Tamoxifen suppresses brain metastasis of estrogen receptor-deficient breast cancer by skewing microglia polarization and enhancing their immune functions

Animals were treated in accordance with the US National Institutes of Health Animal.

Protection Guidelines and approved by the Wake Forest Baptist Health Institutional Animal Care and Use Committee. Five to 6 weeks old of female nude mice were used. The mice were housed (five per cage) with a stable temperature (24 ± 1 °C), a 12-h light/dark cycle, and unrestricted access to food and water. The housing environment and animal health were monitored by the laboratory animal center. To examine the role of estrogen on brain metastasis, nude mice were anesthetized and randomly divided into five groups: (1) tumor transplant only, (2) tumor transplant plus 17β-estradiol (E2), (3) tumor transplant plus ovariectomy (OVX), (4) tumor transplant plus OVX and E2 supplement, (5) tamoxifen (Tamo), and (6) OVX + E2 + Tamo. For OVX, the mouse was anesthetized by inhalation of isofluorane. After wiping the injection site with three times of betadine and 70% alcohol, a midline dorsal incision (approx 1 cm) was made using a sharp scissor. A smaller incision (1 cm) in the muscle layer on either side of midline incision was then made to allow entry into the peritoneal cavity. The ovaries and uterus were identified in a fat pad. The end of bilateral uterus were ligated by 6-0 polyproplene suture and the ovaries were removed. The sham group received the same incision but the ovaries were not removed. For E2 supplement, a 0.5-cm incision was made under the neck skin and implanted with 1.5 mg/pellet E2 (Innovative Research of America, 60 day release) and incision was closed by 6–0 polypropylene suture. Finally, the 6-0 polyproplene suture was used to close the incision site of body wall and skin. One dose of buprenorphine (0.05 mg/kg) was subcutaneously administered and animals were allowed to wake up on the heated pad. The animals were positioned laterally and kept warm for 30 min until they recovered from anesthesia. One week later, mice received luciferase-labeled 231BrM cells by intracardiac injection (i.c.) at a concentration of 2 × 10⁵ cells in 100 μL PBS into the left cardiac ventricle. After 3 days of tumor transplantation, mice in the tamoxifen group received tamoxifen (20 mg/kg) treatment by i.p. injection every 3 days for 30 days. The whole body photon flux of mice was measured immediately after injection to confirm a successful injection using IVIS Xenogen bioimager (Caliper). Tumor growth was monitored by bioluminescence until day 30. For bioluminescent imaging, the mice were injected with D-luciferin intraperitoneally (100 mg/kg), followed by capturing images every week using IVIS Xenogen bioimager. The brain metastasis was monitored and the luminescence was quantified once per week. At the endpoint, whole brain was removed, incubated in PBS with 0.6 mg/mL luciferin for 5 min and ex vivo photon flux was measured by IVIS.

Human breast cancer specimens were obtained from surgical pathology archives of the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC), and Cooperative Human Tissue Network, and Pathology Shared Resource at WFBCCC. All tissue sections were obtained by surgical resection and the patient’s information about age, cancer type, and menstrual period and status were record by Wake Forest Baptist hospital. This study was approved by the Wake Forest School of Medicine Institutional Review Board and written informed consent was obtained from all participants.

Human breast cancer line, MDA-MB231BrM2a (231BrM), was a kind gift from Dr. Massague (Memorial Sloan-Kettering Cancer Center). SKBrM3 cell line was derived from parental SKBr3 cells through three rounds of in vivo selection [24, 25]. SkBrM and 231BrM cell lines were authenticated by using GenePrint® 10 STR System (Promega, # B9510). Human and mouse microglia lines, HMC3 and SIM-A9, were purchased from American Type Culture Collection (ATCC) and were authenticated by ATCC. The SkBrM and 231BrM were cultured in DMEM supplemented with 10% FBS, streptomycin (100 mg/mL) and penicillin (100 U/mL) and HMC3 and SIM-A9 cells were cultured in DMEM/F12 medium supplemented with 5% FBS. All cells were grown at 37 °C under 5% CO2. The HMC3 cells were seeded in a 10-cm dish. After reaching 70% confluence, cells were incubated with DMEM/F12 medium supplemented with 2% FBS or in medium containing 1 nM E2 (Sigma) or in 1 nM E2 plus the STAT3 inhibitor, STATTIC (Selleckchem.​com) at 0.5 μM concentration or in 1 nM E2 plus the estrogen receptor antagonist, tamoxifen (Sigma), at 1 μM concentration. We also knocked down the expression of ERα and β in the human microglia cell line using shRNA and examined the effect of estrogen on modulating M2 microglia-mediated brain metastasis. After 24 h, cells were washed twice with PBS and then incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h. The conditioned medium (CM) harvested from the cell culture was centrifuged at 300×g for 10 min to remove the cells and stored at − 80 °C. All cell lines were ensured to be mycoplasma negative by using a universal mycoplama detection kit (ATCC, #30-1012 k, Lot: 70008746). The cells were collected and qRT-PCR and western blotting were used to quantify protein and mRNA levels. In another round of cell culturing, E2-treated cells were washed again and cultured for an additional 24 h in fresh medium. The CM were collected to identify cytokines using the cytokines array (Raybio). To examine the effect of microglia on T cell proliferation, the E2-treated SIM-A9 cells were cultured with fluorescent-labeled (CFSE Cell Proliferation Kit; Thermo Fisher) primary mouse T cell for 24 h, and the cell proliferation was measured by flow cytometry.

The human brain sections were stained using goat anti-CD206 (1:200, R&D systems) for M2 microglia and anti-CD47 (1:100, Invitrogen) for tumor cells and anti-SIRP (1:500, Cell signaling). Brain sections were then incubated with appropriate HRP-conjugated secondary antibodies using diaminobenzidine as the substrate. The signals were evaluated based on their intensities after subtracting the signals of the primary antibody-omitted negative controls. In some cases, the primary antibodies were replaced by isotype antibodies to control for non-specific binding of the antibodies. To determine the area of CD206⁺ cells in brain metastasis, we chose 3 randomly selected fields in each tumor and measured staining intensity by using the Image-Pro software. We also chose and measured staining intensities of 3 non-tumor areas in the consecutive slide, and this background intensity was subtracted to normalize the tumor intensity in the specimens. The average normalized intensity of 3 fields was used as the score of that patient. The scoring range was set from 0 (lowest) to 3 (highest).

The cultured microglia cells were homogenized (1:3, in cultured cells) in the RIPA buffer, and then centrifuged at 17,000g for 30 min at 4 °C. The protein concentrations of the supernatants were determined and adjusted to the same concentration. Supernatants (30 g of total protein) were mixed with sample buffer containing 0.5 M of dithiothreitol, heated to 80 °C for 10 min, loaded into each well of 4% polyacrylamide gel and resolved at 120 V for 2 ;h. The separated proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA), blocked in 5% milk, and probed with respective primary antibodies: JAK (1:1000, Cell signaling), STAT3 (1:1000, Cell signaling), p-STAT3 (1:3000, Cell signaling), GAPDH (1:10000) (Cell Signaling), and PD-L1 (1:2000) (Cell Signaling). The bound antibodies were detected using an enhanced chemiluminescence detection kit (PerkinElmer, Boston, MA, USA). The band densities were measured using an imaging system (BioChemi; UVP, Upland, CA, USA) and analyzed using ImageJ (1.43u) (http://​rsb.​info.​nih.​gov/​ij/​). For gel loading control, membranes were stained with monoclonal-GAPDH antibody (1:50000, Cell signaling).

Nude mice were anesthetized with an overdose of isoflurane and their brains were removed and placed in ice-cold PBS containing 2.5 mg/ml of trypsin and dissociated using mechanical shearing. The cell suspension homogenates were passed through a 40-μm nylon membrane (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and then centrifuged at 500 g for 10 min. The pellets were suspended in PBS, and microglial cells were isolated using a combination of protocols of differential density (stepwise Percoll) gradient centrifugation and immunomagnetic Iba1⁺ cell separation. Purified microglial cells were fixed in 4% paraformaldehyde at 4 °C for 1 h and then incubated with F4/80 and CD86 antibody (1:250) (eBioscience), Tmem119 (1:250) (eBioscience), CD206 (1:250) (eBioscience), CD44 (1:10000) (BioLegend), and ESA (1:250) (Invitrogen) at 4 °C for 16 h. After they were washed twice with PBS, the stained cells were subjected to flow cytometric analysis (FACScan; Becton-Dickinson, Mountain View, CA, USA). The isotype antibodies were be used as control. The percentage of cells was calculated using Cell-QuestTM software (Becton-Dickinson).

231BrM cells were labeled with PKH26 dye (Sigma, USA). The labeled cells were then washed three times and cultured overnight to reduce nonspecific leakage of dye during the assay. Labeled 231BrM cells were mixed with HMC3 human microglia cells that were pre-treated with or without E2 (1 nM) or E2 plus tamoxifen at 1 μM concentration. Co-cultured 231BrM and HMC3 cells in the culture slides were harvested after 8 h and fixed with 4% paraformaldehyde. Microglial cells were counterstained with anti-Iba1 antibody overnight. Secondary antibody conjugated with fluorescent dye Alexa-fluor 488 (1:1000, Invitrogen) was used to detect the microglia. The phagocytic activities were measured by immunofluorescence microscope and flow cytometry. For quantification of phagocytosis, the phagocytic percentage was calculated as: 100 × [percent Iba1⁺/PKH26⁺ cells/(percent Iba1⁺/PKH26⁻ cells + percent Iba1⁺/PKH26⁺ cells)].

To examine the estrogen-induced M2 microglial polarization and examine the effect of tamoxifen on estrogen-induced microglial polarization, HMC3 cells were first infected with lentivirus containing green fluorescent protein (GFP) gene and GFP⁺ cells were sorted by FACS. The GFP⁺ HMC3 cells were seeded in 96-well plates for one day and were transfected with the Arginase-1 promoter reporter plasmid (Addgene) using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA). After 24h of incubation, the cells were treated with only E2 or E2 plus tamoxifen in presence of 2% FBS and cultured for another 24h. Cells were washed twice and were treated with 100μL D-luciferin for 5min. The expression of luciferase was detected by using IVIS Xenogen bioimager. For arginase-1 promoter luciferase normalization, the photon flux was divided by GFP signal which was measured by Multi-Mode Reader (Biocompare). Each experiment was conducted a minimum of three times.

The total number (n) of observations in each group is indicated in figure captions. Data are represented as mean ± standard deviation (SD). Significance was set at p0.05. All studies with more than two groups were analyzed using one-way ANOVA and then Bonferroni post-hoc tests if the overall effect was significant. Two-way ANOVA was used to analyze the effect of E2, OVX, and tamoxifen on brain metastasis of breast cancer in vivo. Bonferroni post-hoc tests were done if the main effects or interactions were significant. One-way ANOVA was used to analyze the effect of estrogen, OVX, STATTIC or tamoxifen treatment on M1/M2 gene polarization and tumor progression, effect of different concentrations of TNF-α or CCL5 on cancer progression. Significance between groups was represented as *, p0.05; **, p0.01; ***, p0.001.

To test the effect of female hormones on brain metastasis of ER-negative breast cancer and to understand how tamoxifen modulate the metastatic growth in the brain, we first performed bilateral ovariectomy (OVX) in 5–6 week-old female mice. One week after the surgery, mice were intracardially implanted with luciferase-labeled human ER-negative breast cancer cells, 231BrM. The mice were then implanted with or without 17β-estradiol (E2) by subcutaneous implantation of a disc or received tamoxifen treatment (20 mg/kg) by i.p. injection every 3 days for 1 month (Fig. 1a). Tamoxifen is known to be blood brain barrier (BBB) permeable [26]. As shown in Fig. 1a–d, E2 significantly increased the incidence and growth of brain metastasis [brain metastasis: Veh (33%) vs. E2 (89%)]. On the other hand, OVX dramatically suppressed brain metastasis progression [brain metastasis: E2 (89%) vs. OVX (11%)]. However, the OVX-mediated suppression of brain metastasis was rescued by E2 treatment [brain metastasis: OVX (11%) vs. E2 + OVX (33%)]. Interestingly, tamoxifen alone or OVX-treated with E2 plus tamoxifen significantly repressed brain metastasis growth to the similar level observed in the OVX group (Fig. 1b–d). Since MDA-MB-231 cells are resistant to tamoxifen [27], we sought of a possibility that the tumor-suppressive effect is due to the tumor microenvironment. Previous studies showed that activation of microglia, especially pro-tumor M2 microglia, promoted the progression of glioma [28‐31]. Therefore, we examined M1 and M2 microglia in the brain of OVX-, E2-, tamoxifen-, OVX plus tamoxifen-, and OVX plus E2 and tamoxifen-treated mice by flow cytometry. The result of FACS analysis revealed that the population of M1 microglial cell (F4/80⁺) was significantly decreased (Fig. 1e and Supplemental Figure 1A) and high levels of M2 microglial cells (CD206⁺) accumulated in the lesions of E2-treated mice (Fig. 1f and Supplemental Figure 1B), suggesting that E2 induced microglia polarization to M2 phenotype in the brain metastatic lesions. In contrast, the effect of E2 in microglia polarization was abrogated by OVX, tamoxifen, or OVX with E2 plus tamoxifen (Fig. 1e, f and Supplemental Figure 1A and B). These results indicate that E2 promotes brain metastasis by skewing microglia polarization to M2 phenotype. To validate this notion in a clinical setting, we examined the status of M2 microglia activation in brain metastatic lesions of pre- and post-menopausal breast cancer patients (Fig. 1g). We observed that M2 microglia (CD206⁺ cells) abundantly infiltrated in the brain lesions of pre-menopause patients compared to post-menopause patients (Fig. 1g). Microglia and peripheral macrophages are known to be recruited by brain metastasis lesion. Although microglia and macrophage share many common surface markers and similar physiological functions, recent studies have demonstrated that microglia and macrophages are two distinct myeloid populations with different developmental origins [32, 33], and they can be distinguished by CD14 macrophage marker [34‐36]. We found that the macrophages were recruited in the brain metastasis lesion of breast cancer patients (Fig. 1g).

However, the population of CD45⁺ macrophage was significantly less than that of CD206⁺ M2 microglia. (Fig. 1g). We also examined the infiltrated microglia and macrophage in the mouse brain using the macrophage marker CD45 and the microglia marker Tmem119. We found that the microglia (CD45⁻/Tmem119⁺) are more abundantly infiltrated than macrophages (CD45⁺/Tmem119⁻) in the brain metastatic lesions (Supplemental Figure 1C). We also observed the increase in CD45⁺ cells in the metastatic mice brain after E2 treatment (Supplemental Figure 1D), which is consistent with the previous studies [37, 38].

Because M2 microglia are abundantly infiltrated in brain metastatic lesions of pre-menopause patients, we examined whether estrogen promotes brain metastasis by polarizing microglia to M2 microglia. We found that the expression of estrogen receptor (ER) on microglia was upregulated in the mice brain after E2 supplement (Supplemental Figure 2A). We examined the expression of ERs in HMC3 (Human) and SIM-A9 (Mouse) microglia cells and found that the expressions of estrogen receptor α and β are upregulated by E2 treatment (Supplemental Figure 2B). To further investigate the effect of estrogen on microglia polarization, HMC3 and SIM-A9 cells were treated with E2 followed by examining the expression of M1 and M2 markers. Our results showed that E2 significantly increased the mRNA expression of M2-related genes, arginase-1 (Arg1), arginase-2 (Arg2), and CD206, in both human and mouse microglial cells lines (Fig. 2a and b). On the other hand, blocking estrogen receptor (ER) signaling by tamoxifen or knockdown of ERα or ERβ by shRNA significantly suppressed E2-induced M2 microglia polarization (Fig. 2c, d, and Supplemental Figure 2C and D). Phenol red in tissue culture media is a weak estrogen [39], we also examined the microglia’s polarization by incubating human microglia cells with or without Phenol red and with or without 2% of FBS or charcoal-stripped FBS for 1 and 3 days. The cell viability (Supplemental Figure 3A) and M1/M2 phenotype (Supplemental Figure 3B, 3C) had no significant difference between the groups. This result is consistent with other published studies [40]. As the JAK-STAT3 pathway is known to induce M2 microglia polarization [41], we examined the status of phospho-STAT3 and JAK expression in microglia after E2 treatment. Indeed, E2 treatment strongly enhanced the activation of JAK/STAT3 in microglia, while tamoxifen suppressed E2-induced activation of JAK and STAT3 (Fig. 2e). We then treated microglia with E2 in the presence or absence of the STAT3 inhibitor (STATTIC) or tamoxifen and examined their effects on polarization of M1/M2 microglia using the Arg1 promoter reporter [42]. We found that E2 upregulated the Arg1 gene promoter activity, and this effect was significantly suppressed by STATTIC and/or tamoxifen treatment (Fig. 2f). Moreover, inhibition of STAT3 and estrogen receptor reversed the E2-mediated suppression of M1 (CD86⁺/Iba1⁺) and promotion of M2 (CD206⁺/Iba1⁺) polarization (Fig. 2g, h). These results suggest that the E2 promotes M2 polarization of microglia via activation of the STAT3 pathway.

To examine how E2 promotes brain metastasis by upregulating M2 microglia polarization, we treated breast cancer brain metastatic cells, SKBrM (ER⁻/Her2⁺) and 231BrM (ER⁻/PR⁻/Her2⁻), with conditioned medium (CM) that was generated from human HMC3 microglia treated with or without E2. To avoid direct effect of E2 on tumor growth, microglia were treated with E2 (1 nM) for 24 h, washed with PBS to remove E2, and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h. The CM was then collected and added to SKBrM and 231BrM cells. We found that the CM derived from E2-treated microglia significantly increased cell viability (Fig. 3a) and colony forming ability (Fig. 3b) of both SKBrM and 231BrM cells. The expression of stemness-inducing genes, SOX2, OCT4, and NANOG, were also significantly upregulated when the brain metastatic cells were treated with the CM derived from E2-treated microglia (Fig. 3c and d). Furthermore, treatment of SKBrM and 231BrM cells with CM generated from E2-treated microglia showed significant increase in their sphere forming ability (Fig. 3e) and CD44⁺/ESA⁺ cancer stem cell (CSC) population (Fig. 3f). In contrast, direct treatment of cancer cells with the same dose of E2 (1 nM) did not affect the stem cell renewal or expression of stemness genes (Supplemental Figure 4A-D). Furthermore, E2-mediated promotion of the CSC population (CD44⁺/ESA⁺) was also significantly suppressed by STATTIC treatment (Fig. 3g). These results suggest that E2 induces secretory factor(s) from microglia, which in turn promotes tumor cell growth and self-renewal. We also examined the direct cytotoxic effect of tamoxifen on tumor cell viability in vitro (Fig. 3h). Our results showed that tamoxifen suppressed cell viability at the concentration of 100 μΜ, but not at 1 μΜ (the dose used throughout our microglia experiments) (Fig. 3h), indicating that the anti-tumor effect of tamoxifen is not mediated by direct cytotoxic effect on tumor cells, and it is rather mediated by the effect on microglia. To further investigate the effect of estrogen on microglia-derived tumor progression, 231BrM cells were treated with CM from microglia that were pre-treated with E2 or E2 plus tamoxifen or E2 plus shERα/β. We found that CM from E2 plus tamoxifen or shERα/β treated microglia significantly suppressed the tumor promoting effect of E2 treated microglia CM (Fig. 3i, j, Supplemental Figure 4E and F). Similarly, we found that CM derived from microglia that were treated with E2 plus tamoxifen or shERα/β significantly suppressed the population of CSC (Supplemental Figure 4G) and the expression of stemness-related genes SOX2 and NANOG (Fig. 3k-m, Supplemental Figure 4H and I).

To identify the factor(s) induced by E2 in microglia, we performed cytokine array analysis (consisting of 99 cytokines and growth factors) and found that E2 treatment significantly increased the secretion of C-C motif chemokine ligand 5 (CCL5) whereas it decreased tumor necrosis factor-α (TNF-α) secretion in the microglial CM (Fig. 4a). The effect of E2 in increasing CCL5 and decreasing TNF-α secretion was further verified in human microglia by performing ELISA (Fig. 4b and c). In contrast, blocking estrogen receptor by tamoxifen or shERα/β significantly suppressed E2-mediated secretion of CCL5 and upregulated the production of TNF-α (Fig. 4b, c and Supplemental Figure 5A-D). TNF-α and CCL5 are known to be associated with M1 and M2 phenotypes, respectively [43‐45]. To examine the functional effects of TNF-α and CCL5, we treated breast cancer cells with recombinant TNF-α or CCL5 and examined their effect on these cells. We found that TNF-α treatment significantly decreased cell viability while CCL5 significantly increased viability of both SKBrM and 231BrM cells (Fig. 4d, e). It should be noted that TNF-α has been shown to kill some tumor cells through activation of apoptotic signaling [46, 47]. We also found that the mRNA expression of CSC markers, SOX2, OCT4, and NANOG, was increased in 231BrM cells when they were treated with recombinant CCL5 (Fig. 4f). Similarly, CCL5 augmented the population of ESA⁺/CD44⁺ CSC (Fig. 4g). To examine whether the upregulation of stemness genes by the CM is indeed mediated by CCL5, CM of E2-treated microglia was treated with CCL5 neutralizing antibody followed by measuring the expression of CSC genes. Our result indicates that depletion of CCL5 in the CM significantly suppressed the expression of stemness genes SOX2, OCT4, and NANOG (Fig. 4h). Moreover, the stimulatory effect of CM on stem cell population (CD44⁺/ESA⁺ CSC) was also suppressed after the CCL5 depletion (Fig. 4i). In addition, the E2-mediated increase in the population of CSC (CD44⁺/ESA⁺) was significantly suppressed by tamoxifen treatment (Fig. 4i). Because cancer cells may also directly affect polarization of microglia [48, 49], we examined the effect of tumor cell-derived conditioned medium. Our results indicated that the direct treatment of microglia with E2 promoted M2 microglia polarization significantly more than the treatment with tumor conditioned medium (Supplemental Figure 5E and F). These results further support our notion that E2 stimulates the secretion of CCL5 from microglia, which in turn promotes self-renewal of tumor cells.

It is known that tumor cells express programmed death-ligand 1 (PD-L1) that binds PD-1 on T cell, resulting in dysfunction of cytotoxic T cell activity [50]. Several other studies have also shown that microglia expresses PD-L1, which interacts with T cell PD-1 to suppress adaptive immune function and tumor infiltration of T cells [50‐52]. We therefore examined whether E2 affects PD-L1 expression in microglia. As shown in Fig. 5a, both mRNA and protein expression of PD-L1 in microglia were significantly upregulated by E2, and inhibiting STAT3 activation by STATTIC or blocking estrogen receptor by tamoxifen treatment significantly suppressed E2-induced upregulation of PD-L1 (Fig. 5a). Moreover, the expression of PD-L1 was elevated on the membrane of microglia after E2 treatment (Fig. 5b). To investigate how upregulation of PD-L1 on microglia by E2 affects T cell function, T cells were incubated with microglia cells that were pre-treated with E2. We found that the proliferation of T cells was significantly suppressed when T cell were incubated with E2-treated microglia (Fig. 5c). Blocking STAT3 activation in microglia by STATTIC treatment restored E2-mediated suppression of T cell proliferation (Fig. 5c).

Microglia cells are known to exhibit anti-tumor functions by phagocytosis and by releasing cytotoxic factors [53]. However, tumor cells express CD47 that interacts with signal regulatory protein α (SIRPα) on microglia [54] and triggers the ‘do not eat me’ signal which enables cancer cells to evade microglia’s phagocytic ability. To examine if E2 functionally affects the “do not eat me” signal, we measured the expression of SIRPα on microglia and CD47 on tumor cells. We found that SIRPα was significantly upregulated in human and mouse microglia upon E2 treatment (Fig. 5d). Tamoxifen dramatically decreased E2-mediated upregulation of SIRPα (Fig. 5d). We also found that E2 significantly increased the expression of CD47 in both SKBrM and 231BrM cells (Fig. 5e). Although the SKBrM and 231BrM cells are considered to be “ER-negative cells”, several studies also have found that 30% of TNBCs express ERα and β by an antibody validation analysis [55‐58]. Therefore, we suspected that upregulation of CD47 by E2 was still through the activation of the ER signaling. To test this possibility, we infected 231BrM with lentivirus including shRNA to ERα and β and examined CD47 expression in the presence and absence of E2. We found that the effect of E2 on CD47 expression was indeed nullified by the shRNAs. (Supplemental Figure 6A). The next question is how ER upregulates CD47. E2 is known to upregulate the PI3K/AKT signaling [59, 60], and previous study have shown that CD47 expression is controlled by PI3K/Akt phosphorylation [61, 62]. Therefore, to examine whether E2 upregulates the expression of CD47 by activating PI3K, 231BrM cells were treated with PI3K inhibitor, LY294002. We found that blocking PI3K activation in tumor cells impaired the E2-induced upregulation of CD47 (Supplemental Figure 6B), suggesting that activation of CD47 by E2 is likely to be mediated through the interaction with ER followed by activation of the PI3K signaling. Importantly, the expression of CD47 was shown to be strongly upregulated in brain metastatic lesions of pre-menopause patients (Fig. 5f). We then examined the effect of E2 on phagocytic ability of microglia. 231BrM cells were pre-labeled with the red-fluorescent dye, PKH26 [63], and co-cultured with microglia that were treated with or without E2 or E2 plus tamoxifen. After 24 h of incubation, small pieces of PKH26⁺ cells were accumulated inside of microglia in the control group, suggesting that tumor cells were rapidly degraded by microglia through phagocytosis (Fig. 5g). However, the microglia suppressed phagocytic activities toward tumor cells after E2 pre-treatment (Fig. 5g and h). In contrast, tamoxifen reversed E2-mediated suppression of phagocytic activities (Fig. 5g and h). These results suggest that E2 suppresses microglia’s immune function by blocking the phagocytic ability and upregulating PD-L1, which in turn promotes brain metastasis progression.