Fig. 5

Estrogen suppresses innate anti-tumor immune function of microglia. a PD-L1 expression was examined in human microglia (HMC3) cells after treating them with or without E2, STAT3 inhibitor (0.5 μM STATTIC), or tamoxifen (1 μΜ) by RT-PCR and western blot. One-way ANOVA, *: p < 0.05; ***: p < 0.001. (n = 3). b Representative images of immunohistochemical analysis of PD-L1 in human microglia (HMC3) cells that were treated with or without E2 for 24 h. Scale bar: 50 μm. c T cells were incubated with E2− or E2 plus STATTIC-treated microglia. Proliferation of T cells was measured by flow cytometry. One-way ANOVA, ***: p < 0.001. (n = 3). d Human (HMC3) microglia cells were treated with or without E2 (1 nM) or E2 plus tamoxifen (1 μM) for 24 h, and the expression of SIRPα was examined by qRT-PCR. One-way ANOVA, **: p < 0.01; ***: p < 0.001. (n = 3). e SKBrM and 231BrM cells were treated with or without E2 for 24 h, and the expression of CD47 was examined by qRT-PCR. t test, ***: p < 0.001. (n = 3). f The brain metastatic lesions from pre- or post-menopause patients in Fig. 1g were sectioned and they were subjected to immunohistochemical analysis for CD47⁺. Arrows indicate CD47⁺ cells (brown) in pre-menopause (n = 7) or post-menopause (n = 7) patients. Scale bar: 100 μm. g Phagocytic activities of HMC3 human microglial cells (green labeled) with or without E2 or E2 plus tamoxifen treatment were examined using PKH26-1 labeled 231BrM cells (red labeled). Both cells were incubated together for 24 h, photographed (left panels), and the number of microglia that phagocytosed tumor cells was counted to quantify microglial phagocytic activity as shown in the right panel. h A parallel set of cells in (g) was analyzed by flow cytometry to quantify phagocytic abilities of microglia after E2 or E2 plus tamoxifen treatment. One-way ANOVA, ***: p < 0.001. (n = 3). The data are presented as the mean ± SD