MiR-183-5p-enriched Endothelial Extracellular Vesicles: Key Players in Pre-Metastatic Niche Formation in Breast Cancer?

Breast cancer (BC) remains one of the most prevalent cancers globally. While primary tumors are often treatable, metastatic BC is incurable and the leading cause of death. Our group previously discovered that endothelial cell-derived extracellular vesicles (EVs), enriched in miR-142-5p, miR-183-5p, and miR-222-3p—collectively termed “miR-TAM”—promote macrophage polarization to a pro-tumorigenic M2-like state, enhancing tumor growth in murine BC models. However, the role of miR-TAM in metastasis, particularly in EV-driven pre-metastatic niche (PMN) formation, remains unclear.

Mouse endothelial cell-derived EVs were isolated via differential ultracentrifugation and characterized by western blot and nanoparticle tracking analysis (NTA). EV uptake was assessed in RAW264.7, bone marrow-derived macrophages (BMDMs), and mouse embryonic fibroblasts (MEFs) via confocal microscopy. Changes in PMN-related gene expression following miR-TAM-EV treatment were evaluated using qPCR. To assess the in vivo effects of miR-TAM, peritumoural administration of miR-TAM- or control miRNA-enriched EVs was conducted in 4T1 tumor-bearing mice, followed by flow cytometry and immunohistochemistry.

Isolated EVs were enriched for EV markers CD63, CD81, and syntenin, but lacked the negative marker cytochrome C. Endothelial EVs showed a major peak in the small EV size range (<200 nm) and were efficiently internalized by recipient cells. Treatment with miR-TAM-enriched EVs upregulated pro-tumorigenic genes—Csf3, Cxcl1, Col3a1, Il-1β, and Ccl3—in macrophages and fibroblasts, key players in PMN formation. Repeated peritumoural delivery of miR-183-5p-enriched EVs significantly increased monocytes and reduced CD4⁺ and CD8⁺ T cell levels in the lungs of 4T1 tumour-bearing mice, promoting an immunosuppressive environment conducive to metastasis.

We uncover a previously unrecognized role for miR-TAM-enriched endothelial EVs in promoting metastasis in vitro and in vivo, identifying them as modulators of PMN formation. These findings highlight their potential as therapeutic targets to prevent metastatic progression in BC.