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Understanding and Predicting Cancer Cell Aggressiveness from Their Migratory Phenotypes • Advisor: Konstantinos Konstantopoulos

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Understanding and Predicting Cancer Cell Aggressiveness from Their Migratory Phenotypes • Advisor: Konstantinos Konstantopoulos UNDERSTANDING AND PREDICTING CANCER CELL AGGRESSIVENESS FROM THEIR MIGRATORY PHENOTYPES By Bin Sheng Wong A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland April 2019 © 2019 Bin Sheng Wong All Rights Reserved Abstract Cell migration is an integral process for diverse physiological and pathological processes, including embryonic and tissue development, wound healing, immune response, chronic inflammation and cancer metastasis. Cancer metastasis, a coordinated and complex multistep process by which tumor cells disseminate from primary tumors to secondary sites, is responsible for up to 90% of all cancer-related death. During metastasis, tumor cells must interact, migrate and navigate through considerably different microenvironments including tissues at the local and distal sites, as well as within the circulatory system. As such, enhanced cell motility has been widely associated with increased cancer aggressiveness and metastatic potential, and overall poor patient outcome. A thorough understanding of the biochemical and physicomechanical driving forces underlying cell motility can therefore provide critical insights for the development of novel and effective diagnostic, preventative, therapeutic and prognostic strategies against cancer. We start by first examining the role of podocalyxin on the migratory behavior of pancreatic cancer cells. The sialoglycoprotein podocalyxin is absent in normal pancreas but is overexpressed in pancreatic cancer and is associated with poor clinical outcome. Here we investigate the role of podocalyxin in migration and metastasis of pancreatic adenocarcinomas using SW1990 and Pa03c as cell models. Although ezrin is regarded as a cytoplasmic binding partner of podocalyxin that regulates actin polymerization via Rac1 or RhoA, we did not detect podocalyxin-ezrin association in pancreatic cancer cells. Moreover, depletion of podocalyxin did not alter actin dynamics or modulate Rac1 and RhoA activity in pancreatic cancer cells. Using mass spectrometry, bioinformatics analysis, co-immunoprecipitation, and pulldown assays, we discovered a novel, direct binding interaction between the cytoplasmic tail of podocalyxin and ! ""! the large GTPase dynamin-2 at its GTPase, middle, and pleckstrin homology domains. This podocalyxin-dynamin-2 interaction regulated microtubule growth rate, which in turn modulated focal adhesion dynamics and ultimately promoted efficient pancreatic cancer cell migration via microtubule- and Src-dependent pathways. Depletion of podocalyxin in a hemispleen mouse model of pancreatic cancer diminished liver metastasis without altering primary tumor size. We next apply our knowledge of cell migration to develop a device for glioblastoma prognosis. Glioblastoma is the most aggressive form of brain cancer, characterized by high recurrence and dismal prognosis. Presently, there is no effective in vitro platform that can rapidly measure complex cellular phenotypic traits and accurately predict patient-specific clinical outcomes. Here, we employed a proprietary in vitro testing platform, Microfluidic Invasion Network Device (MIND), and screened a panel of 22 patient-derived primary glioblastoma specimens in a blind manner. We evaluated the ability of glioblastoma cells to navigate and squeeze through confined microenvironments that mimic in vivo tight perivascular conduits and white matter tracts in the brain parenchyma, as well as the proliferative capacity of highly motile subpopulations. By combining migratory- and proliferative-based indices, MIND predicts progression-free survival (p=0.008) and time to recurrence (p=0.006) retrospectively with high sensitivity (85%), specificity (89%), and accuracy (86%).! In a pilot prospective study, MIND classified all patients accurately based on their survival outcomes. Overall, this dissertation illustrates the importance of studying cell migration, both at the basic science as well as the translational levels, to better understand the mechanisms of cancer aggressiveness and develop potentially useful clinical applications. ! """! Thesis Committee Professor Konstantinos Konstantopoulos (Advisor) Department of Chemical and Biomolecular Engineering Professor Denis Wirtz Department of Chemical and Biomolecular Engineering Associate Professor Stavroula Sofou Department of Chemical and Biomolecular Engineering Associate Professor Lei Zheng (Chair) Department of Oncology Assistant Professor Luo Gu Department of Materials Science and Engineering ! "#! Acknowledgements First and foremost, I would like to express my deepest gratitude to my Ph.D. advisor Dr. Konstantinos Konstantopoulos for his leadership, mentorship and guidance. It has been an honor working in his laboratory and learning what it takes to become a great scientist. Throughout my time here, he has created and maintained a conducive, collaborative and financially supportive work environment for me to explore interesting scientific questions and acquired various important skills, both scientifically and professionally, that are crucial for my future career. More importantly, I am continuously inspired by his unwavering integrity and passion for science, which has been a major driving force that has kept me motivated and not discouraged when experiments were not working out as expected. Next, I would like to thank all the members, both past and present, of the Konstantopoulos laboratory, whom I have had the pleasure to work with on a daily basis: Dr. Kimberly Stroka, Dr. Zhizhan Gu, Dr. Wei-Chien Hung, Dr. Colin Paul, Dr. Daniel Shea, Dr. Panagiotis Mistriotis, Dr. Alexandros Afthinos, Dr. Christopher Yankaskas, Emily Wisniewski, Robert Law, Runchen Zhao, Soontorn Tuntithavornwat, Yuqi Zhang, Kaustav Bera, Se Jong Lee, Christina Hum, Avery Tran, Elizabeth Orth, Tian Zhu, Nianchao Wang, Pranav Mehta, Siqi Cui, Yao Wang. In addition, I would also like to thank the undergraduate and high school students whom I have had the opportunity to mentor over the years, and have assisted me in various ways: Alec Greenberg, Yuan Hao Wong, Jake Bieber, Deborah Chin and Amber Fang. Among my colleagues, I would like to extend my gratitude specifically to Panos who has introduced and taught me, as well as the entire lab, many important molecular biology techniques ! #! that have entirely transformed how the Konstantopoulos lab approaches complex cell biology problems. His positive energy and enthusiasm for science are always infectious and stimulating. A special thank you to Dan whom I have worked with closely on all the pancreatic cancer related projects. Your efficient and meticulous work ethic has helped pushed our projects forward significantly. I would also like to acknowledge Chris, for not only being a responsible laboratory captain whom I can always rely on and go to if there is anything wrong with the laboratory, but also for his insightful contribution to the glioblastoma prognosis project. Lastly, I would like to thank Soontorn for being such a loyal friend whom has not only been tremendously helpful especially for the in vivo mice work, but also someone whom I look forward to having lunch everyday and confide in with my deepest personable troubles (as well as appointments, don’t worry he will understand what I meant by appointments if he reads this). While the members of the Konstantopoulos lab are all amazing and talented individuals, I will not be able to complete my projects without the constant and enormous support from my collaborators. Thank you to Dr. Lei Zheng for his expertise in pancreatic cancer mice models and for training me to perform sophisticated mice surgeries. Thank you to Dr. Joy Yang who has guided me on the molecular cloning of recombinant podocalyxin probes. Thank you to Dr. Alfredo Quiñones-Hinojosa and Dr. Sagar Shah for providing valuable primary glioblastoma patient samples and the analysis of clinical outcomes of these patients. Outside of work and laboratory, I am lucky to have met many wonderful friends who have immensely enriched my life. I would like to first thank David Raciti for being my first roommate and the first friend I made in the United States. Though only a month older than me, David has ! #"! always been like a big brother to me, teaching me many important life skills including riding a bicycle, swimming and driving a car. His dedication to his research and work has also been an inspiration to me. Next, I would like to express my deepest appreciation for Nash Rochman and Scott Albert, whom I have lived with for the past 4-5 years. They have not only been excellent roommates but also my best friends and family here. Over the years, we have shared many memorable (me!m(")r"b(")l) moments together, including the road trip down to South Carolina, weekly dinner at the Dizz, yearly corn maze and various musical adventures. They are always there for me when I need help or just someone to talk to, and I truly treasure the close friendships that we have built together. Last but certainly not least, I have to thank Mathew Kempske for everything that he has done for me. I will not be the man I am today without him. He is the impetus that made me more open and accepting of myself and my sexuality, which had been a silent battle and constant struggle for years before our encounter. I am also a more caring and better individual because of him. Thank you for tolerating my quirks, occasional nonsense and fervent love of musicals. I really cherish all the good (as well
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