The Establishment Of Liver Metastastic Model Of Gallbladder Cancer And Isolation Of The Subpopulation With High Metastastic Potential

Objective to establish the experimental liver metastastic model of gallbladder cancer and isolate the subpopulation with high metastastic potential through the model, which might serve as the reliable tools in research on liver metastasis of gallbladder cancer in vivo and in vitro.Methods GBC-SD was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells from sub-confluent monolayers were harvested by trypsinization and resuspended in saline to a final concentration of 1×10~7 cells/ml, and subsequently inoculated in the spleen of athymic mice, aged between 4-5 weeks.Once the liver metastasis are developed in experimental animal, the tumor cells will be isolated from metastastic foci and cultured in vitro, and then inoculated in the same way as their parents cells for second round of selection. The similar steps were repeated for the third round of selection for subpopulation with high metastastic potential. The metaststic lesion and isolated cells were observed under the lightmicroscopy for morphologic character and from two cell lines, together with liver of experimental animal, DNA were isolated for PCR amplification of three pairs of human-specific microsatellite.Results We established a metastatic model of human gallbladder cancer in nude mice. Liver metastases were uniformly distributed throughout the liver parenchyma with predominance to the periphery. Despite multiple sites of involvement, we observed that the left lobes were most commonly affected in all experimental animals. Histological examination of metastatic lesion demonstrated an adencarcinoma. The gross hepatic metastases were detected in 10th, 7th, 5th weeks after inoculation respectively in first, second and third round selection while the incident of metastases were averaged 60%, 70% and 90%. From third round metastatic model, we isolated a subpopulation with high metastic potential and designated as GBC-SD/M, which exhibited similar histological character with its parents cell line GBC-SD under inverted light microscopy. In addition, the three bands of PCR products, which were amplified with three pairs of specific primers for three homo-specific microsatellites, were detected in the GBC-SD and GBC-SD/M but not in the liver of tumor-bearing animal.Conclusion The metastatic model established study mimicked the natural process in which the cells of gallbladder cancer in blood stream spread to liver and form metastases. it is a reliable and efficient tool for study on metastasis of gallbladder cancer to liver in vivo. Isolated from hepatic metastases, the GBC-SD/M is a subpopulation with high metastatic potential, retaining histological properties and identification of genetic background while compared with its parent cell GBC-SD.