Cholangiocarcinoma (CCA) is a highly aggressive biliary tumor with a poor prognosis and limited treatment options. Various critical aspects of CCA development remain unclear. Lately, the dynamic process of epithelial-mesenchymal transition (EMT) was highlighted to play a crucial role in the fundamental mechanism of metastatic dissemination. However, in vitro EMT studies are mainly limited to conventional 2D models which lack in vivo tumor physiology. Recently, 3D organoid culture methods provide us new model possibilities. Therefore, our research aim was to establish a representative in vitro organoid-based epithelial-mesenchymal transition model in cholangiocarcinoma including its in vivo pathophysiology and tumor microenvironment. This study focusses on three different aspects of the tumor microenvironment regarding EMT activation in CCA organoids: 1.The effects of EMT promoting growth factors and culture media on EMT activation. Various growth factors are highlighted to play a crucial role in EMT activation in different cancer types. To study EMT activation in CCA, various growth factors were added to CCA organoids. We observed round shaped organoid structures consisting of single layered epithelium for all conditions. Additionally, gene expression levels of EMT related markers were increased for specific growth factor conditions. The use of various culture media combined with transforming growth factor β1 (TGF-β1) showed the most substantial differences in mRNA expression of EMT markers in branching medium (BM). Based on these results, specific growth factors and BM were used to find the optimal culture conditions to induce EMT. Although we did not find significant morphological differences between the various conditions, altered gene expression levels of EMT markers were observed after TGF-β1 and tumor necrosis factor alpha (TNF-α) treatment in BM. 2.The effect of the tumor extracellular matrix on EMT activation. The tumor extracellular matrix (ECM) is an important component of the tumor microenvironment that stimulates the malignant behavior of surrounding cells. The combination of CCA organoids and patient-derived ECM resulted in morphological changes. Cells with a polygonal shape adopted an irregular shape after TGF-β1 treatment. On top of that, our data revealed that patient-specific ECM and CCA organoids altered the gene expression of EMT related markers. 3.The effect of the interstitial fluid flow on EMT activation. The use of a microfluidic platform enabled us to study the effect of fluid flow on EMT in vitro. Cells formed organoid structures in the absence and the presence of a fluid flow. However, cells only migrated to the surrounding culture medium due to the presence of a fluid flow. When cells were seeded directly into the chip, cells formed a tube structure with protrusions growing outwards. The protrusions growth was accelerated by the addition of TGF-β1 and led to increased cell migration. Additionally, fluid flow conditions resulted in altered gene expression levels of EMT related markers. Our data suggest that fluid flow could potentially stimulate EMT in CCA organoids. Overall, our findings have demonstrated that growth factors, native ECM and interstitial fluid flow could be used to induce EMT in vitro. Indicating that the tumor microenvironment plays a crucial role in EMT and tumor progression. Our novel in vitro CCA organoid-based EMT model provides a foundation towards a more representative EMT in vitro model to unravel the underlying mechanism of CCA and its progression.