Extracellular Vesicles from Canine Mammary Tumor Cells Promote Macrophage M2 Polarization and Enhance Tumor Progression

Extracellular vesicles (EVs) derived from tumor cells play a crucial role in modulating and reprogramming the tumor microenvironment (TME), thereby promoting tumor progression. While previous studies have investigated the impact of tumor-derived EVs on macrophages in various cancers, such as bladder cancer and osteosarcoma, and in canines, particularly in melanoma, little is known about the effects of canine mammary tumors influence on macrophage polarization. This study aimed to elucidate the functional role of EVs derived from CIPp cells, a canine mammary tumor cell line, in macrophage polarization and tumor progression. CIPp-derived EVs were isolated and characterized, and their effects on macrophage phenotype were examined using the canine macrophage cell line DH82. Exposure to CIPp-EVs induced DH82 macrophages to adopt a tumor-associated macrophage (TAM)-like M2 phenotype, as confirmed by quantitative RT-PCR, immunofluorescence, and flow cytometry. CIPp-EV treatment significantly upregulated M2-associated markers (CD206, VEGF-A, IL-10, and COX2) while downregulating M1-associated markers (iNOS and IL-6). Furthermore, conditioned media (CM) from CIPp-EV–treated macrophages enhanced CIPp tumor cell migration and induced epithelial–mesenchymal transition (EMT), evidenced by the reduction of E-cadherin and increased expression of vimentin, fibronectin, and α-SMA. Together, these findings demonstrate that CIPp-derived EVs reprogram the TME by driving TAM-like M2 macrophage polarization, which in turn promotes tumor cell migration and EMT, thereby facilitating tumor progression and metastasis.