The Omental Fat Band as an Immunomodulatory Microenvironment for Ovarian Cancer Courtney Alicia Cohen Dissertation submitted t o the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical and Veterinary Sciences Paul C. Roberts, Chair Eva Marie Schmelz William S. Swecker David R. Bevan Liwu Li April 30th, 2013 Blacksburg, VA. Keywords: omentum, ovarian cancer, metastasis, microenvironment, parity, IL-12 The Omental Fat Band as an Immunomodulatory Microenvironment for Ovarian Cancer By Courtney Alicia Cohen (Abstract) Cancer research is evolving. Historically concerned with the mechanisms by which malignant cells circumvent cell death signaling and maintain unchecked proliferation, focus has shifted to the complex interactions between the tumor cell and the surrounding microenvironment. Ovarian cancer has one of the highest incidence-to-death ratios of all cancers, and is typically asymptomatic until the later stages, often resulting in metastasis prior to discovery. Naturally occurring phenotypes like lactation and child-bearing (parity) reduce ovarian cancer incidence, but the mechanisms are not understood. As the primary site for ovarian cacner metastasis, and a secondary lymphoid organ capable of mounting potent innate and adaptive immune responses, we believe the omental fat band (OFB) provides a unique opportunity to study complex interactions within the tumor microenvironment. Additionally, we hypothesize that once understood, leukocyte populations within the OFB could be modulated to disrupt the pro-tumorigenic cascade. Using fluorescence-activated cell sorting (FACS) and quantitative realtime PCR (qRT-PCR), we comparatively evaluated the changes in the compositional immune profile of the OFB as a result of parity and cancer. Parous mice were associated with a reduction in macrophages and neutrophils in the OFB, resulting in an inherent “protective state” that was refractory to metastatic cancer cell growth after intraperitoneal implantation. This indicates that the leukocyte populations within the OFB play an important role in tumor development. Therefore we utilized the potent TH1-type immunomodulatory cytokine IL-12 in a membrane-bound form to circumvent reported side effects, such as hepatic and renal damage, cardiotoxicity and death. Targeted IL-12 delivery to the OFB resulted in delayed disease development, although not protection from subsequent challenge. This was also associated with a reduction tumor-associated macrophages (TAMs) and neutrophils (TANs) within the OFB. Kinetic studies demonstrated that these changes were induced by a significant reduction in neutrophil and macrophage chemoattractants early on in the pro-tumorigenic cascade (7 days post- implantation). This work demonstrates that the OFB is a functionally plastic tissue that can be harnessed and re-mobilized to display an anti-tumorigenic microenvironment. iii Acknowledgements I am truly thankful for all the wonderful memories and experiences since joining this program. To all those faculty, fellow students and friends that touched my life during my time at Virginia Tech, I am extremely grateful. To my mentors, Dr. Roberts and Dr. Schmelz, thank you for taking me on late in the game, and for your time, guidance and expertise. To my committee, Dr. Bevan, Dr. Swecker and Dr. Li, thank you for all the constructive input, and for your incredible patience. To Dr. Ward Kirner, thank you for making the trip up from Atlanta to serve as my external examiner, for your advice and good humor. I have had wonderful labmates, both past and present. To Dr. Andrew Herbert, who has always been my example of what a strong scientist should be; your integrity, honesty, and respect have always been the motivation that got me through this program. Your confidence in me gave me confidence in myself; I am incredibly grateful to have gotten the chance to work with you, and I’m honored to call you a friend. To Lynn Heffron, our lab momma, you are a brilliant scientist with extraordinary potential, and I can’t wait to see how high you’ll rise now that you’ve been given the chance. You have always been indispensible in the lab, both during animal studies and as a scientific sound board, and I am truly thankful for the extraordinary amount of help and time you have always given me without a second thought. To Amanda Shea, my co-worker, counterpart, and absolute best friend in the world, there is no way I could have gotten through this program without you. You have been a friend, mentor, counselor and cheering squad throughout this process, and I can only hope that I’ve been the same for you. I can’t wait to watch you turn the world of science policy on its head, there’s no one I’d trust more to take on the job than you! To Dr. Angela Anderson, your constant positive attitude and dedication to your students has been an inspiration over the years, and I’m thankful for all the advice and support. To all the other lab members I have been fortunate to work with, Dr Neeraj Singh, Matthew Kurnick, Julie Karfakis, Elizabeth Strawn, Dr Tila Khan, Amanda Gasser, Joey Mazzei, Dr Binu Velayudhan, and so many others; you have all been a part of getting me to where I am today with your support, guidance, laughter and love. I have been fortunate to work with so many amazing people. iv Thank you to my family, who has never been anything but encouraging; I am very lucky to have such an incredible support system. To my parents, who have been extraordinarily patient and shown an amazing amount of faith, thank you for always believing in me. To my big brother Jim and sister-in-law Michele, thank you for always giving me the confidence to be myself, to be strong, and for showing me how to face my problems with humor and determination. To my little sisters Tessa and Sarah, thank you for always seeing me in the best possible light, for believing in me, and for being such a blast (even if we are stuck at the beach in a monsoon). To my brother-in-law Mario, you have always astounded me with your incredible work ethic, fun-loving nature and compassion; thank you for being a part of our lives, and for taking such good care of my little sister. To my Aunt Pam, thank you for making such a long trip to watch me defend, for igniting my love of reading, and for our ‘adventures’ when I was a kid. Thank you to my grandfather, Dr. Stanley Cohen, who was such an incredible role model, motivator and friend. You taught me the importance of hard work, critical thinking and a wide appreciation for all the wonder in the world. I wish you could have been here to see me accomplish this goal, thank you for always believing in me. To my wonderful grandmother Leanore Cohen, thank you for all your support and guidance, you have always shown me how to face obstacles with grace and a strong will, and I admire those qualities in you very much. To my grandparents Alice and Jim Childs, thank you for your love and for always being so proud of my every little accomplishment. To all the awesome and incredible friends I have made in the last 5 years, I could write pages describing all the little ways that you have improved my life, learning and experiences here. To Andy and Karlie Herbert, Amanda Shea, Josh Nicholson, Anne and Bill Hyman, Josh and Heather Graham, Neeraj Singh, Ashish Ranjan, Brent Sanford, Clifton Cassidy, Jeff and Stacia Alexander, Lauren Thomas, Gade Kimsawatde, Vrushali Chavan, Andrew Fulton, Jason Ridley, Angela Anderson, Tim and Kara Hudson, Megan Duvall, Matthew and Lauren Moskitis, Brian Gehring, Brian Lauby, Jen Morrison, Stephen Lindamood and all the other wonderful people that I’ve had the honor of knowing, you have all been a cherished part of my life, and I thank you for that. v I reserve the biggest thank you for my wonderful boyfriend James. From the moment I met you, my life changed for the better. You have always believed in me without doubt, you’ve been my biggest fan and supporter, and an incomparable force of good and kindness in my life. I have nothing but admiration for your integrity, strength and compassion, and I’m incredibly grateful for all your support, especially in the last few months. I could never have gotten through this without you; you believed in me, so I couldn’t fail. I love you, and I will be grateful for the rest of my life. vi Attributions Several colleagues aided in the writing and research behind the chapters presented as part of this dissertation. A brief description of their contributions is included here. Chapter 3. Intra-abdominal Fat Depots Represent Distinct Immunomodulatory Microenvironments: A Murine Model. Chapter 3 was accepted in PlosOne. Shea, A.A. is a member of the Schmelz lab in the Human Nutrition, Food and Exercise (HNFE) department at VT. As co-author of this paper, she aided in the design and performance of the experiments, collection and analysis of data, generation of figures, interpretation of data and the writing of this paper. Heffron, C.L. is a member of the Roberts lab in the Biomedical Sciences and Pathobiology (DBSP) department at VT. As a co-author of this paper, she aided in experimental planning, animal work and sample collection and processing. Schmelz, E.M. is the Principal Investigator of the Schmelz lab in the HNFE department at VT. As a co- author of this paper, she aided in the design of these experiments, the interpretation of data and the writing of this paper, and provided editorial comments; she was co-principal investigator for one of the grants supporting this research.