Fig. 4

Estrogen induces secretion of CCL5 from microglia and promotes tumor growth and stemness. a Human microglial (HMC3) 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. CM was collected and analyzed by using the cytokine/growth factor array (Raybio). b, c Human microglial (HMC3) cells were treated with or without E2 (1 nM) or E2 plus tamoxifen (1 μM) for 24 h and cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for additional 24 h. Cell lysates (b) and CM (c) were examined for the amount of TNF-α and CCL5 by ELISA (n = 5/group). One-way ANOVA, *: p < 0.05; **: p < 0.01; ***: p < 0.001. d, e SKBrM (d) and 231BrM (e) cells were treated with the indicated concentrations of recombinant TNFα and CCL5 for 24 h. They were then examined for cell viability by the MTS assay. One-way ANOVA, *: p < 0.05; **: p < 0.01; ***: p < 0.001. (n = 4). f 231BrM cells were treated with CCL5 for 24 h followed by measuring the stemness genes by qRT-PCR. t test, ***: p < 0.001. (n = 4). g 231BrM cells were treated with CCL5 for 24 h followed by measuring the population of ESA⁺/CD44⁺ stem cell by flow cytometry One-way ANOVA, ***: p < 0.001. (n = 4). h and i Human microglia were treated with or without E2 (1 nM) for 24 h, and their CM was prepared. The CM was then added with anti-CCL5 antibody to deplete CCL5. 231BrM cells were then treated with the CCL5-depleted CM followed by assaying stemness-related genes by qRT-PCR (h) and ESA⁺/CD44⁺ stem cell population by FACS (i). The data are presented as the mean ± SD. One-way ANOVA, *: p < 0.05; **: p < 0.01; ***: p < 0.001. (n = 4)