Biofabrication of 3D breast cancer models for dissecting the cytotoxic response of human T cells expressing engineered MAIT cell receptors.

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8⁺T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.