) Y C LIM, P Y LOW, "The Role and Contribution of the Endothelium to Formation of the Premetastatic Niche and to Organ Specific Metastasis" (BIT’s 6th Annual World Congress of Cancer, May 23-25 2013, Xi’an, China) 261 ... subunits of β1 integrin family that contain the αA-domain Laminin is also found to bind to non-αA-domain containing integrins such as integrins α3β1, α6β1, α7β1 and α6β4 37,44-46 Integrins also function... adhesion of tumour cells during the process of metastasis 61 Integrin αLβ2 and mucin (MUC1) can bind to ICAM-1 to mediate breast cancer cells adhesion 62,63 Similarly, integrin α4β1 interacts... within 1-2 weeks after the injection of tumour secreted factors 230,235 These tumour- derived factors comprise pro-angiogenic factors, hypoxiadependent factors, inflammatory cytokines, chemokines,