Isolation And Identification Of Cancer Stem Cells In Human Gallbladder Carcinoma And Regulation Of The Malignant Behaviors

Part I Culture of floating tumor spheres from human gallbladder carcinoma in serum-free media and study of the Biological characteristicsObjective To culture and expand the floating tumor spheres from human gallbladder carcinoma cells in vitro and study the biological characteristics of the sphere forming cells, and to determine whether cancer stem cells were contained in the gallbladder carcinoma and filter the cell surface markers preliminary.Methods Human primary gallbladder carcinoma specimens and gallbladder cancer cell line GBC-SD were made into single cancer cells and cultured to grow into tumor spheres in serum-free DMEM/F12 media which contained some cell factors. The tumor spheres were passaged and expanded. Then differentiation analysis was performed by culturing in media containing serum and stem markers including Oct-4 and Nestin were detected using real time RT-PCR. MTT method was used to measure proliferation capacity and sensitivity to chemotherapeutic drugs, and tumorigenicity was measured by transplanted into the nude mice. Then the expression of CD24, CD44 and CD133 in spheres forming cells and adherent cells was tested using flow cytometry.Results Both primary human gallbladder cancer cells and GBC-SD cells were found to grow into spheres in the serum-free environment and the sphere forming cells could reform new spheres. The floating tumor spheres became adherent and cells migrated from the spheres, and the spheres became smaller and disappeared in the differentiating environment. The expression of Oct-4 and Nestin was down-regulated in the adherent cells. Compared with the adherent cells, sphere forming cells showed the higher proliferation capacity, chemoresistance to gemcitabine and 5-fluorouracil and higher tumorigenicity. CD133+cells showed a higher proportion in sphere forming cells than in adherent cells.Conclusion Human gllbladder carcinoma cells contain cancer stem cellst and the cancer stem cells could be expanded to some extent in stem cells environment, and CD 133 protein may be the cell surface marker of the cancer stem cells.Part II Isolation and identification of cancer stem cells in human gallbladder carcinomaObjective To isolate and identify the cancer stem cells in gallbladder carcinoma using CD24,CD44 and CD133 proteins.Methods Fresh tissue samples of human gallbladder carcinoma were cut into smaller pieces and were transplanted into nude mice to established tumor model. Gallbladder carcinoma tissue and gallbladder cancer cell line GBC-SD were made into single cells and labeled anti-human CD24-FITC, CD44-APC and CD133-PE antibody. The CD24+,CD44+ and CD133+subset cells were isolated and the biological characteristics was studied. Then the multiple-marker isolation was performed and the biological characteristics of subset cells including sphere formation, sensitivity to chemotherapeutic drugs, tumorigenicity, self-renewal and differentiation potential were studied.Results A majority of mouse tumor models were successfully established.The proportion of CD24+,CD44+ and CD133+ subset cells were 20.83%～35.72% and 55.73%, 78.19%～93.36% and 96.78%,1.93%～3.43% and 61.28%, in primary gallbladder carcinoma and cell line GBC-SD respectively. For biological characteristics, CD24+ and CD24 cells showed no significant difference in spheroid formation capacity or in tumorigenicity (P>0.05).While CD44+ and CD133+ gallbladder carcinoma cells showed higher spheroid formation capacity and tumorigenicity than their crresponding negative subsets (P<0.05). The proportion of CD44+ CD133+ subset was 2.53% and 60.62% in primary gallbladder carcinoma and cell line GBC-SD respectively. Compared to CD44+ CD 133, CD44+ CD 133+ subset cells showed higher spheroid formation capacity, chemoresistance and tumorigenicity (P<0.05). Both flow cytometry and immunohistochemistry showed that the transplantable tumors derived from CD44+ CD133+ cells contained not only CD44+ CD133+ cells,but CD44-and CD133-subsets.Conclusion CD44+CD133cells exhibited cancer stem cells characteristics in human gallbladder carcinoma and the cancer stem cells were enriched in this subset cells.PartⅢInfluence of CD133-specific siRNA on the biological behaviors of gallbladder cancer cells and expression of transcription factor Glis in gallbladder cancer cellsObjective To study the role of CD133 in gallbladder cancer cells by using CD133-specific siRNA to silence CD 133 expresion in GBC-SD.And to study the expression of of transcription factor Glis in gallbladder cancer cells.Methods CD133-specific siRNA was transfected into gallbladder cancer cell line GBC-SD, and Western blot and flow cytometry were used to analyze and optimize the efficiency and effectiveness of transfection. Then tumor sphere formation capacity was detected in serum-free environment, and proliferation capacity and sensitivity to 5-Fu were tested using MTT method. A cell wounding assay was performed to analyze cell migration. Expression of transcription factors Glil, Gli2, Gli3 in CD44+CD133+cell and others subsets was detected using real time RT-PCR.Results Expression of CD 133 was down-regulated after specific siRNA transfection. Compared with control cells, those transfected specific siRNA showed a lower tumor sphere formation, proliferation and cell migration capacity (P<0.05), while chemoresistance showed no significant change(P>0.05). Nuclear transcription factor Glil expression was upregulated in CD44+CD133+gallbladder cancer cells.Conclusion CD 133 was associated with proliferation and migration of CSCs in GBC and Glil may be involved in the regulation of biological characteristics of gallbladder cancer stem cells.