Isolation of cancer stem cells from primary cervical carcinoma and identification of their biological characteristics / 肿瘤

ABSTRACT

Objective:To isolate cancer stem cells from cervical carcinoma and to identify their biological characteristics. Methods:Tumor specimens were obtained from 19 cervical cancer patients at stages ⅠA-ⅡB. Primary cells were cultured in tumor sphere medium (TSM) after mechanical dissociation combined with enzymatic digestion. A series of assays were used to identify the characteristics of the sphere forming cells derived from primary culture. Colony formation was observed by limiting dilution method. MTT assay was used to assess proliferation inhibition by paclitaxel and doxorubicin. Cell surface markers were analysed by fluorescence-activated cell sorter (FACS). The expression of stemness-related genes, drug resistance-related genes, and oncogenes were detected by RT-PCR and Western blotting. Tumorigenicity was evaluated by subcutaneous injection of 1×10~5 sphere-forming cells into nude mice. The tumor formation capability was recorded and pathological classification was performed.Results:After 10 to 15 d culture, the formation of non-adherent spheres could be observed in 8 out of 19 primary tumor cells. The formation ratio was increased with the increase in clinical staging. Sphere-forming cells had colony formation capability. Paclitaxel (100 nmol/L) and doxorubicin (100 nmol/L) inhibited the proliferation of these cells by (77.65±6.46)% and (48.00±7.15)%, respectively. The difference was significant (P<0.01). FACS detection results indicated the phenotypes of sphere-forming cells were CD34~-CD105~-CD44~+CK17~+. RT-PCR detection indicated that spheres expressed stemness-related genes (Oct4 and Piwil2), drug resistance gene ABCG2, and oncogenes (c-myc, sox-2 and stat3). Western blotting further indicated stemness-related protein (Oct4 and Piwil2) expression in spheres. Tumors appeared in all animals at 12 weeks after subcutaneous injection of 1×10~5 sphere forming cells and exhibited a high degree of similarity with the primary tumor in cervical cancer patients. Conclusion:Human cervical cancer stem cells were successfully isolated,which provided a useful model for individualized therapy and evaluation of the therapeutic efficacy for cervical cancer patients.