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. 5-6 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 and 5) Tamoxifen. 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 1.5mg/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 x 105 cells in 100 μL PBS into the left cardiac ventricle. After 3 days of tumor transplantation, mice in tamoxifen group received tamoxifen (20 mg/kg) treatment by i.p. injection every three 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 minutes 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.
Cell culture and reagents
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 1nM E2 plus the estrogen receptor antagonist, Tamoxifen (Sigma) at 1 μM concentration. After 24 hours, cells were washed twice with PBS and then incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 hours. The conditioned medium (CM) harvested from the cell culture were centrifuged at 300 ×g for 10 minutes to remove the cells and stored at -80 °C. All cell lines were ensured to be mycoplasma negative by using universal mycoplama detection kit (ATCC, #30-1012k, 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 hours 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 hours 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 17,000 g 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), SOX2 (1:500) (Cell Signaling), Oct4 (1:1000) (Cell signaling), Nanog (1:500) (Cell Signaling), 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 antibody (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 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 microglia 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 x [percent Iba1+/PKH26+ cells / (percent Iba1+/PKH26- cells + percent Iba1+/PKH26+ cells)].
CCL5 and TNF-α ELISA
Microglial cells (HMC3) were treated with or without E2 (1 nM) or E2 plus tamoxifen (1 μM) for 24 hours. Cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for additional 24 hours. Conditioned medium was collected and analyzed by the human CCL5 and TNF-α ELISA kit (Sigma, USA). The HMC3 cells were homogenized in RIPA lysis buffer and centrifuged at 13,000g for 30 min at 4 °C. The supernatant were collected, and protein concentrations were measured and adjusted to 1 mg/mL. Briefly, 96-well plates were first coated with anti-CCL5 or TNF-α monoclonal antibodies followed by addition of 100 μL of the microglia conditional medium or the same volume of the CCL5 and TNF-α standards. The plate was incubated at 4 °C overnight on a rocking platform. After washing the plates, the detection antibody and streptavidin solution were added to each well. After incubation, the TMB and stop solution were added to each well. Finally, the plates were examined at an absorbance wavelength of 450 nm. Standard curves were obtained from values generated from known concentrations of mouse and human CCL5 or TNF-α provided by the kit.
Promoter reporter assay for Arginase-1 gene.
To examine the estrogen-induced M2 microglial polarization and 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 then seeded in 96-well plates for one day and followed by tranfecting them with the Arginase-1 promoter reporter plasmid (Addgene) using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA). After 24 hours of incubation, the cells were treated with only E2 or E2 plus tamoxifen in presence of 2% FBS and cultured for another 24 hours. Cells were washed twice and were treated with 100 μL D-luciferin for 5 min. 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.
Data are represented as mean ± standard deviation (SD). Significance was set at p < 0.05. The estrogen effect on M1/M2 gene expression, effect of estrogen-derived microglial CM on tumor growth, and effect of estrogen on microglia phagocytosis ability were analyzed using unpaired Student’s t test. 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 *, p < 0.05; **, p < 0.01; ***, p < 0.001