High-throughput method to analyze the cytotoxicity of CAR T Cells in a 3D tumor spheroid model using image cytometry

Abstract

Abstract Chimeric antigen receptor (CAR)-T cell therapy is an antigen-dependent cellular therapy that has gained considerable traction in the field of cancer immunotherapy. Currently, there are six FDA-approved CAR-T cell therapies, which all target the CD19 or BCMA antigens for hematologic B cell malignancies. In recent years, a strong focus has been placed on CAR T cell therapy discovery for solid tumors, which may better recapitulate physiological conditions, thereby potentially improving the selection of CAR construct candidates. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to better assess CAR-T cell therapies against solid tumors. Recently, plate-based image cytometry has emerged as a method to investigate and characterize CAR T cell functions in a high-throughput manner. Image cytometry has demonstrated capabilities in analyzing transduction efficiency, cell proliferation, and cytotoxicity for CAR T cell therapy. With the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors. In this work, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. Herein, we demonstrate the use of high-throughput image cytometry to characterize PSMA CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids and simultaneously monitor location of the T cells in vitro. The proposed method can effectively assess cell-mediated 3D tumor spheroid cytotoxicity and generate time- and dose-dependent results.