Abstract 3908: Exploring CAR-T cell killing dynamics through live-cell fluorescence microscopy

Abstract Introduction: Harnessing the potent cytotoxicity of chimeric antigen receptor T (CAR-T) cells has revolutionized cancer immunotherapy. A key factor influencing CAR-T cell efficacy is target antigen density on cancer cells. Human epidermal growth factor receptor 2 (HER2), overexpressed in various cancers like ovarian and lung carcinomas, is a promising target for CAR-T cell therapy. Traditional cytotoxicity assays generally provide only static endpoint measurements and often rely on 2D cell culture models, which fail to capture the structural complexity of tumors in vivo—a factor crucial to CAR-T cell efficacy. Live-cell imaging offers a solution by enabling non-invasive, real-time monitoring of cytotoxicity. Therefore, this study aims to explore the dynamics of CAR-T cell-mediated cytotoxicity against cancer cells with varying HER2 expression levels, comparing outcomes between 2D and 3D cell culture models using the Omni platform. Methods: Monolayers and spheroids of SKOV3 (ovarian carcinoma) and A549 (lung adenocarcinoma) cells, tagged with GFP, were exposed to HER2 CAR-T cells at different Effector : Target (E:T) ratios. Using the Omni platform, hourly brightfield and fluorescent images were captured over 72 hours assess cancer cell growth and cytotoxicity and quantify the cytolysis of the fluorescent target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent intensity of treated wells to no treatment control wells. Results: The study demonstrated a dose-dependent cytotoxic effect of HER2 CAR-T cells that correlated with increasing amounts of CAR-T cells. Comparing SKOV3 cells with A549 cells showed higher sensitivity and earlier response for all E:T ratios. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells, while it demonstrated 40.3% cytolysis of A549 cells. In comparing the cytotoxic effect of HER2 CAR-T cells on SKOV3 spheroids versus monolayers, the 3D spheroid model exhibited a more rapid and efficient dose-dependent response. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells in spheroid, while it demonstrated 61.1% cytolysis of SKOV3 cells in monolayer. Conclusion: This study underscores the importance of target antigen density in determining CAR-T cell efficacy, as SKOV3 cells, with higher HER2 expression, were more prone to CAR-T cell killing compared to A549 cells. Additionally, it also highlights the critical role of the assay model type, 2D versus 3D, in accurately assessing cytotoxicity. The Omni platform provided dynamic, real-time insights into cell viability and cytotoxicity, highlighting its potential to optimize immunotherapy strategies. Citation Format: Skylar Dewees, Nathalie Opdam - van de Laar, Stacie Chvatal, Danielle Califano, Ben Streeter. Exploring CAR-T cell killing dynamics through live-cell fluorescence microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3908.