Abstract 3723: Evaluation of <i>ex vivo</i> CAR-T cell cytotoxicity and infiltration using multimodal 2-D and 3-D imaging approaches

Abstract Background: Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized cancer treatment, particularly for hematologic malignancies. Beyond CD19 and CD20, BCMA (B-cell maturation antigen)-targeted CAR-T therapies have become a major focus, especially for multiple myeloma. In fact, BCMA is considered one of the most validated targets in plasma cell malignancies. In pursuit of improving the efficacy and number of applications of this therapy, sensitive and robust cytotoxicity assays are required to efficiently evaluate different CAR constructs, effector-to-target cell ratios, and immune effector cells ex vivo. Furthermore, as CAR-T cell therapy is now under active investigation for the treatment of solid tumors, there is an urgent need for models that mimic the complexity of 3-D tumor environments. Methods and results: In this study, we engineered two luciferase-GFP dual reporter cancer cell lines, Raji-GFP-Luc2 and NCI-H929-GFP-Luc2, and demonstrated their use in a streamlined combined bioluminescence and live imaging assay. Notably, Raji-GFP-Luc2 and NCI-H929-GFP-Luc2 endogenously express high levels of the two FDA-approved CAR-T target antigens, CD19 and BCMA, respectively. Using these lines in co-culture with mock and targeting CAR-T cells, we tracked reporter cancer and CAR-T cell interactions by fluorescence live imaging. Fluorescence quantification clearly demonstrated that CAR-T cells killed cancer cells at higher levels compared to mock controls in both 2- and 3-D co-culture. We then evaluated dose-dependent cancer cell killing by targeting CAR-T cells in 2- and 3-D by luciferase assay. Importantly, we found that quantification of either transgene yields comparative results despite utilizing distinct assays and readouts. Finally, we embedded reporter cancer cell spheroids in 3-D matrices and performed time-lapse imaging of CAR-T cell infiltration. Bioluminescence assay of the embedded spheroids confirmed increased cancer cell killing by targeting CAR-T cells in a solid tumor model. Conclusions: Overall, our dual reporter cancer cell lines are powerful and effective tools for assaying CAR-T cell cytotoxicity. Furthermore, our multimodal imaging platform integrates bioluminescence and live fluorescence imaging to quantitatively and visually assess CAR-T cell cytotoxicity. With scalability and sensitivity, this assay provides an avenue for standardizing CAR-T cytotoxicity testing and will aid in the expansion and improvement of this promising therapy. Citation Format: Catherine McManus, John G. Foulke, Meghan Sikes, Luping Chen, Hyeyoun Chang, Fang Tian. Evaluation of ex vivo CAR-T cell cytotoxicity and infiltration using multimodal 2-D and 3-D imaging approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3723.