This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

Central European Institute of Technology Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

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van Niel G, Carter DRF, Clayton A, Lambert DW, Raposo G, Vader P. Challenges and directions in studying cell–cell communication by extracellular vesicles. Nat Rev Mol Cell Biol. 2022;23:369–382. doi: 10.1038/s41580-022-00460-3. PubMed DOI

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, 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. 2018;7:1535750. doi: 10.1080/20013078.2018.1535750. PubMed DOI PMC

Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404. doi: 10.1002/jev2.12404. PubMed DOI PMC

Jeppesen DK, Fenix AM, Franklin JL, Higginbotham JN, Zhang Q, Zimmerman LJ, et al. Reassessment of Exosome Composition. Cell. 2019;177:428–445.e18. doi: 10.1016/j.cell.2019.02.029. PubMed DOI PMC

Hagiwara K, Ochiya T, Kosaka N. A paradigm shift for extracellular vesicles as small RNA carriers: From cellular waste elimination to therapeutic applications. Drug Deliv Transl Res. 2014;4:31–37. doi: 10.1007/s13346-013-0180-9. PubMed DOI PMC

Abdouh M, Floris M, Gao ZH, Arena V, Arena M, Arena GO. Colorectal cancer-derived extracellular vesicles induce transformation of fibroblasts into colon carcinoma cells. J Exp Clin Cancer Res. 2019;38:1–22. doi: 10.1186/s13046-019-1248-2. PubMed DOI PMC

Elashiry M, Elashiry MM, Elsayed R, Rajendran M, Auersvald C, Zeitoun R, et al. Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo. J Extracell vesicles. 2020;9:1795362. doi: 10.1080/20013078.2020.1795362. PubMed DOI PMC

Ridder K, Sevko A, Heide J, Dams M, Rupp AK, Macas J, et al. Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment. Oncoimmunology. 2015;4:1–8. doi: 10.1080/2162402X.2015.1008371. PubMed DOI PMC

Taverna S, Pucci M, Giallombardo M, Di Bella MA, Santarpia M, Reclusa P, et al. Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway. Sci Rep. 2017;7:1–14. doi: 10.1038/s41598-017-03460-y. PubMed DOI PMC

Cossetti C, Iraci N, Mercer TR, Leonardi T, Alpi E, Drago D, et al. Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells. Mol Cell. 2014;56:193–204. doi: 10.1016/j.molcel.2014.08.020. PubMed DOI PMC

Tkach M, Théry C. Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 2016;164:1226–1232. doi: 10.1016/j.cell.2016.01.043. PubMed DOI

Li YJ, Wu JY, Wang JM, Hu XB, Xiang DX. Emerging strategies for labeling and tracking of extracellular vesicles. J Control Release. 2020;328:141–59. doi: 10.1016/j.jconrel.2020.08.056. PubMed DOI

Lai CP, Kim EY, Badr CE, Weissleder R, Mempel TR, Tannous BA, et al. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nat Commun. 2015;6:1–12. doi: 10.1038/ncomms8029. PubMed DOI PMC

Lázaro-Ibánez E, Al-Jamal KT, Dekker N, Faruqu FN, Saleh AF, Silva AM, et al. Selection of fluorescent, bioluminescent, and radioactive tracers to accurately reflect extracellular vesicle biodistribution in vivo. ACS Nano. 2021;15:3212–3227. doi: 10.1021/acsnano.0c09873. PubMed DOI PMC

Banfai K, Garai K, Ernszt D, Pongracz JE, Kvell K. Transgenic exosomes for thymus regeneration. Front Immunol. 2019;10:1–9. doi: 10.3389/fimmu.2019.00862. PubMed DOI PMC

Naseri Z, Oskuee RK, Jaafari MR, Moghadam MF. Exosome-mediated delivery of functionally active miRNA-142-3p inhibitor reduces tumorigenicity of breast cancer in vitro and in vivo. Int J Nanomedicine. 2018;13:7727–7747. doi: 10.2147/IJN.S182384. PubMed DOI PMC

Grange C, Tapparo M, Bruno S, Chatterjee D, Quesenberry PJ, Tetta C, et al. Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging. Int J Mol Med. 2014;33:1055–1063. doi: 10.3892/ijmm.2014.1663. PubMed DOI PMC

Nicola AM, Frases S, Casadevall A. Lipophilic dye staining of cryptococcus neoformans extracellular vesicles and capsule. Eukaryot Cell. 2009;8:1373–1380. doi: 10.1128/EC.00044-09. PubMed DOI PMC

Reclusa P, Verstraelen P, Taverna S, Gunasekaran M, Pucci M, Pintelon I, et al. Improving extracellular vesicles visualization: From static to motion. Sci Rep. 2020;10:1–9. doi: 10.1038/s41598-020-62920-0. PubMed DOI PMC

Rice BW, Cable MD, Nelson MB. In vivo imaging of light-emitting probes. J Biomed Opt. 2001;6:432. doi: 10.1117/1.1413210. PubMed DOI

Monici M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Annu Rev. 2005;11:227–256. doi: 10.1016/S1387-2656(05)11007-2. PubMed DOI

Karasev MM, Stepanenko OV, Rumyantsev KA, Turoverov KK, Verkhusha VV. Near-infrared fluorescent proteins and their applications. Biochem. 2019;84:32–50. PubMed

Hong G, Antaris AL, Dai H. Near-infrared fluorophores for biomedical imaging. Nat Biomed Eng. 2017;1:0010. doi: 10.1038/s41551-016-0010. DOI

Rautaniemi K, Zini J, Löfman E, Saari H, Haapalehto I, Laukka J, et al. Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles. Nanoscale Adv. 2022;4:226–240. doi: 10.1039/D1NA00755F. PubMed DOI PMC

Pužar Dominkuš P, Stenovec M, Sitar S, Lasič E, Zorec R, Plemenitaš A, et al. PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochim Biophys Acta - Biomembr. 2018;1860:1350–1361. doi: 10.1016/j.bbamem.2018.03.013. PubMed DOI

Takov K, Yellon DM, Davidson SM. Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes. J Extracell Vesicles. 2017;6:1–15. doi: 10.1080/20013078.2017.1388731. PubMed DOI PMC

Collot M, Ashokkumar P, Anton H, Boutant E, Faklaris O, Galli T, et al. MemBright: a family of fluorescent membrane probes for advanced cellular imaging and neuroscience. Cell Chem Biol. 2019;26:600–614.e7. doi: 10.1016/j.chembiol.2019.01.009. PubMed DOI

Loconte L, Arguedas D, El R, Zhou A, Chipont A, Guyonnet L, et al. Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods. J Extracell Vesicles. 2023;12:12384. doi: 10.1002/jev2.12384. PubMed DOI PMC

González MI, González-Arjona M, Santos-Coquillat A, Vaquero J, Vázquez-Ogando E, de Molina A, et al. Covalently labeled fluorescent exosomes for in vitro and in vivo applications. Biomedicines. 2021;9:81. doi: 10.3390/biomedicines9010081. PubMed DOI PMC

Boysen AT, Whitehead B, Stensballe A, Carnerup A, Nylander T, Nejsum P. Fluorescent labeling of helminth extracellular vesicles using an in vivo whole organism approach. Biomedicines. 2020;8:213. doi: 10.3390/biomedicines8070213. PubMed DOI PMC

Gangadaran P, Hong CM, Ahn BC. An update on in vivo imaging of extracellular vesicles as drug delivery vehicles. Front Pharmacol. 2018;9:1–14. doi: 10.3389/fphar.2018.00169. PubMed DOI PMC

Verweij FJ, Balaj L, Boulanger CM, Carter DRF, Compeer EB, D’Angelo G, et al. The power of imaging to understand extracellular vesicle biology in vivo. Nat Methods. 2021;18:1013–1026. doi: 10.1038/s41592-021-01206-3. PubMed DOI PMC

Thorne N, Inglese J, Auld DS. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol. 2010;17:646–657. doi: 10.1016/j.chembiol.2010.05.012. PubMed DOI PMC

Yeh HW, Xiong Y, Wu T, Chen M, Ji A, Li X, et al. ATP-independent bioluminescent reporter variants to improve in vivo imaging. ACS Chem Biol. 2019;14:959–965. doi: 10.1021/acschembio.9b00150. PubMed DOI PMC

Berger F, Paulmurugan R, Bhaumik S, Gambhir SS. Uptake kinetics and biodistribution of 14C-d-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: Impact on bioluminescence based reporter gene imaging. Eur J Nucl Med Mol Imaging. 2008;35:2275–2285. doi: 10.1007/s00259-008-0870-6. PubMed DOI PMC

Iwano S, Sugiyama M, Hama H, Watakabe A, Hasegawa N, Kuchimaru T, et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science. 2018;359:935–939. doi: 10.1126/science.aaq1067. PubMed DOI

Kojima R, Takakura H, Ozawa T, Tada Y, Nagano T, Urano Y. Rational design and development of near-infrared-emitting firefly luciferins available in vivo. Angew Chemie - Int Ed. 2013;52:1175–1179. doi: 10.1002/anie.201205151. PubMed DOI

Harwood KR, Mofford DM, Reddy GR, Miller SC. Identification of mutant firefly luciferases that efficiently utilize aminoluciferins. Chem Biol. 2011;18:1649–1657. doi: 10.1016/j.chembiol.2011.09.019. PubMed DOI PMC

Imai T, Takahashi Y, Nishikawa M, Kato K, Morishita M, Yamashita T, et al. Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice. J Extracell Vesicles. 2015;4:1–8. doi: 10.3402/jev.v4.26238. PubMed DOI PMC

Gupta D, Liang X, Pavlova S, Wiklander OPB, Corso G, Zhao Y, et al. Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging. J Extracell vesicles. 2020;9:1800222. doi: 10.1080/20013078.2020.1800222. PubMed DOI PMC

Hikita T, Miyata M, Watanabe R, Oneyama C. In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter. Sci Rep. 2020;10:1–10. doi: 10.1038/s41598-020-73580-5. PubMed DOI PMC

Wu AYT, Sung YC, Chen YJ, Chou STY, Guo V, Chien JCY, et al. Multiresolution imaging using bioluminescence resonance energy transfer identifies distinct biodistribution profiles of extracellular vesicles and exomeres with redirected tropism. Adv Sci. 2020;7:1–18. doi: 10.1002/advs.202001467. PubMed DOI PMC

Perez GI, Broadbent D, Zarea AA, Dolgikh B, Bernard MP, Withrow A, et al. In vitro and in vivo analysis of extracellular vesicle-mediated metastasis using a bright Red-Shifted Bioluminescent Reporter Protein. Adv Genet. 2022;3:2100055. doi: 10.1002/ggn2.202100055. PubMed DOI PMC

Chu J, Oh Y, Sens A, Ataie N, Dana H, Macklin JJ, et al. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat Biotechnol. 2016;34:760–767. doi: 10.1038/nbt.3550. PubMed DOI PMC

Almeida S, Santos L, Falcão A, Gomes C, Abrunhosa A. In vivo tracking of extracellular vesicles by nuclear imaging: Advances in radiolabeling strategies. Int J Mol Sci. 2020;21:1–13. doi: 10.3390/ijms21249443. PubMed DOI PMC

Gomes CM, Abrunhosa AJ, Ramos P, Pauwels EKJ. Molecular imaging with SPECT as a tool for drug development. Adv Drug Deliv Rev. 2011;63:547–554. doi: 10.1016/j.addr.2010.09.015. PubMed DOI

Khan AA, De Rosales RTM. Radiolabelling of extracellular vesicles for PET and SPECT imaging. Nanotheranostics. 2021;5:256–274. doi: 10.7150/ntno.51676. PubMed DOI PMC

Faruqu FN, Wang JTW, Xu L, McNickle L, Chong EMY, Walters A, et al. Membrane radiolabelling of exosomes for comparative biodistribution analysis in immunocompetent and immunodeficient mice – A novel and universal approach. Theranostics. 2019;9:1666–1682. doi: 10.7150/thno.27891. PubMed DOI PMC

Varga Z, Gyurkó I, Pálóczi K, Buzás EI, Horváth I, Hegedus N, et al. Radiolabeling of extracellular vesicles with 99mTc for quantitative in vivo imaging studies. Cancer Biother Radiopharm. 2016;31:168–173. PubMed

Stéen EJL, Edem PE, Nørregaard K, Jørgensen JT, Shalgunov V, Kjaer A, et al. Pretargeting in nuclear imaging and radionuclide therapy: Improving efficacy of theranostics and nanomedicines. Biomaterials. 2018;179:209–245. doi: 10.1016/j.biomaterials.2018.06.021. PubMed DOI

Royo F, Cossío U, Ruiz De Angulo A, Llop J, Falcon-Perez JM. Modification of the glycosylation of extracellular vesicles alters their biodistribution in mice. Nanoscale. 2019;11:1531–7. doi: 10.1039/C8NR03900C. PubMed DOI

Shi S, Li T, Wen X, Wu SY, Xiong C, Zhao J, et al. Copper-64 labeled PEGylated exosomes for in vivo positron emission tomography and enhanced tumor retention. Bioconjug Chem. 2019;30:2675–83. doi: 10.1021/acs.bioconjchem.9b00587. PubMed DOI PMC

Molavipordanjani S, Khodashenas S, Abedi SM, Moghadam MF, Mardanshahi A, Hosseinimehr SJ. 99mTc-radiolabeled HER2 targeted exosome for tumor imaging. Eur J Pharm Sci. 2020;148:105312. doi: 10.1016/j.ejps.2020.105312. PubMed DOI

Britton MM. Magnetic resonance imaging of chemistry. Chem Soc Rev. 2010;39:4036–4043. doi: 10.1039/b908397a. PubMed DOI

Yokoo T, Bae WC, Hamilton G, Karimi A, Borgstede JP, Bowen BC, et al. A quantitative approach to sequence and image weighting. J Comput Assist Tomogr. 2010;34:317–331. doi: 10.1097/RCT.0b013e3181d3449a. PubMed DOI

Hu L, Wickline SA, Hood JL. Magnetic resonance imaging of melanoma exosomes in lymph nodes. Magn Reson Med. 2015;74:266–271. doi: 10.1002/mrm.25376. PubMed DOI PMC

Han Z, Liu S, Pei Y, Ding Z, Li Y, Wang X, et al. Highly efficient magnetic labelling allows MRI tracking of the homing of stem cell-derived extracellular vesicles following systemic delivery. J Extracell Vesicles. 2021;10:e12054. doi: 10.1002/jev2.12054. PubMed DOI PMC

Busato A, Bonafede R, Bontempi P, Scambi I, Schiaffino L, Benati D, et al. Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: A new method to obtain labeled exosomes. Int J Nanomedicine. 2016;11:2481–2490. PubMed PMC

Shaikh S, Rehman FU, Du T, Jiang H, Yin L, Wang X, et al. Real-time multimodal bioimaging of cancer cells and exosomes through biosynthesized iridium and iron nanoclusters. ACS Appl Mater Interfaces. 2018;10:26056–26063. doi: 10.1021/acsami.8b08975. PubMed DOI

Sancho-Albero M, Ayaz N, Sebastian V, Chirizzi C, Encinas-Gimenez M, Neri G, et al. Superfluorinated extracellular vesicles for in vivo imaging by 19 F-MRI. ACS Appl Mater Interfaces. 2023;15:8974–8985. doi: 10.1021/acsami.2c20566. PubMed DOI PMC

Hwang DW, Choi H, Jang SC, Yoo MY, Park JY, Choi NE, et al. Noninvasive imaging of radiolabeled exosome-mimetic nanovesicle using 99m Tc-HMPAO. Sci Rep. 2015;5:1–10. doi: 10.1038/srep15636. PubMed DOI PMC

Choi H, Kim MY, Kim DH, Yun H, Oh BK, Kim SB, et al. Quantitative biodistribution and pharmacokinetics study of GMP-grade exosomes labeled with89Zr radioisotope in mice and rats. Pharmaceutics. 2022;14:1–19. doi: 10.3390/pharmaceutics14061118. PubMed DOI PMC

Cohen O, Betzer O, Elmaliach-Pnini N, Motiei M, Sadan T, Cohen-Berkman M, et al. “Golden” exosomes as delivery vehicles to target tumors and overcome intratumoral barriers: In vivo tracking in a model for head and neck cancer. Biomater Sci. 2021;9:2103–2114. doi: 10.1039/D0BM01735C. PubMed DOI

Lara P, Palma-Florez S, Salas-Huenuleo E, Polakovicova I, Guerrero S, Lobos-Gonzalez L, et al. Gold nanoparticle based double-labeling of melanoma extracellular vesicles to determine the specificity of uptake by cells and preferential accumulation in small metastatic lung tumors. J Nanobiotechnology. 2020;18:1–17. doi: 10.1186/s12951-020-0573-0. PubMed DOI PMC

Arifin DR, Witwer KW, Bulte JWM. Non-Invasive imaging of extracellular vesicles: Quo vaditis in vivo? J Extracell vesicles. 2022;11:e12241. doi: 10.1002/jev2.12241. PubMed DOI PMC

Zomer A, Steenbeek SC, Maynard C, Van Rheenen J. Studying extracellular vesicle transfer by a Cre-loxP method. Nat Protoc. 2016;11:87–101. doi: 10.1038/nprot.2015.138. PubMed DOI

Zomer A, Maynard C, Verweij FJ, Kamermans A, Schäfer R, Beerling E, et al. In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell. 2015;161:1046–1057. doi: 10.1016/j.cell.2015.04.042. PubMed DOI PMC

Luo W, Dai Y, Chen Z, Yue X, Andrade-Powell KC, Chang J. Spatial and temporal tracking of cardiac exosomes in mouse using a nano-luciferase-CD63 fusion protein. Commun Biol. 2020;3:1–9. doi: 10.1038/s42003-020-0830-7. PubMed DOI PMC

Horodecka K, Düchler M. CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles. Int J Mol Sci. 2021;22:6072. doi: 10.3390/ijms22116072. PubMed DOI PMC

de Jong OG, Murphy DE, Mäger I, Willms E, Garcia-Guerra A, Gitz-Francois JJ, et al. A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA. Nat Commun. 2020;11:1–13. PubMed PMC

Ye Y, Shi Q, Yang T, Xie F, Zhang X, Xu B, et al. In vivo visualized tracking of tumor-derived extracellular vesicles using CRISPR-Cas9 system. Technol Cancer Res Treat. 2022;21:153303382210853. doi: 10.1177/15330338221085370. PubMed DOI PMC

Han C, Qin G. Reporter systems for assessments of extracellular vesicle transfer. Front Cardiovasc Med. 2022;9:1–6. doi: 10.3389/fcvm.2022.922420. PubMed DOI PMC

Verweij FJ, Revenu C, Arras G, Dingli F, Loew D, Pegtel DM, et al. Live tracking of inter-organ communication by endogenous exosomes in vivo. Dev Cell. 2019;48:573–589.e4. doi: 10.1016/j.devcel.2019.01.004. PubMed DOI

Scott A, Sueiro Ballesteros L, Bradshaw M, Tsuji C, Power A, Lorriman J, et al. In Vivo characterization of endogenous cardiovascular extracellular vesicles in larval and adult zebrafish. Arterioscler Thromb Vasc Biol. 2021;41:2454–2468. doi: 10.1161/ATVBAHA.121.316539. PubMed DOI PMC

Blavier L, Nakata R, Neviani P, Sharma K, Shimada H, Benedicto A, et al. The capture of extracellular vesicles endogenously released by xenotransplanted tumours induces an inflammatory reaction in the premetastatic niche. J Extracell Vesicles. 2023;12:12326. doi: 10.1002/jev2.12326. PubMed DOI PMC