Olation and characterisation of urinary exosomes Eline Oeyen1, Geert Baggerman2, Hanny Willems2 and Inge MertensUniversity of Antwerp/VITO, Antwerp, Belgium; 2University of Antwerp /VITO, Antwerp, Belgium; 3University of Antwerp, BelgiumPF03.Analysis of extracellular vesicles from plasma of advanced (IIIc or IV stages) melanoma sufferers during kinase inhibitor and/or immunotherapy therapies Pascal Colosetti1, Henri Montaudi, Philippe Bahadoran2, Robert Ballotti3, Sophie Rome4 and Corine BertolottoIntroduction: Exosomes are nanosized extracellular vesicles which can be secreted by normal, diseased and tumour cells in all body fluids (i.e. plasma, breast milk and urine). Given that their origin, molecular content material and function, exosomes are appropriate as a supply of diagnostic biomarkers. Within this way, urine exosomes present a targeted view into the urogenital tract to boost the ability to detect urological ailments or tumours and their Ubiquitin-Conjugating Enzyme E2 D1 Proteins Species progression. Techniques: Isolation of exosomes from urine was optimised to obtain a pure exosome fraction. Size exclusion chromatography (SEC) combined having a preprocessing step (SAE1 Proteins custom synthesis ultrafiltration or a industrial kit) was employed. The urine sample collection was approved by the Ethics committee on the University of Antwerp, comply together with the Declaration of Helsinki. Isolated extracellular vesicles were characterised by tactics like nanoparticle tracking analysis, western blotting, electron microscopy and assymetrical-flow field-flow fractionation coupled with UV and multiangle light scattering detectors (AF4/UV-MALS). Isolated vesicles have been applied in down stream proteomic analysis. Outcomes: Western blot data demonstrated the presence of EV-specific proteins Flotillin-1, CD9, CD63 and CD81 within the EV-relevant fractions of each isolation procedures. NTA final results and electron microscopy images showed a higher enrichment of EVs utilizing ultrafiltration and SEC. Proteomic analysis also demonstrated that this isolation approach offers extra identifications of EV-relevant proteins. The results of AF4/UVMALS demonstrated that fraction 9 soon after SEC was most enriched in EVs. The size of your isolated vesicles ranged from 40 to 160 nm. Conclusion: In conclusion, ultrafiltration combined with SEC is preferred as isolation process of EVs from urine. AF4/UV-MALS proved to be a great high quality control strategy for urinary EVs to identify their purity and size.PF03.Diagnostic and prognostic prospective of miRNA alterations in blood based extracellular vesicles from clear cell renal cell carcinoma patients Joana Heinzelmann, Diana Kuhn, Sophie Baumgart, Sebastian Hoelters, Michael Stoeckle and Kerstin Junker Department of Urology and Paediatric Urology, Saarland University, Saarbrucken, GermanyUMR Inserm U1060/INRA 1397; Hospital; INSERM; INRAIntroduction: Extracellular vesicles (EVs) shaping tumour microenvironment, contribute to pre-metastatic niche formation, favour tumour dissemination and mediate resistance to treatment options. We have previously shown that senescent melanoma cells, in response to treatment with chemotherapeutic drugs, release soluble (e.g. chemokine CCL2) and insoluble aspects. The origin and roles of these insoluble factors remain to become elucidated nevertheless it is admitted that the quantity of circulating EVs can be a issue of poor prognosis in melanoma. Furthermore, escalating line of evidences indicate that the composition of these vesicles exhibits tissue specificity. Within this study we propose a pilot clinical study on patients with sophisticated non-resectab.
