Dakovad, Lubos Minarc, Zbynek Zdrahalb, V zslav Bryjaa and Vendula Posp halovaa Division of Experimental Biology, Faculty of αvβ5 custom synthesis Science, Masaryk University, Brno, Czech Republic; bCore Facility Proteomics, Central European Institute of Technologies, Masaryk University, Brno, Czech Republic; cDepartment of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic; dDepartment of Pathology, University Hospital Brno, Brno, Czech RepublicaIntroduction: Extracellular vesicles (EVs) function in bidirectional cell ell communication and contribute towards the sustained development, invasion, and metastasis of cancer cells inside the tumour microenvironment (TME). EVs comprise two most important classes exosomes and shed microvesicles (sMVs, also termed microparticles and ectosomes) with distinct modes of biogenesis. Inside each EV class, subtypes exist that may be distinguished by their distinct protein/RNA signatures. Whilst considerably is recognized about exosome cargo content and functionality, sMVs are poorly understood. Procedures: Right here, we compare protein/RNA profiles and functionality of sMVs and exosomes secreted from human key (SW480) and metastatic (SW620) colorectal cancer cell lines. Milligram amounts of EVs have been purified from cell culture media utilizing a combination of differential ultracentrifugation/isopycnic iodixanol density centrifugation. Label-free quantitative mass spectrometry was performed to receive protein profiles for SW480-derived and SW620-derived sMVs. Results: We show that sMVs, in contrast to exosomes, are ALIX-, TSG101-, CD63- and CD9- and include a different suite of key cancer progression modulators. Protein/RNA signatures for SW480-derived sMVs and exosomes differ from every other as well as from their SW620-derived counterparts. SW480-derived sMVs are enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, although SW620derived sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Fibroblast invasion capabilities of SW480-derived and SW620-derived sMVs are comparable. Summary/conclusion: Additionally, we report for the very first time a extensive biochemical/functional analysis of a hitherto undescribed subpopulation of sMVs. We anticipate our in-vitro findings might be a beginning point for extra sophisticated studies aimed at elucidating the PI3Kγ web biochemical and functional properties of EV subtypes in vivo. The emerging roles of precise EV subtypes within the TME we believe will alter our view of cancer biology and may present new targets for therapeutic intervention. Funding: Funding assistance from La Trobe University, Melbourne, Australia.Introduction: High-grade serous carcinoma (HGSC) from the ovaries, fallopian tube and peritoneum would be the deadliest gynaecological malignancy with 5-year survival price below 30 . HGSC is frequently accompanied by ascites, a pathological accumulation of fluid in the peritoneum, which could be exploited as a liquid biopsy containing not simply cancer cells but in addition the tumour microenvironment such as extracellular vesicles (EVs). Tumour cells create substantially more EVs than healthy cells, hence malignant ascites could be the source of enriched pool of EVs of HGSC origin. Methods: Ascitic fluids depleted of cells were fractioned using size-exclusion chromatography and two fractions containing and not containing EVs have been further analysed. In parallel, smaller EVs have been also isolated from ascitic fluids using differential ultracentrifugation followed by purification step in sucrose/D2O cushion.