Abstract CAR T therapies have revolutionized hematologic cancer treatment but face significant barriers in solid tumors, primarily due to the tumor microenvironment. Dense extracellular matrix (ECM) regions impede lymphocyte migration and infiltration, reducing therapeutic efficacy. To model these clinical challenges, we developed a high-throughput imaging assay to evaluate CAR T cytotoxicity in three-dimensional (3D) ECM-embedded tumor spheroids. The epithelial cell adhesion molecule (EpCAM), typically overexpressed in epithelial cancers, provides a clinically relevant target for CAR T therapy. EpCAM-targeted CAR T cells were tested against T-47D spheroids embedded in ECM to mimic solid tumor architecture. Our imaging-based approach revealed dose-dependent cytotoxicity and deep infiltration of spheroids by CAR T cells, despite delayed cytopathic responses compared to suspension cultures. Furthermore, this cytotoxicity is distinct to EpCAM-engineered CAR T cells when compared to nonengineered T lymphocytes. Importantly, elevated IC50 values under embedded conditions underscore the impact of ECM on therapeutic kinetics. These results offer actionable insights for optimizing CAR T design and dosing strategies to overcome microenvironmental barriers. By quantifying migration, infiltration, and killing in physiologically relevant models, this work supports translational efforts to improve CAR T efficacy in solid tumors and informs clinical development pathways for next-generation immunotherapies. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ernest Heimsath, Paul Held, Peter Brescia, Joe Clayton. High-throughput imaging approaches to characterize CAR T therapies for solid tumor microenvironments 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 2130.
Heimsath et al. (2026) studied this question.