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Cited by 574Extracellular vesicles in diseaseCellular transport and secretionRNA Interference and Gene Delivery

Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

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Lai, Charles P., Kim, Edward Y., Badr, Christian E., Weissleder, Ralph, Mempel, Thorsten R., Tannous, Bakhos A., Breakefield, Xandra O.·2015·Nature Communications
DOI 10.1038/ncomms8029PMC PMC4435621
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Abstract

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo . Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH 2 -termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.

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  • The presence of GFP in microglia indicates tumor-derived EV uptake.

    “The presence of GFP in microglia thus indicates tumor-derived EV uptake (Figure 2C).”

    From Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21 (2019) · cites this paper as Lai et al., 2015

    The Results section, “Visualizing tumour-derived EVs in vivo,” identifies membrane-targeted PalmGFP as a label for tumour-derived vesicles, supporting the use of that signal to track EVs. But the Discussion says the identity of non-tumour cells taking up EVs remains to be established; the paper does not show uptake by microglia specifically. It also distinguishes membrane-targeted PalmGFP from soluble GFP, so GFP presence alone is not established as proof of EV uptake. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

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Research works printed but not linked: 61

  1. Colombo M. et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J. Cell Sci. 126, 5553– 5565 (2013). 24105262
  2. Lai C. P.-K. & Breakefield X. O. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front. Physiol. 3, 228 (2012). 22754538 PMC3384085
  3. Camussi G., Deregibus M. C., Bruno S., Cantaluppi V. & Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int. 78, 838– 848 (2010). 20703216
  4. Peinado H. et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883– 891 (2012). 22635005 PMC3645291
  5. Vader P., Breakefield X. O. & Wood M. J. A. Extracellular vesicles: emerging targets for cancer therapy. Trends Mol. Med. 20, 385– 393 (2014). 24703619 PMC4082760
  6. EL Andaloussi S., Mäger I., Breakefield X. O. & Wood M. J. A. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347– 357 (2013). 23584393
  7. Skog J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470– 1476 (2008). 19011622 PMC3423894
  8. 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). 17486113
  9. Koumangoye R. B., Sakwe A. M., Goodwin J. S., Patel T. & Ochieng J. Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading. PLoS ONE 6, e24234 (2011). 21915303 PMC3167827
  10. Mittelbrunn M. I. A. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011). 21505438 PMC3104548
  11. Suetsugu A. et al. Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude mouse models. Adv. Drug Deliv. Rev. 65, 1– 8 (2012). 22921594
  12. Sharma S. et al. Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 4, 1921– 1926 (2010). 20218655 PMC2866049
  13. Lai C. P. et al. Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter. ACS Nano 8, 483– 494 (2014). 24383518 PMC3934350
  14. Triola G., Waldmann H. & Hedberg C. Chemical biology of lipidated proteins. ACS Chem. Biol. 7, 87– 99 (2012). 22148864
  15. Zacharias D. A. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913– 916 (2002). 11988576
  16. Zuber M. X., Strittmatter S. M. & Fishman M. C. A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature 341, 345– 348 (1989). 2797153
  17. Kobayashi T., Gu F. & Gruenberg J. Lipids, lipid domains and lipid-protein interactions in endocytic membrane traffic. Semin. Cell Dev. Biol. 9, 517– 526 (1998). 9835639
  18. Dolo V. et al. Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles. Biochim. Biophys. Acta 1486, 265– 274 (2000). 10903477
  19. Cocucci E., Racchetti G., Podini P. & Meldolesi J. Enlargeosome traffic: exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both. Traffic 8, 742– 757 (2007). 17488290
  20. Badr C. E. & Tannous B. A. Bioluminescence imaging: progress and applications. Trends Biotechnol. 29, 624– 633 (2011). 21788092 PMC4314955
  21. Tannous B. A., Kim D.-E., Fernandez J. L., Weissleder R. & Breakefield X. O. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11, 435– 443 (2005). 15727940
  22. Bertrand E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell. 2, 437– 445 (1998). 9809065
  23. Lee Y. S., Kim S. H., Cho J. A. & Kim C. W. Introduction of the CIITA gene into tumor cells produces exosomes with enhanced anti-tumor effects. Exp. Mol. Med. 43, 281– 290 (2011). 21464590 PMC3104250
  24. Thery C. et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 166, 7309– 7318 (2001). 11390481
  25. Witwer K. W. et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles 2, (2013). PMC3760646 24009894
  26. Zhuang X. et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol. Ther. 19, 1769– 1779 (2011). 21915101 PMC3188748
  27. Alvarez-Erviti L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341– 345 (2011). 21423189
  28. Silva A. K. A. et al. Magnetic and photoresponsive theranosomes: translating cell-released vesicles into smart nanovectors for cancer therapy. ACS Nano 7, 4954– 4966 (2013). 23641799
  29. Ohno S.-I. et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21, 185– 191 (2013). 23032975 PMC3538304
  30. Wallace P. K. et al. Tracking antigen-driven responses by flow cytometry: Monitoring proliferation by dye dilution. Cytometry 73A, 1019– 1034 (2008). 18785636
  31. Davis D. M. & Sowinski S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nat. Rev. Mol. Cell Biol. 9, 431– 436 (2008). 18431401
  32. Di Vizio D., Kim J., Hager M., Morello M. & Yang W. Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res. 69, 5601– 5609 (2009). 19549916 PMC2853876
  33. Atai N. A. et al. Heparin blocks transfer of extracellular vesicles between donor and recipient cells. J. Neurooncol. 115, 343– 351 (2013). 24002181 PMC3856724
  34. Alieva M., Ritsma L., Giedt R. J., Weissleder R. & van Rheenen J. Imaging windows for long-term intravital imaging. IntraVital 3, e29917 (2014). 28243510 PMC5312719
  35. Marangoni F., Murooka T. T., Manzo T. & Kim E. Y. The transcription factor NFAT exhibits signal memory during serial T-cell interactions with antigen-presenting cells. Immunity 38, 237– 249 (2013). 23313588 PMC3582823
  36. Bauer C. A. et al. Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction. J. Clin. Invest. 124, 2425– 2440 (2014). 24812664 PMC4089459
  37. Baud V. & Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat. Rev. Drug Discov. 8, 33– 40 (2009). 19116625 PMC2729321
  38. Badr C. E. et al. Suicidal gene therapy in an NF-κB-controlled tumor environment as monitored by a secreted blood reporter. Gene Ther. 18, 445– 451 (2011). 21150937 PMC3776578
  39. Badr C. E. et al. Real-time monitoring of nuclear factor kappaB activity in cultured cells and in animal models. Mol. Imaging 8, 278– 290 (2009). 19796605 PMC2856067
  40. Mittelbrunn M. I. A. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nature Communications 2, 282– 10 (2011). PMC3104548 21505438
  41. Sharma S., Das K., Woo J. & Gimzewski J. K. Nanofilaments on glioblastoma exosomes revealed by peak force microscopy. J. R. Soc. Interface 11, 20131150 (2014). 24402921 PMC3899882
  42. Rieck B. Unexpected durability of PKH 26 staining on rat adipocytes. Cell Biol. Int. 27, 445– 447 (2003). 12758093
  43. Im H. et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat. Biotechnol. 32, 490– 495 (2014). 24752081 PMC4356947
  44. Escrevente C., Keller S., Altevogt P. & Costa J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer 11, 108 (2011). 21439085 PMC3072949
  45. Lionnet T. et al. A transgenic mouse for in vivo detection of endogenous labelled mRNA. Nat. Method 8, 165– 170 (2011). PMC3076588 21240280
  46. Malone R. W., Felgner P. L. & Verma I. M. Cationic liposome-mediated RNA transfection. Proc. Natl Acad. Sci. USA 86, 6077– 6081 (1989). 2762315 PMC297778
  47. Parolini I. et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J. Biol. Chem. 284, 34211– 34222 (2009). 19801663 PMC2797191
  48. Svensson K. J. et al. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1. J. Biol. Chem. 288, 17713– 17724 (2013). 23653359 PMC3682571
  49. Morelli A. E. et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104, 3257– 3266 (2004). 15284116
  50. Nanbo A., Kawanishi E., Yoshida R. & Yoshiyama H. Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J. Virol. 87, 10334– 10347 (2013). 23864627 PMC3753980
  51. Fitzner D. et al. Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. J. Cell Sci. 124, 447– 458 (2011). 21242314
  52. Feng D. et al. Cellular internalization of exosomes occurs through phagocytosis. Traffic 11, 675– 687 (2010). 20136776
  53. Théry C., Ostrowski M. & Segura E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9, 581– 593 (2009). 19498381
  54. Valenti R. et al. Tumor-released microvesicles as vehicles of immunosuppression. Cancer Res. 67, 2912– 2915 (2007). 17409393
  55. Webber J., Steadman R., Mason M. D., Tabi Z. & Clayton A. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res. 70, 9621– 9630 (2010). 21098712
  56. Coussens L. M. & Werb Z. Inflammation and cancer. Nature 420, 860– 867 (2002). 12490959 PMC2803035
  57. McCabe J. B. & Berthiaume L. G. Functional roles for fatty acylated amino-terminal domains in subcellular localization. Mol. Biol. Cell 10, 3771– 3786 (1999). 10564270 PMC25678
  58. Matsuda T. & Cepko C. L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. U.S.A. 104, 1027– 1032 (2007). 17209010 PMC1764220
  59. Waldner C., Roose M. & Ryffel G. U. Red fluorescent Xenopus laevis: a new tool for grafting analysis. BMC Dev. Biol. 9, 37 (2009). 19549299 PMC2706234
  60. Sena-Esteves M., Tebbets J. C., Steffens S., Crombleholme T. & Flake A. W. Optimized large-scale production of high titer lentivirus vector pseudotypes. J. Virol. Methods 122, 131– 139 (2004). 15542136
  61. Boussif O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297– 7301 (1995). 7638184 PMC41326

Cited by

Sorted by citations

Authors
Lai, C. P., Kim, E. Y., Badr, C. E., Weissleder, R., Mempel, T. R., Tannous, B. A., & Breakefield, X. O.
Affiliations
Harvard University (US)
Corresponding author
  • Lai, Charles P.
  • Breakefield, Xandra O.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2015-05-13
Volume
6
Issue
1
Pages
7029-7029
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/ncomms8029
PMC
PMC4435621
OpenAlex
W1872071454
Cited by
574
References
4
Citations by year
2026: 33 · 2025: 36 · 2024: 48 · 2023: 49 · 2022: 61 · 2021: 80 · 2020: 80 · 2019: 57 · 2018: 49 · 2017: 43 · 2016: 32 · 2015: 6
FWCI
18.97
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Voices Against Brain Cancer
  • National Institutes of Health (CA069246, CA179563)
  • Canadian Institutes of Health Research
  • NIH Office of the Director (U19 CA179563, CA179563)
  • National Cancer Institute (P01CA069246, CA069246, R01CA150975, U19CA179563, CA150975, CA179563)
  • National Institute of Allergy and Infectious Diseases (R01AI111595, K99AI073457, P01AI112521, R00AI073457, AI073457)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30DK043351)
  • National Institute of Neurological Disorders and Stroke (P30NS045776)
  • National Center for Research Resources (S10RR023440)
  • Office of Strategic Coordination
  • Common Fund
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
extracellular vesicles, RNA translation, live-cell imaging, MS2 coat protein, horizontal transfer, EV uptake, intercellular communication, bioluminescence reporter, cancer cells
MeSH terms
Extracellular Vesicles, Animals, Gene Expression Regulation, Humans, Neoplasms, Experimental, RNA, Genes, Reporter, GAP-43 Protein, Cell Line, Tumor, Green Fluorescent Proteins, Mice

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