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  • br protective e ect of OVX Further the

    2020-08-12

    
    protective effect of OVX. Further, the survival benefit of OVX was not observed in immune-deficient as compared to wild-type mice, sug-gesting that immune activity is critical in the antitumor effect of E2 depletion [7]. We confirm in our experiments that OVX reduces the progression of 4T1 TNBCs as compared to that of intact animals in a murine model, thus suggesting that ovarian E2 may play a role in sti-mulating TNBC growth in vivo (Fig. 8A) P < 0.0001. To determine if estrogens have a direct effect on 4T1 TNBC (ERα-negative) cell pro-liferation, we used the Incucyte™ system as described in methods to investigate 4T1 cell progression in vitro. No growth stimulation of Deoxycholic acid as monitored by cell confluence was observed when tumor cells were grown in the presence of E2 as compared to control-treated 4T1 cells over a time course of 5 days (Fig. 8B). Furthermore, treatment with SERD JD128 at doses ranging from 10 to 1000 nM did not elicit any significant effect on cell growth in vitro as shown in Fig. 8B. Together, these data indicate that effects of sex steroids on progression of 4T1 tumors in vivo are likely due to interactions with cells in the TME.
    Furthermore, in this in vivo study, we assessed antitumor efficacy of JD128 alone and combined with an anti-PD-L1 checkpoint antibody. The 4T1 tumor cells implanted in mammary glands exhibit highly ag-gressive behavior and are generally found to metastasize widely to cause early mortality. In contrast to a lack of effects of either estrogens or antiestrogens on 4T1 tumor progression in vitro, we find that the
    Fig. 4. Synthesis pathways for analogues. Analogues 21-32. See text for more details.
    antiestrogens fulvestrant and JD128 are each effective in inhibition of 4T1 tumor growth in vivo in syngeneic BALB/c mouse models (Fig. 8C). Since these mice are immune-intact, we next assessed the effect of treatment with an anti-PD-L1 checkpoint inhibitor alone and in com-bination with either fulvestrant or JD128. As shown in Fig. 8C, anti-PD-
    L1 antibody alone elicited no significant effect on 4T1 tumor progres-sion, while JD128 and fulvestrant were each able to induce significant suppression of 4T1 tumor progression in vivo. These results appear to be consistent with the notion that antiestrogens interact with immune cells in the TME and may play an important role in stopping tumor
    Fig. 5. Biologic activity of selected SERD can-didates. A) Downregulation of ER protein. ER-positive MCF-7 cells were treated in phenol-red free RPMI 1640 without FBS and containing vehicle control (CON) or 100 nM concentra-tions of either fulvestrant (FX) or antiestrogens 105, 109, 121, 140, 151, 160 or JD128 in vitro. After 4 h, cells were harvested and processed for PAGE and Western immunoblots using ERα antibody (1D5, Thermofisher Scientific). RPL13A was used as a loading control. B) Specific [3H]estradiol-17β (E2) binding and competition for binding by antiestrogen JD128 or fulvestrant (FX) at 10 nM was assessed in human MCF-7 breast cancer cells using methods as described before [36,41]. C) Re-sponse of the ERE-luciferase T47D reporter construct to estrogen antagonists fulvestrant (10 nM) or JD128 (10 nM) in combination with 2 nM 17β-estradiol as compared to treatment with 17β-estradiol alone (E2; 2 nM). Cells were dosed with either E2 alone or with SERDs combined with E2 in phenol red-free medium with 0.1% dextran-coated charcoal-treated FBS in luminometer plates. Data are presented as relative light units (RLU) relative to that of E2 alone in three replicate assays (4 wells per re-plicate) + SEM. Treatment with E2 alone in-duced a 12-fold induction of ER-dependent lu-ciferase activity quantified as RLU relative to vehicle control-treated samples.
    Fig. 6. Steroid-like SERD JD128 inhibits estrogen-induced BC cell proliferation in vitro and in vivo. A) ER-positive MCF-7, T47D and ZR75 cells were grown in phenol red-free media with 1% DCC-FBS for 48 h., then treated 48 h. with 2 nM estradiol-17β alone (control) or in combination with 10 nM doses of JD128. Note that MCF-7 cell popu-lations included cells with no HER2-overexpression (MCF-7/PAR), cells with HER2-overexpression (MCF-7/HER2) and MCF-7 cells with tamoxifen resistance (MCF-7/TMR). Cell proliferation is shown as % of that in estradiol-treated controls (n = 3 experiments). Inhibition of cell prolifera-tion in MCF-7/PAR, MCF-7/HER2, MCF-7/TMR, T47D and ZR75 cells averaged 98%, 85%, 94%, 97% and 98% as compared to estradiol-treated controls. JD128 sig-nificantly blocked proliferation in all BC cell models in vitro (P < 0.001). Of note, E2 alone stimulated cell pro-liferation several-fold in each cell line as compared to cells treated only with vehicle (not shown). B) JD128 inhibits