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The power of imaging to understand extracellular vesicle biology in vivo

Abstract

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

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Fig. 1: Timeline of EV imaging milestones and broad overview of microscopy techniques to resolve EVs at different scales.
Fig. 2: Tagging strategies to image EV production.
Fig. 3: Imaging EV propagation in vivo.
Fig. 4: Tagging strategies to image EV interaction, uptake, and fate.

References

  1. 1.

    Van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  2. 2.

    Jiang, D. et al. Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation. Nat. Cell Biol. 21, 966–977 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Huang, Y. et al. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat. Cell Biol. 21, 991–1002 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Nicolás-Ávila, J. A. et al. A network of macrophages supports mitochondrial homeostasis in the heart. Cell 183, 94–109.e23 (2020).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  5. 5.

    Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367–371 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Bálint et al. Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells. Science 368, 897–901 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Marki, A. et al. Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis. J. Exp. Med. 218, e20200551 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Schubert, D. A brief history of adherons: the discovery of brain exosomes. Int. J. Mol. Sci. 21, 1–9 (2020).

    Google Scholar 

  10. 10.

    Harding, C., Heuser, J. & Stahl, P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol. 97, 329–339 (1983).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Raposo, G. et al. B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Yáñez-Mó, M. et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066 (2015).

    PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Budnik, V., Ruiz-Cañada, C. & Wendler, F. Extracellular vesicles round off communication in the nervous system. Nat. Rev. Neurosci. 17, 160–172 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Stahl, P. D. & Raposo, G. Extracellular vesicles: exosomes and microvesicles, integrators of homeostasis. Physiology 34, 169–177 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Boulanger, C. M., Loyer, X., Rautou, P.-E. & Amabile, N. Extracellular vesicles in coronary artery disease. Nat. Rev. Cardiol. 14, 259–272 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Verweij, F. J. et al. Live tracking of inter-organ communication by endogenous exosomes in vivo. Dev. Cell 48, 573–589.e4 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    van der Vos, K. E. et al. Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain. Neuro. Oncol. 18, 58–69 (2016).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  19. 19.

    Hyenne, V. et al. Studying the fate of tumor extracellular vesicles at high spatiotemporal resolution using the zebrafish embryo. Dev. Cell 48, 554–572.e7 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Fazeli, G., Trinkwalder, M., Irmisch, L. & Wehman, A. M. C. elegans midbodies are released, phagocytosed and undergo LC3-dependent degradation independent of macroautophagy. J. Cell Sci. 129, 3721–3731 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Ridder, K. et al. Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation. PLoS Biol. 12, e1001874 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Zomer, A. et al. In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell 161, 1046–1057 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    de Jong, O. G. et al. A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA. Nat. Commun. 11, 1113 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. 24.

    Gonçalves, M. S. T. Fluorescent labeling of biomolecules with organic probes. Chem. Rev. 109, 190–212 (2009).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  25. 25.

    Pužar Dominkuš, P. et al. PKH26 labeling of extracellular vesicles: characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochim. Biophys. Acta Biomembr. 1860, 1350–1361 (2018).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  26. 26.

    Corso, G. et al. Systematic characterization of extracellular vesicles sorting domains and quantification at the single molecule–single vesicle level by fluorescence correlation spectroscopy and single particle imaging. J. Extracell. Vesicles 8, 1663043 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Collot, M. et al. MemBright: a family of fluorescent membrane probes for advanced cellular imaging and neuroscience. Cell Chem. Biol. 26, 600–614.e7 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Dehghani, M., Gulvin, S. M., Flax, J. & Gaborski, T. R. Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size. Preprint at bioRxiv https://doi.org/10.1101/532028 (2019).

  29. 29.

    Kuffler, D. P. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J. Comp. Neurol. 302, 729–738 (1990).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Gray, W. D., Mitchell, A. J. & Searies, C. D. An accurate, precise method for general labeling of extracellular vesicles. MethodsX 2, 488–496 (2015).

    Article  Google Scholar 

  31. 31.

    Chuo, S. T.-Y., Chien, J. C.-Y. & Lai, C. P.-K. Imaging extracellular vesicles: current and emerging methods. J. Biomed. Sci. 25, 91 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Mckinnon, K. M. et al. Labeling extracellular vesicles for nanoscale flow cytometry. Sci. Rep. 7, 1878 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. 33.

    Liao, Z. et al. Acetylcholinesterase is not a generic marker of extracellular vesicles. J. Extracell. Vesicles 8, 1628592 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Lai, C. P. et al. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nat. Commun. 6, 7029 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Mittelbrunn, M. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  36. 36.

    Mathieu, M., Martin-Jaular, L., Lavieu, G. & Théry, C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 21, 9–17 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Badr, C. E. & Tannous, B. A. Bioluminescence imaging: progress and applications. Trends Biotechnol. 29, 624–633 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Takahashi, Y. et al. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. J. Biotechnol. 165, 77–84 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Lai, C. P. et al. Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter. ACS Nano. 8, 483 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Wu, A. Y. T. et al. Multiresolution imaging using bioluminescence resonance energy transfer identifies distinct biodistribution profiles of extracellular vesicles and exomeres with redirected tropism. Adv. Sci. 7, 2001467 (2020).

    CAS  Article  Google Scholar 

  41. 41.

    Hoshino, A. et al. Tumour exosome integrins determine organotropic metastasis. Nature 527, 329–335 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Chen, G. et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature 560, 382–386 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Ghoroghi, S. et al. Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes. eLife 10, 61539 (2021).

    Article  Google Scholar 

  44. 44.

    Zaborowski, M. P. et al. Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles. Sci. Rep. 9, 17387 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. 45.

    Shinoda, H., Shannon, M. & Nagai, T. Fluorescent proteins for investigating biological events in acidic environments. Int. J. Mol. Sci. 19, 1548 (2018).

    PubMed Central  Article  CAS  Google Scholar 

  46. 46.

    Fan, S. et al. Glutamine deprivation alters the origin and function of cancer cell exosomes. EMBO J. 39, e103009 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Sung, B. H., Ketova, T., Hoshino, D., Zijlstra, A. & Weaver, A. M. Directional cell movement through tissues is controlled by exosome secretion. Nat. Commun. 6, 7164 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Sung, B. H. et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells. Nat. Commun. 11, 2092 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Verweij, F. J. et al. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. J. Cell Biol. 217, 1129–1142 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Bebelman, M. P. et al. Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells. Nat. Protoc. 15, 102–121 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Beer, K. B. et al. Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures. Nat. Commun. 10, 3490 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. 52.

    Mustonen, A. M. et al. First in vivo detection and characterization of hyaluronan-coated extracellular vesicles in human synovial fluid. J. Orthop. Res. 34, 1960–1968 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Choudhuri, K. et al. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature 507, 118–123 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Saliba, D. G. et al. Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells. eLife 8, e47528 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Ambrose, A. R., Hazime, K. S., Worboys, J. D., Niembro-Vivanco, O. & Davis, D. M. Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles. Proc. Natl Acad. Sci. USA. 117, 23717–23720 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Kanwar, S. S., Dunlay, C. J., Simeone, D. M. & Nagrath, S. Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14, 1891–1900 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Icha, J., Weber, M., Waters, J. C. & Norden, C. Phototoxicity in live fluorescence microscopy, and how to avoid it. BioEssays 39, 1700003 (2017).

    Article  Google Scholar 

  58. 58.

    Spikes, J. D. Photosensitization in mammalian cells. in Photoimmunology (eds Parrish, J. A. et al.) 23–49 (Springer, 1983).

  59. 59.

    Elgamal, S., Colombo, F., Cottini, F., Byrd, J. C. & Cocucci, E. Imaging intercellular interaction and extracellular vesicle exchange in a co-culture model of chronic lymphocytic leukemia and stromal cells by lattice light-sheet fluorescence microscopy. Methods Enzymol. 645, 79–107 (2020).

    PubMed  Article  PubMed Central  Google Scholar 

  60. 60.

    Buschow, S. I. et al. MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways. Traffic 10, 1528–1542 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  61. 61.

    Hurbain, I. et al. Microvilli-derived extracellular vesicles govern morphogenesis in Drosophila wing epithelium. Preprint at bioRxiv https://doi.org/10.1101/2020.11.01.363697 (2020).

  62. 62.

    González‐Méndez, L. et al. Polarized sorting of Patched enables cytoneme‐mediated Hedgehog reception in the Drosophila wing disc. EMBO J. 39, e103629 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  63. 63.

    Gross, J. C., Chaudhary, V., Bartscherer, K. & Boutros, M. Active Wnt proteins are secreted on exosomes. Nat. Cell Biol. 14, 1036–1045 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  64. 64.

    Matusek, T. et al. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog. Nature 516, 99–103 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  65. 65.

    Gradilla, A. C. et al. Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion. Nat. Commun. 5, 5649 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  66. 66.

    Tassetto, M., Kunitomi, M. & Andino, R. Circulating immune cells mediate a systemic rnai-based adaptive antiviral response in drosophila. Cell 169, 314–325.e13 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. 67.

    Wang, J. et al. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr. Biol. 24, 519–525 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. 68.

    Wehman, A. M., Poggioli, C., Schweinsberg, P., Grant, B. D. & Nance, J. The P4-ATPase TAT-5 inhibits the budding of extracellular vesicles in C. elegans embryos. Curr. Biol. 21, 1951–1959 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. 69.

    Hyenne, V. et al. hRAL-1 controls multivesicular body biogenesis and exosome secretion. J. Cell Biol. 211, 27–37 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. 70.

    Härkönen, K. et al. CD44s Assembles hyaluronan coat on filopodia and extracellular vesicles and induces tumorigenicity of MKN74 gastric carcinoma. Cells Cells 8, 276 (2019).

    Article  CAS  Google Scholar 

  71. 71.

    Verweij, F. J., Hyenne, V., Van Niel, G. & Goetz, J. G. Extracellular vesicles: catching the light in zebrafish. Trends Cell Biol. 29, 770–776 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  72. 72.

    Abels, E. R. et al. Glioblastoma-associated microglia reprogramming is mediated by functional transfer of extracellular miR-21. Cell Rep. 28, 3105–3119.e7 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    Gupta, D. et al. Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging. J. Extracell. vesicles 9, 1800222 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. 74.

    Men, Y. et al. Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS. Nat. Commun. 10, 4136 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  75. 75.

    Baumeister, R. & Ge, L. The worm in us - Caenorhabditis elegans as a model of human disease. Trends Biotechnol. 20, 147–148 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  76. 76.

    Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498–503 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  77. 77.

    Fortini, M. E., Skupski, M. P., Boguski, M. S. & Hariharan, I. K. A survey of human disease gene counterparts in the Drosophila genome. J. Cell Biol. 150, F23–F30 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  78. 78.

    Santoriello, C. & Zon, L. I. Hooked! Modeling human disease in zebrafish. J. Clin. Invest. 122, 2337–2343 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  79. 79.

    Caygill, E. E. & Brand, A. H. The GAL4 system: a versatile system for the manipulation and analysis of gene expression. in Drosophila: Methods and Protocols 2nd edn (ed. Dahmann, C.) 33–52 (Humana Press, 2016).

  80. 80.

    Port, F. et al. A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila. eLife 9, e53865 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  81. 81.

    Albadri, S., De Santis, F., Di Donato, V. & Del Bene, F. CRISPR/Cas9-mediated knockin and knockout in zebrafish. in Genome Editing in Neurosciences. Research and Perspectives in Neurosciences (eds. Jaenisch, R., Zhang, F. & Gage, F.) 41–49 (2017).

  82. 82.

    Muntasell, A., Berger, A. C. & Roche, P. A. T cell-induced secretion of MHC class II-peptide complexes on B cell exosomes. EMBO J. 26, 4263–4272 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  83. 83.

    Lachenal, G. et al. Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity. Mol. Cell. Neurosci. 46, 409–418 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  84. 84.

    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  Article  PubMed Central  Google Scholar 

  85. 85.

    Rocha, S. et al. 3D cellular architecture affects microRNA and protein cargo of extracellular vesicles. Adv. Sci. 6, 1800948 (2019).

    Article  CAS  Google Scholar 

  86. 86.

    Thippabhotla, S., Zhong, C. & He, M. 3D cell culture stimulates the secretion of in vivo like extracellular vesicles. Sci. Rep. 9, 13012 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  87. 87.

    Cao, J. et al. Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury. Stem Cell Res. Ther. 11, 206 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  88. 88.

    Kim, M., Yun, H.-W., Park, D. Y., Choi, B. H. & Min, B.-H. Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells. Tissue Eng. Regen. Med. 15, 427–436 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. 89.

    Lehrich, B. M., Liang, Y. & Fiandaca, M. S. Foetal bovine serum influence on in vitro extracellular vesicle analyses. J. Extracell. Vesicles 10, e12061 (2021).

    PubMed  PubMed Central  Article  Google Scholar 

  90. 90.

    Chen, L., Ma, L. & Yu, L. WGA is a probe for migrasomes. Cell Discov. 5, 13 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  91. 91.

    Ma, L. et al. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 25, 24–38 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  92. 92.

    Addi, C. et al. The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis. Nat. Commun. 11, 1941 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. 93.

    Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  94. 94.

    Cashikar, A. G. & Hanson, P. I. A cell-based assay for CD63-containing extracellular vesicles. PLoS One 14, e0220007 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  95. 95.

    Wegner, C. S. et al. Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5. Histochem. Cell Biol. 133, 41–55 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  96. 96.

    Mathieu, M. et al. Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nat. Commun. https://doi.org/10.1038/s41467-021-24384-2 (2021).

  97. 97.

    Lenzini, S., Bargi, R., Chung, G. & Shin, J. W. Matrix mechanics and water permeation regulate extracellular vesicle transport. Nat. Nanotechnol. 15, 217–223 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  98. 98.

    Mu, W., Rana, S. & Zöller, M. Host matrix modulation by tumor exosomes promotes motility and invasiveness. Neoplasia 15, 875-IN4 (2013).

    Article  CAS  Google Scholar 

  99. 99.

    Wiklander, O. P. B. et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J. Extracell. Vesicles 4, 26316 (2015).

    PubMed  Article  PubMed Central  Google Scholar 

  100. 100.

    Ridder, K. et al. Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment. Oncoimmunology 4, e1008371 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  101. 101.

    Riau, A. K., Ong, H. S., Yam, G. H. F. & Mehta, J. S. Sustained delivery system for stem cell-derived exosomes. Front. Pharmacol. 10, 1368 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. 102.

    Rilla, K. et al. Extracellular vesicles are integral and functional components of the extracellular matrix. Matrix Biol. s 75–76, 201–219 (2019).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  103. 103.

    Pastuzyn, E. D. et al. The neuronal gene arc encodes a repurposed retrotransposon gag protein that mediates intercellular RNA transfer. Cell 172, 275–288.e18 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  104. 104.

    Ashley, J. et al. Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell 172, 262–274.e11 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  105. 105.

    Edgar, J. R., Manna, P. T., Nishimura, S., Banting, G. & Robinson, M. S. Tetherin is an exosomal tether. eLife 5, e17180 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  106. 106.

    Morad, G. et al. Tumor-derived extracellular vesicles breach the intact blood-brain barrier via transcytosis. ACS Nano. 13, 13853 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  107. 107.

    Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341–345 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  108. 108.

    Denzer, K. et al. Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J. Immunol. 165, 1259–1265 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  109. 109.

    Gao, L. et al. Tumor-derived exosomes antagonize innate antiviral immunity. Nat. Immunol. 19, 233–245 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  110. 110.

    Vilcaes, A. A., Chanaday, N. L. & Kavalali, E. T. Interneuronal exchange and functional integration of synaptobrevin via extracellular vesicles. Neuron 109, 971–983.e5 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  111. 111.

    Ko, S. Y. et al. Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake. Commun. Biol. 2, 386 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  112. 112.

    Neumann, C. J. & Cohen, S. M. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 871–880 (1997).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  113. 113.

    Tkach, M. et al. Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. EMBO J. 36, 3012–3028 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  114. 114.

    Heusermann, W. et al. Exosomes surf on filopodia to enter cells at endocytic hot spots and shuttle within endosomes to scan the ER. J. Cell Biol. 213, 173–184 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  115. 115.

    Arasu, U. T., Härkönen, K., Koistinen, A. & Rilla, K. Correlative light and electron microscopy is a powerful tool to study interactions of extracellular vesicles with recipient cells. Exp. Cell. Res. 376, 149–158 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  116. 116.

    Thomou, T. et al. Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature 542, 450–455 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  117. 117.

    Sterzenbach, U. et al. Engineered exosomes as vehicles for biologically active proteins. Mol. Ther. 25, 1269–1278 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  118. 118.

    Kur, I.-M. et al. Neuronal activity triggers uptake of hematopoietic extracellular vesicles in vivo. PLoS Biol. 18, e3000643 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  119. 119.

    Frühbeis, C. et al. Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication. PLoS Biol. 11, e1001604 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  120. 120.

    Khmelinskii, A. et al. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers. Mol. Biol. Cell 27, 360–370 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  121. 121.

    Joshi, B. S., De Beer, M. A., Giepmans, B. N. G. & Zuhorn, I. S. Endocytosis of extracellular vesicles and release of their cargo from endosomes. ACS Nano. 14, 32 (2020).

    Google Scholar 

  122. 122.

    Cao, H. et al. In vivo real-time imaging of extracellular vesicles in liver regeneration via aggregation-induced emission luminogens. ACS Nano. 13, 3522–3533 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  123. 123.

    Webber, J. P. et al. Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes. Oncogene 34, 290–302 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  124. 124.

    Lener, T. et al. Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper. J. Extracell. vesicles 4, 30087 (2015).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  125. 125.

    Fais, S. et al. Evidence-Based clinical use of nanoscale extracellular vesicles in nanomedicine. ACS Nano. 10, 3886–3899 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  126. 126.

    Kalluri, R. & LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 367, eaau6977 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  127. 127.

    Androuin A., Verweij F.J. & Van Niel, G. Zebrafish as a preclinical model for extracellular vesicle-based therapeutic development. Adv. Drug Deliv. Rev. https://doi.org/10.1016/j.addr.2021.05.025 (2021).

  128. 128.

    Liégeois, S., Benedetto, A., Garnier, J.-M., Schwab, Y. & Labouesse, M. The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J. Cell Biol. 173, 949–961 (2006).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  129. 129.

    Koles, K. et al. Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons. J. Biol. Chem. 287, 16820–16834 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  130. 130.

    Corrigan, L. et al. BMP-regulated exosomes from male reproductive glands reprogram female behavior. J. Cell Biol. 206, 671–688 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  131. 131.

    Wolf, P. The nature and significance of platelet products in human plasma. Br. J. Haematol. 13, 269–288 (1967).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  132. 132.

    Nunez, E. A., Wallis, J. & Gershon, M. D. Secretory processes in follicular cells of the bat thyroid. III. The occurrence of extracellular vesicles and colloid droplets during arousal from hibernation. Am. J. Anat. 141, 179–201 (1974).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  133. 133.

    Trams, E. G., Lauter, C. J., Norman Salem, J. & Heine, U. Exfoliation of membrane ecto-enzymes in the form of micro-vesicles. Biochim. Biophys. Acta - Biomembr. 645, 63–70 (1981).

    CAS  Article  Google Scholar 

  134. 134.

    Johnstone, R. M., Bianchini, A. & Teng, K. Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions. Blood 74, 1844–1851 (1989).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  135. 135.

    Heijnen, H. F., Schiel, A. E., Fijnheer, R., Geuze, H. J. & Sixma, J. J. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 94, 3791–3799 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  136. 136.

    Yang, T. et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm. Res. 32, 2003–2014 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

The authors acknowledge financial support from the International Society for Extracellular Vesicles, the French Society of Extracellular vesicles, the Société Française des Microscopies, the ITMO BCDE for their support for the organization of the ISEV workshop ‘EV imaging in vivo’ that provided the basis for this review. We thank the workshop organizing committee members G.D.’A., V.H., E.-M.K.-A., X.L., and K.W. for their help. We thank P. Stahl (Washington University School of Medicine, USA) for stimulating discussions and insight. F.J.V. is supported by INCa 2019-125, E.B.C. thanks M. Dustin for support through ERC AdG 670930. D.R.F.C. is supported by the BBSRC (BB/P006205/1) and Cancer Research UK (A28052). V.H. and J.G.G. are funded by La Ligue contre le Cancer, by INCa (PLBIO19-291), by Plan Cancer (Nanotumor) and through institutional funds from University of Strasbourg and INSERM. K.R. is supported by the UEF Cell and Tissue Imaging Unit, Biocenter Kuopio and Biocenter Finland. We apologize to colleagues for any relevant work that could not be cited due to space restrictions.

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Correspondence to Frederik J. Verweij or Guillaume van Niel.

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D.R.F.C. is employed by Evox Therapeutics. S.E.A. serves on the Scientific Advisory Board of EVOX Therapeutics. All other authors have no competing interests.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Verweij, F.J., Balaj, L., Boulanger, C.M. et al. The power of imaging to understand extracellular vesicle biology in vivo. Nat Methods 18, 1013–1026 (2021). https://doi.org/10.1038/s41592-021-01206-3

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