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Isolation and characterization of extracellular vesicle subpopulations from tissues

Abstract

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

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Fig. 1: Representative electron microscopy section of human colon cancer tissue.
Fig. 2: Schematic overview of the isolation procedure for tissue-derived EVs.
Fig. 3: EV characterization (Step 40).
Fig. 4: Iodixanol cushion separation instead of discontinuous iodixanol gradient (Step 40B).
Fig. 5: Step-by-step visualization of the tissue EV isolation procedure.

Data availability

All identified proteins and their expression are provided in the supporting primary research article by Crescitelli et al35. The full-length, unprocessed blots and gels related to Extended Data Fig. 5 have been submitted to the figshare repository (https://figshare.com/articles/figure/Isolation_and_characterization_of_extracellular_vesicles_from_tissues_WesternBlot_Extended_data_Fig_4_pdf/12854744). All the information regarding flow cytometry experiments is available as supplementary information (Supplementary Tables 1 and 2) to this article. All parameters have been submitted to EVtrack (http://evtrack.org/) in the original publication35 (http://evtrack.org/search_results.php?evtraqid=EV190108&pmid=&author=&compl_score=&biofluid=&study_aim=&isolation_method=&species=&prot_methods=&keyword=&part_methods=&year=&submit=1).

The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE90 partner repository with the dataset identifier PXD021694.

References

  1. 1.

    Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    CAS  PubMed  Google Scholar 

  2. 2.

    Xiao, H. et al. Mast cell exosomes promote lung adenocarcinoma cell proliferation—role of KIT-stem cell factor signaling. Cell Commun. Signal. 12, 64 (2014).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Colombo, M., Raposo, G. & Théry, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).

    CAS  PubMed  Google Scholar 

  4. 4.

    Kim, C. W. et al. Extracellular membrane vesicles from tumor cells promote angiogenesis via sphingomyelin. Cancer Res. 62, 6312–6317 (2002).

    CAS  PubMed  Google Scholar 

  5. 5.

    Al-Nedawi, K. & Read, J. Analysis of extracellular vesicles in the tumor microenvironment. Methods Mol. Biol. 1458, 195–202 (2016).

    CAS  PubMed  Google Scholar 

  6. 6.

    Ji, Q. et al. Primary tumors release ITGBL1-rich extracellular vesicles to promote distal metastatic tumor growth through fibroblast-niche formation. Nat. Commun. 11, 1211 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Nishida-Aoki, N. et al. Disruption of circulating extracellular vesicles as a novel therapeutic strategy against cancer metastasis. Mol. Ther. 25, 181–191 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Gangadaran, P., Hong, C. M. & Ahn, B. C. An update on in vivo imaging of extracellular vesicles as drug delivery vehicles. Front. Pharmacol. 9, 169 (2018).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Luga, V. et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151, 1542–1556 (2012).

    CAS  PubMed  Google Scholar 

  10. 10.

    Atay, S. et al. Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion. Proc. Natl Acad. Sci. U. S. A. 111, 711–716 (2014).

    CAS  PubMed  Google Scholar 

  11. 11.

    Qu, J. L. et al. Gastric cancer exosomes promote tumour cell proliferation through PI3K/Akt and MAPK/ERK activation. Dig. Liver Dis. 41, 875–880 (2009).

    CAS  PubMed  Google Scholar 

  12. 12.

    Urabe, F. et al. Extracellular vesicles as biomarkers and therapeutic targets for cancer. Am. J. Physiol. Cell Physiol. 318, C29–C39 (2020).

    CAS  PubMed  Google Scholar 

  13. 13.

    Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Crescitelli, R. et al. Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J. Extracell. Vesicles 2, 1 (2013).

    Google Scholar 

  15. 15.

    Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad. Sci. U. S. A. 113, E968–E977 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Lässer, C. et al. Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. RNA Biol. 14, 58–72 (2017).

    PubMed  Google Scholar 

  17. 17.

    Zabeo, D. et al. Exosomes purified from a single cell type have diverse morphology. J. Extracell. Vesicles 6, 1329476 (2017).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Willms, E. et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci. Rep. 6, 22519 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lässer, C. et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J. Transl. Med. 9, 9 (2011).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Keller, S., Ridinger, J., Rupp, A. K., Janssen, J. W. & Altevogt, P. Body fluid derived exosomes as a novel template for clinical diagnostics. J. Transl. Med. 9, 86 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Höög, J. L. & Lötvall, J. Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J. Extracell. Vesicles 4, 28680 (2015).

    PubMed  Google Scholar 

  22. 22.

    Domcke, S., Sinha, R., Levine, D. A., Sander, C. & Schultz, N. Evaluating cell lines as tumour models by comparison of genomic profiles. Nat. Commun. 4, 2126 (2013).

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Chen, B., Sirota, M., Fan-Minogue, H., Hadley, D. & Butte, A. J. Relating hepatocellular carcinoma tumor samples and cell lines using gene expression data in translational research. BMC Med. Genomics 8,, S5 (2015).

    Google Scholar 

  24. 24.

    Allen, M., Bjerke, M., Edlund, H., Nelander, S. & Westermark, B. Origin of the U87MG glioma cell line: good news and bad news. Sci. Transl. Med. 8, 354re353 (2016).

    Google Scholar 

  25. 25.

    Perez-Gonzalez, R., Gauthier, S. A., Kumar, A. & Levy, E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J. Biol. Chem. 287, 43108–43115 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Gallart-Palau, X., Serra, A. & Sze, S. K. Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR. Mol. Neurodegener. 11, 41 (2016).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Vella, L. et al. A rigorous method to enrich for exosomes from brain tissue. J. Extracell. Vesicles 6, 1348885 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Hurwitz, S. N. et al. An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease. J. Neurosci. Methods 307, 210–220 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hurwitz, S. N., Olcese, J. M. & Meckes, D. G., Jr. Extraction of extracellular vesicles from whole tissue. J. Vis. Exp. (144), e59143 (2019).

  30. 30.

    Huang, Y. et al. Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles. J. Extracell. Vesicles 9, 1 (2020).

    Google Scholar 

  31. 31.

    Yelamanchili, S. V. et al. MiR-21 in extracellular vesicles leads to neurotoxicity via TLR7 signaling in SIV neurological disease. PloS Pathog. 11, e1005032 (2015).

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Banigan, M. G. et al. Differential expression of exosomal microRNAs in prefrontal cortices of schizophrenia and bipolar disorder patients. PLoS One 8, e48814 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Polanco, J. C., Scicluna, B. J., Hill, A. F. & Götz, J. Extracellular vesicles isolated from the brains of rTg4510 mice seed tau protein aggregation in a threshold-dependent manner. J. Biol. Chem. 291, 12445–12466 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Jang, S. C. et al. Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma. J. Extracell. Vesicles 8, 1635420 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Crescitelli, R. et al. Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. J. Extracell. Vesicles 9, 1722433 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lunt, S. J., Chaudary, N. & Hill, R. P. The tumor microenvironment and metastatic disease. Clin. Exp. Metastasis 26, 19–34 (2009).

    PubMed  Google Scholar 

  37. 37.

    Su, M.-J., Parayath, N. N. & Amiji, M. M. Exosome-mediated communication in the tumor microenvironment. In Diagnostic and Therapeutic Applications of Exosomes in Cancer (eds. Amiji, M. & Ramesh, R.) 187–218 (Academic Press, London, UK, 2018).

  38. 38.

    Skalnikova, H. K. et al. Isolation and characterization of small extracellular vesicles from porcine blood plasma, cerebrospinal fluid, and seminal plasma. Proteomes 7, 17 (2019).

    CAS  PubMed  Google Scholar 

  39. 39.

    Carnino, J. M., Lee, H. & Jin, Y. Isolation and characterization of extracellular vesicles from Broncho-alveolar lavage fluid: a review and comparison of different methods. Respir. Res. 20, 240 (2019).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Monguió-Tortajada, M. et al. Extracellular-vesicle isolation from different biological fluids by size-exclusion chromatography. Curr. Protoc. Stem Cell Biol. 49, e82 (2019).

    PubMed  Google Scholar 

  41. 41.

    Momen-Heravi, F. et al. Current methods for the isolation of extracellular vesicles. Biol. Chem. 394, 1253–1262 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Balaj, L. et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2, 180 (2011).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Steenbeek, S. C. et al. Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles. Embo J. 37,, e98357 (2018).

    Google Scholar 

  44. 44.

    Cianciaruso, C. et al. Molecular profiling and functional analysis of macrophage-derived tumor extracellular vesicles. Cell Rep. 27, 3062–3080.e11 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445.e18 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Yousef, H., Czupalla, C. J., Lee, D., Butcher, E. C. & Wyss-Coray, T. Papain-based single cell isolation of primary murine brain endothelial cells using flow cytometry. Bio Protoc. 8, e3091 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Angyal, A. et al. CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin-FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner. Int. Immunol. 22, 71–80 (2010).

    CAS  PubMed  Google Scholar 

  48. 48.

    Fan, Y. et al. Low intraprostatic DHT promotes the infiltration of CD8+ T cells in BPH tissues via modulation of CCL5 secretion. Mediators Inflamm. 2014, 397815 (2014).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Benck, C. J., Martinov, T., Fife, B. T. & Chatterjea, D. Isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation. J. Vis. Exp. (107), e53638 (2016).

  50. 50.

    Uchea, C., Owen, S. F. & Chipman, J. K. Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures. Toxicol. Res. (Camb.) 4, 494–507 (2015).

    CAS  PubMed Central  Google Scholar 

  51. 51.

    Autengruber, A., Gereke, M., Hansen, G., Hennig, C. & Bruder, D. Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function. Eur. J. Microbiol. Immunol. (Bp 2, 112–120 (2012).

  52. 52.

    Möller, K., Stahl, T., Boltze, J. & Wagner, D. C. Isolation of inflammatory cells from rat brain tissue after stroke. Exp. Transl. Stroke Med. 4, 20 (2012).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Takeuchi, Y., Higuchi, K., Yatabe, T., Miwa, M. & Yoshizaki, G. Development of spermatogonial cell transplantation in Nibe croaker, Nibea mitsukurii (Perciformes, Sciaenidae). Biol. Reprod. 81, 1055–1063 (2009).

    CAS  PubMed  Google Scholar 

  54. 54.

    Legroux, L. et al. An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry. J. Neurosci. Methods 247, 23–31 (2015).

    CAS  PubMed  Google Scholar 

  55. 55.

    Berdnikovs, S., Abdala-Valencia, H. & Cook-Mills, J. M. Endothelial cell PTP1B regulates leukocyte recruitment during allergic inflammation. Am. J. Physiol. Lung Cell. Mol. Physiol. 304, L240–L249 (2013).

    CAS  PubMed  Google Scholar 

  56. 56.

    Filtjens, J. et al. Expression of the inhibitory Ly49E receptor is not critically involved in the immune response against cutaneous, pulmonary or liver tumours. Sci. Rep. 6, 30564 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Welte, Y., Davies, C., Schäfer, R. & Regenbrecht, C. R. Patient derived cell culture and isolation of CD133+ putative cancer stem cells from melanoma. J. Vis. Exp. (73), e50200 (2013).

  58. 58.

    Dunleavey, J. M. et al. Vascular channels formed by subpopulations of PECAM1+ melanoma cells. Nat. Commun. 5, 5200 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Quintana, E. et al. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell 18, 510–523 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Zhou, J. et al. Colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing. J. Vis. Exp. (55), 3322 (2011).

  61. 61.

    Weigmann, B. et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat. Protoc. 2, 2307–2311 (2007).

    CAS  PubMed  Google Scholar 

  62. 62.

    Lunavat, T. R. et al. BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells. Proc. Natl Acad. Sci. U. S. A. 114, E5930–E5939 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Jeurissen, S. et al. The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. Cell Adh. Mig.r 11, 196–204 (2017).

    CAS  Google Scholar 

  64. 64.

    Jingushi, K. et al. Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin. Int. J. Cancer 142, 607–617 (2018).

    CAS  PubMed  Google Scholar 

  65. 65.

    Van Deun, J. et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J. Extracell. Vesicles 3, 1 (2014).

    Google Scholar 

  66. 66.

    Chen, C., Lin, B. R., Hsu, M. Y. & Cheng, C. M. Paper-based devices for isolation and characterization of extracellular vesicles. J. Vis. Exp. (98), e52722 (2015).

  67. 67.

    Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22 (2006).

    PubMed  Google Scholar 

  68. 68.

    Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V. & Laktionov, P. P. Isolation of extracellular vesicles: general methodologies and latest trends. Biomed. Res. Int. 2018, 8545347 (2018).

    PubMed  PubMed Central  Google Scholar 

  69. 69.

    Jang, S. C. et al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano 7, 7698–7710 (2013).

    CAS  PubMed  Google Scholar 

  70. 70.

    Hyatt, G. W. & Wilber, M. C. The storage of human tissues for surgical use. Postgrad. Med. J. 35, 338–343 (1959).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    World MedicalAssociation. World medical association declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310, 2191–2194 (2013).

    Google Scholar 

  72. 72.

    Haukaas, T. H. et al. Impact of freezing delay time on tissue samples for metabolomic studies. Front. Oncol. 6, 17 (2016).

    PubMed  PubMed Central  Google Scholar 

  73. 73.

    Bæk, R., Søndergaard, E. K. L., Varming, K. & Jørgensen, M. M. The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray. J. Immunol. Methods 438, 11–20 (2016).

    PubMed  Google Scholar 

  74. 74.

    Yuana, Y. et al. Handling and storage of human body fluids for analysis of extracellular vesicles. J. Extracell. Vesicles 4, 29260 (2015).

    PubMed  Google Scholar 

  75. 75.

    Virues Delgadillo, J., Delorme, S., El-Ayoubi, R., DiRaddo, R. & Hatzikiriakos, S. Effect of freezing on the passive mechanical properties of arterial samples. J. Biomed. Sci. Eng. 3, 645–652 (2010).

    Google Scholar 

  76. 76.

    Han, B. & Bischof, J. C. Engineering challenges in tissue preservation. Cell Preserv. Technol. 2, 91–112 (2004).

    Google Scholar 

  77. 77.

    Nayar, S., Campos, J., Steinthal, N. & Barone, F. Tissue digestion for stromal cell and leukocyte isolation. Methods Mol. Biol. 1591, 225–234 (2017).

    CAS  PubMed  Google Scholar 

  78. 78.

    Kusuma, G. D. et al. To Protect and to Preserve: Novel Preservation Strategies for Extracellular Vesicles. Front Pharmacol 9, 1199 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Jayachandran, M., Miller, V. M., Heit, J. A. & Owen, W. G. Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J. Immunol. Methods 375, 207–214 (2012).

    CAS  PubMed  Google Scholar 

  80. 80.

    Szatanek, R., Baran, J., Siedlar, M. & Baj-Krzyworzeka, M. Isolation of extracellular vesicles: determining the correct approach (review). Int. J. Mol. Med. 36, 11–17 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Théry, 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, 1535750 (2018).

    PubMed  PubMed Central  Google Scholar 

  82. 82.

    Lunavat, T. R. et al. Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells—evidence of unique microRNA cargos. RNA Biol. 12, 810–823 (2015).

    PubMed  PubMed Central  Google Scholar 

  83. 83.

    Cvjetkovic, A. et al. Detailed analysis of protein topology of extracellular vesicles—evidence of unconventional membrane protein orientation. Sci. Rep. 6, 36338 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. 84.

    Tian, Y. et al. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. J. Extracell. Vesicles 9, 1697028 (2020).

    CAS  PubMed  Google Scholar 

  85. 85.

    Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Google Scholar 

  86. 86.

    McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Kim, D.-K. et al. EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles. J. Extracell. Vesicles 2, 1 (2013).

    CAS  Google Scholar 

  88. 88.

    Keerthikumar, S. et al. ExoCarta: a web-based compendium of exosomal cargo. J. Mol. Biol. 428, 688–692 (2016).

    CAS  PubMed  Google Scholar 

  89. 89.

    Kalra, H. et al. Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. PLoS Biol. 10, e1001450 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Deutsch, E. W. et al. The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. Nucleic Acids Res. 45, D1100–D1106 (2016).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Centre for Cellular Imaging at the University of Gothenburg and the National Microscopy Infrastructure (VR-RFI 2016-00968)) for microscopy support and, in particular, M. Micaroni for his assistance in buffer preparation for sample preparation and image acquisition. We thank the Proteomic Core Facility at the University of Gothenburg for performing the LC-tandem MS analysis, in particular, A. Thorsell, J. Fuchs and B.-M. Olsson. The Proteomics Core Facility is grateful to the Inga-Britt and Arne Lundbergs Forskningsstiftlese for the donation of the Orbitrap Fusion Tribrid MS instrument. We thank R. Cattaneo for his assistance with figure preparation. We thank the VBG Group Herman Krefting Foundation for Asthma and Allergy Research for supporting the Krefting Research Centre at the University of Gothenburg. This work was funded by the Swedish Research Council (K2014-85X-22504-01-3), the Swedish Heart and Lung Foundation (20120528), the Swedish Cancer Foundation (CAN2014/844) and the Knut and Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden).

Author information

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Authors

Contributions

R.C. performed most of the experiments and analyzed the data. R.C., C.L. and J.L. designed the study. C.L. performed the flow cytometry and proteomics data analysis. The data were interpreted collectively, and the manuscript was written by R.C. with the support of C.L. and J.L. All authors have read, commented on and given approval of the final version of the manuscript.

Corresponding author

Correspondence to Rossella Crescitelli.

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Competing interests

The authors have developed multiple EV-associated patents for putative clinical utilization: US20200088734A1, United States, R.C., J.L.; WO2020146390A1, WIPO (PCT), R.C., J.L.; GB2574785A, United Kingdom, C.L., J.L.; US20070298118A1, United States, J.L.; CA3017586A1, Canada, J.L.; US20200163998A1, United States, J.L.; and WO2014076137A1, WIPO (PCT), J.L. J.L. owns equity in Codiak BioSciences Inc. and Exocure Biosciences Inc. and consults in the field of EVs through Vesiclebio AB. R.C. and C.L. own equity in Exocure Bioscience Inc.

Additional information

Peer review information Nature Protocols thanks Rienk Nieuwland and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Crescitelli, R. et al. J. Extracell. Vesicles 9, 1722433 (2020): https://doi.org/10.1080/20013078.2020.1722433

Jang, S. C. et al. J. Extracell. Vesicles 8, 1635420 (2019): https://doi.org/10.1080/20013078.2019.1635420

Extended data

Extended Data Fig. 1 Representative sections of mouse and human tissue and EVs isolated from these tissues.

a and b, Sections of mouse tissues from subcutaneous melanoma tissue (a) and colon cancer (b) are shown. EVs isolated from subcutaneous melanoma and colon cancer tissue shown in a1–3 and b1–3 are as follows: lEVs (1), sEVs (2) and a mixture of both lEVs and sEVs isolated by using the iodixanol cushion separation (3). c and d, Sections of tissues isolated from human colon mucosa (c) and colon cancer (d) are shown. EVs in the extracellular space are indicated with red arrows. Mixtures of both lEVs and sEVs isolated by using iodixanol cushion separation from colon mucosa and tumor tissue are shown in c1 and d1 (scale bars: a = 1 µm, a1–a3 = 500 nm, b = 1 µm, b1–b3 = 200 nm, c = 2 µm, the higher-magnification panel in c = 500 nm, c1 = 200 nm, d = 1 µm, the higher-magnification panel in d = 500 nm, d1 = 200 nm). Human samples were collected and processed in alignment with the Regional Ethical Review Board at the University of Gothenburg, Sweden (melanoma metastases #096-12 and #995-16; colon cancer and colon mucosa #118-15) and according to institutional animal use and care guidelines (Directive 2010/63/EU on the protection of animals used for scientific purposes).

Extended Data Fig. 2 Protocol optimization: enzymatic treatment applied at different steps of the procedure.

a, The 2018 Protocol was used to isolate EVs without using collagenase D and DNase I (blue arrows). In the 2019 Protocol, collagenase and DNase I treatment was applied to the re-suspended vesicle-enriched pellets (green arrows). In the Final Protocol, collagenase D and DNase I treatment were applied directly to the tissue pieces after the dissociating step (red arrows). b and c, Micrograms of proteins isolated from lEVs (b) and sEVs (c) normalized to the grams of tissue for each protocol; 2018 Protocol, n = 4; 2019 Protocol, n = 2; Final Protocol 3, n = 13. Human samples were collected and processed in alignment with the Regional Ethical Review Board at the University of Gothenburg, Sweden (#096-12 and #995-16). The patient provided informed written consent. The image is modified from Crescitelli et al.35.

Extended Data Fig. 3 Evaluation of the molecular effects of collagenase D and DNase I on CD9, CD63 and CD81.

a and b, Flow cytometry analysis showing the gating and the expression of CD9, CD63 and CD81 after collagenase D and DNase I treatment of HMC-1 cells (a) and lEVs and sEVs (b). The EVs were bound to anti-CD63 beads before analysis. The viability of HMC-1 cells after enzymatic treatment was evaluated with flow cytometry and 7-AAD. The information about the antibodies, cell line, EV-binding beads, instruments and software used to perform flow cytometry experiments is listed in Supplementary Table 1. The information about the cell and EV populations analyzed in the flow cytometry experiments is given in Supplementary Table 2. c, The expression of CD9, CD63 and CD81 on EVs after collagenase D and DNase I treatment was investigated by ExoView. Enzymatic treatment was performed on HMC1 cells and their conditional media (containing EVs), and the media were loaded onto the chips. The results are presented as the mean, but individual values from three different experiments are shown as well. n = 3. FSC-A, forward scatter area; K, thousand; PE, phycoerithrin; SSC-A, side scatter area. The image is modified from Crescitelli et al.35.

Extended Data Fig. 4 Quantitative proteomic analysis (tandem mass tag (TMT)) of the EV subpopulations isolated from cutaneous melanoma metastatic tissues.

Lists of the top 100 proteins identified from three online EV databases (EVpedia, ExoCarta and VesiclePedia87,88,89) were used to construct a list of common EV proteins. Furthermore, new markers suggested by Kowal and colleagues15 and the protein AGO2 were added to the list. After the duplicates were removed, a multi-group comparison was performed in Qlucore. Proteins differentially expressed in our dataset (P = 0.01, Q = 0.02) are shown in the heat map. Human samples were collected and processed in alignment with the Regional Ethical Review Board at the University of Gothenburg, Sweden (#096-12 and #995-16). The patient provided informed written consent. The image is modified from Crescitelli et al.35.

Extended Data Fig. 5 Validation of EV proteins by western blot.

a, Western blot was used to investigate the presence of vesicle markers in lEVs and sEVs, including CD63, flotillin-1, CD9 and CD81, as well as the endoplasmatic reticulum protein calnexin. 10 μg of proteins was loaded per sample. b, Western blot was performed to validate a selection of proteins from the TMT analysis in all six EV subpopulations from two cutaneous melanoma metastatic tissues, including ADAM10, mitofillin, flotillin-1 and CD81. 4 μg of total protein was loaded per sample except for the western blot of large HD EVs from tumor 1 and 2 and the western blot of small HD EVs from tumor 2. The EV yield was insufficient; thus, 36 µl (maximum volume) of EVs was loaded instead, and they corresponded to 0.6, 0.7 and 2.5 µg of proteins. Human samples were collected and processed in alignment with the Regional Ethical Review Board at the University of Gothenburg, Sweden (#096-12 and #995-16). The patient provided informed written consent. The image is modified from Crescitelli et al.35.

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Supplementary Tables 1 and 2 supporting Extended Data Fig. 3.

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Crescitelli, R., Lässer, C. & Lötvall, J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc 16, 1548–1580 (2021). https://doi.org/10.1038/s41596-020-00466-1

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