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 by Axion BioSystems. 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 to assess cancer cell growth and cytotoxicity and quantify the cytolysis of 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 As expected, A549 cells treated with CAR T-cells exhibited dose-dependent decrease in fluorescence confluency (%), with near-complete cell lysis observed in the 5:1 E:T ratio group at 160 hours. Immune cell-mediated killing was assessed by applying HER2-targeted CAR T-cells to both SKOV3-GFP and A549-GFP spheroids. SKOV3-GFP spheroids treated with CAR T-cells showed a dose-dependent decrease in fluorescence intensity, with higher E:T ratios (2:1, 5:1, and 10:1) resulting in approximately 75% reduction in fluorescence intensity by 72 hours. In contrast, A549 spheroids only exhibited significant killing at 5:1 and 10:1 E:T ratios, while minimal decreases in intensity were observed at other ratios. Conclusion The Omni platform enables real-time monitoring of CAR T-cell interactions with target cells, providing key insights into the cytotoxic potential of these engineered immune cells. Fluorescent metrics were used to track immune cell-mediated killing, with changes in fluorescence confluency and intensity reflecting the extent of target cell death. Results showed that the potency of CAR T-cell-mediated cytolysis was dose-dependent, with higher E:T ratios leading to greater target cell killing. SKOV3-GFP spheroids, which overexpress HER2, were more susceptible to HER2 CAR T-cell-mediated killing, while A549-GFP spheroids with low HER2 expression required higher E:T ratios for effective cytolysis. Citation Format: Danny Flanigan, Nathalie Opdam - van de Laar, Denise Sullivan, DANIEL MILLARD, STACIE CHVATAL, . Fluorescence potency assays for evaluating HER2 CAR T-cell-mediated killing in cancer cells 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 3713.
Flanigan et al. (Fri,) studied this question.