Exercise boost after surgery improves survival in model of metastatic breast cancer

Abstract

Introduction: Despite advances in breast cancer diagnosis and treatment of the primary tumor, metastatic breast cancer tumors remain largely incurable, and their growth is responsible for the majority of breast cancer-related deaths. There is therefore a critical need to identify ways to reduce metastatic tumor burden and increase breast cancer patient survival. While surgery and pharmacological treatments are the cornerstones of breast cancer intervention, epidemiological data suggests that physical activity can lower the risk of breast cancer development, improve adjuvant treatment tolerance, reduce the risk of disease recurrence and lower breast cancer-related death.

Methods: In this preclinical study, we set out to examine the impact of exercise on metastatic development in triple negative breast cancer (TNBC), using different 4T1 metastasis models, voluntary wheel running and surgical interventions. Tumors were analyzed for hypoxia and immune cell infiltration.

Results: Voluntary wheel running was observed to significantly increase metastasis-free survival, doubling the median survival time. However, these improvements were only observed when a boost in physical exercise occurred following surgery. To investigate this, we performed mock surgeries and confirmed surgical stress was needed to enable the positive effects of the boost in exercise on reducing metastatic tumor burden in mice with either spontaneous metastasis or experimentally-induced metastasis. These changes occurred in the absence of alterations in tumor growth, hypoxia and immune cell infiltration.

Discussion: Taken together, our results suggest that having a boost of physical activity following surgery may be beneficial to delay breast cancer metastatic development.

Keywords: breast cancer; metastasis; physical exercise; surgery; tumor microenvironment.

Figure 1

Exercise initiated after surgical tumor removal improves metastasis-free survival in an orthotopic model. (A) Visual representation of the experimental design. (B) The survival of mice subjected to surgical removal of mammary fat pad tumors with or without access to running wheels were analyzed with Kaplan-Meier analysis. The survival curves were compared using a Log-rank (Mantal-Cox) test. Pair-wise comparisons of the survival curves, revealed that the survival of the mice in the Post-sur EX group was significant longer than the EX group (p=0.03), while the rest of the pair-wise comparisons revealed no significant difference in survival between the groups (Control vs. Post-sur EX p= 019, Control vs. Ex p= 0.51). (C) The median time it took for mice to develop clinical signs of metastatic disease that necessitated euthanasia was examined by performing a Kaplan-Meier survival analysis combined with a Log-rank (Mantal-Cox) test on data from mice that developed metastatic disease. Pair-wise comparisons of the survival curves revealed that the development of metastatic disease in the mice in the Post-sur EX group was significant delayed compared to the Control group (p=0.02, dark blue *) and EX group (p=0.03, orange *).

Figure 2

Analysis of lung metastases reveals no significant differences between the groups in orthotopic model. (A) Visual representation of the experimental design and highlighting the focus of the data – i.e. examinations of collected lungs. (B) Representative images of the degree of lung metastasis. (C) Number of metastatic lung lesions (Control= 21, Post-sur EX= 17, EX= 22). (D) Total area of metastatic lung lesions. (E) Representative images of Ki-67 positive stained tumor cells in the lungs of mice (F) Percentage of Ki-67 positive stained tumor cells per cells in the lungs (Control= 7, Post-sur EX= 6, EX= 7). (G) Flow cytometry analysis of the immune landscape in the collected lungs (Control= 12, Post-sur EX= 10, EX= 11). NK = natural killer cells, CD4 = CD4 positive T cells, CD8 = CD8 positive T cells, DC= dendritic cells, MP= Macrophages, NP=Neutrophils. Parametric data was analyzed by a one-way ANOVA paired with Holm-Šídák’s multiple comparisons test (F, G: DC) and nonparametric data with a Kruskal-Wallis test with Dunn’s multiple comparisons test (C, D, G: NK cells, CD4, CD8, MP, and NP). A two-way ANOVA was used to analyzed the difference in MP and NP between mice with and without metastatic disease (G).

Figure 3

A boost of exercise alone does not affect the development of metastatic disease in an experimental metastasis model. (A) Visual representation of the experimental design. (B) Representative images of the degree of lung metastasis. (C) Number of metastatic lung lesions. (D) Total area of metastatic lung lesions. The data in C and D were analyzed by a one-way ANOVA paired with Šídák’s multiple comparisons test (Sedentary= 12, Pre-EX=10, Post-sur EX= 15, EX= 14).

Figure 4

Initiating exercise following surgery significantly reduces development of metastatic disease. (A) Visual representation of the experimental design. (B) Representative images of the degree of lung metastasis. (C) Number of metastatic lung lesions. (D) Total area of metastatic lung lesions. The data were analyzed with an ordinary one-way ANOVA paired with Holm-Šídák’s multiple comparisons test (Sham= 16, Control= 17, Post-sur EX= 16).