Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations.
Antigens, Surface
/ metabolism
Biomarkers
/ metabolism
Cell Line, Tumor
Chromatography, Gel
/ methods
Extracellular Vesicles
/ metabolism
Glutamate Carboxypeptidase II
/ metabolism
Humans
Immunomagnetic Separation
/ methods
Male
Proof of Concept Study
Prostatic Neoplasms
/ metabolism
Ultracentrifugation
/ methods
Ultrafiltration
/ methods
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
02 06 2021
02 06 2021
Historique:
received:
10
12
2020
accepted:
18
05
2021
entrez:
3
6
2021
pubmed:
4
6
2021
medline:
9
11
2021
Statut:
epublish
Résumé
Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.
Identifiants
pubmed: 34079007
doi: 10.1038/s41598-021-91129-y
pii: 10.1038/s41598-021-91129-y
pmc: PMC8172572
doi:
Substances chimiques
Antigens, Surface
0
Biomarkers
0
FOLH1 protein, human
EC 3.4.17.21
Glutamate Carboxypeptidase II
EC 3.4.17.21
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
11585Références
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell. Biol. 10(12), 1470–1476 (2008).
pubmed: 19011622
pmcid: 3423894
doi: 10.1038/ncb1800
Minciacchi, V. R., Freeman, M. R. & Di Vizio, D. Extracellular vesicles in cancer: Exosomes, microvesicles and the emerging role of large oncosomes. Semin. Cell Dev. Biol. 40, 41–51 (2015).
pubmed: 25721812
pmcid: 4747631
doi: 10.1016/j.semcdb.2015.02.010
Zitvogel, L. et al. Eradication of established murine tumors using a novel cell-free vaccine: Dendritic cell-derived exosomes. Nat. Med. 4(5), 594–600 (1998).
pubmed: 9585234
doi: 10.1038/nm0598-594
Colombo, M., Raposo, G. & Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).
pubmed: 25288114
doi: 10.1146/annurev-cellbio-101512-122326
van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell. Biol. 19(4), 213–228 (2018).
pubmed: 29339798
doi: 10.1038/nrm.2017.125
Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell. Biol. 9(6), 654–659 (2007).
pubmed: 17486113
doi: 10.1038/ncb1596
Yanez-Mo, M. et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066 (2015).
pubmed: 25979354
doi: 10.3402/jev.v4.27066
Heidrich, I. et al. Liquid biopsies: Potential and challenges. Int. J. Cancer 148, 528–545 (2020).
pubmed: 32683679
doi: 10.1002/ijc.33217
Minciacchi, V. R. et al. Extracellular vesicles for liquid biopsy in prostate cancer: Where are we and where are we headed?. Prostate Cancer Prostatic DIS 20(3), 251–258 (2017).
pubmed: 28374743
pmcid: 5569339
doi: 10.1038/pcan.2017.7
Quandt, D. et al. Implementing liquid biopsies into clinical decision making for cancer immunotherapy. Oncotarget 8, 48507–48520 (2017).
pubmed: 28501851
pmcid: 5564665
doi: 10.18632/oncotarget.17397
Gould, S. J. & Raposo, G. As we wait: Coping with an imperfect nomenclature for extracellular vesicles. J. Extracell. Vesicles 2, 20389 (2013).
doi: 10.3402/jev.v2i0.20389
Thery, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7(1), 1535750 (2018).
pubmed: 30637094
pmcid: 6322352
doi: 10.1080/20013078.2018.1535750
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. 35(2), 256–263 (1984).
pubmed: 6151502
Pan, B. T. et al. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J. Cell. Biol. 101(3), 942–948 (1985).
pubmed: 2993317
doi: 10.1083/jcb.101.3.942
Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20(3), 332–343 (2018).
pubmed: 29459780
pmcid: 5931706
doi: 10.1038/s41556-018-0040-4
Akers, J. C. et al. Biogenesis of extracellular vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J. Neurooncol. 113(1), 1–11 (2013).
pubmed: 23456661
pmcid: 5533094
doi: 10.1007/s11060-013-1084-8
Tricarico, C., Clancy, J. & D’Souza-Schorey, C. Biology and biogenesis of shed microvesicles. Small GTPases 8(4), 220–232 (2017).
pubmed: 27494381
doi: 10.1080/21541248.2016.1215283
Willms, E. et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci. Rep. 6, 22519 (2016).
pubmed: 26931825
pmcid: 4773763
doi: 10.1038/srep22519
Zabeo, D. et al. Exosomes purified from a single cell type have diverse morphology. J. Extracell. Vesicles 6(1), 1329476 (2017).
pubmed: 28717422
pmcid: 5505001
doi: 10.1080/20013078.2017.1329476
Konoshenko, M. Y. et al. Isolation of extracellular vesicles: General methodologies and latest trends. Biomed. Res. Int. 2018, 8545347 (2018).
pubmed: 29662902
pmcid: 5831698
doi: 10.1155/2018/8545347
Tauro, B. J. et al. 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 (2012).
pubmed: 22285593
doi: 10.1016/j.ymeth.2012.01.002
Kalra, H. et al. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics 13(22), 3354–3364 (2013).
pubmed: 24115447
doi: 10.1002/pmic.201300282
Thery, C. et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 30, 3–22 (2006).
doi: 10.1002/0471143030.cb0322s30
Witwer, K. W. et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles 2, 20360 (2013).
doi: 10.3402/jev.v2i0.20360
Consortium, E.-T. et al. EV-TRACK: Transparent reporting and centralizing knowledge in extracellular vesicle research. Nat. Methods 14(3), 228–232 (2017).
doi: 10.1038/nmeth.4185
Van Deun, J. et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J. Extracell. Vesicles 3, 24858 (2014).
doi: 10.3402/jev.v3.24858
Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).
pubmed: 26194179
doi: 10.3402/jev.v4.27031
Mistry, K. & Cable, G. Meta-analysis of prostate-specific antigen and digital rectal examination as screening tests for prostate carcinoma. J. Am. Board Fam. Pract. 16(2), 95–101 (2003).
pubmed: 12665174
doi: 10.3122/jabfm.16.2.95
Andreoiu, M. & Cheng, L. Multifocal prostate cancer: Biologic, prognostic, and therapeutic implications. Hum. Pathol. 41(6), 781–793 (2010).
pubmed: 20466122
doi: 10.1016/j.humpath.2010.02.011
Andriole, G. L. et al. Prostate cancer screening in the randomized Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial: Mortality results after 13 years of follow-up. J. Natl. Cancer Inst. 104(2), 125–132 (2012).
pubmed: 22228146
pmcid: 3260132
doi: 10.1093/jnci/djr500
Silver, D. A. et al. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin. Cancer Res. 3(1), 81–85 (1997).
pubmed: 9815541
O’Keefe, D. S., Bacich, D. J. & Heston, W. D. W. Prostate Specific Membrane Antigen, in Prostate Cancer: Biology, Genetics, and the New Therapeutics (Humana Press, 2001).
Duijvesz, D. et al. Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer. PLoS ONE 8, e82589 (2013).
pubmed: 24391718
pmcid: 3876995
doi: 10.1371/journal.pone.0082589
Krishn, S. R. et al. Prostate cancer sheds the alphavbeta3 integrin in vivo through exosomes. Matrix Biol. 77, 41–57 (2019).
pubmed: 30098419
doi: 10.1016/j.matbio.2018.08.004
Di Cristofano, A. & Pandolfi, P. P. The multiple roles of PTEN in tumor suppression. Cell 100(4), 387–390 (2000).
pubmed: 10693755
doi: 10.1016/S0092-8674(00)80674-1
Bebelman, M. P. et al. Biogenesis and function of extracellular vesicles in cancer. Pharmacol. Ther. 188, 1–11 (2018).
pubmed: 29476772
doi: 10.1016/j.pharmthera.2018.02.013
Uzoh, C. C. et al. PTEN-mediated pathways and their association with treatment-resistant prostate cancer. BJU Int. 104(4), 556–561 (2009).
pubmed: 19220271
doi: 10.1111/j.1464-410X.2009.08411.x
Milella, M. et al. PTEN: Multiple functions in human malignant tumors. Front. Oncol. 5, 24 (2015).
pubmed: 25763354
pmcid: 4329810
doi: 10.3389/fonc.2015.00024
Gabriel, K. et al. Regulation of the tumor suppressor PTEN through exosomes: A diagnostic potential for prostate cancer. PLoS ONE 8(7), e70047 (2013).
pubmed: 23936141
pmcid: 3723640
doi: 10.1371/journal.pone.0070047
Borcherding, N. et al. ROR1, an embryonic protein with an emerging role in cancer biology. Protein Cell 5(7), 496–502 (2014).
pubmed: 24752542
pmcid: 4085287
doi: 10.1007/s13238-014-0059-7
Yamada, T. et al. Comparison of methods for isolating exosomes from bovine milk. J. Vet. Med. Sci. 74(11), 1523–1525 (2012).
pubmed: 22785357
doi: 10.1292/jvms.12-0032
Linares, R. et al. High-speed centrifugation induces aggregation of extracellular vesicles. J. Extracell. Vesicles 4, 29509 (2015).
pubmed: 26700615
doi: 10.3402/jev.v4.29509
Maas, S. L. et al. Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J. Control Release 200, 87–96 (2015).
pubmed: 25555362
pmcid: 4324667
doi: 10.1016/j.jconrel.2014.12.041
Geeurickx, E. et al. The generation and use of recombinant extracellular vesicles as biological reference material. Nat. Commun. 10(1), 3288 (2019).
pubmed: 31337761
pmcid: 6650486
doi: 10.1038/s41467-019-11182-0
Gorgens, A. et al. Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. J. Extracell. Vesicles 8(1), 1587567 (2019).
pubmed: 30949308
pmcid: 6442110
doi: 10.1080/20013078.2019.1587567
Taylor, D. D. & Shah, S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87, 3–10 (2015).
pubmed: 25766927
doi: 10.1016/j.ymeth.2015.02.019
Dhondt, B. et al. Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. J. Extracell. Vesicles 9(1), 1736935 (2020).
pubmed: 32284825
pmcid: 7144211
doi: 10.1080/20013078.2020.1736935
Hopkins, B. D. et al. A secreted PTEN phosphatase that enters cells to alter signaling and survival. Science 341(6144), 399–402 (2013).
pubmed: 23744781
pmcid: 3935617
doi: 10.1126/science.1234907
Cheruvanky, A. et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am. J. Physiol. Renal Physiol. 292(5), F1657–F1661 (2007).
pubmed: 17229675
doi: 10.1152/ajprenal.00434.2006
Vergauwen, G. et al. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Sci. Rep. 7(1), 2704 (2017).
pubmed: 28577337
pmcid: 5457435
doi: 10.1038/s41598-017-02599-y
Nordin, J. Z. et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11(4), 879–883 (2015).
pubmed: 25659648
doi: 10.1016/j.nano.2015.01.003
Osteikoetxea, X. et al. Differential detergent sensitivity of extracellular vesicle subpopulations. Org. Biomol. Chem. 13(38), 9775–9782 (2015).
pubmed: 26264754
doi: 10.1039/C5OB01451D
Yoshioka, Y. et al. Comparative marker analysis of extracellular vesicles in different human cancer types. J. Extracell Vesicles. 2, 20424 (2013).
doi: 10.3402/jev.v2i0.20424
Li, J. et al. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles. J. Extracell. Vesicles 4, 26883 (2015).
pubmed: 26022510
doi: 10.3402/jev.v4.26883
Hurwitz, S. N. et al. Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers. Oncotarget 7(52), 86999–87015 (2016).
pubmed: 27894104
pmcid: 5341331
doi: 10.18632/oncotarget.13569
Sharp, A. et al. Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer. J. Clin. Invest. 129(1), 192–208 (2019).
pubmed: 30334814
doi: 10.1172/JCI122819
Jorgensen, M. et al. Extracellular Vesicle (EV) Array: Microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J. Extracell. Vesicles 2, 20920 (2013).
doi: 10.3402/jev.v2i0.20920