Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients

Abstract

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

Methods to isolate extracellular vesicles for diagnosis

“…Profiles of miRNAs within serum EVs from ovarian cancer patients are significantly different than those of normal patients and of patients with benign cancer [30]. Urinary EVs may be a valuable source of diagnostically-useful miRNAs in prostate cancer [31]. Proteins in urinary EVs may provide biomarkers of acute kidney injury; for example, fetuitin-A in urinary EVs may be useful in diagnosing structural renal injury [32,33], and decrease in the level of podocalyxinlike protein 1 (PODXL) in urinary EVs may provide a biomarker to classify renal disease [34].…”

“…Two prostate cancer biomarkers, PCA3 and TMPRSS2-ERG, have been detected in urinary EVs from prostate cancer patients [53]. Also, urinary EVs are enriched with miRNAs, so isolation and analysis of urinary EVs can help to identify miRNA biomarkers for diseases such as prostate cancer [31]. Results can provide a diagnostic standard for comparison of patients with healthy individuals [54].…”

The role of extracellular vesicle microRNAs in cancer biology

“…Exosomal lincRNA and miRNA are also detected in the urine of prostate and bladder cancer patients [77][78][79]. Bryzgunova et al [77] reported that the level of miR-19b, normalized to that of miR-16, in urine-derived exosomes was significantly lower in prostate cancer patients than in healthy donors .…”

“…Bryzgunova et al [77] reported that the level of miR-19b, normalized to that of miR-16, in urine-derived exosomes was significantly lower in prostate cancer patients than in healthy donors . Armstrong et al also reported that in bladder cancer, high levels of miR-21 and miR-4454 are commonly detected in whole blood cell, tumor, and urine.…”

Microdevices for Non-Invasive Detection of Bladder Cancer

“…Indeed, the protein and miRNA composition of tumor-derived EVs found in urine have been observed to reflect the presence of cancer [43]. For example, differential protein and miRNA profiles were observed in patients with prostate cancer [105,106]. From 24 proteins known to be differentially expressed in bladder cancer patients, one (TACSTD2) was found to also be present at significantly elevated levels in EVs isolated from urine [107].…”