Assessing Large-scale CAR-T Cell Cytotoxicity Ex Vivo Using 2-D and 3-D Multimodal Imaging Techniques for Industrial Standards

CAR-T cell therapy is a revolutionary and highly effective treatment for hematologic malignancies, with rapid advancements particularly in applications to solid tumors. The development and refinement of this innovative therapy rely on efficient ex vivo assays to evaluate CAR-T cell cytotoxicity. Furthermore, advancement in the treatment of solid tumors using CAR-T cell therapy requires the development of ex vivo assays specifically using solid tumor models. Here, we report the development of luciferase reporter cancer cell lines that endogenously express high levels of key CAR-T target antigens such as CD19, CD20, BCMA, or HER2. Using targeted and mock-engineered CAR-T cells, we evaluated cytotoxicity against these reporter cells via a bioluminescence assay and phase contrast/fluorescence live imaging assays. Our multimodal imaging approach demonstrated significantly higher cancer cell killing by targeted CAR-T cells compared to mock CAR-T cells. To enhance our live imaging capabilities and enable assays for CAR-T cell infiltration into 3-D tumor models, we engineered GFP-Luc2 dual reporter cancer cell lines. Using 3-D confocal live imaging, we observed reduced spheroid size and decreased luciferase signal in Raji-GFP-Luc2 spheroids co-cultured with CD19 CAR-T cells versus mock CAR-T controls, indicating superior cancer cell killing when using targeted CAR-T cells. Upon embedding Raji-GFP-Luc2 spheroids in 3-D matrices to mimic a more physiologically relevant tumor microenvironment, we captured CAR-T cell infiltration into spheroids by time-lapse 3-D imaging. Our results highlight the utility of combining luciferase bioluminescence and live fluorescence imaging to assay CAR-T cell cytotoxicity and infiltration in 2-D and 3-D co-culture models. The luciferase assay provides sensitive, quantitative measurements, while live fluorescence imaging reveals the spatial and temporal dynamics of CAR-T cell-cancer cell interactions. These scalable assays hold significant potential for large-scale industrial applications in CAR-T cell therapy development.