br growth of human breast tumor xenografts
growth of human breast tumor xenografts in vivo. MCF-7 human breast cancer cells were subcutaneously inoculated in nude mice previously primed with estradiol pellets. When animals developed tumors of comparable size they were randomized to treatment with vehicle control (control) or JD128 at 15 and 75 mg/kg once a day by oral gavage for 28 days. Tumors were measured every 3 days, and tumor volume was calculated as V= (l ×w × w)/2). Results are expressed as mean ± SEM. *** P < 0.001 as compared to control group.
progression in vivo. To investigate eﬀects of antiestrogens and immune checkpoint inhibitors when administered in combination, we next used mass cytometry to study the immune cell subpopulations present in the TME in vivo.
3.6. Mass cytometry analyses show that antiestrogens reduce MDSC in murine 4T1 tumors in syngeneic mice
To explore mechanistic pathways that underlie the antitumor eﬀects of antiestrogens alone and combined with an ICI (Fig. 8C), we used mass cytometry by time of flight analyses (cyTOF) with a panel of se-lected labeled Pam3CSK4 to track the levels and activities of immune cell subsets in the TME.
Single cell suspensions were prepared from 4T1 tumors grown in BALB/c mice that were treated for 12 days as detailed in Fig. 8C. Cells were then labeled and analyzed by cyTOF. Results of these analyses are
summarized in Fig. 9. Of the two major MDSC subsets that have been described in humans and mice based on their phenotypic, morpholo-gical and functional characteristics (e.g. G-MDSC and M-MDSC), both G-MDSC and M-MDSC subsets are significantly reduced on treatments with either antiestrogens alone or when given in combination with anti-PD-L1 antibody as compared to appropriate controls (Fig. 9D, E), with a somewhat enhanced eﬀect on the G-MDSC population. The results in-dicate that this biologic eﬀect of antiestrogens may be due to expression of ERα in both G-MDSC and M-MDSC subsets (Fig. 9F).
3.7. Eﬀects of antiestrogens on tumor-infiltrating lymphocytes and cytokines in 4T1 tumors in vivo
In order to gain a better understanding of all tumor infiltrating leukocytes, we analyzed single cell suspensions from tumors (Figs. 8C and 9) by looking at CD8+ and CD4 + TILs. An adaptive T-cell
Fig. 7. Expansion of MDSC is dependent on estrogen signaling and reversed by antiestrogens. E2 increases total numbers of MDSC, with total numbers of human MDSC derived from bone marrow (BM) of BC patients. Retrospectively-collected BM cells from de-identified BC patients were purified by established methods including red blood cell lysis and Ficoll gradients and then incubated with GM-CSF and IL-6 for 6 days in either regular RPMI medium + 15% FBS (NM) (contains estrogens), NM with antiestrogens (FULV or JD128) or in phenol red-free medium with 15% charcoal coated-dextran treated FBS (EFM) (estrogen-depleted) with or without the addition of 100 nM estradiol-17β (EFM + E2). Normal cell culture medium (NM) drives E2-dependent signaling due to the presence of various estrogens in FBS as well as estrogenic properties of phenol red. These eﬀects were significantly inhibited by fulvestrant (FULV) and JD128 at 1 μM concentrations in normal medium. A) The gating strategy used to identify MDSC is shown. The figure shows total MDSC populations (CD45+CD3−CD19−CD20−CD56−CD11b+) identified by following the gating strategy of Ruﬀell et al.  and modified by Svoronos et al. . B) Top panel: graph showing quantification of total MDSC cultivated as described. Lower panel: JD128 blocks phosphorylation/activation of STAT3 in G-MDSC subpopulations under the same conditions described in A. Results show median fluorescence intensity for p-STAT3 in G-MDSC subsets (CD45+HLA-DR−CD11b+CD14−CD15+) after expansion of human total MDSC. Of note, the eﬀect of JD128 is similar to that achieved with E2 depletion (EFM).
Fig. 8. Estrogen eﬀects on ERα negative tumor growth in vitro and in vivo. A) Ovariectomy reduces progression of 4T1 TNBC in syngeneic mice in vivo. Female 6-wk-old BALB/c mice, either ovariectomized (ovx) or sham-operated (intact), were inoculated s.c. with 2 × 105 4T1 TNBC cells. Tumor growth was then assessed every 3 days, with tumor volume calculated as V= (l × w × w)/ 2. **** P ≤ 0.0001. B) 4T1 triple negative breast cancer cells do not respond to estrogen or antiestrogens in vitro. 4T1 cells were grown in the presence (+E2) or absence (-E2) of estradiol-17β and increasing concentrations of JD128 at 10 nM (128-10), 100 nM (128-100) or 1000 nM (128–1000). Cell proliferation was assessed using the Incucyte S3 Live-Cell Analysis system with pictures obtained every 4–6 h. Graph shows average cell proliferation expressed as phase object confluence measured for 5 days. No significant diﬀerences were observed in cell proliferation. C) Antiestrogens combined with anti-PD-L1 checkpoint antibody inhibit TNBC cell growth. Female BALC/c mice (6-wk-old) received intra-mammary implants with 2 × 105 murine 4T1 TNBC cells. After tumors reached approx. 200 mm3 mice were randomized and treated with ve-hicle (CON), 100 μg of anti-PD-L1 antibody every 3rd day (Ab), 50 mg/kg SERD128 via oral gavage (JD128), 5 mg fulvestrant s.c. (FULV) and combina-tions of antibody and fulvestrant (FULV + Ab) or antibody and JD128 (JD128+Ab). Tumors were measured every 3 days, and tumor volume was calculated as V= (l × w × w)/2. ** P < 0.01, **** P < 0.0001 as compared to control group.