Extracellular vesicles (EVs) are nano sized vesicles that are secreted by normal, diseased and tumour cells in all body fluids. They contain a variety of molecules such as proteins, RNAs and lipids. They are involved in both physiological and pathophysiological processes ranging from immune regulation, cell-to-cell communication, tumor progression and metastasis. Since their origin, molecular content and function, EVs are suitable as source of biomarkers. EV-based diagnostics using liquid biopsies could be a great alternative to surgical biopsies and cystoscopies. In this way, urinary EVs provide a targeted view into the urogenital tract to enhance the ability to detect urological diseases or tumours and their progression as they are released by the epithelia of the complete urogenital tract.
Although EVs hold promising potential, their isolation and characterization from different body fluids is still not optimal and standardized due to their small size. We optimized the isolation of EVs from urine. Our method involves ultrafiltration in combination with size exclusion chromatography. Cell-free urine is up concentrated using an ultrafiltration filter with a molecular weight cut-off of 100 kDa. The filtrate is placed on a size exclusion chromatography column and soluble proteins and aggregates are separated from the urinary EVs.
The isolated EVs were characterized by different techniques. Western blotting was used to confirm the presence of EV-specific proteins such as Flotillin-1, CD9 and HSP70. Transmission electron microscopy was used to visualize the EVs. The particle size and number of particles were also determined by nanoparticle tracking analysis. Asymmetrical-flow field-flow fractionation in combination with a multi-detection system UV and multi-angle light-scattering (AF4/UV-MALS) was also performed. This technique is very recent in EV research and overcomes some drawbacks of other currently used techniques. It determines the amount of EVs and their size. Finally, downstream proteomic analysis was performed to obtain insight in the protein content of the isolated urinary EVs. Of the 670 identified proteins in the EV fraction, 575 were EV related according to the gene ontology annotation.
In conclusion, urinary EVs are a potential source of biomarkers for the diagnosis and follow-up of urological diseases or tumours. These results demonstrated that ultrafiltration in combination with size exclusion chromatography is a suitable method for the enrichment of EVs from urine. We also demonstrated that AF4/UV-MALS is a good quality control method for urinary EVs.