Physical activity modulates mononuclear phagocytes in mammary tissue and inhibits tumor growth in mice

Abstract

The risk for breast cancer is significantly reduced in persons who engage in greater amounts of physical activity, and greater physical activity before or after diagnosis associates with reduced disease-specific mortality. Previous mechanistic studies indicate that components of innate immunity can mediate an inhibitory effect of physical activity on several types of tumor. However, in breast cancer specifically, the myeloid compartment of innate immunity is thought to exhibit high propensity for an immunosuppressive role that obstructs anti-tumor immunity. Thus, we tested the notion that greater physical activity alters mononuclear phagocytes in mammary tissue when inhibiting nascent tumor in a murine model of breast cancer. To model greater physical activity, we placed an angled running wheel in each mouse's home cage for two weeks before tumor engraftment with EO771 mammary cancer cells that express luciferase for bioluminescent detection. Fully immunocompetent mice and mice with compromised adaptive immunity showed significantly less mammary tumor signal when given access to running wheels, although the effect size was smaller in this latter group. To investigate the role of the myeloid compartment, mononuclear phagocytes were ablated by systemic injection of clodronate liposomes at 24 h before tumor engraftment and again at the time of tumor engraftment, and this treatment reversed the inhibition in wheel running mice. However, clodronate also inhibited mammary tumor signal in sedentary mice, in conjunction with an expected decrease in gene and protein expression of the myeloid antigen, F4/80 (Adgre1), in mammary tissue. Whole transcriptome digital cytometry with CIBERSORTx was used to analyze myeloid cell populations in mammary tissue following voluntary wheel running and clodronate treatment, and this approach found significant changes in macrophage and monocyte populations. In exploratory analyses, whole transcriptome composite scores for monocytic myeloid-derived suppressor cell (M-MDSC), macrophage lactate timer, and inflammation resolution gene expression programs were significantly altered. Altogether, the results support the hypothesis that physical activity inhibits nascent mammary tumor growth by enhancing the anti-tumor potential of mononuclear phagocytes in mammary tissue.

Keywords: Breast cancer; Exercise oncology; Innate immunity; Macrophages; Monocytes; Myeloid cells; Transcriptomics; Tumor immunology; Voluntary wheel running.

Figure 1. Effect of voluntary wheel running on mammary tumor in the C57BL/6-EO771 model of breast cancer.

(A) Trajectory of tumor signal (mean ± SEM on logarithmic scale) from engraftment on Day 0 to Day 1 and Day 7 in control mice and voluntary wheel running mice. (B) Representative images of bioluminescent tumor signal in C57BL/6 mice; radiance color scale is photos/s/cm²/sr × 10⁷; radiance scale minimum is one order of magnitude lower for Day 0 and Day 1. (C–D) Mean ± SEM and individual data points of three independent experiments with n = 4–6 mice per group per experiment for (C) Day 1 (×10⁶) and (D) Day 7 (×10⁸). ***P < 0.001 for bars under horizontal line.

Figure 2. Effect of voluntary wheel running on EO771 mammary cancer in SCID mice.

(A) Trajectory of tumor signal (mean ± SEM on logarithmic scale) from engraftment on Day 0 to Day 1 and Day 7 in control mice and voluntary wheel running mice. (B) Representative images of bioluminescent tumor signal in SCID mice; radiance color scale is photos/s/cm²/sr × 10⁷; radiance scale minimum is one order of magnitude lower for Day 0 and Day 1. (C–D) Mean ± SEM and individual data points of three independent experiments with n = 6 mice per group per experiment for (C) Day 1 (×10⁶) and (D) Day 7 (×10⁸). *P < 0.05 for bars under horizontal line.

Figure 3. Effects of voluntary wheel running × clodronate treatment in the immunocompetent C57BL/6-EO771 model of breast cancer.

(A) Effects on tumor signal in mammary tissue at Day 1. Mean ± SEM and individual data points of three independent experiments, with n = 5–6 mice per group per experiment. (B) Concurrent effects on Adgre1 gene expression in mammary tissue at Day 1. Mean ± SEM and individual data points of three independent experiments, with n = 5–6 mice per group per experiment. (C) Concurrent effects on F4/80 protein expression in mammary tissue at Day 1. Mean ± SEM and individual data points of two independent experiments, with n = 5–6 mice per group per experiment. (D) Matrix of representative images of F4/80 immunofluorescence (red) in mammary tissue sections with nuclei (blue) at Day 1, scale bar 50 µm. ***P < 0.001, *P < 0.05 for bars or groups of bars under horizontal lines. Clodro: Clodronate.

Figure 4. Mononuclear phagocyte proportions in mammary tissue as estimated by CIBERSORTx with the ImmuCC reference gene set at Day 1 in the immunocompetent C57BL/6-EO771 model of breast cancer.

Mean ± SEM and individual data points, with n = 5–6 mice per group. In (F), only three subjects exhibited non-zero values. *P < 0.05 for bars or groups of bars under horizontal lines. Clodro: Clodronate.

Figure 5. Mammary macrophage phenotype among groups from the wheel running × clodronate factorial paradigm in the immunocompetent C57BL/6-EO771 model of breast cancer.

The multivariate composite variable that maximally discriminated among voluntary wheel running-only mice (Wheel), clodronate-only mice (Clodronate), and control mice (Control) on measures of M1-like and M2-like macrophage percentages at Day 1 was re-constructed. Positive side of the composite variable is defined by relatively more M1-like and less M2-like macrophages while the negative side is defined by less M1-like and more M2-like. Mean ± SD indicated by red point with vertical range line; individual data points in black, with n = 5–6 mice per group. *P < 0.05 for Wheel vs. Control.

Figure 6. TRA composite scores in the immunocompetent C57BL/6-EO771 model of breast cancer as a function of voluntary wheel running × clodronate treatment at Day 1.

(A) Monocytic myeloid-derived suppressor cell (M-MDSC) transcriptome. (B) Granulocytic myeloid-derived suppressor cell (G-MDSC) transcriptome. (C) Macrophage lactate timer transcriptome. (D) Inflammation resolution transcriptome. Mean ± SEM and individual data points, with n = 5–6 mice per group. Scores on log2 scale centered at zero. See text for references for genes sets. Defining genes for each transcriptome are listed in Data S3–S5. ** P < 0.01, * P < 0.05 for bars or groups of bars under horizontal lines. Clodro: Clodronate.