Abstract
Abstract CD47 is a trans-membrane “don’t-eat-me” signaling protein that enables tumor cells to evade clearance by neighboring phagocytes. Blocking CD47 allows phagocytes to clear tumor cells and is a promising new approach for cancer immunotherapy. In this study, we characterized anti-CD47 antibody-mediated engulfment of living tumor cells (CCRF-CEM) by mouse bone-marrow derived macrophages (BMDMs) or immortalized mouse macrophages (J774A.1). Phagocytosis was quantified using a pH-sensitive cell-labeling fluorescent probe, pHrodo, and automated kinetic live-cell analysis (IncuCyte). CCRF-CEM cells were labeled, washed and then treated with antibody. Target cells were then added to BMDMs or J774A.1 cells in 96-well plates. Phase- and fluorescence images were captured and quantified over time. Anti-CD47 antibody, but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30’-4h), as evidenced by a significant increase in intracellular fluorescence. Close inspection of the time-lapse images revealed clear cellular engulfment coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 macrophages. Interestingly, the rate and degree of engulfment was effector cell-dependent. The mechanism of engulfment was not via induction of target cell apoptosis since anti-CD47 did not induce PS externalization (Annexin V) or activate caspase 3/7. Our experimental findings substantiate the known pro-phagocytic effects of anti-CD47 antibodies, and provide a model system and method for quantitative functional analysis and mechanistic insight of CD47 modulators as cancer therapeutics.
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