Establishment and characterization of cell lines from liver fluke-associated cholangiocarcinoma induced in a hamster model.

Cholangiocarcinoma (CCA) is a relatively rare tumor that occurs primarily in tropical countries and particularly in those with a high incidence of liver fluke infection. A hamster model for a liver fluke-associated CCA has been described previously. In the present study, hamster cholangiocarcinoma cell lines were established and characterized in order to obtain information regarding diagnostically useful tumor marker which could shed light for a future investigation for human cholangiocarcinoma. Two related cell lines, one from the original intrahepatic bile duct tumor and one from an allotransplanted tumor, were established. The established cell lines were found to have population doubling times of 31 and 26 hours respectively, and were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum for over 80 passages. The cell monolayers were subjected to scanning and transmission electron microscopic study and found to have ultrastructural characteristics, including cytoplasmic lumens, consistent with those of adenocarcinoma cells of epithelial origin. An immunoperoxidase study using monoclonal antibodies (MAbs) specific for tumor antigens showed the cytoplasm and membrane of both cell lines to be positive. These antigens were also secreted in soluble form into the culture medium, judging from polyacrylamide gel electrophoresis in the presence of SDS and from immunoblot analyses. Different lines of evidence presented suggested that a 200 kDa glycoprotein produced and secreted by the tumor cell lines could be considered a cholangiocarcinoma-associated marker which has diagnostic potential.

Identification of tumor markers for cholangiocarcinoma and evaluation of their diagnostic potential.

Results obtained from studies using experimental animal model clearly showed that (1) A marker(s) for CCA does exist; 2) This marker is a glycoprotein with a molecular weight of 200 kDa; (3) It is produced and secreted in vitro by tumor cell lines; (4) It is highly immunogenic in mice and the MAb specific for this antigen is directed against the carbohydrate moiety; (5) This tumor antigen can be detected in serum and bile of tumor-bearing animals by a sandwich ELISA employing this MAb; (6) Kinetic studies show a gradual elevation of this antigen during tumor development; and (7) The elevation of this antigen can be detected at a time when no pathological changes have yet taken place, as judged by microscopic examination. Preliminary work from the human counterpart using human cholangiocarcinoma cell line showed promising results. CCA-specific antigen could be similarly identified and the MAbs produced were highly specific for this 160 kDa antigen.

Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer.

A new human cholangiocarcinoma cell line (HuCCA-1) was established from cholangiocarcinoma (CCA) tissue fragments surgically removed from a Thai patient with intrahepatic bile duct cancer. The growth medium used for the primary cell culture was Ham's F12 supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml epithelial growth factor (EGF). Approximately one month later, the cells were subcultured in Ham's F12 supplemented with only 10% FBS. The population doubling time was approximately 55 hr. Staining of the cells for cytokeratin and mucin confirmed that the cells were mucin-secreting tumor of epithelial cell origin. The supernatant fluid secreted a number of non-specific tumor markers including CA125 and traces of MCA and AFP. The ability of the HuCCA-1 cell line to synthesize specific marker that may have potential in the diagnosis of cholangiocarcinoma is now being investigated.