Isolation and characterization of urine microvesicles from prostate cancer patients: different approaches, different visions

Abstract

Background Because of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result. Methods The aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors. After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis, transmission electron microscopy, AlphaScreen Technology, and whole miRNA Transcriptome. Results Our results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC. Conclusions Our study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.

Capturing the Kidney Transcriptome by Urinary Extracellular Vesicles—From Pre-Analytical Obstacles to Biomarker Research

“…Obviously, this represents a problem for study comparisons, even if reporting guidelines now help to identify differences, facilitate replication and/or explain the lack of it [12,172]. Prior studies have explored the effect of uEV isolation workflows on uEV RNA sequencing profiles focusing on miRNA sequencing [15,[20][21][22]. We have previously demonstrated that the uEV isolation workflow (UC, HFD and NG) has a surprisingly variable impact on the miRNA and mRNA profiles [13].…”

“…Moreover, only few studies have characterized the effect of pre-analytical variables on the uEV, especially regarding the end-point biomolecular level used in biomarker studies, e.g., the transcriptome [14]. Equally, only a few studies have comprehensively characterized the effect of EV isolation methods on transcriptomics [13,15,16,[20][21][22]. Thus, it is difficult to compare results between dissimilar studies.…”

Prostate cancer-derived small extracellular vesicle proteins: the hope in diagnosis, prognosis, and therapeutics

“…A study successfully isolated sEVs from normal prostate RWPE1 cells using ultrafiltration and characterized their associated proteins [ 37 ]. Another study found that SEC demonstrated advantages of high purity and low protein contamination in the extraction of sEVs from the plasma and urine of PCa patients, ensuring the quality requirements for subsequent downstream analysis [ 38 , 39 ]. Chen et al developed a highly efficient sEV isolation platform called Exosome detection via the ultrafast-isolation system (EXDUS) that is based on ultrafiltration, which achieves ultrafast and efficient purification of sEVs through negative pressure oscillation and double-coupled harmonic oscillator-enabled membrane vibration.…”

Extracellular vesicles in human milk

“…EVs carry DNA, RNA, microRNA (miRNA), proteins and lipids, which play important roles in intercellular communication, which modulates and regulates adjacent recipient cells. Exosomes have been found in physiologic fluids such as bronchoalveolar lavage [2], serum [3,4], urine [5] and are most abundant in human milk [6], as well as in bovine, yak, goat and porcine sources [7][8][9][10][11].…”