Comparative analysis of EV isolation method for urinary extracellular vesicles proteomics in cancer

Urinary extracellular vesicles (uEVs) have emerged as a promising source of biomarkers for non-invasive diagnostic applications. To this end, a robust, scalable and reproducible uEV isolation method is required. In recent years several methods were reported, however a comprehensive comparison of uEV isolation methods is missing. In this study, we optimized and compared four EV isolation methods for uEV profiling by mass spectrometry-based proteomics. Furthermore, as a proof of concept, we applied the best performing method to profiling urine from lung cancer patients. Finally, to explore urinary pellet as an alternative sample source for cancer detection, uEV protein profiles were compared to urine fractions.

The following EV isolation methods were compared using 15 mL and 2 mL urine: VN96 peptide-based capture, Strong Anion Exchange (SAX) beads, Titanium Dioxide (TiO₂), and Size Exclusion Chromatography (SEC). Comparison was performed using Western Blot for EV markers, Coomassie staining and proteomics using a Tims-TOF HT platform as read-out.

Our results so far showed that EV markers are best captured using the VN96 method with 2 mL as input. For 15 mL input, VN96-based EV capture and TiO₂ method performed equally well. Proteomics results will be presented.

Profiling of uEVs isolated using VN96 from cancer patients (n=60; prostate, bladder, colorectal, cervical and lung cancer) and healthy controls (n=13) demonstrated that cancer type-specific biology and proteins can be detected in urinary EV proteomes.

Finally, we are currently exploring whether a simpler protocol could provide similar uEV-carried information or more comprehensive data for cancer biomarker discovery. The comparative analysis of uEV, urine pellet, supernatant and full urine will be reported using samples from prostate and kidney cancer patients and healthy donors.

In conclusion, our study provides a comprehensive comparison of four uEV isolation methods and their applications to cancer urine, underscoring the potential of uEVs as a rich source for noninvasive protein biomarkers for cancer detection.