Integrin-DefinedPlasma Extracellular Vesicles forEarly Breast Cancer Detection and Bone Metastasis Association

Abstract Sensitive detection of breast cancer and metastatic disease remains critical for improving patient outcomes. Plasma extracellular vesicles (EVs) carrying integrins have shown strong promises for characterizing metastatic progression and organotropism, but their clinical potential remains largely unexplored, particularly for early detection. Here, we applied a gold nanoparticle-based single vesicle imaging method to investigate the feasibility of plasma EV-associated integrins as biomarkers for early breast cancer detection and bone metastasis association. EVs were immunocaptured via the tetraspanin CD81 and labeled with membrane dyes and antibody/AuNPs targeting integrin monomers αV, β3, α6, or β5, enabling quantification of total captured EVs with fluorescence imaging and the target-specific EV subpopulations via dark field imaging. Single vesicle analysis revealed that the fraction of EVs that were positive for integrin αV was significantly elevated in Stage I patients and was further enriched in patients with bone metastasis relative to healthy donors. A similar pattern was observed for integrin β3-positive EVs. In contrast, integrins α6 and β5 showed no significant differences across groups. Receiver operating characteristic analysis revealed strong diagnostic performance for αV and β3, with area under the curve (AUC) values exceeding 0.95 for both markers in distinguishing bone-metastatic disease from healthy controls. Further, integrin αV demonstrated higher sensitivity (AUC = 0.88) for early stage detection than integrin β3 (AUC = 0.74), while both showed similar performance in distinguishing bone-metastatic disease. These findings suggest that EV-associated integrins αV and β3 are associated with breast cancer presence and progression, with integrin αV showing potential as a biomarker for early detection. This proof-of-concept feasibility study establishes a foundation for mechanistic and translational validation of EV-associated integrins as promising biomarkers in liquid biopsy assays for clinical applications.