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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.

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., BN/Gijsje Koenderink Lab

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Lung function is highly dependent on the structure of the lung parenchyma. Remodeling of the alveolar compartment is a central part of the pathology of chronic lung disease such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Pathological lung remodeling occurs as an interplay between cells and the extracellular matrix (ECM) which in addition to its structural role influence most aspects of cell behavior. This thesis aimed to improve on the available in vitro methods for studies of cell-ECM interaction in the alveolar compartment of the human lung. A technique to produce acellular lung slices for cell culture from clinical lung resection material with preservation of native 3-dimensional structure and ECM composition was established and evaluated. Protein turnover in cell cultures in decellularized lung slices (DLS) was analyzed by mass spectrometry implementing amino acids labeled with stable heavy isotopes to distinguish between cell and DLS derived ECM proteins. The work entailed study of ECM production of primary human lung fibroblasts and primary human alveolar epithelial type II cells (AECII) in DLS from derived from human lungs. In addition, a device for mechanical stretching of lungs slice cultures have been designed and evaluated. Fibroblasts and AECII cultured in DLS secreted ECM components that were incorporated in structure of the lung slices. Fibroblasts showed to be highly responsive to their extracellular milieu and when cultured in IPF derived ECM, they produced ECM recapitulating pathological alterations of the ECM scaffold. In a comparison with fibroblast culture on standard plastic tissue culture surfaces the deposition of ECM proteins was hampered compared DLS culture. DLS culture of AECII showed that these cells produced a wide array of ECM proteins including interstitial matrix components mostly associated with cells of mesenchymal origin. AECII from explanted healthy and end stage COPD lungs were almost