SMAC mimetics induce human macrophages to phagocytose live cancer cells

Macrophages engulf apoptotic bodies and cellular debris as part of homeostasis, but they can also phagocytosis live cells such as aged red blood cells. Pharmacologic reprogramming with the SMAC mimetic LCL-161 in combination with T cell-derived cytokines can induce macrophages to phagocytosis live cancer cells in mouse models. Here we extend these findings to encompass a wide range of monovalent and bivalent SMAC mimetic compounds, demonstrating that live cell phagocytosis is a class effect of these agents. We demonstrate robust phagocytosis of live pancreatic and breast cancer cells by primary human macrophages across a range of healthy donors. Unlike mouse macrophages where combination of SMAC mimetics with lymphotoxin enhanced phagocytosis, human macrophages were more efficiently polarized to phagocytose live cells by the combination of SMAC mimetics and IFNg. We profiled phagocytic macrophages by transcriptional and proteomic methodologies, uncovering a positive feedback loop of autocrine TNFa production. PBMCs were isolated from eight individual donors and differentiated into macrophages. For each donor, macrophages were treated for 24 hours with either DMSO, rhLTα1/β2, IFN-γ, LCL-161 (500nM), LCL-161 + rhLTα1/β2, or LCL-161 + IFN-γ. After treatment, macrophages were harvested, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat# 74104) according to the manufacturer’s protocol. (company). Additionally, phagocytosis assays were performed on treated macrophages from all donors to ensure treatment response.