Identification of cathepsin L and urokinase-type plasminogen activator in plasma-derived small extracellular vesicles of breast cancer patients

Small extracellular vesicles (sEVs) are nanosized vesicles released by both normal and cancer cells, carrying biomolecular cargo that may serve as disease biomarkers, including cancer. We investigated protease signatures of plasma-derived sEVs in chemotherapy-naïve stage I-III breast cancer (BC) patients. sEVs were enriched from plasma of BC patients (n = 49), healthy controls (n = 30), and benign cases (fibroadenoma; n = 15) using a total exosome isolation kit and their enrichment was verified by different approaches. Protease profiling using a Proteome Profiler array identified cathepsin L (CTSL) and urokinase-type plasminogen activator (PLAU/uPA) in BC-derived sEVs. Western blotting detected CTSL and PLAU/uPA in BC-derived sEVs, but not in healthy controls or benign cases. Gelatin zymography revealed significantly higher CTSL-associated gelatinolytic activity in BC-derived sEVs relative to non-cancer groups, with the highest activity in stage III patients. ELISA confirmed elevated PLAU/uPA levels in BC-derived sEVs compared with their corresponding plasma. STRING protein-protein interaction analysis linked CTSL and PLAU with uPAR, IL-6, and heparanase (HPSE), all detected in BC-derived sEVs. Public mRNA database analysis showed that higher CTSL and PLAU/uPA expressions were associated with poorer overall survival, while higher CTSL, PLAU/uPA, and uPAR expressions were associated with poorer distant metastasis-free and relapse-free survival. ROC Plotter online database analysis revealed elevated CTSL and PLAU/uPA mRNA levels were associated with improved chemotherapy response, whereas higher IL-6 mRNA levels correlated with chemoresistance. Together, these findings identify candidate sEV-associated proteins whose gene expression profiles in public databases are associated with survival outcomes and treatment response in BC.