Skip to main content
SpringerLink
Log in

Isolation and Characterization of Salivary Exosomes for Cancer Biomarker Discovery

  • Protocol
  • First Online:
Extracellular Vesicles in Diagnosis and Therapy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2504))

Abstract

Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2(8):569–579. https://doi.org/10.1038/nri855

    Article  CAS  PubMed  Google Scholar 

  2. Bunggulawa EJ, Wang W, Yin T, Wang N, Durkan C, Wang Y, Wang G (2018) Recent advancements in the use of exosomes as drug delivery systems. J Nanobiotechnology 16(1):81. https://doi.org/10.1186/s12951-018-0403-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. LeBleu VS, Kalluri R (2020) Exosomes as a multicomponent biomarker platform in cancer. Trends Cancer 6(9):767–774. https://doi.org/10.1016/j.trecan.2020.03.007

    Article  CAS  PubMed  Google Scholar 

  4. Colombo M, Raposo G, Théry C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289. https://doi.org/10.1146/annurev-cellbio-101512-122326

    Article  CAS  PubMed  Google Scholar 

  5. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304. https://doi.org/10.1016/j.ymeth.2012.01.002

    Article  CAS  PubMed  Google Scholar 

  6. Lässer C, Alikhani VS, Ekström K, Eldh M, Paredes PT, Bossios A, Sjöstrand M, Gabrielsson S, Lötvall J, Valadi H (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9. https://doi.org/10.1186/1479-5876-9-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang C-Z, Cheng X-Q, Li J-Y, Zhang P, Yi P, Xu X, Zhou X-D (2016) Saliva in the diagnosis of diseases. Int J Oral Sci 8(3):133–137. https://doi.org/10.1038/ijos.2016.38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kaufman E, Lamster IB (2002) The diagnostic applications of saliva—a review. Crit Rev Oral Biol Med 13(2):197–212. https://doi.org/10.1177/154411130201300209

    Article  PubMed  Google Scholar 

  9. Lim Y, Wan Y, Vagenas D, Ovchinnikov DA, Perry CFL, Davis MJ, Punyadeera C (2016) Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers. BMC Cancer 16(1):749. https://doi.org/10.1186/s12885-016-2785-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ovchinnikov DA, Wan Y, Coman WB, Pandit P, Cooper-White JJ, Herman JG, Punyadeera C (2014) DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers. Biomark Insights 9:53–60. https://doi.org/10.4137/BMI.S16199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhan C, Yang X, Yin X, Hou J (2019) Exosomes and other extracellular vesicles in oral and salivary gland cancers. Oral Dis 26(5):865–875. https://doi.org/10.1111/odi.13172

    Article  PubMed  Google Scholar 

  12. Wang Z, Li F, Rufo J, Chen C, Yang S, Li L, Zhang J, Cheng J, Kim Y, Wu M, Abemayor E, Tu M, Chia D, Spruce R, Batis N, Mehanna H, Wong DTW, Huang TJ (2020) Acoustofluidic salivary exosome isolation: a liquid biopsy compatible approach for human papillomavirus–associated oropharyngeal cancer detection. J Mol Diagn 22(1):50–59. https://doi.org/10.1016/j.jmoldx.2019.08.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cao J, Zhang M, Xie F, Lou J, Zhou X, Zhang L, Fang M, Zhou F (2020) Exosomes in head and neck cancer: roles, mechanisms and applications. Cancer Lett 494:7–16. https://doi.org/10.1016/j.canlet.2020.07.005

    Article  CAS  PubMed  Google Scholar 

  14. Han Y, Jia L, Zheng Y, Li W (2018) Salivary exosomes: emerging roles in systemic disease. Int J Biol Sci 14(6):633–643. https://doi.org/10.7150/ijbs.25018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lau C, Kim Y, Chia D, Spielmann N, Eibl G, Elashoff D, Wei F, Lin Y-L, Moro A, Grogan T, Chiang S, Feinstein E, Schafer C, Farrell J, Wong DTW (2013) Role of pancreatic cancer-derived exosomes in salivary biomarker development. J Biol Chem 288(37):26888–26897. https://doi.org/10.1074/jbc.M113.452458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sun Y, Huo C, Qiao Z, Shang Z, Uzzaman A, Liu S, Jiang X, Fan L-Y, Ji L, Guan X, Cao C-X, Xiao H (2018) Comparative proteomic analysis of exosomes and microvesicles in human saliva for lung cancer. J Proteome Res 17(3):1101–1107. https://doi.org/10.1021/acs.jproteome.7b00770

    Article  CAS  PubMed  Google Scholar 

  17. Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, Vered M (2015) Human saliva-derived exosomes: comparing methods of isolation. J Histochem Cytochem 63(3):181–189. https://doi.org/10.1369/0022155414564219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Maas SLN, Breakefield XO, Weaver AM (2017) Extracellular vesicles: unique intercellular delivery vehicles. Trends Cell Biol 27(3):172–188. https://doi.org/10.1016/j.tcb.2016.11.003

    Article  CAS  PubMed  Google Scholar 

  19. Zhang HG, Grizzle WE (2014) Exosomes: a novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions. Am J Pathol 184(1):28–41. https://doi.org/10.1016/j.ajpath.2013.09.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ageta H, Ageta-Ishihara N, Hitachi K, Karayel O, Onouchi T, Yamaguchi H, Kahyo T, Hatanaka K, Ikegami K, Yoshioka Y, Nakamura K, Kosaka N, Nakatani M, Uezumi A, Ide T, Tsutsumi Y, Sugimura H, Kinoshita M, Ochiya T, Mann M, Setou M, Tsuchida K (2018) UBL3 modification influences protein sorting to small extracellular vesicles. Nat Commun 9(1):3936. https://doi.org/10.1038/s41467-018-06197-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Andreu Z, Yáñez-Mó M (2014) Tetraspanins in extracellular vesicle formation and function. Front Immunol 5:442. https://doi.org/10.3389/fimmu.2014.00442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Villarroya-Beltri C, Gutiérrez-Vázquez C, Sánchez-Cabo F, Pérez-Hernández D, Vázquez J, Martin-Cofreces N, Martinez-Herrera DJ, Pascual-Montano A, Mittelbrunn M, Sánchez-Madrid F (2013) Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun 4:2980. https://doi.org/10.1038/ncomms3980

    Article  CAS  PubMed  Google Scholar 

  23. Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH (2014) Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1. Blood 124(25):3748–3757. https://doi.org/10.1182/blood-2014-05-576116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sandfeld-Paulsen B, Jakobsen KR, Bæk R, Folkersen BH, Rasmussen TR, Meldgaard P, Varming K, Jørgensen MM, Sorensen BS (2016) Exosomal proteins as diagnostic biomarkers in lung cancer. J Thorac Oncol 11(10):1701–1710. https://doi.org/10.1016/j.jtho.2016.05.034

    Article  PubMed  Google Scholar 

  25. Chen IH, Xue L, Hsu CC, Paez JS, Pan L, Andaluz H, Wendt MK, Iliuk AB, Zhu JK, Tao WA (2017) Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc Natl Acad Sci U S A 114(12):3175–3180. https://doi.org/10.1073/pnas.1618088114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Madhavan B, Yue S, Galli U, Rana S, Gross W, Müller M, Giese NA, Kalthoff H, Becker T, Büchler MW, Zöller M (2015) Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity. Int J Cancer 136(11):2616–2627. https://doi.org/10.1002/ijc.29324

    Article  CAS  PubMed  Google Scholar 

  27. Theodoraki MN, Yerneni SS, Hoffmann TK, Gooding WE, Whiteside TL (2018) Clinical significance of PD-L1(+) exosomes in plasma of head and neck cancer patients. Clin Cancer Res 24(4):896–905. https://doi.org/10.1158/1078-0432.CCR-17-2664

    Article  CAS  PubMed  Google Scholar 

  28. Li C, Li C, Zhi C, Liang W, Wang X, Chen X, Lv T, Shen Q, Song Y, Lin D, Liu H (2019) Clinical significance of PD-L1 expression in serum-derived exosomes in NSCLC patients. J Transl Med 17(1):355. https://doi.org/10.1186/s12967-019-2101-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wan Y, Vagenas D, Salazar C, Kenny L, Perry C, Calvopiña D, Punyadeera C (2017) Salivary miRNA panel to detect HPV-positive and HPV-negative head and neck cancer patients. Oncotarget 8(59):99990–100001. https://doi.org/10.18632/oncotarget.21725

    Article  PubMed  PubMed Central  Google Scholar 

  30. Zhang X, Wan Y, Chata R, Brazzale A, Atherton JJ, Kostner K, Dimeski G, Punyadeera C (2016) A pilot study to demonstrate diagnostic potential of galectin-3 levels in saliva. J Clin Pathol 69(12):1100–1104. https://doi.org/10.1136/jclinpath-2016-203631

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Figure 1 was created using Servier Medical Art templates, licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com. CP is funded by Cancer Australia (APP 1145657) and NHMRC Ideas Grant (APP 2002576).

Author information

Author notes

  1. Lucas Trevisan França de Lima and Juliana Müller Bark contributed equally as first authors.

Authors and Affiliations

  1. Queensland University of Technology, The School of Biomedical Sciences, Saliva and Liquid Biopsy Translational Laboratory, Kelvin Grove, QLD, Australia

    Lucas Trevisan França de Lima, Juliana Müller Bark, Mohammad Rasheduzzaman, Chameera Ekanayake Weeramange & Chamindie Punyadeera

  2. Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery (GRIDD) and Menzies Health Institute Queensland (MIHQ), Griffith University, Brisbane, QLD, Australia

    Chamindie Punyadeera

Authors
  1. Lucas Trevisan França de Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juliana Müller Bark
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Rasheduzzaman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chameera Ekanayake Weeramange
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chamindie Punyadeera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chamindie Punyadeera .

Editor information

Editors and Affiliations

  1. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Maurizio Federico

  2. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Barbara Ridolfi

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Trevisan França de Lima, L., Müller Bark, J., Rasheduzzaman, M., Weeramange, C.E., Punyadeera, C. (2022). Isolation and Characterization of Salivary Exosomes for Cancer Biomarker Discovery. In: Federico, M., Ridolfi, B. (eds) Extracellular Vesicles in Diagnosis and Therapy. Methods in Molecular Biology, vol 2504. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2341-1_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2341-1_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2340-4

  • Online ISBN: 978-1-0716-2341-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation