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  • br fold molar excess of unlabeled estradiol was


    100-fold molar excess of unlabeled estradiol-17β was present in paired samples to determine displaceable binding [41]. Competitive ligand binding to ER-positive MCF-7 LY-500307 is detected by the ability of a test compound to displace labeled estradiol-17β from the cells in vitro.
    2.6. Estrogen receptor-dependent transcriptional activity
    A stable ER-positive T47D ERE luciferase reporter cell line, in which the ERE and the reporter luciferase gene are consistently expressed in the cell line were used in this study (Signosis). The cell line was es-tablished by transfection of luciferase reporter vector along with neo-mycin expression vector followed by neomycin selection, with neo-mycin-resistant clones subsequently screened for E2 induced luciferase activity or for measurement of potential antiestrogenic activity. Early passages of cells were cultured in complete medium containing RPMI supplemented with penicillin (100 units/mL), streptomycin (100 μg/ ml), 10% FBS and G418 (75μg/ml). At 24 h prior to assays, cells were trypsinized, washed and plated in each well of a 96-well plate with 5 × 104 cells in 100 μl with phenol-red-free medium containing 0.1% dextran-coated charcoal-treated FBS [42,43]. Cells were then treated with 17β-estradiol alone or combined with fulvestrant or JD128 for 24 h. Thereafter, media was removed by aspiration and 100 μl of PBS was added to each well, followed by aspiration of medium and addition of 50 μl of lysis buffer to each well. Cells were incubated in lysis buffer for 30 min at room temperature. Lysate was mixed 1:1 with luciferase substrate (Promega), and luminescence was measured using a MLX microtiter plate luminometer (Dynex) and quantified as relative light units (RLU) according to established procedures [42,43]. Total protein was quantified using BioRad Protein Assay (BioRad).
    2.7. In vivo breast tumor models
    Animals were housed in a pathogen-free environment with con-trolled light and humidity and received food and water ad libitum. All studies were approved by the UCLA Animal Research Protection Committee.
    For experiments using human BC cells as subcutaneous xenografts, ovariectomized female nude mice at 6 weeks of age were obtained from Charles River. MCF-7 human BC cells (2 × 107) were implanted in the flanks of mice who had been primed three days before cell injections with estradiol-17β (0.36 mg, 60 days slow-release pellets, Innovative Research of America) as before [35,36,44]. When tumors grew to 50-100 mm3, animals were randomized to different treatment groups in-cluding a) vehicle control, b) JD128 at 15 mg/kg (by oral gavage daily for 28 days) and c) JD128 at 75 mg/kg (by oral gavage daily for 28 days). Tumor volumes for mice in experimental and control groups were measured every 3–4 days, with tumor volume calculated by (l × w
    × w) / 2, with tumor length l, and tumor width w in mm. Data were presented as the mean ± SEM for tumor volumes measured in cubic mm. Data were analyzed by use of ANOVA and student’s t-test statis-tical approaches as before [35,36,44]. To determine the potential effect of estrogen depletion on the pro-gression of tumors in vivo, 4T1 murine TNBC cells (ATCC) were injected in the 4th mammary fat pad (2 × 105 cells) of either ovariectomized or sham-operated 6-week-old syngeneic female BALB/c mice (Jackson Laboratory). Tumors were measured every 3–4 days, and tumor volume was calculated as (l × w × w) / 2 as above.
    In further studies to determine the effects of antiestrogen treatment alone or in combination with anti-PD-L1 antibody on murine tumor progression in vivo, ovariectomized 6-week-old female syngeneic BALB/ c mice were used (Jackson Laboratory). Three days prior to tumor cell inoculation, mice were injected with estradiol-17β (0.36 mg, 60 days slow-release pellets, Innovative Research of America). 4T1 cells were inoculated in the 4th mammary fat pad (2 × 105 cells), and mice were randomized after tumors reached an average size of 200-250 mm3. For treatment, mice were divided into 6 groups: a) vehicle control or
    f) JD128 and anti-PD-L1 antibody at doses as described for treatment as single agents. Tumors were measured every 3–4 days, and tumor vo-lume was calculated as above. After 10–12 days, mice were anesthe-tized by established methods, with blood collected by cardiac puncture in BD vacutainer vials with EDTA (terminal procedure). An approved secondary method of euthanasia was then used to ensure animals were deceased. Tumors were harvested, with final tumor weights and sizes compared among groups. Mass cytometry studies to assess selected immune cell populations and biomarkers were performed as detailed below as well as IHC to detect CD8+ TILs.