LABELING EXTRACELLULAR VESICLES FOR HIGHLY SENSITIVE FLOW CYTOMETRY ANALYSES

P. Simeone^1,2, D. Brocco³, F. D’Ascanio^1,2,4, D. De Bellis^1,2, G. Colasante^1,2, A. Aquilini-Mummolo^1,2, T. Esmail^1,2, A. Younas², M. Filoso², C. Cichella², M. C. Cufaro⁵, A. Di Sebastiano⁵, D. Pieragostino⁶, S. Burattini⁷, M. Battistelli⁷, P. Del Boccio⁵, A. Fontana⁸, R. Di Pietro¹, P. Lanuti^1,2 | ¹Department of Medicine and Aging Sciences, University “G. d’Annunzio” of Chieti-Pescara, Italy; ²Flow Cytometry Core Facility, Center for Advanced Studies and Technology (CAST), University “G. d’Annunzio” of Chieti-Pescara, Italy; ³Department of Medical, Oral and Biotechnological Sciences, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy; ⁴Department of Humanities, Law and Economics, Leonardo da Vinci University, Torrevecchia Teatina, Italy: ⁵Department of Science, University “G. d’Annunzio”, Chieti Pescara, Italy; ⁶Department of Innovative Technologies in Medicine and Dentistry, G. d’Annunzio University of Chieti-Pescara, Italy; ⁷Department of Biomolecular Sciences, University of Urbino, Urbino, Italy; ⁸Department of Pharmacy, University “G. d’Annunzio”, Chieti Pescara, Italy

Extracellular vesicles (EVs) are released by shedding during many different processes by all cell types. They cross all biological barriers and have been detected in all body fluids. For these reasons EVs are increasingly thought to be new potential dynamic biomarkers useful for liquid biopsy. However, the application of pre-analytical processing phases before any EV flow cytometry measurement is mandatory, even if their impact on results is not predictable. For this reason, the translation of basic research into clinical practice has been precluded. We have optimized a simple flow cytometry method, which, avoiding any pre-enrichment step, allows the identification, classification, enumeration, and separation, by fluorescence activated cell sorting, circulating EVs stemming from different parental cells. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs, which has been combined with fluorescent phalloidin to exclude damaged elements and different mixes of specific monoclonal antibodies. The application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples and many others biological fluids, as well as cell supernatants. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo, as well as for in vitro and in vivo functional assays. Therefore, LCD staining of EVs may open new routes for the EV clinical translation.