CAR-Tmigration andcytotoxicity in atumor-vasculature-on-chipmodel

CAR-T cell therapies have shown remarkable success in hematologic malignancies; however, their efficacy in solid tumors remains limited. This is primarily due to the complex tumor microenvironment (TME), which creates immunosuppressive conditions and physical barriers that hinder CAR-T infiltration and function. Current in vitro models fail to replicate these critical parameters, limiting the ability to predict clinical performance. To address this gap, we developed a tumor-vasculature-on-chip model using the OrganoPlate platform, enabling perfusion of CAR-T cells through a functional endothelial vessel adjacent to a tumor compartment. This setup allows assessment of key processes such as extravasation, migration, and killing kinetics in a physiologically relevant context. EpCAM-positive HT-29 colorectal cancer cells and EpCAM-negative A375 melanoma cells were co-cultured with an endothelial tubule to evaluate EpCAM-targeting CAR-T cells. CAR-T cells selectively killed EpCAM-positive HT-29 cells while sparing EpCAM-negative A375 cells, demonstrating antigen-specific activity. Killing was dose-dependent, and endothelial integrity was maintained at most CAR-T concentrations but disrupted at the highest doses, revealing a therapeutic window. We further compared CAR-T constructs with different co-stimulatory domains (CD28 vs. 4-1BB) and observed reduced potency with 4-1BB. Addition of IL-2 enhanced CAR-T cytotoxicity. Cytokine profiling showed increased IFN-gamma, TNF-alpha, and IL-6 over time, and morphometric analysis confirmed endothelial disruption at high effector-to-target ratios. The platform was also used to study combination strategies, including immune checkpoint inhibitors and temozolomide. This modular, scalable organ-on-chip system enables phenotypic and functional characterization of CAR-T cells under relevant conditions.