Abstract

Phagocytosis is a complex set of processes for the internalization, destruction, and removal of pathogens and unwanted cells from the body. One key phagocytic function is clearance of apoptotic cells, termed efferocytosis, which is crucial in resolving inflammatory events. Also, tumor cells can be targeted with monoclonal antibodies (mAbs) that promote engulfment via antibody-dependent cellular phagocytosis (ADCP), which holds therapeutic promise in oncology. Here we provide in vitro functional assays to accurately assess phagocytotic mechanisms. Using Live-Cell Analysis phagocytosis is kinetically quantified using a pH-sensitive fluorophore pHrodo® which fluoresces at low pH. pHrodo® labeled target cells were added to effector cells. To induce ADCP target cells were first incubated with mAbs. Images were acquired using the Incucyte® and fluorescence automatically quantified with integrated software. The iQue® Advanced Flow Cytometer and iQue® Human ADCP Kit enables quantification via co-localization of labeled target and CD14+ effector cells. Target cells were incubated with mAbs prior to immune cell addition and cells analyzed using integrated software. Anti-CD47 promoted macrophage-mediated phagocytosis of CCRF-CEM tumor cells by blockade of CD47 “don’t-eat-me” signal. Also, clinical anti-CD20 mAbs Truxima and Rituximab promoted ADCP of Ramos target cells by BMDM in a density- and concentration-dependent manner. iQue® data showed Truxima induced ADCP of Ramos and Raji target cells by PBMCs with similar EC50 values (29.1 ng/mL and 24.8 ng/mL, respectively). Polarized macrophages had differential ADCP capability of Trastuzumab treated AU565 target cells, with a concentration-dependent response for M2 but not M1 macrophages, consistent with M2 anti-inflammatory functions. These data exemplify that live-cell analysis, alongside advanced flow cytometry, is a powerful approach that enables highthroughput quantification of phagocytosis amenable to antibody screening.