Abstract:

Liquid biopsy is a promising alternative to traditional tissue biopsies for diagnosing cancer because it offers advantages such as minimal invasiveness, accessibility, and ease of operation. Extracellular vesicles (EVs) are lipid bilayer vesicles that contain proteins, DNA, and RNA and are secreted by cells. Indeed, urinary EVs are important sources of cancer biomarkers. The lipid bilayer protects EV proteins from degradation by enzymes present in bodily fluids. Prostate cancer (PCa) is among the most prevalent malignancies in developed countries and is the second-leading cause of cancer-related mortality in men. Current screening methods commonly used to initially evaluate patients with suspected PCa include serum prostate-specific antigen (PSA) testing and digital rectal examination (DRE), with magnetic resonance imaging (MRI) and transrectal ultrasound often recommended for further assessment. However, both PSA testing and DRE have limited specificities, which results in a substantial number of unnecessary prostate biopsies. Consequently, additional reliable biomarkers need to be urgently discovered for rapidly diagnosing PCa more accurately. Prostate-derived secretions, including those associated with malignancies, are detectable in urine owing to the anatomical proximity of the prostate to the urethra; hence urine is a promising liquid-biopsy medium for discovering PCa biomarkers, which is a topic that has been the focus of extensive research efforts in recent years. However, isolating EVs from biofluids in sufficient yields for proteomics analysis remains challenging.

In this study, functional magnetic beads EVlent (extracellular vesicles isoLated efficiently, naturally, and totally) with high-affinity capabilities were developed for selectively enriching EVs from biological fluids.The surfaces of the beads were modified with three antibodies that target CD9, CD63, and CD81, which enables the specific recognition of EV surface proteins. The isolation performance of EVlent was validated by comprehensively characterizing urinary EVs using Western blotting (WB), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). WB revealed prominent bands for EV markers (CD9, TSG101, and HSP70) in EVlent-enriched samples, whereas weaker bands were observed following ultracentrifugation (UC). NTA revealed that the EVs isolated by EVlent are predominantly in the 50-400 nm size range, with a content of 4.1×10⁹ particles/mL, which is significantly higher than the value of 1.8×10⁹ particles/mL obtained by UC. TEM confirmed that the isolated EVs have characteristic elliptical or cup-shaped vesicular structures. These findings demonstrate that EVlent outperforms UC in terms of enrichment efficiency and purity, delivering a separation efficiency of 87.2% compared to the value of 30.3% obtained by UC. We used proteomics to analyze urinary EVs isolated from 15 healthy volunteers and 15 patients with prostate cancer using EVlent affinity magnetic beads with the aim of identifying potential biomarkers for prostate cancer. On average, 2039 proteins and 14490 peptides were identified in the control group, while 1982 proteins and 13100 peptides were identified in the patient group. Further analysis revealed 91 proteins commonly found in the Vesiclepedia database (Top 100). Compared with the healthy volunteers, 88 proteins were upregulated and 90 proteins were downregulated in patients with prostate cancer. Gene ontology (GO) analysis showed that these upregulated proteins are enriched in extracellular exosomes, extracellular space, extracellular region, collagen-containing extracellular matrix, proteolysis and protein-binding. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted associations between ribosomes, protein digestion and absorption, complement and coagulation cascades, prostate cancer, transcriptional misregulation in cancer, aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, and pancreatic secretion. Notably, four proteins including plasminogen activator urokinase (PLAU), platelet-derived growth factor subunit A (PDGFA), matrix metalloproteinase 3 (MMP3), and neuroblastoma RAS viral oncogene homolog (NRAS) were identified within the prostate cancer pathway, highlighting their potential as biomarkers for the early diagnosis and prognosis of prostate cancer. In conclusion, this study introduced EVlent as a robust platform for the efficient isolation and proteomics analysis of EVs, providing valuable insight into urinary EV biomarkers and their clinical prostate-cancer applications.

Key words: extracellular vesicles, urine, proteomics, prostate cancer, affinity purification