Annexin A2-dependent extracellular vesicle proteome modulates pre-metastatic stromal fibroblast behavior in triple-negative breast cancer

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subclass of breast cancer with limited treatment options and a strong tendency to metastasize to the lung. Tumor-derived extracellular vesicles (EVs) are key mediators of stromal reprogramming in the pre-metastatic niche. Annexin A2 (AnxA2) is overexpressed in TNBC and linked to signal transduction, cytoskeletal remodeling, and poor prognosis, but its role in shaping EV cargo composition and consequent stromal behavior remains unclear. METHODS: EVs were isolated from the conditioned media of MDA-MB-4175 (LM2) TNBC cells with stable AnxA2 knockdown and control cells using differential ultracentrifugation. EVs were characterized and validated using nanoparticle tracking analysis, Western blotting, and cryo-electron microscopy. EV proteomes were profiled using quantitative proteomics, followed by bioinformatic analyses. Functional and pathway enrichment was performed to identify proteome signatures altered by AnxA2 depletion. The internalization of EVs by recipient lung fibroblasts (MRC-5 and WI-38 cells) was assessed using confocal imaging, and EV-mediated effects on fibroblast behavior were evaluated through migration assays and immunoblotting for proteins associated with fibroblast activation. RESULTS: AnxA2 knockdown reduced pro-metastatic cellular properties in LM2 cells. EVs from AnxA2-deficient cells displayed reduced abundance of proteins involved in cytoskeletal regulation, adhesion, and vesicle trafficking, with enrichment of immune-related proteins. Pathway analyses predicted reduced motility and vesicle trafficking signaling. PKH26 labeled EVs derived from AnxA2 expressing cells exhibited increased internalization by lung fibroblasts as detected by confocal imaging. Functionally, AnxA2-enriched EV-mediated interactions enhanced fibroblast migration, proliferation, and activation marker expression compared with AnxA2-deficient EVs, consistent with the predicted proteomic changes. CONCLUSIONS: AnxA2 emerges as a regulator of the protein composition of TNBC-derived EVs, enhancing EV-mediated interactions with lung fibroblasts and modulating fibroblast motility and activation, linking tumor cell protein expression to stromal remodeling in the lung microenvironment. This work establishes a framework suggestive of an EV cargo-driven mechanism through which EVs derived from AnxA2-expressing cells may influence lung-specific metastatic colonization in TNBC and highlights the importance of evaluating EV-mediated communication in cancer progression.