Establishment and Characterization of a New Intrahepatic Cholangiocarcinoma Cell Line, ICC-X2

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor of the biliary tract that is prone to recurrence and metastasis and is characterized by poor sensitivity to chemotherapy and overall prognosis. For these reasons, there is an urgent need to understand its pathological mechanisms and develop effective treatments. To address this challenge, the establishment of suitable preclinical models is critical.

Methods: Fresh ICC tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, and short tandem repeat (STR) analysis. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-fluorouracil (5-FU) were evaluated by CCK-8 assay. Subcutaneous injection of 1 × 10⁶ cells to three BALB/c nude mice was conducted for xenograft studies. The hematoxylin and eosin (H&E) staining was used to detect the pathological status of the cell line. The expression of biomarkers CK7, CK19, Ki-67, E-cadherin and vimentin was determined by immunocytochemistry assay.

Results: A new ICC cell line named ICC-X2 was successfully established. Like ICC-X3 established using the same patient's metastatic tumor, the cell line has been continuously cultured in vitro for more than a year and has been passaged more than 100 times. ICC-X2 retained the typical biliary epithelial morphology. The population doubling time of ICC-X2 is 48 h. The cells demonstrated an abnormal nearly tetraploid karyotype. The STR analysis confirmed that ICC-X2 was highly consistent with the primary tumor tissue and not cross-contaminated by existing cell lines. ICC-X2 cells positively expressed CK7, CK19, E-cadherin, and vimentin, and the positive expression of Ki-67 in ICC-X2 cells was 40%. The ICC-X2 cells exhibited a strong clonogenic ability. The drug sensitivity test indicated that ICC-X2 was sensitive to oxaliplatin and paclitaxel, but naturally resistant to gemcitabine and 5-FU. ICC-X2 was rapidly able to form transplanted tumors in vivo after subcutaneous inoculation in nude mice.

Conclusions: ICC-X2 is an excellent experimental model that can be used for studying the occurrence, development, and metastasis of ICC and investigating the mechanism of tumor drug resistance.

Keywords: Animal model; Cell line; Establishment; Intrahepatic cholangiocarcinoma; Xenograft.