Histopathological and Ki-67 Immunohistochemical Evaluation of Macrophage-Derived Extracellular Vesicles Loaded with 4T1 Tumor Lysate in Prophylactic and Therapeutic Murine Breast Cancer Models

Background: Extracellular vesicles (EVs) derived from macrophages have emerged as promising carriers for tumor-associated antigens in cancer immunotherapy. This study evaluated the prophylactic and therapeutic efficacy of macrophage-derived EVs loaded with 4T1 breast cancer cell lysate in a murine breast cancer model and explored their translational relevance from a One Health perspective.

Methods: RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) and passively loaded by overnight incubation with 4T1 tumor cell lysate prior to EV isolation. Female BALB/c mice received antigen-loaded EVs either prophylactically (three injections before tumor challenge) or therapeutically after tumors became palpable. A control group received free 4T1 tumor cell lysate without EVs. Tumor growth, survival, histopathological alterations, lung metastasis, and Ki-67 expression were evaluated.

Results: Prophylactic EV administration markedly inhibited tumor growth compared with the control group (day 35 tumor volume: 0.0543 vs. 24.8 mm³, p = 0.001). Ki-67 expression was significantly reduced from 29% in controls to 14% in the prophylactic group (p = 0.001). Histopathological examination demonstrated fewer mitotic figures, reduced nuclear pleomorphism, and no histologically detectable metastatic foci in the examined lung sections of prophylactically treated mice. Therapeutic administration produced intermediate pathological improvement. Although survival was higher in the prophylactic group, the difference was not statistically significant.

Conclusions: Macrophage-derived EVs loaded with 4T1 tumor lysate significantly suppressed tumor growth and proliferation and reduced histological evidence of pulmonary metastasis, particularly when administered prophylactically. These findings support further investigation of antigen-loaded EVs as a translational strategy for comparative oncology and One Health, where naturally occurring cancers in animals and experimental murine models can provide insights relevant to human breast cancer.