Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.The authors also acknowledge the Genomics Resource Core facility (WCM) for their high-quality service. The authors thank C. Ghajar and J. Weiss for feedback on the manuscript and members of the Lyden laboratory for discussions. Our study was supported by the National Cancer Institute (U01-CA169538 to D.L.), the National Institutes of Health (NIH; R01-CA169416 to D.L. and H.P.; R01-CA218513 to D.L. and H.Z.), the US Department of Defense (W81XWH-13-10249 to D.L.), W81XWH-13-1-0425 (to D.L., J.Br.), the Sohn Conference Foundation (D.L., I.M., H.P. and H.Z.), the Children’s Cancer and Blood Foundation (D.L.), The Manning Foundation (A.H. and D.L.), The Hartwell Foundation (D.L.), The Nancy C. and Daniel P. Paduano Foundation (D.L.), The Starr Cancer Consortium (H.P. and D.L.; D.L. and H.Z.), the Pediatric Oncology Experimental Therapeutic Investigator Consortium (POETIC; D.L.), the James Paduano Foundation (D.L. and H.P.), the NIH/WCM CTSC (NIH/NCATS: UL1TR00457 to H.M. and H.Z.; UL1TR002384 to D.L., H.M. and H.Z.), the Malcolm Hewitt Wiener Foundation (D.L.), the Champalimaud Foundation (D.L.), the Thompson Family Foundation (D.L., R.S.), U01-CA210240 (D.L.), the Beth Tortolani Foundation (J.Br.), the Charles and Marjorie Holloway Foundation (J.Br.), the Sussman Family Fund (J.Br.), the Lerner Foundation (J.Br.), the Breast Cancer Alliance (J.Br.), the Manhasset Women’s Coalition Against Breast Cancer (J.Br.), the National Institute on Minority Health and Health Disparities (NIMHD) of the NIH (MD007599 to H.M.), NIH/NCATS (UL1TR00457 to H.M.). C.R., A.M., D.F., A.F., A.S. and H.O. acknowledge FEDER (Fundo Europeu de Desenvolvimento Regional funds through COMPETE 2020) POCI, Portugal 2020 (NORTE-01-0145-FEDER-000029) and FCT – Fundação para a Ciência e a Tecnologia in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and the FCT project POCI-01-0145-FEDER-016585 (PTDC/BBB-EBI/0567/2014). The authors acknowledge FCT for grants to A.M. (SFRH/BPD/75871/2011) and A.F. (SFRH/BPD/111048/2015). D.F. acknowledges FCT (SFRH/BD/110636/2015), the BiotechHealth PhD Programme (PD/0016/2012) and the American Portuguese Biomedical Research Fund.S