Exercise training inhibits the progression of breast cancer in vivo
To optimize the anti-cancer effect of exercise, resistance training was combined with aerobic exercise. The specific modeling techniques and intervention strategies are presented in Fig. 1. Following successful modeling, the mice did not exhibit a decrease in appetite; however, their weight tended to increase (Fig. 2A). In these experiments, with increasing exercise training, tumor volume in the Ex group (p < 0.05) and tumor weight (p < 0.01) were significantly lower than in the SED group. We also selected Dox as a positive control, and both Dox and Dox + Ex groups significantly reduced tumor volume and mass (p < 0.001), and the decreasing trend in the Dox + Ex group was more obvious than that in the Dox group alone, and the therapeutic effect may be better. The results are shown in Fig. 2B, 2C, and 2D. To elucidate the mechanism behind this synergistic effect, we performed H&E staining on mouse tumor tissues. The results of the histological analysis of the tumors also showed that there was no necrotic area in the tumor tissue of the SED group. In addition, the nuclei were deeply stained, the cell morphology was polygonal, and the cell proliferation was vigorous. Compared with the SED group, the density of tumor cells in the Ex group and the Dox group was significantly reduced, among which the Dox + Ex group could cause structural changes of tumor, mild staining of nuclei, bleeding, inflammatory cell infiltration and cell coagulation or formation of apoptotic bodies (Fig. 2E).
Exercise training inhibits splenomegaly in a breast cancer mouse model
In addition, at the end of the experiment, the mice were sacrificed and the samples were collected. Tumor progression is known to be positively associated with the promotion of splenic aggregation (splenomegaly) of tumor immune cells [16]. According to Fig. 2E, it was found that the tumor growth process was easy to cause spleen enlargement involving the spleen immune organ, and Ex group alone had little effect on reducing the function of spleen enlargement. Compared with the SED group, the Dox group, and the Dox + Ex group were found to reduce splenomegaly after chemotherapy, and the synergistic exercise was associated with increased efficacy (p = 0.0127 and p = 0.0005) (Fig. 3B). Histopathological changes in the spleen were observed by H&E staining. As shown in (Fig. 3C), swelling of splenocytes and accumulation of a large number of immune-related cells were observed in the spleen in the SED group and Ex group. In the Dox group, no obvious evidence of morphological damage was found in splenocytes with clear nuclei. The Dox + Ex group significantly attenuated the morphological changes of splenocytes and spleen quality caused by tumor. Although exercise training alone could not reduce spleen compensation, exercise training could enhance the reduction of splenomegaly after Dox administration.
Exercise training activates and redistributes immune cells
Natural killer (NK) and T cells play important roles in the immune system and contribute to tumor killing during tumor progression. During exercise, NK cells exhibit the most sensitivity and are quickly recruited into circulation [17]. However, it remains unclear whether combining chemotherapeutic agents with exercise can enhance immune system response. In this study, we extracted cells from lymphoid, spleen, thymus tissues, and peripheral blood to analyze the effects of exercise, chemotherapy, and their combination on NK cells NKT cell and T cells, the specific flow gate strategy is shown in (Fig. 4A). Flow cytometry results showed that compared with SED group, NK cells increased significantly after exercise training, especially in peripheral blood (p < 0.05) (Fig. 4E). In spleen, lymph, and peripheral blood, the proportion of NK cells increased after exercise in the Ex group compared with the Dox group. (Fig. 4B-D). NKT cells are a group of special T cell subsets with both T cell receptor TCR and NK cell receptor on the cell surface [18]. It also increased after exercise. But Ex has little effect in peripheral blood (Fig. 4I). However, compared with SED and Dox groups, the increase of Dox + Ex group was significantly higher (p < 0.05) (Fig. 4F-H). Similar conclusions were also obtained in T cell subsets, with the proportion of T cells in thymus, lymph and peripheral blood increased after exercise compared with SED group (p < 0.05) (Fig. 4J-M). The proportion of NK cells, NKT cells and T cells also increased after exercise training, and were stimulated to mature in various immune organs, showing antitumor effects. Overall, our cytometry analysis reveals that exercise significantly enhances the activation of immune cells.
Exercise training modifies the tumor immune microenvironment
Next, immunohistochemical staining was used to measure the content of NK cells and T cells in tumor tissues, and the results showed that the NK cells in the Ex group increased significantly (p > 0.05) (Fig. 5A). Compared with the Dox group alone, the number of NK cells in the Ex group could be increased after exercise intervention, and Dox killed mainly by inhibiting the tumor cell cycle. After exercise intervention, the vitality and number of immune cells can be activated to carry out the killing effect and increase the anticancer effect. A similar conclusion was also obtained for another group of CD8 T cells, which mainly perform killing functions (p > 0.05) (Fig. 5B-C).
Exercise training improves cardiotoxicity induced by Doxorubicin
Clinically, most chemotherapy drugs indiscriminately attack normal cells while killing tumor cells, often causing a range of side effects. Doxorubicin is a cell cycle non-specific anti-breast cancer drug, but its most serious side effect is cardiotoxicity [19]. To evaluate the potential of ameliorating the cardiotoxicity caused by doxorubicin after aerobic exercise intervention, immunohistochemical detection of heart tissues showed that the morphology of myocardial cells in the SED group and the Ex group was normal, and the myocardial fibers were neatly arranged with uniform cytoplasmic staining. In contrast, in the Dox group alone, the myocardial fibers were disorganized, the diameter of the myocardial fibers increased, the cytoplasmic staining was uneven, and the myocardial fibers were swollen. In the Dox group, cavitation and degeneration were observed, and some myocardial fibers were broken and necrotic. Moreover, exercise intervention prevented Dox-induced cardiac tissue damage in the Dox + Ex group, with reduced vacuolation and degeneration, as shown in (Fig. 6).
Exercise training inhibits tumor angiogenesis
To study the regulatory effect of aerobic exercise intensity on tumor angiogenesis, we detected the expression of CD31 in blood vessels within tumor tissues. Immunohistochemistry and Western blot detection were utilized to stain and quantify CD31 and VEGF, evaluate tumor angiogenesis, and determine whether exercise inhibits tumor growth by affecting angiogenesis. The results indicated that compared to the SED group, exercise can reduce angiogenesis in tumor tissues (p < 0.0001) (Fig. 7A-B). Furthermore, angiogenesis was significantly reduced in the Dox group alone (p < 0.0001), and the Dox + Ex group showed better angiogenic inhibition after combined exercise training (p < 0.0001) (Fig. 7C-D).
Exercise training enhances the chemotherapeutic effect of Doxorubicin
We found that the combination of exercise and doxorubicin can effectively inhibit tumor growth by regulating the distribution and activation of NK cells. Western blotting was used to analyze the expression of apoptosis-related proteins in mouse tumors. Compared to the SED group, the expression level of cleaved caspase-3 protein was significantly increased in the tumor tissues of the Ex group. In addition, the expression of apoptosis-related proteins induced by the pro-apoptotic protein Bax and the expression level of the anti-apoptotic protein Bcl2 were significantly decreased (p < 0.05). The Dox group alone also activated the apoptotic pathway of tumor cells, while the Ex + Dox group had a more obvious activation effect, significantly increasing the expression of the cleaved caspase-3 protein and causing a decrease in the expression of the anti-apoptotic protein Bcl2 (Fig. 8A-E). Studies have shown that exercise can not only promote the occurrence of tumor cell apoptosis but also increase or decrease the level of inflammatory cytokines [20]. The use of chemotherapy drugs can also lead to the occurrence of inflammation in the body. q-PCR detection of inflammatory cytokines showed that the Ex group could reduce the release of IL-6, IL-1β, and other inflammatory factors (p < 0.05). In the Dox group, chemotherapy drug treatment will cause an inflammatory response in the body, and intervention combined with exercise can reduce the occurrence of inflammation. The results showed that exercise can significantly reduce the expression of pro-inflammatory cytokines IL-6, IL-1β, and other markers. Through the analysis of flow cytometry and immunohistochemistry results, we know that after exercise, NK cells mainly activate the secretion of TNF-α and IFN-γ to execute the killing function. Through q-PCR results, it is found that the Ex group activates TNF-α to execute the killing function, while the Dox + Ex group inhibits the killing function mainly by secreting INF-γ and its receptor (Fig. 8F-K).