Fig. 3
Estrogen-mediated M2 microglial polarization promotes tumor growth and stemness. a SKBrM and 231BrM cells were incubated with the CM derived from E2-treated microglia for 24 h. They were then examined for cell viability. Veh CM or E2 CM: microglia cells were treated with PBS or E2 (1 nM) for 24 h, and they were washed with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h. t test, *: p < 0.05; **: p < 0.01. (n = 3). b Colony forming abilities of SKBrM and 231BrM cells were measured in the presence or absence of the CM derived from E2-treated human microglia. t test, **: p < 0.01. (n = 3). c, d SKBrM and 231BrM cells were incubated with the CM derived from E2-treated microglia for 24 h, and the expressions of stemness genes were examined by qRT-PCR., t test, **: p < 0.01; ***: p < 0.001. (n = 3). e Brain metastatic breast cancer cells (SKBrM and 231BrM) were treated with the non-E2 or E2 CM for 24 h and their sphere forming abilities were measured. t test, *: p < 0.05. (n = 3). f Effect of the CM derived from E2-treated microglia on cancer stem cell markers, CD44+/ ESA+ were examined using flow cytometry. t test, *: p < 0.05. (n = 3). g SKBrM (Left) and 231BrM (Right) cells were examined for CD44+/ESA+ cancer stem cell population by FACS. One-way ANOVA, **: p < 0.01. (n = 3). h 231BrM cells were incubated with tamoxifen at different concentrations for 24 h. They were then examined for cell viability. One-way ANOVA, ***: p < 0.001. (n = 3). i 231BrM cells were incubated with the CM derived from microglia that were treated with E2 alone or E2 plus tamoxifen for 24 h. They were then examined for cell viability. E2 or E2 + Tamo CM: microglia cells were treated with E2 (1 nM) or E2 plus tamoxifen (1 μM) for 24 h, and they were washed with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h. One-way ANOVA, *: p < 0.05. (n = 3). j Colony forming abilities of 231BrM cells were measured in the presence or absence of the CM derived from E2− or E2 plus tamoxifen-treated human microglia. One-way ANOVA, **: p < 0.01. (n = 3). k–m 231BrM cells were incubated with the CM derived from E2− or E2 plus tamoxifen-treated microglia for 24 h, and the expressions of stemness genes were examined by qRT-PCR. The data are presented as the mean ± SD. One-way ANOVA, **: p < 0.01; ***: p < 0.001. (n = 3)