Abstract 2107: A novel real-time cell imaging assay to quantify macrophage efferocytosis

Abstract

Tumor-associated macrophages (TAMs) are found in the microenvironment of solid tumors, representing one of the most abundant immune cell types within tumor stroma. Studies have demonstrated that the presence of TAMs correlates with tumor progression and poor prognosis. The engulfment of apoptotic tumor cells by TAMs (a phagocytic process known as efferocytosis) results in clearance of neoplastic antigens, which prevents antigen presentation and activation of effector T cells. Additionally, it has been shown that TAMs produce cytokines that create an immunosuppressive environment that facilitates tumor evasion of immune surveillance and promotes tumor growth. Furthermore, TAMs have been reported to be key players in the promotion of tumor angiogenesis and metastasis. Since TAMs play such an important role in cancer progression, inhibition of their function in the tumor microenvironment becomes an attractive approach of cancer immunotherapy. Traditionally, quantifying efferocytosis in vitro has been a technically challenging task. Our lab has established a novel high-throughput assay that can be used to screen drug candidates that inhibit TAM efferocytosis. Our new assay uses the IncuCyte ZOOM real-time imaging platform to visualize macrophage efferocytosis of apoptotic cells that are labeled with a pH-sensitive probe (pHrodo) that only emits fluorescence in acidic enviroments. This technology enables us to quantify phagocytic events of apoptotic cells that have been processed into the phagolysosome. By using this method, we quantitatively characterized the efferocytosis activity of TAM-like macrophages that were differentiated from primary human monocytes. We tested several agents (including small-molecule compounds and antibodies) and quantified their activities in blocking efferocytosis of TAMs via IC50 concentrations and potency. This new high-throughput assay platform yields highly robust and reproducible data (Z'=0.69), is fully automated, noninvasive (no washing, fixing or lifting of cells), and works with low cell numbers. Our results show that our efferocytosis assay facilitates high-throughput functional screening and can be utilized to identify and characterize new cancer drug candidates, as well as enables research of efferocytosis mechanisms and functions of phagocytic cells. Citation Format: Daniel Bravo, Jianyong Wang, Yongchang Shi. A novel real-time cell imaging assay to quantify macrophage efferocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2107.