Sympathetic nerve–derived extracellular vesicles promote metastatic traits in breast cancer via EMT-related microRNAs

Emerging evidence suggests that the autonomic nervous system contributes to tumor progression, although the molecular mechanisms underlying nerve–tumor communication remain poorly understood. Extracellular vesicles (EVs) are key mediators of intercellular communication within the tumor microenvironment, by transferring bioactive molecules. In this study, we investigated the roles of EVs released from sympathetic nerves in inducing metastasis-related traits in breast cancer (BCa). Sympathetic nerve distribution was evaluated in clinical BCa specimens by histopathological analysis. To investigate the underlying mechanisms, EVs derived from differentiated PC12 cells (dPC12-EVs), a sympathetic neuron-like model, were applied to RMT-1 rat BCa cells. EV-associated miRNA profiling and transcriptomic analyses were performed, and cell migration and proliferation were subsequently evaluated. Histopathological analysis showed that the proportion of sympathetic nerve fibers was higher in primary tumors from patients with lymph node metastases than in those from patients without metastases. Comparative miRNA profiling revealed 12 miRNA cargos enriched in dPC12-derived EVs. Transcriptomic analyses indicated activation of non-canonical epithelial-mesenchymal transition (EMT)-related programs distinct from the classical TGF-β and NFκB signaling pathways. EMT marker analysis validated reduced E-cadherin expression and increased vimentin and N-cadherin expression. Functional validation showed dPC12-EVs enhanced both migration and proliferation of RMT-1 cells, consistent with a hybrid EMT-like phenotype. Synthetic miRNA cocktails from dPC12-EVs recapitulated these phenotypic effects, whereas individual miRNA mimics did not, supporting a functional role for the coordinated action of multiple EV-associated miRNAs. These findings suggest that sympathetic neuron-derived EVs promote a hybrid EMT-like phenotype in BCa cells by enhancing both motility and proliferation through miRNA-mediated regulation of non-canonical EMT signaling.