A microphysiological assay for studying T-cell chemotaxis, trafficking and tumor killing

Abstract Tumors in patients non-responsive to immunotherapy harbor a series of barriers that impede the efficacy of effector T-cells. Consequently, therapeutically modulating the chemotaxis machinery to enable effector T cell infiltration and function in the tumor could result in more successful therapeutic outcomes. Complex in-vitro models allow re-creation of in-vivo tumor complexities in an in-vitro setting, allowing improved translatability to patient biology at the laboratory scale. We identified a gap in available industrial scale microphysiological (MPS) assays for faster validation of targets and strategies that enable T-cell chemotaxis and effector function within tumor microenvironments. Using a commercially available, 96 -chip 2-lane microfluidic assay system, we present a novel, scalable, complex in vitro microphysiological assay to study 3D T-cell chemotaxis and function within native, extracellular matrix (ECM)-rich multicellular tumor environments. Activated or naïve CD3+ T-cells stained with far-red nuclear stain responded to the chemokine gradients generated within the matrigel-collagen ECM by migrating into the microfluidic channel (~5 mm horizontal window), in a concentration- and cell type-dependent manner. Furthermore, we observed and tracked chemotaxis and cancer cell killing function of antigen-specific CD4.CD8.CAR-T cells (chimeric antigen receptor (CAR)-T cells) that responded to CXCR3 agonist gradient built through the expansive 5 mm of cancer cell colony containing stroma. The 2-lane assay system yielded useful information regarding donor and dose-dependent differences in CAR-T cell chemotaxis and tumor killing. The scalable assay system allows a granular window into immune cell migration and function in tissue spaces beyond endothelium, addressing a missing gap in studying tissue-specific immune cell chemotaxis and function to bring forward advancements in cancer immunotherapy.