Breast cancer brain metastasis (BCBM) remains a significant clinical challenge, with limited understanding of how tumor-derived extracellular vesicles contribute to its progression. Large extracellular vesicles (lEVs) represent a distinct EV subtype capable of carrying molecular cargo that reflects and potentially influences the metastatic behavior of tumor cells. We conducted a comprehensive quantitative proteomic analysis of lEVs from four breast cancer cell lines, including brain-tropic MDA-MB-231BR, to elucidate the role of lEVs in BCBM. Label-free LC-MS/MS identified 1,708 proteins (FDR < 1%). Analysis revealed 145 proteins consistently altered in the 231BR line compared with the other cell lines. Notably, CLIC4, MSN, LDHB, RPLP2, EEF1G, and PSMD3 were upregulated. Pathway analysis predicted activation of CXCR4 signaling and inhibition of RhoGDI signaling, pathways consistent with enhanced motility. Comparative analysis with whole-cell membrane (WCM) proteomes suggested that lEVs represent a distinct vesicular cargo compartment rather than random membrane sampling. lEVs were enriched for adhesion-related proteins and showed stronger predicted activation of Ephrin receptor and Integrin signaling compared with the WCM proteome. These findings support lEVs as a biologically informative vesicular compartment for studying tumor-associated cargo and provide a panel of protein candidates for future investigation of BCBM mechanisms and biomarker development.
Oluokun et al. (2026) studied this question.