Abstract 2648: CD47 antibody-induced engulfment of human T-cell leukemia cells by bone marrow-derived macrophages

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 identify and 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 first labeled using pHrodo (250ng ml-1 for 1h), washed and then treated with antibody for 1 h. Target cells were then added to BMDMs or J774A.1 that had been seeded overnight on 96-well plates. Phase- and fluorescence images were captured and quantified every 15min. Anti-CD47 antibody (B6H12.2, 0.04-5μg ml-1), but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30’-4h), as evidenced by an increase in intracellular fluorescence as the label accumulates in the acidic phagosome. After 4h the red fluorescence area was increased by 25-fold (1975 ± 391 μm2 vs 80 ± 41 μm2). From close inspection of the time-lapse images cellular engulfment could be clearly observed, coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 as the effector cell. Interestingly, the rate and degree of engulfment appeared 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. Anti-CD47 had no direct effect on CCRF-CEM proliferation for the first 4 h but upon longer exposures (>8 h) cell growth was attenuated. 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. Citation Format: Gillian Lovell, Clare Szybut, Kalpana Patel, Hinnah Campwala, Nicola Bevan, Dan Appledorn, Tim James Dale, Derek John Trezise. CD47 antibody-induced engulfment of human T-cell leukemia cells by bone marrow-derived macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2017-2648