Exercise-induced extracellular vesicles delay tumor development by igniting inflammation in an immunologically cold triple-negative breast cancer mouse model

Background: Preclinical studies demonstrate that exercise reduces tumor incidence and growth. Rapid release of extracellular vesicles (EVs) during exercise suggests their potential role as mediators of exercise-induced systemic effects and physiological adaptation. This study investigated the impact of exercise-induced plasma EVs on tumor growth and immune tumor microenvironment in murine models of triple-negative breast cancer (TNBC): EO771 (a C57BL/6-derived TNBC cell line) and 4T1 (a BALB/c-derived TNBC cell line). Methods: Size exclusion chromatography was used to isolate exercise-induced EVs from plasma of healthy female mice (BALB/c and C56BL/6, n = 30 per strain) that underwent ten 30-min moderate-intensity treadmill running sessions over 2 weeks. Nanoparticle tracking analysis, Western blot, and electron microscopy confirmed the presence of EVs in the samples. Tumor-bearing mice (n = 72 per strain) were administered with exercise-induced EVs before or/and after tumor implantation. Local and systemic immune responses were assessed using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR). Results: Administration of exercise-induced EVs, particularly before tumor implantation, significantly suppressed tumor growth and reduced tumor burden in both TNBC models. In EO771, endpoint tumor volumes were 278–330 mm³ in treated groups compared to 799 mm³ in untreated (p < 0.0001), while in 4T1, treated groups showed volumes of 287–564 mm³ vs. 696 mm³ in untreated (p = 0.0002). Notable differences in tumor-infiltrating lymphoid and myeloid cell subpopulations indicated immunomodulatory effects of exercise-induced EVs, particularly in the 4T1 model, where their continuous administration significantly increased intratumoral cluster of differentiation 8 (CD8) T lymphocyte proportion (5.77% vs. 0.90% in untreated, p < 0.0001). Similarly, in the EO771 model, exercise-induced EVs administered before tumor implantation led to a marked rise in intratumoral CD8 T lymphocytes (2.24% vs. 1.08% in untreated, p = 0.0181). Conclusion: Our findings indicate that exercise-induced EV treatment elicits a pro-inflammatory antitumor immune response, suggesting a shift of immunologically cold TNBC tumors towards a more inflamed phenotype associated with better outcomes. Our study supports the further investigation of EVs as modulators of antitumor immunity and their potential utility in enhancing the efficacy of immunotherapy.