Bioprinted cancer-stromal in-vitro models in a decellularized ECM-based bioink exhibit progressive remodeling and maturation.

Continuous extracellular matrix (ECM) remodeling and cellular heterogeneity are key contributors to cancer development and can both profoundly affect treatment efficacy. Developing in-vitro models that recapitulate matrix and cellular heterogeneity of the tumor microenvironment (TME) can aid in observations that are currently challenging to acquire with conventional 2D cultures and preclinical animal models. Here we report an extrusion bioprinted co-culture model of head and neck cancer and stromal fibroblasts using a composite bioink containing a reinforced decellularized extracellular matrix hydrogel. Fibroblasts have a significant role in remodeling and matrix deposition. When cultured in the bioactive extracellular matrix ink, they provide the cellular elements typically found in the tumor stroma. Head and neck squamous carcinoma cells (UM-SCC-38) were integrated into the bioink, and in the presence of fibroblasts (HVFFs), they began to proliferate into cell-cell interactive spheroids. As the co-culture model is capable of remodeling, we evaluated the ultrastructure of the bioink. We observed a fibrous collagenous network retained from the ECM of the source tissue containing nanometer-scale pores. Following the deposition of the co-culture model, we observed UM-SCC-38 spheroid formation that began during the first week in culture and continued over a three-week period in which the fibroblasts migrated to regions directly surrounding each spheroid. Using a Luminex assay to quantify matrix metalloproteases in co-cultures compared to monocultures, we observed significant differences in the presence of MMP-9 and MMP-10 expression corresponding to periods of the culture in which collagen underwent remodeling. Time-dependent characterization of collagen synthesis, protease activity, and spheroid growth rates are developed to characterize the system as an advanced co-culture model to evaluate tumor-stromal interactions and remodeling.