Activated Platelets and Antibody Opsonization as Tumor Markers: Novel Avenues for Cancer Diagnosis, Targeted Therapy and Monitoring Therapeutic Outcomes May Lin Yap Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy September 2018 Department of Clinical Pathology Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne Declaration This is to certify that I. This thesis comprises only of my original work towards the Doctor of Philosophy degree, except where indicated in the preface; II. Due acknowledgement has been made in the text to all other materials used; III. The thesis is fewer than 100,000 words in length, exclusive of tables, bibliographies and appendices as approved by the Research Higher Degrees Committee. May Lin Yap September 2018 I Acknowledgement To say my journey towards completing this dissertation was one filled with reward and gratification is, truthfully speaking, understating the uplifting sense of self-worth and purpose that I feel as a researcher. What is even more meaningful and of significance to me however is that I did not make this journey alone: I therefore must express my heartfelt gratitude to the people who have contributed to this endeavour. To my PhD supervisors, Mark Hogarth and Karlheinz Peter, thank you for the opportunity to undertake this PhD under both your guidance. It is such a virtuous task to be a supervisor, and I am honored to have had the opportunity to learn under such passionate leaders who continuously motivate me to be a better person. My sincerest gratitude to Geoff Pietersz and James McFadyen for your patient guidance, expertise and motivation. Also, a very special thanks to Bruce Wines for your mentorship and encouragement over the many years. To Sandra Esparon, Halina Trist, the Hogarth Lab and the Atherothrombosis group: it has been such a joy working alongside each and every one of you. To my committee members Uwe Ackerman and Graham Mackay: thank you both for the valuable insight and advice throughout my PhD journey. I would also like to acknowledge special individuals who have played a vital role in starting my academic journey: Denise Jackson and Michael Lovelace, who first introduced me to the world of medical research. Valentina Puntmann, Ehsan Sharif-Paghaleh and Greg Mullen for their supervision which inspired my interest in the world of molecular imaging. To my family: my mom and dad, who sacrificed so much to give me everything that I have ever needed in life and so much more. To May Hon and Sang Yung, my most loyal cheerleaders. Thank you for your unconditional love and support. To Allan, who has stood patiently beside me throughout this journey, thank you for believing in me. This achievement is as much yours as it is mine. To my friends Damien and Ash, Kee Hong, Jaclyn, Grace, Sheanee, Stephanus, CC, Ken, CK, Jason, Wooby, Daniel, Elisa, Jeannie, Jeannie and Aaron. To my London girls, Nicole and Elena: thank you for the fun times and for always checking up on me. And to Joe, who will always be the better half of me. Thank you for inspiring me to be the best that I can be. II Peer-reviewed articles arising from this candidature 1. M. L. Yap, J. D. McFadyen, X. Wang, M. Ziegler, Y. C. Chen, A. Willcox, P. M. Hogarth, G. A. Pietersz, B. Lim, K. Peter, Activated platelets in the tumor microenvironment for targeting of antibody-drug-conjugates to tumors and metastases, Theranostics. 9, 1154– 1169 (2019). 2. M. L. Yap and K. Peter, Molecular positron emission tomography in cardiac ischemia/reperfusion. Providing a path to personalised medicine!, Circ Res, (2019). (Accepted for publication) 3. M. Ziegler, X. Xu, M. L. Yap, H. Hu, J. Zhang, K. Peter, A Self-Assembled Fluorescent Nanoprobe for Imaging and Therapy of Cardiac Ischemia/Reperfusion Injury, Advanced Therapeutics 0, 1800133. 4. M. Ziegler, K. Haigh, T. Nguyen, X. Wang, B. Lim, M. L. Yap, E. M. Eddy, J. J. Haigh, K. Peter, The pulmonary microvasculature entraps induced vascular progenitor cells (iVPCs) systemically delivered after cardiac ischemia-reperfusion injury: Indication for preservation of heart function via paracrine effects beyond engraftment, Microcirculation, e12493 (2018). 5. M. L. Yap, X. Wang, G. A. Pietersz, K. Peter, Mesoscale Nanoparticles: An Unexpected Means for Selective Therapeutic Targeting of Kidney Diseases!, Hypertension 71, 61–63 (2018). 6. M. L. Yap, J. D. McFadyen, X. Wang, N. A. Zia, J. D. Hohmann, M. Ziegler, Y. Yao, A. Pham, M. Harris, P. S. Donnelly, P. M. Hogarth, G. A. Pietersz, B. Lim, K. Peter, Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging, Theranostics 7, 2565–2574 (2017). 7. G. A. Pietersz, X. Wang, M. L. Yap, B. Lim, K. Peter, Therapeutic targeting in nanomedicine: the future lies in recombinant antibodies, Nanomedicine 12, 1873–1889 (2017). 8. B. Lim, Y. Yao, A. L. Huang, M. L. Yap, U. Flierl, J. Palasubramaniam, M. T. K. Zaldivia, X. Wang, K. Peter, A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel ThreeDimensional Fluorescence Emission Computed Tomography (FLECT) Technology, Theranostics 7, 1047–1061 (2017). III 9. B. D. Wines, M. L. Yap, M. S. Powell, P.-S. Tan, K. K. Ko, E. Orlowski, P. M. Hogarth, Distinctive expression of interleukin-23 receptor subunits on human Th17 and γδ T cells, Immunol Cell Biol 95, 272–279 (2017). IV Awards and Presentations 1. Recipient of Poster Prize at the Eradicate Cancer World Congress 2018 Poster Title - Imaging Activated Platelets in Cancer – An avenue for early cancer detection and targeted treatment. 2. Recipient of the Baker Heart and Diabetes Institute 2018 Travel Award. 3. Presented at the European Society of Molecular Imaging 2018 Poster Title - Imaging Activated Platelets in Cancer: A Novel Approach for Cancer Imaging. 4. Presented at the Australian Society of Molecular Imaging (ASMI) Scientific Meeting 2017 Oral Presentation Title - Targeting Activated Platelets: A Novel Approach for Cancer Imaging. 5. Presented at the Australian Vascular and Biology Society (AVBS) 2017 Poster Title - Targeting Activated Platelets: A Novel Approach for Cancer Imaging. 6. Recipient of the Bright Sparks Travel Award from the Baker Heart and Diabetes Institute 2017. 7. Recipient of FASMI Young Investigator’s Award at the Federation of Asian Society for Molecular Imaging (FASMI) 2017 Oral Presentation Title - Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging. 8. Presented at the Australian Society of Medical Research (ASMR) Victorian Student Symposium 2017 Oral Presentation Title - Targeting Activated Platelets: A Novel Approach for Cancer Imaging. 9. Recipient of Student Poster 1st Prize at the Australian Society of Molecular Imaging (ASMI) Scientific Meeting 2015 Poster Title - Non-invasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post Cardiac Transplant. V Table of Contents Declaration ............................................................................................................ I Acknowledgement ............................................................................................... II Peer-reviewed articles arising from this candidature .......................................... III Awards and Presentations .................................................................................... V Abstract ...............................................................................................................IX Chapter 1. Literature Review ................................................................................ 1 1.0 Platelets ....................................................................................................................... 1 1.1 Platelet Structure ......................................................................................................... 1 1.2 Platelet Function .......................................................................................................... 3 1.3 The Role of Platelets in Cancer ................................................................................... 4 1.3.1 Platelet Mediated Epithelial-Mesenchymal Transition ........................................ 5 1.3.2 Platelets Dependent Immune Evasion of Tumor Cells ........................................ 6 1.3.3 Platelets Effect on Tumor Cell Arrest and Extravasation .................................... 6 1.3.4 Platelets Role in Tumor Growth and Angiogenesis ............................................. 7 2.0 Molecular Imaging ...................................................................................................... 8 2.1 Radionuclide Imaging - PET and SPECT ................................................................... 9 2.1.1 Positron Emission Tomography (PET) Imaging................................................ 10 2.1.2 Single-photon Emission Computed Tomography (SPECT) Imaging ................ 11 2.2 Fluorescence Imaging ............................................................................................... 12 2.2.1 The Role of Fluorescence Imaging in Cancer.................................................... 13 2.3 Ultrasound ................................................................................................................. 14 2.3.1 The Role of Ultrasound in Cancer ..................................................................... 15 2.4 Computed Tomography (CT) ...................................................................................