Abstract
CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With the FDA's initiative to advance alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and inability to automate, among others. To address these limitations, we optimized the assay using multimodality imaging methods, including bioluminescence, impedance tracking, phase contrast, and fluorescence, to track CAR-T cells co-cultured with CD19, CD20, and HER2 luciferase reporter cancer cells in real-time. Additionally, we varied the ratio of CAR-T cells to cancer cells to determine optimal cytotoxicity readouts. Our findings demonstrated that the CAR-T cell group effectively attacked cancer cells, and the optimized assay provided superior temporal and spatial precision measurements of ex vivo CAR-T killing of cancer cells, confirming the reliability, consistency, and high throughput of the optimized assay.