In Vivo Tracking of Extracellular Vesicles in Mice Using Fusion Protein Comprising Lactadherin and Gaussia Luciferase

  • Yuki TakahashiEmail author
  • Makiya Nishikawa
  • Yoshinobu Takakura
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)


Extracellular vesicles (EVs) are cell-derived vesicles comprising a lipid bilayer and are found in body fluids, such as blood, sweat, and urine. As EVs, especially exosomes, function as endogenous intercellular delivery tools, their roles in various biological events have been extensively investigated. In addition, they are expected to become safe and effective drug delivery systems (DDS) because of their intrinsic nature. In the development of EV-based DDS, as well as in the investigation of the biological functions of EVs, it is important to analyze the in vivo behavior of EVs by tracking them. Therefore, we have developed a sensitive EV-labeling method to track EVs in vivo by designing a fusion protein comprising lactadherin (LA) (alias milk fat globule-EGF factor 8), a protein that binds to EV membranes through interaction with phosphatidylserine, and Gaussia luciferase (gLuc), a chemiluminescent protein. gLuc-LA-labeled EVs are easily obtained by transfecting EV-producing cells with a gLuc-LA-encoding plasmid vector. Here, we describe methods to label EVs with the fusion protein and to track the labeled EVs in vivo.

Key words

Gaussia luciferase Lactadherin In vivo imaging Extracellular vesicles Exosomes 


  1. 1.
    Yáñez-Mó M, Siljander PR, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066. doi: 10.3402/jev.v4.27066 CrossRefPubMedGoogle Scholar
  2. 2.
    Boukouris S, Mathivanan S (2015) Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl 9:358–367. doi: 10.1002/prca.201400114 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Akers JC, Gonda D, Kim R et al (2013) Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neuro-Oncol 113:1–11. doi: 10.1007/s11060-013-1084-8 CrossRefGoogle Scholar
  4. 4.
    Valadi H, Ekström K, Bossios A et al (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659. doi: 10.1038/ncb1596 CrossRefPubMedGoogle Scholar
  5. 5.
    Turpin D, Truchetet ME, Faustin B et al (2016) Role of extracellular vesicles in autoimmune diseases. Autoimmun Rev 15:174–183. doi: 10.1016/j.autrev.2015.11.004 CrossRefPubMedGoogle Scholar
  6. 6.
    Fernández-Messina L, Gutiérrez-Vázquez C, Rivas-García E et al (2015) Immunomodulatory role of microRNAs transferred by extracellular vesicles. Biol Cell 107:61–77. doi: 10.1111/boc.201400081 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Miller IV, Grunewald TG (2015) Tumour-derived exosomes: tiny envelopes for big stories. Biol Cell 107:287–305. doi: 10.1111/boc.201400095 CrossRefPubMedGoogle Scholar
  8. 8.
    Das S, Halushka MK (2015) Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc Pathol 24:199–206. doi: 10.1016/j.carpath.2015.04.007 CrossRefPubMedGoogle Scholar
  9. 9.
    Alenquer M, Amorim MJ (2015) Exosome biogenesis, regulation, and function in viral infection. Virus 7:5066–5083. doi: 10.3390/v7092862 CrossRefGoogle Scholar
  10. 10.
    Milbank E, Martinez MC, Andriantsitohaina R (2016) Extracellular vesicles: pharmacological modulators of the peripheral and central signals governing obesity. Pharmacol Ther 157:65–83. doi: 10.1016/j.pharmthera.2015.11.002 CrossRefPubMedGoogle Scholar
  11. 11.
    Porro C, Trotta T, Panaro MA (2015) Microvesicles in the brain: biomarker, messenger or mediator? J Neuroimmunol 288:70–78. doi: 10.1016/j.jneuroim.2015.09.006 CrossRefPubMedGoogle Scholar
  12. 12.
    Zhu YG, Feng XM, Abbott J et al (2014) Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin-induced acute lung injury in mice. Stem Cells 32:116–125. doi: 10.1002/stem.1504 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Heldring N, Mäger I, Wood MJ et al (2015) Therapeutic potential of multipotent mesenchymal stromal cells and their extracellular vesicles. Hum Gene Ther 26:506–517. doi: 10.1089/hum.2015.072 CrossRefPubMedGoogle Scholar
  14. 14.
    Yuyama K, Sun H, Mitsutake S et al (2012) Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia. J Biol Chem 287:10977–10989. doi: 10.1074/jbc.M111.324616 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yuyama K, Sun H, Sakai S et al (2014) Decreased amyloid-β pathologies by intracerebral loading of glycosphingolipid-enriched exosomes in Alzheimer model mice. J Biol Chem 289:24488–24498. doi: 10.1074/jbc.M114.577213 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hao S, Moyana T, Xiang J (2007) Review: cancer immunotherapy by exosome-based vaccines. Cancer Biother Radiopharm 22:692–703. doi: 10.1089/cbr.2007.368-R CrossRefPubMedGoogle Scholar
  17. 17.
    Pitt JM, Charrier M, Viaud S et al (2014) Dendritic cell-derived exosomes as immunotherapies in the fight against cancer. J Immunol 193:1006–1011. doi: 10.4049/jimmunol.1400703 CrossRefPubMedGoogle Scholar
  18. 18.
    Vader P, Mol EA, Pasterkamp G et al (2016) Extracellular vesicles for drug delivery. Adv Drug Deliv Rev 106:148–156. doi: 10.1016/j.addr.2016.02.006 CrossRefPubMedGoogle Scholar
  19. 19.
    Takahashi Y, Nishikawa M, Shinotsuka H et al (2013) Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. J Biotechnol 165:77–84. doi: 10.1016/j.jbiotec.2013.03.013 CrossRefPubMedGoogle Scholar
  20. 20.
    Imai T, Takahashi Y, Nishikawa M et al (2015) Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice. J Extracell Vesicles 4:26238. doi: 10.3402/jev.v4.26238 CrossRefPubMedGoogle Scholar
  21. 21.
    Yamashita T, Takahashi Y, Nishikawa M et al (2016) Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation. Eur J Pharm Biopharm 98:1–8. doi: 10.1016/j.ejpb.2015.10.017 CrossRefPubMedGoogle Scholar
  22. 22.
    Morishita M, Takahashi Y, Nishikawa M et al (2015) Quantitative analysis of tissue distribution of the B16BL6-derived exosomes using a streptavidin-lactadherin fusion protein and iodine-125-labeled biotin derivative after intravenous injection in mice. J Pharm Sci 104:705–713. doi: 10.1002/jps.24251 CrossRefPubMedGoogle Scholar
  23. 23.
    Yeo RW, Lai RC, Zhang B et al (2013) Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 65:336–341. doi: 10.1016/j.addr.2012.07.001 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Yuki Takahashi
    • 1
    Email author
  • Makiya Nishikawa
    • 1
  • Yoshinobu Takakura
    • 1
  1. 1.Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical SciencesKyoto UniversityKyotoJapan

Personalised recommendations