Characterization of a hybrid hydrogel for the studying of lymphocyte infiltration and migration in a 3-dimensional <i>in vitro</i> tumor stroma device

Solid tumors reprogram their surrounding microenvironment to develop a tumor promoting and immunosuppressive niche of cells and extracellular matrix known as the tumor stroma. While many successful immunotherapies modulating the use of cytotoxic lymphocytes have been established, success in solid tumors has been limited, in part due to the stroma disrupting T cell infiltration and/or migration into the tumor boundary, thereby obstructing their contact with cancer cells. There exists a need for the development of three-dimensional (3D) tissue engineered models to better understand the mechanisms of infiltration and migration of solid tumors by cytotoxic lymphocytes. Here we present the validation of a 3D hydrogel system, implemented into a tumor-on-a-chip device, allowing for the observation of infiltration and migration of T cells, in addition to the influence that tumor cells have on stromal fibroblasts. This hydrogel allows for greater infiltration of T cells compared to other formulations. Differences in migration are observed depending on lymphocyte type. Fibroblasts are influenced towards a cancer-associated fibroblast morphology with increased alpha-smooth muscle actin expression, and are seen to modify their spatial orientation relative to A375 melanoma cells. Finally fibroblast presence in the hydrogel inhibits infiltration of T cells. Combined, these results validate the application of this model to study lymphocyte infiltration and migration into solid tumors. Future modulation of cell populations, along with the integration of patient derived samples, can provide a system to test immunotherapy effectiveness for cancer patients.