Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

Abstract

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…Consistently, also in liver-flukeassociated CCAs, the vast majority of neoplastic cells co-express CK19 and albumin, a feature characterizing hepatobiliary stem or progenitor cells 63 . Interestingly, the CSC profile is similar between pCCA and the bile duct (mucinous) type iCCA with high representation 47 . Notably, the original human CCA can be reproduced by injecting selected human CSCs into liver of a mouse model of cirrhosis 47 .…”

“…62 , the entity of expression correlating with the worst prognosis. Although in most solid cancers CSCs represent <3% of the total cell population, in CCAs >30% of the tumour mass express CSC markers, pointing to the potential role of CSCs in CCA 47 . Indeed, all CSC markers characterize normal stem cells located in canals of Hering or bile ductules and/or peribiliary glands, further indicating these structures as the site of origin of most CCAs 46 .…”

“…The bile duct (mucinous) type iCCAs arise in large intrahepatic bile ducts that share anatomical (mucin-secreting cholangiocytes and peribiliary glands) and embryological similarities with the extrahepatic biliary tree and pancreatic duct system 45,46 . This iCCA subtype displays immunohistochemistry, gene expression and a clinicopathological profile that can be super imposed on pCCA 17 and, in addition, shows (together with pCCA) large similarities to pancreatic ductal adenocarcinoma [47][48][49] . Consistently, the pattern of growth and the presence of preneoplastic lesions in cholangiocytes and peribiliary glands lining large intrahepatic bile ducts seem to indicate that these cells are candidate cells of origin 40,43 .…”

Epithelial-mesenchymal transition in cholangiocarcinoma: From clinical evidence to regulatory networks

“…The acquisition of CSC properties by tumor mesenchymal-like cells in response to EMT could have major consequences, in particular by fostering tumor heterogeneity and by contributing to resistance to anticancer drugs in CCA, a tumor containing a strong contingent of CSC [55]. In fact, Shuang et al have recently found, both in iCCA and eCCA, a significant correlation between TGF-β1 and ALDH1 (aldehyde dehydrogenase 1), a functional CSC marker.…”

“…Tumor cells from human iCCA expressing both the mesenchymal marker SNAIL in the nucleus and the biliary epithelial marker CK19 were identified by double immunofluorescence staining, providing conclusive evidence for the presence of EMT traits in malignant cholangiocytes [55]. While not detected in normal biliary epithelium, SNAIL mRNA and protein levels are markedly expressed in CCA, with a sub-cellular localization of the protein in the nuclei of iCCA and eCCA tumor cells [24,32,37,39,48,56].…”