Cholangiocarcinoma (CCA) is a rare but aggressive type of primary liver cancer with a dismal prognosis. CCA commonly metastasizes to the lungs and lymph nodes, significantly reducing overall survival. However, a mechanistic understanding of how CCA invades these metastatic sites remains lacking. This is partly due to the failure of current models to recapitulate the complexity of tissue-specific environments for metastatic CCA. Patient-derived tumor organoids are promising models for studying cancer in vitro, including CCA. However, organoids are frequently cultured in basement membrane extract (BME), which does not represent the native microenvironment. Decellularized scaffolds can serve as a culture environment for investigating tumor-extracellular matrix (ECM) interactions. Nonetheless, decellularized scaffolds are often utilized in conjunction with cancer cell lines, lacking critical characteristics of a growing tumor in vivo. Therefore, this study aims to decellularize and characterize human-derived lung (n=3) and lymph nodes (n=17), and recellularize these with patient-derived CCA organoids (CCAOs) (n=3) to establish an in vitro model for studying the interaction between epithelial tumor cells and the microenvironment of the metastatic site. Decellularization resulted in acellular scaffolds with preserved ECM components. Local ECM heterogeneity was shown by the macro- and micro-scale mechanical properties as determined by rheology and micro-indentation. The CCAOs showed adherence and growth when combining them with the decellularized lung ECM (dLECM). The metabolic activity of CCAOs was diminished when cultured in dLECM compared to those cultured in BME, and was dictated both by the organoid line and the dLECM donor. Distinct CCAO gene expression profiles were observed between the two culture environments (BME and dLECM). This elucidates the effect of the culture environment on the behavior of CCAOs in an in vitro model. In conclusion, the convergence of CCAOs with their organ-specific location of metastasis obtained by decellularization, provides a valuable tool for integrating the ECM of the metastatic location in an in vitro model for the mechanistic study of cancer metastasis.

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.

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