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.

Fig 1. Appearance and size of urine EVs.

(A) TEM images of EVs from urine of HD and PCa patients before and after 0.1 μm filtration. (B) Size distribution of EVs before and after 0.1 μm filtration. Mean size with error bars for range. Additional TEM images can be found in S1 Fig.

Fig 2. Protein concentration in urine EVs.

Tukey box plots of concentration of total protein in preparations of EVs isolated from the urine of PCa patients and HD were determined using NanoOrange fluorescent dye and presented as mg per ml of probe (A) or ng per ml of urine (B).

Fig 3. DNA in preparations of urine EVs.

Tukey box plots of DNA concentration in preparations of EVs isolated from the urine of PCa patients and HD was determined using quantitative multiplex TaqMan PCR for LINE1 and α-satellite human DNA repeats.

Fig 4. Total RNA in preparations of urine EVs.

Bioanalyzer traces of total RNA from ERV fraction of the urine of HD (A) and PCa patient (B). The data from Agilent 2100 Bioanalyzer with 25 nt RNA fragment as an internal standard.

Fig 5. Relative miRNA expression in urine EVs.

Tukey box plots of miRNA expression in all fractions of urine. Asterisks (*) indicate statistical significance. *P<0.0001; **P = 0.0150.

Fig 6. Example miR-19b correlation plots.

Correlation plots of miR-19b concentration in TEV and ERV fractions of healthy individuals (A) and prostate cancer patients (B). AU—arbitrary units of concentration.