Engineering a multilayered 3D stromal barrier model for quantitative analysis of T cell infiltration and cytotoxicity.

The development of immunocompetent three-dimensional (3D) culture systems is critical for advancing in vitro models that enable precise analysis of immune-tumor interactions. Here, we report a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues (CAViTs) approach. This system enables spatial compartmentalization of cancer cells and stromal components, including fibroblasts and endothelial cells, thereby mimicking the tumor microenvironment (TME). When co-cultured with tumor-specific cytotoxic T lymphocytes (CTLs), the system permits quantitative analysis of T cell infiltration and cytotoxicity. Moreover, we constructed a cancer-associated fibroblast (CAF)-rich stroma to model immune exclusion. Drug screening using this model identified histone deacetylase (HDAC) inhibitors as agents capable of reducing stromal barrier function by downregulating ECM components, thereby enhancing T cell penetration. This platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma dynamics and accelerating immunotherapeutic discovery. STATEMENT OF SIGNIFICANCE: We present a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues approach. This system enables spatial compartmentalization of cancer cells and stromal components, closely mimicking the tumor microenvironment. Within this model, tumor cell killing by CTLs was successfully observed, resembling a "hot tumor" phenotype. Furthermore, we established a CAF-rich stroma to recapitulate immune exclusion. Drug screening using this platform revealed that HDAC inhibitors enhanced CTL-mediated cytotoxicity. Overall, this platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma interactions and accelerating the discovery of novel immunotherapeutic strategies.