MUG CCArly: A Novel Autologous 3D Cholangiocarcinoma Model Presents an Increased Angiogenic Potential

SIMPLE SUMMARY: Cholangiocarcinoma is a rare, aggressive, and heterogeneous malignancy of the bile duct. A typical feature of cholangiocarcinoma is that the cancer cells are embedded in a dense stroma. However, the functional role of the reactive tumor stroma has not been fully elucidated, which is certainly due to the lack of suitable in vitro models. Tumor stromal cells or tumor-associated fibroblasts play a central role in angiogenesis, metastasis, and the development of resistance to therapy in cholangiocarcinoma. We successfully isolated tumor cells and tumor-associated fibroblasts from a patient, generated cell lines, characterized the cells in detail, and established an innovative autologous tumor model. By detailed characterization of the tumor/tumor-associated fibroblast model we demonstrated that tumor cells interact with tumor-associated fibroblasts. This model can be used to study tumor stromal crosstalk, tumor angiogenesis and invasion, and the development of drug resistance. ABSTRACT: Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops.