Investigation and characterization of MP derived from media conditioned by various cancer cell lines and their effect on human umbilical vein endothelial cells (HUVECs) under static and flow conditions. being a thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Medical Sciences in the University of Hull by Abdulrahman Eid Saeed Algarni February 2020 Dedication Firstly, I dedicate this thesis to my mother, Gharsah. Without her support, advice and pray, I would not complete this work. Secondly, I would like to dedicate this work to my beloved wife, Badriah, for her persistent support and patience. Third, I dedicate this thesis to my beautiful daughter, Danah, for helping me and understand my long hours in University to complete this work. Finally, I dedicate this work to my sweetheart Jasmine thank you for your love and affection. i Acknowledgements First and foremost, I would like to express my sincere gratitude to Allah and praise him for helping me to complete this work. I would like to express my deepest appreciation to my supervisor Dr. Leigh Madden who has supported me through my research. Without his patience, knowledge and guidance, I wouldn’t be able to complete this project. Many thanks also for his significant comments which helped me to finalise the thesis. I am very grateful to him, who has provided insightful comments about the project results and has offered much advice and useful comments. I would like also to extend my gratitude to Professor John Greenman for his support and guidance throughout my research project. Special thanks go to Dr. Alex and Linsey Malcolm for their assistance and training during my project. I must convey my very special thanks to my best friend Dr. Ibrahim Al-Harthi for continuing listening to any complaints I had throughout the project and for his special assistance through my study. Also, I must convey my special thanks to my colleague Dr Farouq Garada, Dr. Meftah Mehdawi, and Dr. Anwar Fanan, and for their support and encouragement throughout my project. I also would like to thank my brother Dr. Saad Alahmari. Also, a very special thanks to Dr. Abdullah Shwish Al-Dwaihy and Dr. Abdullah Aloufi. Finally, I would like to express my deepest gratitude to my family for supporting me during all my studies at University. Special gratitude to my mother and all my brothers specially Abdullah and Ali and sisters and a very special appreciation to my wife Badriah and my daughters Danah and Jasmine for their support and encouragement throughout my studies. Last but not least, I would like to thank the Saudi cultural bureau and higher ministry of education in Saudi Arabia for their continual financial support. ii Publications and Conferences Published Articles: Algarni, A., Greenman, J. & Madden, L. A. (2019) Procoagulant tumour microvesicles attach to endothelial cells on biochips under microfluidic flow. Biomicrofluidics, 13(6), 064124. Algarni, A., Greenman, J. and Madden, L.A., 2020. Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices. Bloods— International Open Access Journal of Hematology, 1(1), pp.23-34. Posters presentations: Algarni, A., Greenman, J. and Madden, L.A., 2016. PO-48-Assessment of the procoagulant potential state of tumour-MP in cancer patients. Thrombosis research, 140, p.S194. Algarni, A., Greenman, J. and Madden, L.A., 2017. Tumour microvesicles interact with endothelial cells under venous flow in vitro. (Poster presented at ISTH2017, 2324—PB, Germany). iii Abstract Microparticles (MP) are procoagulant due to tissue factor and phospholipid exposure on the surface. MP are tumour-derived and can be a beneficial biomarker of cancer to recognize individuals who are susceptible to venous thrombosis. The aim of the presented work was to develop and validate an in vitro microfluidic system consisting of two distinct microfluidic biochips to enable the investigation of the relationship between tumour MP and endothelial cells in vitro. Firstly, a range of tumour cell lines were assessed for procoagulant activity (PCA) of the cells and also MP released into the media. Pancreatic AsPC-1, human glioma U87 ovarian ES-2 and SKOV-3, were found to have the highest PCA in both cell suspension and cell- free media, while pancreatic MIAPaCa-2 and ovarian A2780 had a lower PCA. Cell lines were then investigated as to whether or not they could form stable spheroids in 3D cell culture U87, AsPC-1 and ES-2 produced the most compact spheroids and had the fastest PCA. In contrast, PANC-1, MIAPaCa-2 and A2780 formed loose shaped spheroids and slower PCA. However, SKOV-3 showed small compact spheroid and slower PCA. Following the application of media flow, ES-2 and U87 were selected and transferred into the developed dual microfluidic biochips model. Labelled MP were quantified via flow cytometry and this showed MP concentration reduced over time suggesting attachment of tumour MP to HUVECs. This reduction in MP was further reflected with a loss of PCA associated with the media. The effect of Doxorubicin on tumour spheroids resulted in an increased PCA of an endothelial cell layer under flow condition. In conclusion, in this study a microfluidic two-chip dynamic model mimics the interstitial fluid flow showed that tumour MPs released from tumour spheroids attach to endothelial cells and potentially could be a mechanism of clot formation in cancer patients. iv Contents Dedication ................................................................................................................................... i Acknowledgements ................................................................................................................... ii Publications and Conferences ...................................................................................................iii Abstract ......................................................................................................................................iv Contents .....................................................................................................................................iv List of Figures ........................................................................................................................... xiii List of Tables .......................................................................................................................... xviii List of abbreviations ................................................................................................................ xix Chapter 1 General introduction .................................................................................................... 1 1.1 Cancer-associated Thrombosis (VTE) .................................................................................. 1 1.2 Cancer-induced hypercoagulability ..................................................................................... 2 1.3 VTE risk factors in cancer ..................................................................................................... 4 1.4 Venous Thrombosis ............................................................................................................. 5 Mechanisms of hypercoagulability in cancer ................................................... 5 1.5 Haemostatic system and cancer .......................................................................................... 6 Background ...................................................................................................... 6 Primary haemostasis ........................................................................................ 7 Secondary haemostasis .................................................................................... 8 1.6 Tissue factor (TF) and cancer ............................................................................................. 10 Background .................................................................................................... 10 TF in cancer progression and angiogenesis ................................................... 10 Mechanisms of TF upregulation in cancer ..................................................... 11 1.7 Microparticles (MP) ........................................................................................................... 11 Background, function, and formation ............................................................ 11 v Procoagulant potential of TF bearing MP ...................................................... 13 Procoagulant properties of membrane surfaces ............................................. 14 Methods and measurement of MP in cancer patients .................................... 15 1.8 Chemotherapy increases risk of VTE ................................................................................. 16 Background .................................................................................................... 16 Mechanisms of chemotherapeutic agents increasing VTE in cancer ............. 17 1.9 Lab on a Chip (LOC) ............................................................................................................ 18 Background .................................................................................................... 18 LOC and cancer research ............................................................................... 19 Advantages and disadvantages of LOC ......................................................... 21 Isolation