Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation

Abstract

Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome‐derived vesicles. We propose that the most commonly used protocols for exosome preparations co‐purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles.

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References 37

1. 1.

   Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9:581-93.

   Not in our library10.1038/nri2567

2. 2.

   Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009;19:43-51.

   Not in our library10.1016/j.tcb.2008.11.003

3. 3.

   Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int. 2010;78:838-48.

   Not in our library10.1038/ki.2010.278

4. 4.

   George JN, Thoi LL, McManus LM, Reimann TA. Isolation of human platelet membrane microparticles from plasma and serum. Blood. 1982;60:834-40.

   Not in our library

5. 5.

   Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol. 1983;97:329-39.

   Not in our library10.1083/jcb.97.2.329

6. 6.

   Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985;101:942-8.

   Not in our library10.1083/jcb.101.3.942

7. 7.

   Harding C, Heuser J, Stahl P. Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol. 1984;35:256-63.

   Not in our library

8. 8.

   Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 1987;262:9412-20.

   Not in our library

9. 9.

   Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183:1161-72.

   Not in our library10.1084/jem.183.3.1161

10. 10.

    Ronquist G, Brody I. The prostasome: its secretion and function in man. Biochim Biophys Acta. 1985;822:203-18.

    Not in our library10.1016/0304-4157(85)90008-5

11. 11.

    Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med. 1998;4:594-600.

    Not in our library10.1038/nm0598-594

12. 12.

    Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, et al. Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol. 1999;147:599-610.

    Not in our library10.1083/jcb.147.3.599

13. 13.

    Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006.

    Not in our library10.1002/0471143030.cb0322s30

14. 14.

    Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol. 2010;12:19-30.

    Not in our library10.1038/ncb2000

15. 15.

    Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, Geuze HJ. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem. 1998;273:20121-7.

    Not in our library10.1074/jbc.273.32.20121

16. 16.

    Mathivanan S, Simpson RJ. ExoCarta: a compendium of exosomal proteins and RNA. Proteomics. 2009;9:4997-5000.

    Not in our library10.1002/pmic.200900351

17. 17.

    Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood. 1999;94:3791-9.

    Not in our library

18. 18.

    Aalberts M, van Dissel-Emiliani FM, van Adrichem NP, van Wijnen M, Wauben MH, Stout TA, et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1 and GLIPR2 in humans. Biol. Reprod. 2012;86:82.

    Not in our library10.1095/biolreprod.111.095760

19. 19.

    Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med. 2001;7:297-303.

    Not in our library10.1038/85438

20. 20.

    Tolmachova T, Anders R, Stinchcombe J, Bossi G, Griffiths GM, Huxley C, et al. A general role for Rab27a in secretory cells. Mol Biol Cell. 2004;15:332-44.

    Not in our library10.1091/mbc.e03-07-0452

21. 21.

    Pols MS, Klumperman J. Trafficking and function of the tetraspanin CD63. Exp Cell Res. 2009;315:1584-92.

    Not in our library10.1016/j.yexcr.2008.09.020

22. 22.

    Cramer EM, Berger G, Berndt MC. Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1. Blood. 1994;84:1722-30.

    Not in our library

23. 23.

    Buschow SI, Nolte-‘t Hoen EN, van Niel G, Pols MS, Ten Broeke T, Lauwen M, et al. MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways. Traffic. 2009;10:1528-42.

    Not in our library10.1111/j.1600-0854.2009.00963.x

24. 24.

    Oshima K, Aoki N, Kato T, Kitajima K, Matsuda T. Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles. Eur J Biochem. 2002;269:1209-18.

    Not in our library10.1046/j.1432-1033.2002.02758.x

25. 25.

    Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol. 2001;166:7309-18.

    Not in our library10.4049/jimmunol.166.12.7309

26. 26.

    Veron P, Segura E, Sugano G, Amigorena S, Thery C. Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells. Blood Cells Mol Dis. 2005;35:81-8.

    Not in our library10.1016/j.bcmd.2005.05.001

27. 27.

    Segura E, Nicco C, Lombard B, Veron P, Raposo G, Batteux F, et al. ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood. 2005;106:216-23.

    Not in our library10.1182/blood-2005-01-0220

28. 28.

    Delcayre A, Estelles A, Sperinde J, Roulon T, Paz P, Aguilar B, et al. Exosome Display technology: applications to the development of new diagnostics and therapeutics. Blood Cells Mol Dis. 2005;35:158-68.

    Not in our library10.1016/j.bcmd.2005.07.003

29. 29.

    Zeelenberg IS, Ostrowski M, Krumeich S, Bobrie A, Jancic C, Boissonnas A, et al. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res. 2008;68:1228-35.

    Not in our library10.1158/0008-5472.can-07-3163

30. 30.

    Hartman ZC, Wei J, Glass OK, Guo H, Lei G, Yang XY, et al. Increasing vaccine potency through exosome antigen targeting. Vaccine. 2011;29:9361-7.

    Not in our library10.1016/j.vaccine.2011.09.133

31. 31.

    Rountree RB, Mandl SJ, Nachtwey JM, Dalpozzo K, Do L, Lombardo JR, et al. Exosome targeting of tumor antigens expressed by cancer vaccines can improve antigen immunogenicity and therapeutic efficacy. Cancer Res. 2011;71:5235-44.

    Not in our library10.1158/0008-5472.can-10-4076

32. 32.
    Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects · 2005

    No notice
33. 33.

    Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP, et al. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest. 2010;120:457-71.

    Not in our library10.1172/jci40483

34. 34.

    Taylor DD, Gercel-Taylor C, Lyons KS, Stanson J, Whiteside TL. T-cell apoptosis and suppression of T-cell receptor/CD3-zeta by Fas ligand-containing membrane vesicles shed from ovarian tumors. Clin Cancer Res. 2003;9:5113-9.

    Not in our library

35. 35.

    Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol. 2005;17:879-87.

    Not in our library10.1093/intimm/dxh267

36. 36.

    Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell. 2006;124:1283-98.

    Not in our library10.1016/j.cell.2006.01.040

37. 37.

    Sibarita JB. Deconvolution microscopy. Adv Biochem Eng Biotechnol. 2005;9:201-43.

    Not in our library10.1007/b102215

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