Characterization of Renal Cell Carcinoma Heterotypic 3D Co-Cultures with Immune Cell Subsets

SIMPLE SUMMARY: Three-dimensional cancer models have gained interest from pre-clinical testing of single drugs and drug combinations. The research aim of this study was to develop a heterotypic 3D co-culture harboring selected immune cell subsets to evaluate the efficacy of a drug combination for the treatment of renal cell carcinoma. Heterotypic spheroids containing 70% cancer, 20% fibroblasts, and 10% endothelial cells were cultured in a scaffold-free system. Native or immortalized immune cells were added directly or 24 h post spheroid formation, and their infiltration was observed. This infiltration was found to be modulated by various treatment conditions. Our study revealed that heterotypic short-term 3D spheroids complemented with immune cell subsets represent a valuable tool for tumor-immune cell interaction and treatment screening platforms. ABSTRACT: Two-dimensional cell culture-based platforms are easy and reproducible, however, they do not resemble the heterotypic cell-cell interactions or the complex tumor microenvironment. These parameters influence the treatment response and the cancer cell fate. Platforms to study the efficacy of anti-cancer treatments and their impact on the tumor microenvironment are currently being developed. In this study, we established robust, reproducible, and easy-to-use short-term spheroid cultures to mimic clear cell renal cell carcinoma (ccRCC). These 3D co-cultures included human endothelial cells, fibroblasts, immune cell subsets, and ccRCC cell lines, both parental and sunitinib-resistant. During spheroid formation, cells induce the production and secretion of the extracellular matrix. We monitored immune cell infiltration, surface protein expression, and the response to a treatment showing that the immune cells infiltrated the spheroid co-cultures within 6 h. Treatment with an optimized drug combination or the small molecule-based targeted drug sunitinib increased immune cell infiltration significantly. Assessing the therapeutic potential of this drug combination in this platform, we revealed that the expression of PD-L1 increased in 3D co-cultures. The cost- and time-effective establishment of our 3D co-culture model and its application as a pre-clinical drug screening platform can facilitate the treatment validation and clinical translation.