Advertisement

Analysis of Extracellular Vesicles in the Tumor Microenvironment

  • Khalid Al-Nedawi
  • Jolene Read
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1458)

Abstract

Extracellular vesicles (ECV) are membrane compartments shed from all types of cells in various physiological and pathological states. In recent years, ECV have gained an increasing interest from the scientific community for their role as an intercellular communicator that plays important roles in modifying the tumor microenvironment. Multiple techniques have been established to collect ECV from conditioned media of cell culture or physiological fluids. The gold standard methodology is differential centrifugation. Although alternative techniques exist to collect ECV, these techniques have not proven suitable as a substitution for the ultracentrifugation procedure.

Key words

Exosome Microvesicle Ultracentrifugation Tumor microenvironment 

Notes

Acknowledgments

This work was supported by a Movember Foundation/Prostate Cancer Canada Discovery Grant # D-2014-1 for Dr. K. Al-Nedawi.

References

  1. 1.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lo Cicero A, Stahl PD, Raposo G (2015) Extracellular vesicles shuffling intercellular messages: for good or for bad. Curr Opin Cell Biol 35:69–77CrossRefPubMedGoogle Scholar
  3. 3.
    Muralidharan-Chari V, Clancy JW, Sedgwick A, D’Souza-Schorey C (2010) Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci 123:1603–1611CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579PubMedGoogle Scholar
  5. 5.
    Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659CrossRefPubMedGoogle Scholar
  6. 6.
    Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan MLG, Karlsson JM et al (2012) Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 119:756–766CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Thakur BK, Zhang H, Becker A, Matel I, Huang Y, Costa-Silva B et al (2014) Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res 24:766–769CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Montermini L, Meehan B, Garnier D, Lee WJ, Lee TH, Guha A, Al-Nedawi K, Rak J (2015) Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content. J Biol Chem 290:24534–24546CrossRefPubMedGoogle Scholar
  9. 9.
    Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, Rak J (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624CrossRefPubMedGoogle Scholar
  10. 10.
    Al-Nedawi K, Meehan B, Rak J (2009) Microvesicles: messengers and mediators of tumor progression. Cell Cycle 8:2014–2018CrossRefPubMedGoogle Scholar
  11. 11.
    Al-Nedawi K (2014) The yin-yang of microvesicles (exosomes) in cancer biology. Front Oncol 4:172CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lee TH, D’Asti E, Magnus N, Al-Nedawi K, Meehan B, Rak J (2011) Microvesicles as mediators of intercellular communication in cancer—the emerging science of cellular ‘debris’. Semin Immunopathol 33:455–467CrossRefPubMedGoogle Scholar
  13. 13.
    Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK et al (2015) Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17:816–826CrossRefPubMedGoogle Scholar
  14. 14.
    Al-Nedawi K, Meehan B, Kerbel RS, Allison AC, Rak J (2009) Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR. Proc Natl Acad Sci U S A 106:3794–3799CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Melo SA, Luecke LB, Kahlert C et al (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523(7559):177-182. doi: 10.1038/nature14581Google Scholar
  16. 16.
    Soo CY, Song Y, Zheng Y et al (2012) Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 136(2):192–197. doi:10.1111/j.1365-2567.2012.03569.xGoogle Scholar
  17. 17.
    Webber J, Clayton A (2013) How pure are your vesicles?. J Extracell Vesicles 2:10.3402/jev.v2i0.19861. eCollection 2013. doi:10.3402/jev.v2i0.19861Google Scholar
  18. 18.
    Shelke GV, Lasser C, Gho YS et al (2014) Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles 3:10.3402/jev.v3.24783.eCollection 2014. doi:10.3402/jev.v3.24783Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Nephrology, Department of MedicineMcMaster UniversityHamiltonCanada
  2. 2.Hamilton Centre for Kidney Research (HCKR)St. Joseph’s HospitalHamiltonCanada

Personalised recommendations