Abstract 5309: Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells

Abstract Extracellular vesicles (EVs) are nano-sized (30-1000 nm) membranous particles released by nearly all cell types. EV biology is a rapidly growing field, however its relative novelty and the intrinsic heterogeneity of EVs result in a lack of standardized and reproducible protocols. We sought to develop and validate an EV isolation method using rhabdomyosarcoma cell lines, with multiple characterization/validation methods, to improve the reproducibility and reliability of EV research in pediatric cancer research. EVs were isolated and analyzed in triplicate from 4 rhabdomyosarcoma cell lines (RH4, RH18, RH30, RD), of varying clinical characteristics including subtype, TP53 status and FOXO1 fusion status. Cells were grown to 70% confluency in six 175cm2 flasks, to ensure sufficient EV content for all downstream analyses, then incubated for 48 hours in media containing 10% EV-depleted FBS. The resultant 120mL of conditioned media (CM) per cell line was collected and subject to differential ultracentrifugation to isolate EVs. CM was centrifuged twice, then concentrated to less than 10mL using 3kDa MWCO filter tubes. Concentrated CM was centrifuged twice before passing through a 0.22μm pore-sized syringe filter prior to the final ultracentrifugations. The sample was ultracentrifuged and the EV pellet was carefully washed, re-centrifuged and resuspended in 100μL of PBS. Protein analysis by Western blot confirmed the presence of EV markers (syntenin-1, TSG101) and absence of non-EV markers (calnexin, histone H2Ax). EV quantification and size distributions were evaluated by NanoSight nanoparticle tracking analysis and visualized by transmission electron microscopy. All isolations were positive for EV markers and negative for non-EV markers by Western Blotting. All isolations showed similar size distributions by nanoparticle tracking analysis, within the range of 30-250nm. EVs in each sample with the typical cup-like shape were observed in electron microscope images of EVs. Overall, our method demonstrates good reproducibility of EV isolations from multiple rhabdomyosarcoma cell lines. This methodology can be used as a baseline for other cell lines to provide greater consensus within the field, allowing further discoveries analyzing the secretome of patient-derived cell lines with the potential to translate into more specific diagnostic tools. Citation Format: Paula R. Quaglietta, Ashby Kissoondoyal, Ethan Malkin, Salvador Mejia, Ann Gong, David Malkin, Reto M. Baertschiger. Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5309.