Presentation Authors: Bert Dhondt*, Glenn Vergauwen, Jan Van Deun, Edward Geeurickx, Lien Lippens, Joeri Tulkens, Ghent, Belgium, Ilkka Miinalainen, Pekka Rappu, Jyrki Heino, Oulu, Finland, Piet Ost, Olivier De Wever, An Hendrix, Nicolaas Lumen, Ghent, Belgium
Introduction: Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery. We developed techniques for high-purity isolation of tumor tissue EV (tEV) and urinary EV (uEV), a prerequisite for the identification of EV-specific functions and biomarkers. This study maps the biomarker potential of uEV in prostate cancer (PC).
Methods: Urine samples from PC patients were collected prior to, and 3 months after local treatment. From 5 patients, paired urine samples and tumor tissue were collected. UEV were isolated with a discontinuous Optiprepâ„¢ density gradient (ODG). Tumor tissue was incubated for 3 hours in cell culture medium and tEV were isolated from the conditioned medium by size exclusion chromatography and ODG. UEV and tEV were characterized using nanoparticle tracking analysis (NTA), western blot (WB), transmission electron microscopy (TEM) and mass spectrometry (MS).
Results: NTA, WB and TEM confirmed the enrichment of uEV and tEV in 1.1 g/ml density fractions (EV fraction), and soluble urinary proteins (SP fraction) in high density fractions. MS of paired tEV and uEV samples showed enrichment of tEV proteins in uEV, demonstrating detection of PC-derived EV in urine. MS identified biologically relevant EV-associated proteins with high repeatability. There was a clear differential protein enrichment between EV and SP fractions, of which gene set enrichment analysis showed differential biological functions and cellular origin. Moreover, urine samples before and after local prostate cancer treatment were enriched in different proteins. This applied to both the EV and SP fractions. A significant over-representation of proteins involved in RNA and protein metabolism was found in uEV prior to therapy, while proteins enriched in SP fractions were related to energy metabolism and cytoskeleton dynamics. Proteins enriched in uEV after local prostate cancer treatment were mainly responsible for the classic transport function of EV.
Conclusions: The development of reliable techniques for the isolation of high-purity tEV and uEV enables identification of tumor-specific proteins and functions of EV. This study shows that urine is a source of EV released by PC. UEV of PC patients are enriched in a unique set of proteins with tumor-specific biological functions. This signature disappears after local PC treatment. The analysis of EV offers additional insights on top of the study of the soluble urinary proteome. Taken together, uEV offer a new perspective for the discovery of promising PC biomarkers.
Source of Funding: This work was supported by â€œKom op tegen Kanker (Stand up to Cancer), the Flemisch cancer societyâ€ (Bert Dhondt: Emmanuel Vander Schueren Research Grant).