Isolation And Characterization Of Tumorigenic Extrahepatic Cholangiocarcinoma Cells With Stem Cell-like Properties

Objective:To investigate the expression of CD24, CD44, EpCAM and CD34 in cholangiocarcinoma and determine whether they may be candidate marker isolated cancer stem cells.Methods:Immunocytochemistry staining, RT-PCR, Western blotting and Flow cytometry were employed to examine the expression and rate of CD24, CD44, EpCAM and CD34 at protein and mRNA levels both in Human cholangiocarcinoma cells (QBC-939) and Human gallbladder carcinoma cells (GBC-SD), respectively. The samples resected from patients undergoing surgical resection in our hospital, used for immunohistochemistry included 10 normal bile duct tissues,20 perihilar cholangiocarcinomas, and 20 distal biliary cancers. Of the 40 Extrahepatic cholangiocarcinoma,8 fresh cancer samples were used to perform the expression and rate of CD24, CD44, EpCAM and CD34 by Flow cytometry.Results:In the QBC939 cell line, the mean frequencies of CD24+, CD44+, and EpCAMhigh cells were 57.57%,89.41% and 97.74%, respectively; 51.01% of QBC939 cells were CD24+CD44+EpCAMhigh. In the GBC-SD cell line, the mean frequencies of CD24, CD44 and EpCAMhigh cells were 82.61%,93.56% and 98.36%, respectively; 80.59% of GBC-SD cells were CD24+CD44+EpCAMhigh. In both cell lines, CD34 was weakly detected only by RT-PCR, but not by immunohistochemistry examination,western blot and flow cytometry analysis. On the contrary, CD24, CD44, EpCAM were highly expressed at mRNA and protein level by RT-PCR, Immunocytochemistry staining and western blot. The results were analogue to achieve in cholangiocarcinoma by immunohistochemistry examination, and the frequency of CD24, CD44, EpCAMhigh,CD24+EpCAMhigh, CD44+EpCAMhigh, CD44+CD24+were 5.19-21.39%,3.79-42.32%,70.83-94.48%,4.27-19.73%, 1.88-21.61%,0.43-6.60%. Furthermore, the expression rate of CD24+CD44+EpCAMhigh subpopulation was 0.39-2.27%(mean:0.94%). Conclusion:CD24 combined with CD44 and EpCAM may be a candidate marker isolated cancer stem cells from cholangiocarcinoma.Objective:To isolate CD24+CD44+EpCAMhigh subset cells of human cholangiocarcinoma and identify their cancer stem cell-like properties.Methods:The procedure of subcutaneous implant of NOD/SCID mice was performed to establish the xenografts model with cholangiocarcinoma specimens. CD24+CD44+EpCAMhigh subpopulation of cholangiocarcinoma cells were sorted from xenografts by flow cytometry, and their tumorigenic potential, self-renewal ability and differentiated ability were assessed.Results:CD24+CD44+EpCAMhigh subset cells isolated from 2 xenografts were found to be highly tumorigenic in NOD/SCID mice. CD24+CD44+EpCAMhigh cells consistently formed new tumors at 1×103 cells in three of eight mice tested, In contrast, CD24"CD44"EpCAMlow/- tumor cells were less tumorigenic and gived rise to tumors when 5×104 cells were s.c. inoculated in only one of eight mice animals. Tumors originated from CD24+CD44+EpCAMhigh cells in NOD/SCID mice recapitulated the histological features and heterogeneity of the original patient tumor.Conclusion:CD24+CD44+EpCAMhigh subset cells were discriminated in human cholangiocarcinoma, which had highly tumorigenic, self-renewal ability and differentiated ability. It was first confirmed that CD24+CD44+EpCAMhigh cells may be human cholangiocarcinoma cancer stem cells.