A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

Abstract Most cellular therapies, like CAR T cells, remain ineffective in solid tumors. This is primarily due to a complex tumor microenvironment (TME), which creates biochemically hostile and often immunosuppressive conditions that limit efficacy of immunotherapies. Besides, cellular therapy efficacy is still often established in traditional 2D cultures that fail to simulate relevant aspects of solid tumor biology. Recent advances in three-dimensional (3D) and organ-on-chip culture systems have provided more physiologically relevant models for immunotherapy testing. These microphysiological systems (MPS) not only offer a 3D environment that alters tumor cell sensitivity to therapy but also enable inclusion of TME components and assessment of processes such as extravasation and infiltration, key steps in CAR T cell activity in vivo. This study focuses on applying an advanced culture technique and further building on the use of a scalable on-chip platform, the OrganoPlate, to grow EpCAM-positive and EpCAM-negative tumor cells in co-culture with an endothelial vessel to study EpCAM-targeting CAR T cell migration and killing kinetics. The CAR T cells specifically targeted and killed EpCAM-positive HT-29 tumor cells while EpCAM-negative A375 tumor cells were not affected. In addition, target cell killing was dependent on the ratio between CAR T and tumor cells (E:T ratio) and was enhanced by addition of IL-2. Inflammatory cytokines like INF-γ, TNFα and IL-6 increased overtime in cultures containing CAR T cells. Morphometric analyses of the endothelial compartment showed E:T ratio dependent disruption of endothelial vessels. Additionally, this system was able to distinguish EpCAM ScFv-CD28-CD3z and EpCAM ScFv-TM-4-1BB-CD3z CAR T cells killing abilities and was used for studying the effect of immune checkpoint inhibitors and Temozolomide, a DNA targeting drug, on CAR T cell performance. Altogether, this work adds to the available advanced culture techniques for immunotherapy developers by describing a model that is modular, scalable, and suitable for phenotypic and functional characterization of CAR T cells.