Sex steroid modulation of macrophages within the prostate tumor microenvironment

Abstract

Background: The role of androgens and other sex steroids is known to influence the prognosis and progression of prostate cancer through different disease states. While androgens are generally regarded as immunosuppressive and estrogens as inflammatory, the specific influence of sex steroids on the immune microenvironment of prostate tumors remains incompletely understood.

Material and methods: In this study, we evaluate the link between sex steroids and prostate cancer immune cells, particularly macrophages. Using in vitro and in vivo models, as well as ex vivo culture of patient prostate tissue, we evaluated the influence of androgen, estrogen, and progesterone on immune cells of the prostate microenvironment.

Results: In vitro, we observed sex steroids induced indirect changes on prostate cancer cell proliferation via THP-1 derived macrophages, but no clear changes were induced using human monocyte derived macrophages. Comparing immunohistochemistry for immunosuppressive macrophage marker CD163 with concomitant circulating sex steroids from the same patients, we observed a correlation with higher dehydroepiandrosterone (DHEA)-sulfate and estrone-sulfate levels associated with higher prostate CD163 expression. Similar relationships between DHEA and CD163 levels were observed in ex vivo cultured prostate biopsies. Finally, in a murine prostate cancer model of long-term sex steroids we observed significant differences in tumor growth in mice implanted with estrogen and DHEA diffusion tubes.

Conclusions: Our results highlight the complex influence of sex steroids on the immune cell composition of prostate tumors. Understanding this biology may help to further personalized therapy and improve patient outcomes.

Keywords: Prostate cancer; immunosuppressive phenotype; macrophages; sex steroids; tumor microenvironment.

Figure 1

Impact of sex steroids on THP-1 differentiated macrophages. Macrophages were cultured during 5 days with or without sex steroids, then polarized into M1 or M2 macrophages for 6 h. Note that sex steroids were not present during subsequent coculture with indicated PCa cell lines. A. Expression of indicated markers was assessed by qRT-PCR following exposure to sex steroids. B. Proliferation of PCa cell line cocultured with M1 and M2 polarized macrophages after 24 h during 72 h. DHEA = dehydroepiandrosterone; DHT = dihydrotestosterone; R1881 = synthetic androgen methyltrienolone; E1 = estrone; E2 = estradiol; Proge = progesterone. *P<0.05.

Figure 2

Impact of sex steroids on peripheral blood mononuclear cell (PBMC) differentiated macrophages. Differentiated macrophages were cultured during 5 days with or without sex steroids, then polarized into M1 or M2 macrophages for 96 h. (A) Flow cytometry analysis of macrophage phenotypic polarization markers from PBMC of healthy donors after exposure to sex steroids. (B) Proliferation of indicated PCa cell lines cocultured with M1 and M2 polarized macrophages after 24 h following indicate steroid treatment. (C) Migration of PC3 cocultured with M1 and M2 macrophages for 48 h. (D, E) Impact of sex steroids on IL-12 production from MDM (with or without coculture with MR49C PCa cells) (D) and from THP-1 macrophages (E). *P<0.05.

Figure 3

Sex steroids effect on human prostate tissue and correlation with immune cells infiltration. A. Relationship of CD163 expression with DHEA-S (left) and E1-S (right). IHC expression of CD163 in radical prostatectomy specimens was normalized for CD45 expression and plotted according to circulating steroids measured by mass spectroscopy from pre-operative plasma of the same patient. B. Schema of experiments using ex vivo culture of biopsies from fresh radical prostatectomy specimens. C. Ratio of CD163⁺/CD68⁺ expression from ex vivo culture of biopsies of fresh radical prostatectomy specimens in indicated steroids for 72 h prior to fixation and IHC staining. *P<0.05.

Figure 4

Effect of sex steroids on tumor growth and immune infiltrate in TRAMP-C2 murine prostate cancer model. (A, B) Tumor growth (A) and survival (B) analysis of mice bearing TRAMP-C2 prostate cancer. Two-way ANOVA was used for statistical analysis. (C) Flow cytometry analysis of M1 (CD45⁺Ly6C^-CMHII⁺F4/80⁺CD11c^-CD11b⁺ cells) and M2 (CD45⁺ Ly6C^-CMHII⁺F4/80⁺CD11c⁺CD11b⁺ cells) macrophages from dissociated spleens. (D, E) Flow cytometry analysis of immune cells (CD45⁺ cells) infiltration and evaluation of lymphocyte T CD3⁺CD4⁺ cells, CD3⁺CD8⁺ cells, and NK cells (CD49⁺ cells) from dissociated tumors (D) and spleens (E).