High-throughput imaging approaches to characterize CAR T therapies for solid tumor microenvironments

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 IC 50 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.