Tumorigenic potential of cancer stem cells (CSCs) isolated from human cholangiocarcinoma (CCA) subtypes in cirrhotic microenvironment

Background and Aims: Cholangiocarcinomas (CCA) are comprised histologically of a mucin-secreting type, which can be intrahepatic (IHCCA) or perihilar (pCCA), and, of a mixed type located peripherally within the liver. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. Methods: CSC markers (CD90, CD13, EpCAM, CD133, LGR5) were investigated in 25 human CCAs, in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after subcutaneous or intrahepatic injection in normal and cirrhotic (CCL4) mice. Results: CSCs comprise more than 30% of the tumor mass. While the CSC profile was similar between mucin-IHCCA and mucinpCCA, CD13+ CSCs characterize the mixed-IHCCA while LGR5+ characterize the mucin-CCA subtypes. A significant percentage of neoplastic cells express epithelial–mesenchymal transition (EMT) markers (SNAIL, Twist), and co-express mesenchymal and epithelial markers. In primary cultures, EMT markers, mesenchymal markers (vimentin, CD90), and CD13 were largely more expressed than the “epithelial” markers (CD133, EpCAM and LGR5). In vitro, CSCs expressing “epithelial” markers formed a higher number of spheroids than CD13+ or CD90+ CSCs. In subcutaneous tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90+ and CD13+ cells. By contrast, in intrahepatic xenografts, in the cirrhotic livers, tumors were dominated by epithelial traits resembling the original human CCAs. Conclusions: CSCs were rich in human CCAs (>30%), implicating CCAs as stem cell based diseases. Different CSC subpopulations expressed EMT markers and co-expressed mesenchymal and epithelial markers, generating different types of cancers depending on the microenvironment. Remarkably, CSCs may reproduce the original human CCAs, when injected into cirrhotic livers.