Updated project metadata. Cholangiocarcinoma (CCA) is a rare but aggressive type of primary liver cancer, with an overall 5-year survival rate of <10%. The extracellular matrix (ECM) plays a particularly important role in CCA tumor biology, manifested in the desmoplastic tumor environment. This study aim is to design an in vitro CCA model combining patient-derived cells and patient-derived ECM mimicking the native microenvironment. decellularized Patient CCA tissue (CCA-matrix; CCA-M) and tumor-free liver tissue (TFL-M) was decellularized and repopulated with CCA organoids (CCAOs). Culture was performed with amino acids labeled with stable heavy isotopes to enable separation of pre-existing ECM from proteins produced by the cells in the mass spectrometry (MS) analysis. CCAOs on decellularized matrix form complex morphological structures, with environment-dependent proliferation and migration dynamics, driven by the occurrence of EMT. CCAOs in TFL-M or CCA-M can be used to study the initiation or progression of desmoplasia in CCA, respectively. Furthermore, in-depth transcriptomic and proteomic analysis reveals that CCA-M induces a transcriptome more resembling in vivo CCA tumor tissue, with an accompanying protective effect of the desmoplastic environment on tumor survival.