University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Engineered Marrow Macrophages For Cancer Therapy: Engorgement, Accumulation, Differentiation, And Acquired Immunity Cory Alvey University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Pharmacology Commons Recommended Citation Alvey, Cory, "Engineered Marrow Macrophages For Cancer Therapy: Engorgement, Accumulation, Differentiation, And Acquired Immunity" (2017). Publicly Accessible Penn Dissertations. 2164. https://repository.upenn.edu/edissertations/2164 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2164 For more information, please contact [email protected]. Engineered Marrow Macrophages For Cancer Therapy: Engorgement, Accumulation, Differentiation, And Acquired Immunity Abstract The ability of a macrophage to engulf and break down invading cells and other targets provides a first line of immune defense in nearly all tissues. This defining ability ot ‘phagos’ or devour can subsequently activate the entire immune system against foreign and diseased cells, and progress is now being made on a decades-old idea of directing macrophages to phagocytose specific targets such as cancer cells. Physical properties of cancer cells influence phagocytosis and relate via cytoskeleton forces to differentiation pathways in solid tumors. Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of CD47, a ‘marker of self.’ These macrophages were then systemically injected into mice with fluorescent human tumors. Within days, the tumors regressed, and fluorescence analyses showed that the more the SIRPα-inhibited macrophages engulfed, the more they accumulated within tumors. In vitro phagocytosis experiments on transwells revealed that macrophage migration through micropores was inhibited by eating. However, during the 1-2 weeks of tumor residency, donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα tumor associated macrophage. Analyses of macrophages on soft or stiff collagenous gels demonstrated a stiffness-driven upregulation of SIRPα. Tissue stiffness also seems to have a role in the development of cancer. Meta-analyses suggest that genomic variation across tumors scales with the stiffness of the tumor tissue of origin. These genomic changes give rise to neoantigens that are important for the development of monocyte/ macrophage immunotherapies. Indeed, marrow-derived macrophages can phagocytose melanoma cells and present neoantigens leading to the activation of T-cells. Unlike past injections of anti-CD47 which cause anemia, no measurable impact on blood profiles, weight, or development of GvHD was observed from systemic injections of SIRPα-inhibited donor macrophages. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Dennis E. Discher Subject Categories Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2164 ENGINEERED MARROW MACROPHAGES FOR CANCER THERAPY: ENGORGEMENT, ACCUMULATION, DIFFERENTIATION, AND ACQUIRED IMMUNITY Cory Michael Alvey A DISSERTATION In Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation ___________________________ Dr. Dennis E. Discher, Ph.D, Robert D. Bent Professor of Chemical and Biomolecular Engineering Graduate Group Chairperson _________________________________________ Dr. Julie A. Blendy, Ph.D. Professor of Pharmacology Dissertation Committee Dr. Jeffrey M. Field, Ph.D. Professor of Pharmacology Dr. Vladimir Muzykantov, M.D., Ph.D., Professor of Pharmacology Dr. Wenchao Song, Ph.D. Professor of Pharmacology Dr. Ellen Pure, Ph.D. Professor of Biomedical Sciences ENGINEERED MARROW MACROPHAGES FOR CANCER THERAPY: ENGORGEMENT, ACCUMULATION, DIFFERENTIATION, AND ACQUIRED IMMUNITY COPYRIGHT 2017 Cory Michael Alvey ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my mentor and thesis advisor Dr. Dennis Discher for believing in me and taking me in his laboratory to pursue my dissertation. I am fortunate to work with a mentor who is a champion of cross-disciplinary approaches to medical research. Though his approach of, “let’s have three progress updates a day” was intense, it was this tactic that kept me on my toes and allowed me to make great progress with my publications. I am grateful that he was open to many ideas I proposed and allowed me to pursue them. His ability to interact with each of the laboratory members with equal intensity, in addition to his large family, made a strong impression on me. I would also like to thank all members of my thesis committee: Dr. Jeff Fields, Dr. Vladimir Muzykantov, Dr. Wenchao Song, and Dr. Ellen Pure for their guidance and support throughout my graduate career. Each meeting with my committee was productive and helped me significantly with the development of my thesis work. I would like to thank the University of Pennsylvania Pharmacology Program with a specific shout-out to the class of 2017. My first year here was my hardest and you made it easier to push through those difficult times. Within the work environment, I would like to thank my colleagues and friends: Dr. Jerome Irianto, Dr. Lucas Smith, Dr. Manu Tewari, Dr. Dave Dingal, Dr. Nisha Sosale, Dr. Kyle Spinler, Dr. Praful Nair, Charlotte Pfeifer, Yuntao Xia, SangKun Cho, Jake Hsu, and Brandon Hayes for productive discussions on research and other non-work-related matters throughout my time in the Discher laboratory. You made it a pleasure to come iii into work day after day. I couldn’t have asked for better co-workers and friends. Throughout my life, I will never forget the times we shared. I wish you all the best! I would like to give special thanks to my close friend Ian Johnston. I can’t imagine a better person to have shared my hobbies with during my time here in Philadelphia. I hope we stay in contact throughout our lives. I will always consider you family. I would like to express my gratitude to my mentors outside the laboratory for their guidance throughout my professional career. Specifically, I would like to thank Dr. Mas Iimura for his mentorship and friendship throughout my years at Ripon College and beyond. We got to know each other during a time when I made the hardest decisions of my life. I was extremely lucky to have met you and truly believe I would not be where I am or the person I am today without you. I can never thank you enough. Finally, I owe a tremendous amount of gratitude to my family. To one of the most important people in my life, my mom, thank you for believing in me and supporting me throughout my entire life despite being overwhelmed with the dreams I had for myself. You are the strongest person I know and it is your strength that I admire most about you and hope I’ve inherited. You’ve had a hard life and have done far too much for your family with little in return. I will do everything I can to repay you and give you the gratitude you deserve. Lastly, I would like to thank the love of my life, Elizabeth. No matter the sense of accomplishment I feel from my achievements in life, they will never compare to the love I have for you. You will always be the most important person in my life. You are an incredible person and I couldn’t have wished for a better partner with whom to share my life. iv ABSTRACT ENGINEERED MARROW MACROPHAGES FOR CANCER THERAPY: ENGORGEMENT, ACCUMULATION, DIFFERENTIATION, AND ACQUIRED IMMUNITY Cory M. Alvey Dennis E. Discher The ability of a macrophage to engulf and break down invading cells and other targets provides a first line of immune defense in nearly all tissues. This defining ability to ‘phagos’ or devour can subsequently activate the entire immune system against foreign and diseased cells, and progress is now being made on a decades-old idea of directing macrophages to phagocytose specific targets such as cancer cells. Physical properties of cancer cells influence phagocytosis and relate via cytoskeleton forces to differentiation pathways in solid tumors. Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of CD47, a ‘marker of self.’ These macrophages were then systemically injected into mice with fluorescent human tumors. Within days, the tumors regressed, and fluorescence analyses showed that the more the SIRPα-inhibited macrophages engulfed, the more they accumulated within tumors. In vitro phagocytosis experiments on transwells revealed that macrophage migration through micropores was inhibited by eating. However, during the 1-2 weeks of tumor residency, donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα tumor associated macrophage. Analyses of macrophages on soft or stiff collagenous gels demonstrated a stiffness-driven upregulation of SIRPα. Tissue stiffness also seems to have a role in the v development of cancer. Meta-analyses suggest that genomic variation across tumors scales with the stiffness of the tumor tissue of origin. These genomic changes give rise to neoantigens that are important for the development of monocyte/macrophage immunotherapies. Indeed, marrow-derived macrophages can phagocytose melanoma cells and present neoantigens leading to the activation of T-cells. Unlike past injections of anti- CD47 which cause anemia, no measurable