Fan et al. BMC Cancer 2011, 11:320 http://www.biomedcentral.com/1471-2407/11/320 Many previous researches have demonstrated the importance of CD133 as a defining factor of the cancer stem cell phenotype, including human liver cell line [19] and hepatocellular carcinoma [32]. In this study, how- ever, we found that CD133 was commonly expressed on the apical membrane of all normal bile ducts and was frequently expressed in the majority of CC cases, which were clearly at the differentiation stages beyond the stem/progenitor cells. Other studies also find that the overall expression of human CD133 is beyond the rare primitive cells and appears to be a general marker for apical membrane of fully differentiated glandular epithe- lia [4,20,28,33]. These findings suggest that it should be inappropriate to employ CD133 as morphological char- acterization of cancer stem cells and the value of CD133 expression as a marker for cancer stem cells should be critically evaluated in future studies [34]. Recently, several studies have reported that the pre- sence of CD133 in various tumors is correlated with poor prognosis. Zeppernick [35] and Beier [17] have found that CD133 expression indicates shorter survival for adverse gliomas and high-grade oligodendroglial tumors. Similarly, the high expression of CD133 in col- orectal adenocarcinoma is related to poor survival of the patients [21,25]. Furthermore, the high expression of CD133 is correlated with the increased tumor grade, advanced disease stage, elevated serum alpha-fetoprotein levels and poor survival of the patients with hepatocellu- lar carcinoma [21]. In this study, we found that CD133 expression is also significantly associated with the survi- val of the patients with CC. However, there are differ- ences between our findings and the previous reports. Firstly, the patients of CC with positive CD133 expres- sion had significantly better prognosis than those nega- tive ones. Secondly, the patterns of CD133 expression varied in different human CC cases and seemed to be related to differentiation stages of the tumors. Like glandular epithelia that usually show apical membrane expression pattern of CD133 [20,33], normal bile ducts showed the same expression pattern. Interestingly, simi- lar expression pattern of CD133 was found in well-dif- ferentiated CC. In contrast, poorly differentiated CC cases usually showed solid growth pattern and most of them were cytoplasmic positive or even negative for CD133. Nevertheless, in the patients with pancreatic ductal adenocarcinoma, there is no correlation between the level of CD133 expression and patient survival [20]. However, Maeda S et al found that the 5-year survival rate of CD133-positive patients was significantly lower than that of CD133-negative patients [36]. Therefore, the role of CD133 expression in evaluating prognosis of cancer patients needs to be explored in further work. Until now, the physiological role of CD133 is still elusive probably due to the occurrence of Page 6 of 8 many mRNA splice variants and the changing glycosyla- tion status of the protein [37,38]. Moreover, the protein has different subcelluar localizations (membrane, cyto- plasm or both), which may confer different functions. It also remains to investigate why CD133 is usually present on apical membrane of normal bile ducts or other gland lumen, why it is transferred from apical membrane to cytoplasm in cancer cells and what its biological func- tions in normal bile duct epithelium and cholangiocaci- noma are. And the answers to these questions could help us to understand the role of CD133 in the develop- ment of CC and other tumors. Conclusions We found CD133 expressed mainly on the apical mem- brane surface of bile ducts of all sizes and most of malignant ducts. And the subcellular localization of the CD133 protein was investigated and three staining pat- terns, i.e. apical membrane, cytoplasm or both, were presented in CC and the first pattern was frequently found in well-differentiated CC. In addition, the positive expression of CD133 was significantly correlated with well or moderately differentiated CC and vice versa. Moreover, the patients of CC with positive CD133 expression had a significantly better prognosis than those negative ones. In brief, our study demonstrates that CD133 expression correlates with the differentiation of CC and indicates that CD133 is a potential indicator for differentiation and prognosis of human CC, the mechanisms of which, however, need to be investigated in further study. Additional material Additional file 1: The differentiation grade and CD133 expression of 23 follow-up cases. The table contained the details of differentiation grade and CD133 expression of all 23 follow-up cases. The differentiation grade was classified as well-, moderately- and poorly-differentiated, and the CD133 expression was presented with “+"as positive while “-” as negative. Abbreviations CSC: cancer stem cell; CC: cholangiocarcinoma; ICC: intrahepatic cholangiocarcinoma; HCC: hilar cholangiocarcinoma Acknowledgements We wish to thank Dr. Tao Ke for very helpful statistical analysis. We are also grateful to the Department of Medical Records Xijing Hospital for excellent working conditions and for providing access to archival materials. The study was supported by an Opening Project from CBSKL (State Key Laboratory of Caner Biology), the Fourth Military Medical University. Author details 1State Key Laboratory of Cancer Biology and Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China. 2Department of Histopathology, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 2QQ, UK.