Fig. 4.

Fulvestrant modulates RT-induced changes in myeloid immune cell populations. A Schematic of treatment timeline. TC11 cells were transplanted to the caudal mammary fat pad as in Fig. 1. Fulvestrant (Fulv) / oil control treatments were initiated 3 weeks after transplantation, and/or a single dose of RT (8 Gy) was administered 1 week following the first Fulv/ vehicle treatment. TC11 tumors were collected at day 5 after RT for flow cytometry, immunohistochemistry, and qPCR analyses. B Relative proportions of tumor CD45+ immune cells that expressed Ly6C/Ly6G at day 5 after RT, analyzed by flow cytometry (mean ± SEM, n = 9). Differences among treatments were determined by one way ANOVA, followed by Tukey post tests. *, p < 0.05; ***, p < 0.001. C Tumor infiltrating Ly6C+/Ly6G+cells visualized by immunohistochemical staining, representative sections. Original magnifications, × 200; scale bars = 50 µm. See Additional file 2 Fig. S3 for quantitation. D Relative proportions of tumor CD45+ immune cells that expressed F4/80 at day 5 after RT, analyzed by flow cytometry (mean ± SEM, n = 9–10). Differences among treatments were determined by one way ANOVA, followed by Tukey post tests. *, p < 0.05. E Tumor infiltrating F4/80+ cells shown by immunohistochemical staining, representative sections. Original magnifications, × 200; scale bars = 50 µm. F Multispectral immunofluorescence staining of myeloid cells in the TME, labeled as shown. Original magnifications, × 200; scale bars = 50 µm. G CD11B signal in sections shown in (G). Original magnifications, × 200; scale bars = 50 µm. H CD206 and CD86 signals in tumors from animals treated with RT and RT+Fulv in sections shown in (G). Original magnifications, × 200; scale bars = 50 µm. I Relative levels of Nos2 transcripts, an indicator of M1 activity. Mean ± SEM; n = 5. Differences among treatments were determined by one way ANOVA, followed by Tukey post tests. *, p < 0.05. J Relative levels of transcripts for genes associated with a type I interferon response (mean ± SEM, n = 4–5). Fulvestrant reduced activation of a type I IFN response by RT in vivo, in contrast to treatment of isolated TC11 cells in vitro (Fig. 2H), suggesting an effect on either the contents of the TME or on the response of the TME to RT. Differences among treatments were determined by one way ANOVA, followed by Tukey post tests. *, p < 0.05; **, p < 0.01