Modulation of murine breast tumor vascularity, hypoxia and chemotherapeutic response by exercise

Abstract

Exercise has been shown to improve postischemia perfusion of normal tissues; we investigated whether these effects extend to solid tumors. Estrogen receptor-negative (ER-, 4T1) and ER+ (E0771) tumor cells were implanted orthotopically into syngeneic mice (BALB/c, N = 11-12 per group) randomly assigned to exercise or sedentary control. Tumor growth, perfusion, hypoxia, and components of the angiogenic and apoptotic cascades were assessed by MRI, immunohistochemistry, western blotting, and quantitative polymerase chain reaction and analyzed with one-way and repeated measures analysis of variance and linear regression. All statistical tests were two-sided. Exercise statistically significantly reduced tumor growth and was associated with a 1.4-fold increase in apoptosis (sedentary vs exercise: 1544 cells/mm(2), 95% CI = 1223 to 1865 vs 2168 cells/mm(2), 95% CI = 1620 to 2717; P = .048), increased microvessel density (P = .004), vessel maturity (P = .006) and perfusion, and reduced intratumoral hypoxia (P = .012), compared with sedentary controls. We also tested whether exercise could improve chemotherapy (cyclophosphamide) efficacy. Exercise plus chemotherapy prolonged growth delay compared with chemotherapy alone (P < .001) in the orthotopic 4T1 model (n = 17 per group). Exercise is a potential novel adjuvant treatment of breast cancer.

Figure 1.

Tumor response to voluntary aerobic exercise. BALB/c mice bearing 4T1 mammary tumors were randomly assigned to exercise or sedentary controls (n = 11–12/group) starting on day 0. Data are presented as means; error bars represent 95% confidence intervals (CIs). A) Tumor growth rate is statistically significantly slowed by exercise. * P = .004, ANCOVA. B) Representative color composites of immunostaining for apoptosis (cleaved caspase-3, green) in mice that exercised vs sedentary controls. Cellular nuclei are stained with Hoechst 33342 (blue). 5x objective, magnification 100%. Scale bar equals 33 µm. The density of apoptotic cells (cleaved caspase-3+ cells/mm²) is statistically significantly higher in the exercise group compared with sedentary controls. Gray lines = mean; black lines = 95% CI, P = .048, two-sided t test. C) Expression of apoptosis-related proteins in response to exercise. Protein was extracted from 4T1-luc tumors, and expression levels of components of the extrinsic (Fas, Caspase 8) and intrinsic (Bcl-2) pathways, as well as downstream mediators of apoptosis (XIAP, PARP), were visualized on western blots. Band intensity was quantified in comparison with actin loading controls. Both Fas (P < .001) and Caspase 8 (P = .044) expression were statistically significantly increased in mice that exercised, rank sum test. All statistical tests were two-sided.

Figure 2.

Effects of aerobic exercise on angiogenesis and vascular maturity in primary 4T1 breast tumors. Cellular nuclei were stained with Hoechst 33342 (blue). 5x objective, magnification 100%. A-C) Immunostaining for blood vessels shows that microvessel density (CD31, green) was statistically significantly higher in the tumors from exercised mice compared with sedentary controls. Scatter points = individual mice; gray lines = mean; black lines = 95% confidence intervals (CIs), P = .004, two-sided t test. D-E) Representative two-color composite images of vessel pericyte coverage determined by colocalization of CD31 and desmin (red) staining. Scale bar equals 40 µm. F) The percentage of tumor area containing pericyte-covered blood vessels (CD31-desmin colocalized pixels/total tumor area pixels x 100%) is significantly higher in the exercise group. Scatter points = individual mice; gray lines = mean; black lines = 95% CI, P = .006, two-sided t test. G) The percentage of vessel area covered by pericytes (CD31-desmin colocalized pixels/CD31+ pixels x 100%) was also significantly higher in tumors from running mice. Scatter points = individual mice; gray lines = mean; black lines = 95% CIs, P = .024, two-sided t test. H-J) Mice were injected with the hypoxia marker EF5 (red), which was detected by immunohistochemistry. Representative whole tumor images are shown. Quantification of hypoxic tumor percentage shows that exercise significantly reduces tumor hypoxia. Scatter points = individual mice; gray lines = mean; black lines = 95% CIs, P = .012, two-sided t test. K) Tumor response to voluntary aerobic exercise combined with cyclophosphamide chemotherapy. BALB/c mice bearing 4T1 mammary tumors were randomly assigned to voluntary wheel running or sedentary controls (n = 17 per group) starting on Day 0. Mice treated with chemotherapy received the maximal tolerated dose (100mg/kg) cyclophosphamide IP on days 7, 9, and 11. Data are presented as means; error bars are 95% CIs. Tumor growth rate is significantly slowed by exercise and cyclophosphamide monotherapies compared with sedentary controls (P < .001, ANCOVA). The combination of exercise and cyclophosphamide resulted in a statistically significant reduction in tumor growth rate relative to the groups that received no treatment, exercise alone, or cyclophosphamide alone (P < .01, ANCOVA). ANCOVA = Analysis of Covariance.