The Mechanism Of CD133~+ Tumor Initiating Cell And Microenviroment In Gallbladder Carcinoma Progression And New Chemotherapy Regimen Treatment Of Advanced Gallbladder Carcinoma

Objective Gallbladder carcinoma is characterized by its high degree of malignant,low operation resection,resistance to chemotherapy/radiotherapy and poor survival rate.The study aimed at the isolation and property identification of CD133 positive(CD133+)subset in human gallbladder cancer cells.Determine the objective response rate in patients with advanced gallbladder treated with new chenotherapy regimen.Methods Immunomagnetic beads were used for isolation of CD133+ subset.Immunofluorescence was used to detect the localization of CD133.Percentage of CD133+ subset was determined through flow cytometry(FCM).Expression of CD133 mRNA and protein was detected by semi quantitative reverse transcription polymerase chain reaction(RT-PCR)and Western blotting respectively.Meanwhile,in vivo tumor formation ability and colony formation capability of CD133+ subset were also detected.CCK8 assay was used to detect the proliferation ability and resistance to anti-tumor drugs of CD133+ subset.The invasion ability of CD133+ subset was evaluated by Transwell assay.Furthermore,expression of stem cell related genes was detected through semi quantitative RT-PCR.Randomized,open,controlled,multicenter group assignment to evaluate the safety and efficacy of new chemotherapy regimen.Results In the first part,CD133 in these cells was located in cell membrane or cytoplasm.Expression level of CD133 mRNA and protein in CD133+ group was significantly higher than that in CD133-group as determined by the gray level.Colony formation efficiency(CFE)in CD133+ group was significantly higher than that in CD133-group.Tumor formation efficiency in CD133+ group and unsorted cells was 100% and 60% respectively,while no tumor formation was observed in CD133-group.Cell proliferation ability in CD133+ group was significantly higher than that in CD133-group.When treated with fluorouracil or Gemcitabine,cells in CD133+ group showed decreased sensitivity to drugs with statistical significance,compared to that in CD133-group.Number of transmembrane cells was significantly higher in CD133+ group than that in CD133-group.In the second part,expression level of CXCR4,p-Akt and CD133 protein in CD133+ group was significantly higher than that in CD133-group as determined by gray value.In CD133+ human gallbladder cancer cell line GBC-SD,SDF-1α or AMD3100 treatment can promote or suppress SDF-1α/CXCR4 axis,resulting in increased or decreased CD133 expression in GBC-SD cells through Akt signaling pathway.Inhibition of Akt signaling pathway led to decreased CD133 expression in human gallbladder cancer cells.In the third part,New chemotherapy regimen group’s OS and PFS are significantly higher than conventional chemotherapy group.New chemotherapy regimen group’s PR is also significantly higher than conventional chemotherapy group.New chemotherapy regimen group could decrease the level of CA19-9 and CEA Conclusions 1.Immunomagnetic beads were successfully used for isolation and purification of CD133+ subset from human gallbladder cancer cell line GBC-SD.CD133+ subset showed high potential for tumor formation,cell proliferation and invasion,as well as insensitivity to chemotherapeutics,with expression of stem cell related genes.2.In CD133+ human gallbladder cancer cell line GBC-SD,CXCR4/Akt/CD133 signaling pathway might be activated.3.New chemotherapy regimen group could be demonstrated to improve OR/OS and PFS vs conventional chemotherapy group.