Peribiliary glands and biliary tree stem cells are involved in the pathogenesis of cholangiocarcinoma arising in patients affected by primary sclerosing cholangitis
Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic cholangiopathy characterized by inflammation and bile duct fibrosis. Massive proliferation of biliary tree stem/progenitor cell (BTSC), expansion of peribiliary glands (PBG) and dysplasia were observed in PSC. Cholangiocarcinoma (CCA) frequently complicates the course of PSC. The aims of the present study were to evaluate the involvement of PBGs and BTSCs in CCA emerging in PSC patients.
Method: Specimens from normal liver (n = 5), PSC (n = 20), and CCA arising in PSC patients (n = 20) were included. Samples were processed for histology, immunohistochemistry and immunofluorescence. In vitro experiments were performed on human (h) BTSCs isolated from extrahepatic biliary tree from organ donors, on human primary CCA cell cultures obtained from mucin producing CCA specimens and on H69, a normal human cholangiocyte cell line. Cultures were analyzed by immunofluorescence and reverse transcription polymerase chain reaction.
Results: All CCAs emerging in PSC patients were mucin-producing tumors characterized by the involvement of PBGs and high expression of stem/progenitor cell markers. In surrounding tissue, pre-neoplastic and neoplastic lesions affected several bile ducts and the associated PBGs. Ducts with neoplastic lesions showed higher inflammation, wall thickness and PBG activation compared to PSC-affected ducts. CCA showed higher expression of nuclear factor-kappa B, interleukin-6 and vascular endothelial growth factor 1, and displayed higher microvascular density compared to non-cancerous ducts. In tissue samples, CCA cells were characterized by higher expression of epithelial-to-mesenchymal transition (EMT) traits and by the absence of primary cilia compared to controls and PSC. In vitro study demonstrated that CCA cells and hBTSCs, when stimulated with lipopolysaccharide and oxysterols, increased the expression of EMT traits, nuclear factor-kappa B, and interleukin-6, and showed the loss primary cilia compared to control conditions.
Conclusion: CCA arising in PSC patients is characterized by extensive PBG involvement and the activation of BTSC niche. In these patients, the presence of duct lesions at different stages (from inflammation to fibrosis and to neoplastic transformation) suggests a progressive tumorigenesis. Lipopolysaccharide and oxysterols induced, in vitro, in hBTSCs and CCA cell cultures, pathologic features similar to those detected in situ in PSC specimens.