Progesterone promotes immunomodulation and tumor development in the murine mammary gland

Abstract

Background: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation.

Methods: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry.

Results: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice.

Conclusion: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.

Keywords: adaptive immunity; breast neoplasms; immunomodulation; lymphocytes; tumor microenvironment; tumor-infiltrating.

Figure 1

P4 treatment and PR overexpression alters immune cell populations in the murine mammary gland. (A) FVB/n mice were implanted with P4 (30 mg) (n=13) or placebo (n=14) pellets. Twenty-one days later, mice were sacrificed and the right and left mammary glands (#4) were excised; inguinal lymph nodes were removed; and the remaining mammary tissue was digested to single-cell suspension. Immunophenotyping was performed by flow cytometry with respective antibodies. An unpaired two-sample student’s t-test was used to compare the means between treatment groups. (B) Representative IHC images depicting PR staining in the mammary glands from two representative 6-month-old virgin PR-overexpressing (PR/Gal4) and control (GAL4) mice. (C) Cohorts of PR/Gal4 (n=11) and GAL4 (n=7) mice were aged for 6 months and were subsequently sacrificed. Left and right mammary glands (#4) and excised inguinal lymph nodes (D) were collected from the mice; tissue digestion was performed; and immunophenotyping was performed via flow cytometry. Changes in the percentages of various immune cell types observed in the mammary glands of 6-month-old PR/Gal4 mice compared with control (GAL4) mice are shown. (D) Numbers of macrophages and NKT cells present in the excised inguinal lymph nodes (LN) of 6-month-old PR/Gal4 and Gal4 mice. A two-sample t-test with Welch’s correction was used (C, D) to compare means between groups. *P<0.05, **P<0.01. IHC, immunohistochemistry; P4, progesterone; PR, progesterone receptor; NKT, natural killer T cells.

Figure 2

PR overexpression suppresses various immune cell populations present in the spleens of 6-month-old mice. Cohorts of PR-overexpressing (PR/Gal4, n=11) and control (GAL4, n=7) mice were aged for 6 months and were subsequently sacrificed. Spleens were collected from the mice; tissue digestion was performed; and immunophenotyping was performed via flow cytometry. (A) Number of immune cells present per spleen of 6-month-old PR-overexpressing (PR/Gal4) and control (GAL4) mice. (B) Adaptive immune cell populations present in the spleens of 6-month-old mice that were impacted by PR overexpression. (C) Innate immune cell populations present in the spleens of 6-month-old mice that were impacted by PR overexpression. For all data represented, two-sample t-test with Welch’s correction was used to compare means between groups. *P<0.05, **P<0.01. NK, natural killer; PR, progesterone receptor.

Figure 3

PR/Gal4 mice developed significantly more mammary gland tumors than control mice. Cohorts of PR-overexpressing (PR/Gal4, n=17) and control (GAL4, n=14) mice were aged until tumor development (~2 years of age on average). During the aging period, mice were bred three times to mimic normal cycling levels of hormone throughout the lifetime. (A) Percentages of PR-overexpressing (PR/Gal4) and control (GAL4) mice that developed tumors by 750 days of age. The difference in the tumor rates between the groups was assessed using two-sample test of proportions. (B) Kaplan-Meier plot depicting overall survival of PR-overexpressing (PR/Gal4) mice and control (GAL4) mice through 750 days of age. No significant differences were observed between the groups, as determined using a log-rank test. (C) Following RNA sequencing of tumor samples (5 Gal4 tumors and 12 PR/Gal4 tumors), subtype analysis was performed by merging the human PAM50 data and the mouse RNA sequencing data by 47 homolog genes. Merged data were subsequently normalized and samples were assigned to PAM50 subtypes. The graph represents the probability of each PAM50 subtype for representative tumor samples from five control (GAL4) mice and 12 PR-overexpressing (PR/Gal4) mice. *P<0.05. n.s., not significant; PR, progesterone receptor.

Figure 4

Differences in expression of immune-related gene signatures among tumors from PR/Gal4 versus control mice. Cohorts of PR-overexpressing (PR/Gal4, n=17) and control (GAL4, n=14) mice were aged until tumor development (~2 years of age on average). During the aging period, mice were bred three times to mimic normal cycling levels of hormone throughout the lifetime. On sacrifice, sections of each tumor were flash frozen and RNA samples were isolated for subsequent RNA sequencing analysis (5 Gal4 tumors and 12 PR/Gal4 tumors). (A) Representative enrichment plots from GO pathway analysis using GSEA. (B) Select GO gene sets enriched in tumor samples from control (GAL4) mice are represented. The graph represents the log of the p value for each gene set. The complete list of the enriched GO gene sets can be found in online supplemental table 3. (C) CIBERSORT analysis was performed on RNA sequencing data obtained from tumor samples of PR-overexpressing (PR/Gal4) and control (GAL4) mice. The graph represents the immune cell population that was found to significantly differ between groups (activated dendritic cells) by one-tailed Wilcoxon test. *P<0.05. GO, Gene Ontology; GSEA, gene set enrichment analysis; PR, progesterone receptor; FDR, false discovery rate; MHC, major histocompatability complex.

Figure 5

Tumors of PR-overexpressing mice contain fewer infiltrating immune cells compared with controls. Cohorts of PR-overexpressing (PR/Gal4, n=10) and control (GAL4, n=7) mice were aged until tumor development (~2 years of age on average). During the aging period, mice were bred three times to mimic normal cycling levels of hormone throughout the lifetime. On sacrifice, tissue digestion was performed on sections of each tumor and a single-cell suspension was obtained, which was used for immunophenotyping by flow cytometry. (A) Comparison of average tumor weights and number of total infiltrating immune cells (CD45+) per gram of tumor among PR-overexpressing (PR/Gal4) and control (GAL4) mice. (B) Changes in numbers of various immune cell population observed between tumor samples of PR-overexpressing (PR/Gal4) and control (GAL4) mice. For all data represented, a Mann-Whitney test was used to compare groups. *P<0.05, **P<0.01. NK, natural killer; PR, progesterone receptor.

Figure 6

Comparison of immune cell populations in the spleens of PR-overexpressing and control mice that developed mammary gland tumors. Cohorts of PR-overexpressing (PR/Gal4, n=10) and control (GAL4, n=7) mice were aged until tumor development (~2 years of age on average). During the aging period, mice were bred three times to mimic normal cycling levels of hormone throughout the lifetime. On sacrifice, spleens were collected from each animal; tissue digestion was performed; and a single-cell suspension was obtained, which was used for immunophenotyping by flow cytometry. (A) Total number of live cells and immune cells (CD45+) isolated from spleens of PR-overexpressing (PR/Gal4) and control (GAL4) mice that developed mammary gland tumors. (B) Numbers of various immune cell populations per gram of spleen present in the spleens of PR-overexpressing (PR/Gal4) and control (GAL4) mice that developed mammary gland tumors. For all data represented, a Mann-Whitney test was used to compare groups. *P<0.05. NK, natural killer; PR, progesterone receptor.

Figure 7

P4/PR promote immunosuppression and growth of syngeneic PR+ mammary tumors. (A) Left: E0771-vec and E0771-mPR (expressing mPR) cells were injected into the mammary fat pads of C57BL/6 mice pretreated with placebo or P4 (30 mg, 30 days) pellets. Right: tumor weights at the time of sacrifice. (B) Flow cytometry was performed on digested tumors using respective antibodies. (C) Tumor experiments were performed as in A, except cells were injected into the mammary fat pads of SCID(severe combined immunodeficiency)-beige mice. Significance between groups was determined using a two-sided t-test. *P≤0.05, **P≤0.01. mPR, mouse progesterone receptor; P4, progesterone; PR, progesterone receptor.