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

Haiying Zhang; Daniela Freitas; Han Sang Kim; Kristina Fabijanic; Zhong Li; Haiyan Chen; Milica Tesic Mark; Henrik Molina; Alberto Benito Martin; Linda Bojmar; Justin Fang; Sham Rampersaud; Ayuko Hoshino; Irina Matei; Candia M. Kenific; Miho Nakajima; Anders Peter Mutvei; Pasquale Sansone; Weston Buehring; Huajuan Wang; Juan Pablo Jimenez; Leona Cohen-Gould; Navid Paknejad; Matthew Brendel; Katia Manova-Todorova; Ana Magalhães; José Alexandre Ferreira; Hugo Osório; André M. Silva; Ashish Massey; Juan R. Cubillos-Ruiz; Giuseppe Galletti; Paraskevi Giannakakou; Ana Maria Cuervo; John Blenis; Robert Schwartz; Mary Sue Brady; Héctor Peinado; Jacqueline Bromberg; Hiroshi Matsui; Celso A. Reis; David Lyden

doi:10.1038/s41556-018-0040-4

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

Haiying Zhang, Daniela Freitas, Han Sang Kim, Kristina Fabijanic, Zhong Li, Haiyan Chen, Milica Tesic Mark, Henrik Molina, Alberto Benito Martin, Linda Bojmar, Justin Fang, Sham Rampersaud, Ayuko Hoshino, Irina Matei, Candia M. Kenific, Miho Nakajima, Anders Peter Mutvei, Pasquale Sansone, Weston Buehring, Huajuan WangJuan Pablo Jimenez, Leona Cohen-Gould, Navid Paknejad, Matthew Brendel, Katia Manova-Todorova, Ana Magalhães, José Alexandre Ferreira, Hugo Osório, André M. Silva, Ashish Massey, Juan R. Cubillos-Ruiz, Giuseppe Galletti, Paraskevi Giannakakou, Ana Maria Cuervo, John Blenis, Robert Schwartz, Mary Sue Brady, Héctor Peinado, Jacqueline Bromberg, Hiroshi Matsui, Celso A. Reis, David Lyden

Developmental & Molecular Biology

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430 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	332-343
Number of pages	12
Journal	Nature Cell Biology
Volume	20
Issue number	3
DOIs	https://doi.org/10.1038/s41556-018-0040-4
State	Published - Mar 1 2018

ASJC Scopus subject areas

Cell Biology

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10.1038/s41556-018-0040-4

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Zhang, H., Freitas, D., Kim, H. S., Fabijanic, K., Li, Z., Chen, H., Mark, M. T., Molina, H., Martin, A. B., Bojmar, L., Fang, J., Rampersaud, S., Hoshino, A., Matei, I., Kenific, C. M., Nakajima, M., Mutvei, A. P., Sansone, P., Buehring, W., ... Lyden, D. (2018). Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nature Cell Biology, 20(3), 332-343. https://doi.org/10.1038/s41556-018-0040-4

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. / Zhang, Haiying; Freitas, Daniela; Kim, Han Sang; Fabijanic, Kristina; Li, Zhong; Chen, Haiyan; Mark, Milica Tesic; Molina, Henrik; Martin, Alberto Benito; Bojmar, Linda; Fang, Justin; Rampersaud, Sham; Hoshino, Ayuko; Matei, Irina; Kenific, Candia M.; Nakajima, Miho; Mutvei, Anders Peter; Sansone, Pasquale; Buehring, Weston; Wang, Huajuan; Jimenez, Juan Pablo; Cohen-Gould, Leona; Paknejad, Navid; Brendel, Matthew; Manova-Todorova, Katia; Magalhães, Ana; Ferreira, José Alexandre; Osório, Hugo; Silva, André M.; Massey, Ashish; Cubillos-Ruiz, Juan R.; Galletti, Giuseppe; Giannakakou, Paraskevi; Cuervo, Ana Maria; Blenis, John; Schwartz, Robert; Brady, Mary Sue; Peinado, Héctor; Bromberg, Jacqueline; Matsui, Hiroshi; Reis, Celso A.; Lyden, David.

In: Nature Cell Biology, Vol. 20, No. 3, 01.03.2018, p. 332-343.

Research output: Contribution to journal › Article › peer-review

Zhang, H, Freitas, D, Kim, HS, Fabijanic, K, Li, Z, Chen, H, Mark, MT, Molina, H, Martin, AB, Bojmar, L, Fang, J, Rampersaud, S, Hoshino, A, Matei, I, Kenific, CM, Nakajima, M, Mutvei, AP, Sansone, P, Buehring, W, Wang, H, Jimenez, JP, Cohen-Gould, L, Paknejad, N, Brendel, M, Manova-Todorova, K, Magalhães, A, Ferreira, JA, Osório, H, Silva, AM, Massey, A, Cubillos-Ruiz, JR, Galletti, G, Giannakakou, P, Cuervo, AM, Blenis, J, Schwartz, R, Brady, MS, Peinado, H, Bromberg, J, Matsui, H, Reis, CA & Lyden, D 2018, 'Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation', Nature Cell Biology, vol. 20, no. 3, pp. 332-343. https://doi.org/10.1038/s41556-018-0040-4

Zhang, Haiying ; Freitas, Daniela ; Kim, Han Sang ; Fabijanic, Kristina ; Li, Zhong ; Chen, Haiyan ; Mark, Milica Tesic ; Molina, Henrik ; Martin, Alberto Benito ; Bojmar, Linda ; Fang, Justin ; Rampersaud, Sham ; Hoshino, Ayuko ; Matei, Irina ; Kenific, Candia M. ; Nakajima, Miho ; Mutvei, Anders Peter ; Sansone, Pasquale ; Buehring, Weston ; Wang, Huajuan ; Jimenez, Juan Pablo ; Cohen-Gould, Leona ; Paknejad, Navid ; Brendel, Matthew ; Manova-Todorova, Katia ; Magalhães, Ana ; Ferreira, José Alexandre ; Osório, Hugo ; Silva, André M. ; Massey, Ashish ; Cubillos-Ruiz, Juan R. ; Galletti, Giuseppe ; Giannakakou, Paraskevi ; Cuervo, Ana Maria ; Blenis, John ; Schwartz, Robert ; Brady, Mary Sue ; Peinado, Héctor ; Bromberg, Jacqueline ; Matsui, Hiroshi ; Reis, Celso A. ; Lyden, David. / Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. In: Nature Cell Biology. 2018 ; Vol. 20, No. 3. pp. 332-343.

@article{20397879b59b4728aba46fe4c5bf5987,

title = "Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation",

abstract = "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.",

author = "Haiying Zhang and Daniela Freitas and Kim, {Han Sang} and Kristina Fabijanic and Zhong Li and Haiyan Chen and Mark, {Milica Tesic} and Henrik Molina and Martin, {Alberto Benito} and Linda Bojmar and Justin Fang and Sham Rampersaud and Ayuko Hoshino and Irina Matei and Kenific, {Candia M.} and Miho Nakajima and Mutvei, {Anders Peter} and Pasquale Sansone and Weston Buehring and Huajuan Wang and Jimenez, {Juan Pablo} and Leona Cohen-Gould and Navid Paknejad and Matthew Brendel and Katia Manova-Todorova and Ana Magalh{\~a}es and Ferreira, {Jos{\'e} Alexandre} and Hugo Os{\'o}rio and Silva, {Andr{\'e} M.} and Ashish Massey and Cubillos-Ruiz, {Juan R.} and Giuseppe Galletti and Paraskevi Giannakakou and Cuervo, {Ana Maria} and John Blenis and Robert Schwartz and Brady, {Mary Sue} and H{\'e}ctor Peinado and Jacqueline Bromberg and Hiroshi Matsui and Reis, {Celso A.} and David Lyden",

note = "Funding Information: The authors acknowledge technical support from Wyatt Technology and especially J. Champagne. 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{\textquoteright}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{\textquoteright}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 – Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia in the framework of the project {\textquoteleft}Institute for Research and Innovation in Health Sciences{\textquoteright} (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. Publisher Copyright: {\textcopyright} 2018 The Author(s).",

year = "2018",

month = mar,

day = "1",

doi = "10.1038/s41556-018-0040-4",

language = "English (US)",

volume = "20",

pages = "332--343",

journal = "Nature Cell Biology",

issn = "1465-7392",

publisher = "Nature Publishing Group",

number = "3",

}

TY - JOUR

T1 - Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

AU - Zhang, Haiying

AU - Freitas, Daniela

AU - Kim, Han Sang

AU - Fabijanic, Kristina

AU - Li, Zhong

AU - Chen, Haiyan

AU - Mark, Milica Tesic

AU - Molina, Henrik

AU - Martin, Alberto Benito

AU - Bojmar, Linda

AU - Fang, Justin

AU - Rampersaud, Sham

AU - Hoshino, Ayuko

AU - Matei, Irina

AU - Kenific, Candia M.

AU - Nakajima, Miho

AU - Mutvei, Anders Peter

AU - Sansone, Pasquale

AU - Buehring, Weston

AU - Wang, Huajuan

AU - Jimenez, Juan Pablo

AU - Cohen-Gould, Leona

AU - Paknejad, Navid

AU - Brendel, Matthew

AU - Manova-Todorova, Katia

AU - Magalhães, Ana

AU - Ferreira, José Alexandre

AU - Osório, Hugo

AU - Silva, André M.

AU - Massey, Ashish

AU - Cubillos-Ruiz, Juan R.

AU - Galletti, Giuseppe

AU - Giannakakou, Paraskevi

AU - Cuervo, Ana Maria

AU - Blenis, John

AU - Schwartz, Robert

AU - Brady, Mary Sue

AU - Peinado, Héctor

AU - Bromberg, Jacqueline

AU - Matsui, Hiroshi

AU - Reis, Celso A.

AU - Lyden, David

N1 - Funding Information: The authors acknowledge technical support from Wyatt Technology and especially J. Champagne. 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. Publisher Copyright: © 2018 The Author(s).

PY - 2018/3/1

Y1 - 2018/3/1

N2 - 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.

AB - 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.

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UR - http://www.scopus.com/inward/citedby.url?scp=85042193942&partnerID=8YFLogxK

U2 - 10.1038/s41556-018-0040-4

DO - 10.1038/s41556-018-0040-4

M3 - Article

C2 - 29459780

AN - SCOPUS:85042193942

VL - 20

SP - 332

EP - 343

JO - Nature Cell Biology

JF - Nature Cell Biology

SN - 1465-7392

IS - 3

ER -

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

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