Physical activity regulates the immune response to breast cancer by a hematopoietic stem cell-autonomous mechanism

Abstract

Physical activity is a modifiable lifestyle factor that is associated with a decreased risk for the development of breast cancer. While the exact mechanisms for the reduction in cancer risk due to physical activity are largely unknown, it is postulated that the biological reduction in cancer risk is driven by improvements in inflammation and immune function with exercise. Hematopoietic stem cells (HSCs) are the progenitor for all of the cells of the immune system and are involved in cancer immunosurveillance through differentiation into cytotoxic cell population. In this study, we investigate the role of physical activity (PA) in a spontaneously occurring model of breast cancer over time, with a focus on tumor incidence, circulating and tumor-infiltrating immune cells as well gene expression profiles of tumors and hematopoietic stem cells. Furthermore, we show that, in addition to a direct effect of PA on the immune cells of tumor-bearing mice, PA reduces the oxidative stress in HSCs of wildtype and tumor-bearing mice, and by doing so, alters the differentiation of the HSCs towards T cells in order to enhance cancer immunosurveillance.

Figure 1.. Physical activity decreases oxidant stress in hematopoietic stem cells and leads to increased circulating neutrophils in wildtype mice.

A. Average running distance per mouse (n=5). B. Representative fluorescence analysis of DCF-DA assay by flow cytometry in HSCs from WT SED and PA mice. C. Quantification of the DCF-DA mean fluorescence intensity (MFI) in HSCs from WT SED and PA mice (n=5). D. Gene expression quantification by qPCR of oxidative stress enzymes in PA HSCs, normalize to SED HSC levels (n=4). E. Gene expression quantification by qPCR of epigenetic enzymes in PA HSCs, normalize to SED HSC levels (n=4). F. Quantification of circulating neutrophils in WT SED and PA mice, as a % of CD45+ cells (n=5). G. Cytokine concentration in serum from WT SED and PA mice (n=6–9). H. Representative flow cytometry analysis of neutrophils (CD45+ Ly6C+ Ly6G+) derived from in vitro differentiation of HSCs from WT SED and PA mice. I. Quantification of neutrophils derived from in vitro differentiation of HSCs from WT SED and PA mice (n=3). (Error bars=SEM; *, p,0.05; 2-tailed Student’s t-test).

Figure 2.. Physical activity regulates the immune response in a murine model of breast cancer.

A. Schematic of study hypothesis (Generated with Biorender). B. Tumor incidence in 12-, 15- and 18-week-old SED and PA MMTV-PyMT (MP) mice. C. Average tumor weights in 12-, 15- and 18-week-old MP SED and PA mice. D. Average tumor volumes in 12-, 15- and 18week-old MP SED and PA mice. (MP SED mice, n=23, 10 and 9; MP PA mice, n=28, 11 and 8; SED tumors, n= 42, 26 and 29; PA tumors, n= 30, 26 and 23; at 12, 15 and 18 weeks, respectively). E-K. Quantification of flow cytometry analysis of tumor-infiltrating immune cells from 12-, 15- and 18-week-old MP SED and MP PA mice, as a % of CD45+ cells. E. Macrophages (CD45+ Ly6C+F4/80+)). F. Neutrophils (CD45+CD11c-Ly6C+Ly6G+). G. Natural Killer (NK) (CD45+CD49b+NKp46+). H. Dendritic cells (DC) (CD45+CD11c+MHCII+). I. B cells (CD45+TCRb-CD19+). J. CD4+ T cells (CD45+TCRb+CD4+). K. CD8+ T cells (CD45+TCRb+CD8+). (Error bars=SEM; *, p,0.05; 2-tailed Student’s t-test).

Figure 3.. Physical activity regulates the immune response in a murine model of breast cancer via a hematopoietic stem cell-autonomous mechanism.

A. Schematic of experimental design (Generated with Biorender). B. Quantification of the DCF-DA mean fluorescence intensity (MFI) in HSCs from MP SED and PA mice (n=5). C. Tumor incidence in 12-week-old MP SED, MP PA, SED→ SED and PA→ SED mice. (MP SED mice, n=23; MP PA mice, n=28; SED→ SED mice, n=12; PA→ SED mice, n=13; SED tumors, n= 42; PA tumors, n= 30; SED→ SED tumors, n=20; PA→ SED tumors n=16). D-J. Quantification of flow cytometry analysis of tumor-infiltrating immune cells from, MP SED, MP PA and PA→ SED mice, as a % of CD45+ cells. D. Macrophages (CD45+ Ly6C+F4/80+). E. Neutrophils (CD45+CD11c− Ly6C+Ly6G+). F. Natural Killer (NK) (CD45+CD49b+NKp46+). G. Dendritic cells (DC) (CD45+CD11c+MHCII+). H. B cells (CD45+TCRb-CD19+). I. CD4+ T cells (CD45+TCRb+CD4+). J. CD8+ T cells (CD45+TCRb+CD8+). (Error bars=SEM; *, p,0.05; 2-tailed Student’s t-test).

Figure 4.. Physical activity modulates gene expression in murine breast cancer tumors.

A. Principal component analysis (PCA) was performed on differentially expressed genes in tumors from SED and PA 12-week-old mice (n=3 tumors per condition). B. Volcano plot of differentially expressed genes in tumors from SED and PA 12-week-old mice. C. Heatmap representative of 186 downregulated and 8 upregulated genes in tumors of 12-week-old PA mice. D. Pathway enrichment analysis of differentially expressed genes in PA 12-week-old mice tumors, p value cut off <0.05 and fold change >2. E. Principal component analysis (PCA) was performed on differentially expressed genes in tumors from SED and PA 18-week-old mice (n=3 tumors per condition). F. Volcano plot of differentially expressed genes in murine tumors from SED and PA 18-week-old mice. G. Heatmap representative of 30 downregulated and 5 upregulated genes in tumors of 18-week-old PA mice, p value cut off <0.05 and fold change >2.

Figure 5.. Physical activity modulates gene expression in HSCs.

A. Principal component analysis (PCA) was performed on differentially expressed genes in HSCs from SED and PA mice (n=3 samples per condition). B. Volcano plot of differentially expressed genes in HSCs from PA and PA→ SED mice. C. Heatmap representative of 37 upregulated genes in HSCs of PA mice, p value cut off <0.01 and fold change >2. D. Pathway enrichment analysis of differentially expressed genes in HSCs from SED and PA mice, p value cut off <0.05 and fold change >2. E. Principal component analysis (PCA) was performed on differentially expressed genes in HSCs from PA and PA→ SED mice (n=3 samples per condition). F. Volcano plot of differentially expressed genes in HSCs from PA and PA→ SED mice. G. Heatmap representative of 26 upregulated genes and 12 downregulated genes in HSCs from PA→ SED mice, p value cut off <0.005 and fold change >2. H. Pathway enrichment analysis of differentially expressed genes in PA→ SED mice, p value cut off <0.05 and fold change >2.