Screening Experiments Of Tongue Cancer Stem Cells By Monoclonal Culture

Objectives:1. To study whether the human tongue cancer cell line Tca-8113 intervened with adriamycin can form holoclone, meroclone and paraclone after unicellular culture, and detect the tumorigenicity of holoclone cells. 2. To enrich tongue cancer stem cell-like cells through single cell clone culture and tumor formation in nude mice based on drug-resistance of tongue cancer stem cells.Methods: In the first part, 3 groups of Tca-8113 cells were set up to cultivate and research. Group A, which the Tca-8113 cells were cultured by using single cell clone method with limited dilution system, would be observed for the characteristics of three different clonal types, including the clone formation rate, clone constituent ratio and clone growth curve. Group B,intervened in vitro by three different concentrations of adriamycin (0.01?g/ml ADM, 0.05?g/ml ADM and 0.10?g/ml ADM) for 48 hours before single cell clone cultivation, would be observed for the clonal types' characteristics as well as group A. Group C, which Tca-8113 had been cultured by the single cell clone method with limited dilution system for 2 weeks, would be observed for the survival rate and morphological changes of holoclone cells after a 48-hour intervention with three different concentrations of adriamycin (0.01?g/ml ADM,0.05?g/ml ADM and 0.1?g/ml ADM). In the second part, the survival holoclone cells of group A, group B and group C were continuously sub-cultured and brought into immunodeficiency tumor formation in nude mice when accumulating up to 2×106 quantity in order to detect clonal cells'tumorigenic potential.Results: The results of the first part demonstrated that in group A,Tca-8113 cells cultured by the single cell clone method can generate holoclone,meroclone and paraclone cells in constituent ratios of (49.02±4.44)%,(26.25±1.89)% and (24.72±4.27)%, respectively. The total clone formation rate was (57.28±1.81)%. In group B, intervened in vitro by three different concentrations of adriamycin (0.0?g/ml ADM, 0.05?g/ml ADM and 0.10?g/ml ADM) for 48 hours, Tca-8113 intervention cells all can form clones. The cells intervened by 0.01?g/ml ADM can form holoclone, meroclone and paraclone cells in constituent ratios of (52.20±1.92)%, (25.31±2.30)% and (22.49±1.82)%,respectively. Its total clone formation rate reached (65.98±3.12)%. On the other hand, the Tca-8113 cells intervened by 0.05?g/ml ADM or 0.10?g/ml ADM can only form holoclone but not meroclone and paraclone. The total clone formation rates were(0.693±0.140)‰ and (0.053±0.032)‰. In group C, the Tca-8113 holoclone cells intervened by 0.01?g/ml ADM, 0.05?g/ml ADM and 0.10?g/ml ADM showed survival rates of (91.15±1.63)%,(61.14±5.35)% and(32.47± 1.48)%,respectively. In the second part, the results demonstrated that among group A, B and C, all proliferative cells of survival holoclone cells were able to form tumors in the nude mice, and the tumor formation rate was 100%.The time of tumor formation was shortened when the concentration of adriamycin increased high.Conclusions: 1. Tca-8113 cells and Tca-8113 cells intervened with adriamycin both have the ability of forming holoclone. 2. Tca-8113 holoclone is surposed to be the clone of cancer stem cells and had stronger drug-resistance characteristic to adriamycin. 3. It can be effectively enrich tongue cancer stem cell-like cells with high tumorigenic potential by intervening Tca-8113 holoclone cells with adriamycin.