Abstract
Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33(3):967–978
Colombo M, Raposo G, Thery 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
van Niel G, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, Heyman M (2001) Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 121(2):337–349
Faure J, Lachenal G, Court M, Hirrlinger J, Chatellard-Causse C, Blot B, Grange J, Schoehn G, Goldberg Y, Boyer V, Kirchhoff F, Raposo G, Garin J, Sadoul R (2006) Exosomes are released by cultured cortical neurones. Mol Cell Neurosci 31(4):642–648. https://doi.org/10.1016/j.mcn.2005.12.003
Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, Flament C, Pouzieux S, Faure F, Tursz T, Angevin E, Amigorena S, Zitvogel L (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 7(3):297–303. https://doi.org/10.1038/85438
Paggetti J, Haderk F, Seiffert M, Janji B, Distler U, Ammerlaan W, Kim YJ, Adam J, Lichter P, Solary E, Berchem G, Moussay E (2015) Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts. Blood 126(9):1106–1117. https://doi.org/10.1182/blood-2014-12-618025
Bobrie A, Krumeich S, Reyal F, Recchi C, Moita LF, Seabra MC, Ostrowski M, Thery C (2012) Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression. Cancer Res 72(19):4920–4930. https://doi.org/10.1158/0008-5472.CAN-12-0925
Filipazzi P, Burdek M, Villa A, Rivoltini L, Huber V (2012) Recent advances on the role of tumor exosomes in immunosuppression and disease progression. Semin Cancer Biol 22(4):342–349. https://doi.org/10.1016/j.semcancer.2012.02.005
Zhang HG, Zhuang X, Sun D, Liu Y, Xiang X, Grizzle WE (2012) Exosomes and immune surveillance of neoplastic lesions: a review. Biotech Histochem 87(3):161–168. https://doi.org/10.3109/10520291003659042
Steinbichler TB, Dudas J, Riechelmann H, Skvortsova II (2017) The role of exosomes in cancer metastasis. Semin Cancer Biol 44:170–181. https://doi.org/10.1016/j.semcancer.2017.02.006
Viry E, Paggetti J, Baginska J, Mgrditchian T, Berchem G, Moussay E, Janji B (2014) Autophagy: an adaptive metabolic response to stress shaping the antitumor immunity. Biochem Pharmacol 92(1):31–42. https://doi.org/10.1016/j.bcp.2014.07.006
Li P, Kaslan M, Lee SH, Yao J, Gao Z (2017) Progress in exosome isolation techniques. Theranostics 7(3):789–804. https://doi.org/10.7150/thno.18133
Taylor DD, Shah S (2015) Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87:3–10. https://doi.org/10.1016/j.ymeth.2015.02.019
Görgens A (2016) Analysis of extracellular vesicles including exosomes by imaging flow cytometry. Science/AAAS Custom Publishing Office, Washington, DC http://www.sciencemag.org/custom-publishing/webinars/analysis-extracellular-vesicles-including-exosomes-imaging-flow-cytometry
Acknowledgments
This work was supported by grants from FNRS “Télévie” (7.4563.15; 7.4508.16), Luxembourg National Research Fund (FNR, PRIDE15/10675146/CANBIO and INTER/DFG/16/11509946), and Luxembourg Institute of Health (LECR-TSI HEMATEXO).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wierz, M., Pierson, S., Gargiulo, E., Guerin, C., Moussay, E., Paggetti, J. (2019). Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation. In: LĂłpez-Soto, A., Folgueras, A. (eds) Cancer Immunosurveillance. Methods in Molecular Biology, vol 1884. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8885-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8885-3_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8884-6
Online ISBN: 978-1-4939-8885-3
eBook Packages: Springer Protocols