Abstract 4575: High-throughput method to analyze the cytotoxicity of CAR T cells in a 3D tumor spheroid model using image cytometry
Authors: Patel Samir , Zurowski David , Hui Daniel , Ka Mignane , Hernandez Charles , Love Andrea , Lin Bo , Moore Andrea , Chan Leo Li-Ying 
Published: April 2023

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. CAR-T cell therapy involves specifically engineering T cells to attack tumor cells by binding a tumor antigen and inducing T cell activation resulting in intracellular signaling and cytokine release. Currently, there are six FDA-approved CAR-T cell therapies, which all target the CD19 or BCMA antigens for hematologic B cell malignancies. In the 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. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. 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 plate-based 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. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. Furthermore, the T cells are fluorescently labeled with a tracer dye to visually locate the cells on the tumor spheroids. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.
Citation Format: Samir Patel, David Zurowski, Daniel Hui, Mignane Ka, Charles Hernandez, Andrea Love, Bo Lin, Andrea Moore, Leo Li-Ying Chan. High-throughput method to analyze the cytotoxicity of CAR T cells in a 3D tumor spheroid model using image cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4575.