In Vitro Experimental Study On Cancer Stem-like Cell In Humn Breast Cancer Cell Lines

1. Preliminary Study on Cancer Stem-like Cell in Human Breast Cancer Cell LinesObjectives To investigate the content of cancer stem-like cells in three human breast cancer cell lines and to explore the methods for enriching cancer stem-like cells in MCF-7.Methods The percentage of SP cells and CD44⁺CD24^-/low cells in human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB435s were detected with flow cytometry. MCF-7 was separated by Percoll gradient centrifugation into five subpopulations. In order to screening which subpopulation contained the highest percent of SP cells, each subpopulation was detected with flow cytometry. Then MCF-7 was cultured in serum-free DMEM/F12 containing EGF, bFGF, BSA and insulin. The percentage of CD44⁺CD24^-/low cells was detected with flow cytometry after 1 week, 2 weeks and 3 weeks.Results The percentage of SP cells was 3.61% in MCF-7 and 2.72% in MDA-MB-435s. There was no SP cell detected in MDA-MB-231. The percentage of CD44⁺CD24^-/low cells was 0.87% in MCF-7, 0.59% in MDA-MB-231 and 94.04% in MDA-MB-435s. MCF-7 was separated by Percoll gradient centrifugation into five different subpopulations. Heterogeneity was observed in SP content, cell cycle distribution, grow curve and colony formation ability among five different subpopulations. Subpopulation D was rich in SP cells, which reached up to 25.23%. CD44⁺CD24^-/low cells in MCF-7 increased after cultured in serum-free medium. The cancer stem-like cells in MCF-7 grew as clonal nonadherent spherical clusters. The percentage of CD44⁺CD24^-/low cells reached up to 29.23% after cultured 3 weeks in serum-free medium.Conclusions SP cell detection and CD44⁺CD24^-/low cell detection are two commonly used methods in detecting breast cancer stem cell. But while detecting in the same cell line the results of two methods often disagrees. Each method has its own limitation. MCF-7 was heterogeneous. The cancer stem-like cells in MCF-7 can enrich after separated by Percoll gradient centrifugation or cultured in serum-free medium. Methods E₂ group added 10^-7mol/L, 10^-8mol/L, 10^-9mol/L estradiol respectively. E₂+TAM group rejoined 10^-6mol/L tamoxifen. The control group added aequales drug solvent (ethanol). The percentage of CD44⁺ CD24^-/low cells in each group was detected with flow cytometry after cultured ten days and twenty days.Results After cultured ten days, the percentage of CD44⁺CD24^-/low cells in each E₂ group was (2.28±0.38)%,(6.35±0.57)%,(2.86±0.29)%, which increased significantly (P＜0.05) compared to the control group. The percentage of CD44⁺CD24^-/low cells in each E₂+TAM group was (1.85±0.19)%,(4.11±0.41)%,(1.73±0.33)%, which decreased significantly(P＜0.05) compared to the homologous E₂ group. After cultured twenty days, the percentage of CD44⁺CD24^-/low cells was (2.41±0.25)%,(6.18±0.41)%,(2.47±0.15)% in each E₂ group and (1.93±0.21)%,(4.26±0.39)%,(1.34±0.23)% in each E₂+TAM group. Compared to E₂ group, the percentage of CD44⁺CD24^-/low cells in each E₂+TAM group decreased significantly(P＜0.05) after added 10^-6mol/L tamoxifen.Conclusions The percentage of cancer stem-like cell (CD44⁺CD24^-/low cell) in MCF-7 increases after treated with estradiol. And tamoxifen can decrease the percentage of cancer stem-like cell in MCF-7.