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A Modified Differential Centrifugation Protocol for Isolation and Quantitation of Extracellular Heat Shock Protein 90 (eHsp90)

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Chaperones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2693))

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Abstract

Studies of the past 15 years have revealed a critical role for extracellular heat shock protein 90alpha (eHsp90α) in the development of several human disorders, including wound healing, cachexia (muscle wasting), inflammatory diseases, and cancers. The two established functions of highly purified eHsp90α protein are to promote cell survival and to stimulate cell migration. However, the mechanism of secretion and the method of isolation of eHsp90α remained to be standardized. Among the half a dozen reported methodologies, differential centrifugation is considered the “gold standard” largely for its quantitative recovery of eHsp90α from a conditioned medium of cultured cells. Herein, we describe a revised protocol that isolates three fractions of extracellular vesicles with distinct ranges of diameters and the leftover vesicle-free supernatant for biochemical analyses, especially eHsp90α, from tumor cell-conditioned media. Quantitation of the relative amount of eHsp90α can be carried out with known amounts of recombinant Hsp90α protein on the same SDS-PAGE. We believe that this modified methodology will prove to be a useful tool for studying eHsp90α in cultured cells and beyond.

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References

  1. El Andaloussi S, Mäger I, Breakefield XO, Wood MJ (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12:347–357

    Article  PubMed  Google Scholar 

  2. Zaborowski MP, Balaj L, Breakefield XO, Lai CP (2015) Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience 65:783–797

    Article  PubMed  PubMed Central  Google Scholar 

  3. Van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64:676–705

    Article  PubMed  Google Scholar 

  4. Tkach M, Théry C (2016) Communication by extracellular vesicles: where we are and where we need to go. Cell 164:1226–1232

    Article  CAS  PubMed  Google Scholar 

  5. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ (1999) Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and α-granules. Blood J Am Soc Hematol 94:3791–3799

    Google Scholar 

  6. Cocucci E, Meldolesi J (2015) Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol 25:364–372

    Google Scholar 

  7. Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579

    Article  PubMed  Google Scholar 

  8. Kalluri R (2016) The biology and function of exosomes in cancer. J Clin Invest 126:1208–1215

    Article  PubMed  PubMed Central  Google Scholar 

  9. Akers JC, Gonda D, Kim R, Carter BS, Chen CC (2013) Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neuro-Oncol 113:1–11

    Article  Google Scholar 

  10. Becker A, Thakur BK, Weiss JM, Kim HS, Peinado H, Lyden D (2016) Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell 30:836–848

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nishida-Aoki N, Tominaga N, Takeshita F, Sonoda H, Yoshioka Y, Ochiya T (2017) Disruption of circulating extracellular vesicles as a novel therapeutic strategy against cancer metastasis. Mol Ther 25:181–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kogure A, Yoshioka Y, Ochiya T (2020) Extracellular vesicles in cancer metastasis: potential as therapeutic targets and materials. Int J Mol Sci 21:4463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ (2018) Extracellular vesicles in cancer—implications for future improvements in cancer care. Nat Rev Clin Oncol 15:617–638

    Article  CAS  PubMed  Google Scholar 

  14. Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A et al (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624

    Article  CAS  PubMed  Google Scholar 

  15. Skog J, Würdinger T, Van Rijn S, Meijer DH, Gainche L, Curry WT et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10:1470–1476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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 106:3794–3799

    Article  PubMed  PubMed Central  Google Scholar 

  17. Gatti S, Bruno S, Deregibus MC, Sordi A, Cantaluppi V, Tetta C et al (2011) Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia–reperfusion-induced acute and chronic kidney injury. Nephrol Dial Transplan 26:1474–1483

    Article  CAS  Google Scholar 

  18. Rak J, Guha A (2012) Extracellular vesicles–vehicles that spread cancer genes. BioEssays 34:489–497

    Article  CAS  PubMed  Google Scholar 

  19. Wieckowski EU, Visus C, Szajnik M, Szczepanski MJ, Storkus WJ, Whiteside TL (2009) Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol 183:3720–3730

    Article  CAS  PubMed  Google Scholar 

  20. Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL (2005) Fas ligand–positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res 11:1010–1020

    Article  CAS  PubMed  Google Scholar 

  21. Cai Z, Yang F, Yu L, Yu Z, Jiang L, Wang Q et al (2012) Activated T cell exosomes promote tumor invasion via Fas signaling pathway. J Immunol 188:5954–5961

    Article  CAS  PubMed  Google Scholar 

  22. Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30:3–22

    Google Scholar 

  23. Momen-Heravi F, Balaj L, Alian S, Trachtenberg AJ, Hochberg FH, Skog J et al (2012) Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol 3:162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Momen-Heravi F, Balaj L, Alian S, Mantel PY, Halleck AE, Trachtenberg AJ et al (2013) Current methods for the isolation of extracellular vesicles. Biol Chem 394:1253–1262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cvjetkovic A, Lötvall J, Lässer C (2014) The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles 3:23111

    Article  Google Scholar 

  26. Clos-Sansalvador M, Monguió-Tortajada M, Roura S, Franquesa M, Borràs FE (2022) Commonly used methods for extracellular vesicles’ enrichment: Implications in downstream analyses and use. Eur J Cell Biol 101:151227

    Article  CAS  PubMed  Google Scholar 

  27. Nordin JZ, Lee Y, Vader P, Mäger I, Johansson HJ, Heusermann W et al (2015) Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11:879–883

    Article  CAS  PubMed  Google Scholar 

  28. Benedikter BJ, Bouwman FG, Vajen T, Heinzmann AC, Grauls G, Mariman EC et al (2017) Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Sci Rep 7:1–13

    Article  CAS  Google Scholar 

  29. Cheruvanky A, Zhou H, Pisitkun T, Kopp JB, Knepper MA, Yuen PS et al (2007) Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292:1657–F1661

    Article  Google Scholar 

  30. Lobb RJ, Becker M, Wen Wen S, Wong CS, Wiegmans AP, Leimgruber A et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031

    Article  PubMed  Google Scholar 

  31. Konoshenko MY, Lekchnov EA, Vlassov AV (2018) Laktionov PP (2018) isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res Int 2018:1

    Article  Google Scholar 

  32. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM et al (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293–304

    Article  CAS  PubMed  Google Scholar 

  33. Tang X, Chang C, Guo J, Lincoln V, Liang C, Chen M et al (2019) Tumour-secreted Hsp90α on external surface of exosomes mediates tumour-stromal cell communication via autocrine and paracrine mechanisms. Sci Rep 9:1–13

    Article  Google Scholar 

  34. Stone KR, Smith RE, Joklik WK (1974) Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses. Virology 58:86–100

    Article  CAS  PubMed  Google Scholar 

  35. Eustace BK, Sakurai T, Stewart JK, Yimlamai D, Unger C, Zehetmeier C et al (2004) Functional proteomic screens reveal an essential extracellular role for hsp90α in cancer cell invasiveness. Nat Cell Biol 6:507–514

    Article  CAS  PubMed  Google Scholar 

  36. Li W, LiY GS, Fan J, Cheng CF, Bright AM et al (2007) Extracellular heat shock protein-90α: linking hypoxia to skin cell motility and wound healing. EMBO J 26:1221–1233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang X, Song X, Zhuo W, Fu Y, Shi H, Liang Y et al (2009) The regulatory mechanism of Hsp90α secretion and its function in tumor malignancy. Proc Natl Acad Sci 106:21288–21293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Burgess EF, Ham AJL, Tabb DL, Billheimer D, Roth BJ, Chang SS et al (2008) Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes. PROTEOMICS Clin Appl 2:1223–1233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wei W, Liu M, Ning S, Wei J, Zhong J, Li J et al (2020) Diagnostic value of plasma HSP90α levels for detection of hepatocellular carcinoma. BMC Cancer 20:1–9

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Dermatology the Norris Comprehensive Cancer Centre, University of Southern California Keck Medical Center, Los Angeles, CA, USA

    Cheng Chang, **n Tang & Wei Li

Authors
  1. Cheng Chang
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  2. **n Tang
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  3. Wei Li
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Corresponding author

Correspondence to Cheng Chang .

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Editors and Affiliations

  1. Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Stuart K. Calderwood

  2. Danville, PA, USA

    Thomas L. Prince

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Chang, C., Tang, X., Li, W. (2023). A Modified Differential Centrifugation Protocol for Isolation and Quantitation of Extracellular Heat Shock Protein 90 (eHsp90). In: Calderwood, S.K., Prince, T.L. (eds) Chaperones. Methods in Molecular Biology, vol 2693. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3342-7_19

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  • DOI: https://doi.org/10.1007/978-1-0716-3342-7_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3341-0

  • Online ISBN: 978-1-0716-3342-7

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