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.
Highlights
Prostate cancer (PCa) is the second most common cancer worldwide in males, with more than 1.1 million new cases diagnosed in 2012
The exosome-rich fraction of vesicles (ERV) obtained by 0.1 μm filtration of total EV (TEV) fraction is enriched in smaller vesicles, including exosomes
The composition of extracellular vesicles (EVs) was studied in TEV and enriched fraction of vesicles (ERV) fractions, except miRNA concentration, which was measured in all fractions
Summary
Prostate cancer (PCa) is the second most common cancer worldwide in males, with more than 1.1 million new cases diagnosed in 2012 (global cancer statistics, http://www.cancerresearchuk. org/). Prostate cancer (PCa) is the second most common cancer worldwide in males, with more than 1.1 million new cases diagnosed in 2012 Despite five-year survival rate reaching 98% in developed countries, early PCa detection and accurate post-therapy monitoring for tumor recurrence, proliferation and metastatic potential is demanded. It can increase the quality of life for PCa patients, ensure timely diagnosis and survival of patients diagnosed at an advance stage. Men with a high PSA are required to undergo additional tests such digital rectal exam or prostate biopsy, which are both uncomfortable and may cause adverse after-effects i.e. a needle biopsy can result in infection or prolonged bleeding afterwards. A non-invasive test for PCa is still desired
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