Isolation and characterization of prostate-specific extracellular vesicles from prostate cancer patients

Background: Plasma-derived extracellular vesicles (EV) represent a complex mixture of vesicles originating from diverse tissues. The selective isolation of prostate-specific extracellular vesicles (PSEV) can offer improved specificity for diagnostic and prognostic biomarker development as well as monitoring therapeutic response of prostate cancer (PCa). This study aimed to compare the yield and quality of PSEV enriched from total EV preparations with those isolated directly from plasma. Materials and methods: Twelve (12) plasma samples (6 from patients with PCa, and 6 from non-cancer subjects) were used in this study. The total EV were isolated with SmartSEC TM columns following the manufacturer's instructions. The PSEV were enriched with anti-PSMA-conjugated magnetic beads from isolated total EV and also directly from plasma samples. Nanoparticle tracking analysis (NTA) was performed to examine the concentration (particles/mL), average diameter (nm), and volume (nm³) for total EV and PSEV. The ultrastructure of isolated PSEV was imaged under a JOEL JEM1400 transmission electron microscope. The mean values were subjected to an unpaired t-test analysis, and a p-value of less than 0.05 was considered statistically significant. Results: NTA analysis showed that 1) Total extracellular vesicles isolated with the column resulted in a significant yield from each plasma sample (1.4E+11 to 7.52E+11 particles/ml, 149.8 to 182 nm in diameter, and 752 to 1,360 nm 3 in volume); 2) PSEV concentration was higher in plasma from African American (AA) population (178.9x10 9 /ml) than in plasma from Caucasian American (CA) population (166.6x10 9 /ml); 3) the PSEV concentration was higher in method using anti-PSMA-conjugated magnetic beads from plasma directly (13.4x10 9 /ml) as compared to that enriched from total extracellular vesicles obtained via column (10.98x10 9 /ml); 4) the average PSEV diameter was greater in method using anti-PSMA-conjugated magnetic beads directly from plasma (198.7nm) as compared to that from total extracellular vesicles obtained via column (149.3nm, p=0.002); and 5) PSEV was about 3.1% of total EV in non-cancer subjects, and 4.7% of total extracellular vesicles in PCa. The TEM analysis demonstrated the existence of electron-dense oval or round particles in diameters ranging from 30-258 nm. However, PSEV showed some spikes on the particle surface. Conclusion: PSEV enrichment using anti-PSMA-conjugated magnetic beads is a feasible approach that could enhance the specificity of potential EV-associated biomarkers, aiding in improved diagnosis, prognosis, risk prediction, and monitoring therapeutic responses in prostate cancer.