Targeting the CD47-SIRPα pathway promotes macrophage-mediated trogocytosis of ovarian cancer cells

Objectives: CD47 is a ubiquitously expressed transmembrane protein that ligates signal regulatory protein alpha (SIRPa), which is expressed on myeloid cells and inhibits phagocytosis (‘Don't eat me signal) and other effector functions. High CD47 expression in metastatic ovarian tumor at diagnosis was associated with worse prognosis. Anti-CD47 antibodies have shown promising results in clinical trials when paired with an opsonizing antibody (e.g. ritixumab for lymphoma) to enhance tumor cell phagocytosis by macrophages. In ovarian cancer, during the period of minimal residual disease after debulking and chemotherapy, ovarian tumor cells are expected to be surrounded by recruited monocyte-derived macrophages and peritoneal self-renewing resident macrophages that could be targets for anti-CD47 therapy. Our goal is to conduct proof-of-principle experiments to test whether anti-CD47 could enhance primary macrophage uptake and/or injury of EOC tumor cells. Given the large size of tumor cells, our initial studies focused on macrophage-mediated trogocytosis (uptake of cell membranes) versus phagocytosis of tumor cells as a mode of tumor cell injury. Download : Download high-res image (92KB) Download : Download full-size image Download : Download high-res image (481KB) Download : Download full-size image Methods: Monocytes were isolated from the PBMC fraction of remnant platelet donor apheresis samples by CD14 microbeads and differentiated into macrophages with M-CSF. Expression of CD47 in SKOV-3 cells, a human epithelial ovarian cancer cell line, was evaluated by flow cytometry. To evaluate trogocytosis of tumor cells by macrophages, SKOV-3 cells were stained with a membrane-bound fluorescent dye (PKH26). CD47 was blocked with anti-CD47 mAb and CD47-blocked or control SKOV-3 cells were co-cultured with macrophages (1:1 or 2:1 ratio) in serum-free media or cell-free ascites (50% final volume) for 2h. Ascites was collected from newly diagnosed ovarian cancer patients prior to debulking surgery. After co-culture, cells were stained with anti-CD33 or anti-CD14 mAb (as macrophage markers) and evaluated by flow cytometry. Total cells, excluding doublets, were gated to determine PKH+CD33+ or PKH+CD14+ cell population as indicator of tumor membrane uptake by macrophages. Similar experiments were performed using undifferentiated monocytes. Live cell images were captured using similarly processed SKOV3 cells and macrophages in serum-free media or ascites to visualize tumor cell trogocytosis. Results: SKOV3 ovarian cancer cells highly express CD47 (Figure 1). Monocytes did not trogocytose SKOV3 cells (± CD47 blockade) in ascites or serum-free media (Figure 2). Preliminary data shows that CD47 blockade enhanced PKH+CD33+ cell populations (0.86% to 7.5% in isotype vs. anti-CD47, respectively) in ovarian cancer ascites compared to serum-free media (0.8% to 2.07%) (Figure 3). Preliminary live cell imaging showed macrophage-mediated trogocytosis of tumor cells (Figure 4). Conclusions: These data suggest that blockade of CD47 may enhance macrophage mediated tumor cell injury via trogocytosis. Further studies will the effect of CD47 blockade on tumor cell viability and proliferation. Promising ex vivo results will be followed by testing CD47 blockade in murine syngeneic ovarian cancer.