title: Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.
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 NH2-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.
authors.affiliation: Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA.
authors.forename: Charles P
authors.initials: CP
authors.lastname: Lai
authors.affiliation: Center for Immunology and Inflammatory Diseases and Department of Medicine, Massachusetts General Hospital, and Program in Immunology, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
authors.forename: Edward Y
authors.initials: EY
authors.lastname: Kim
authors.affiliation: Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA.
authors.forename: Christian E
authors.initials: CE
authors.lastname: Badr
authors.affiliation: Center for Systems Biology, Department of Radiology, Massachusetts General Hospital, and Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
authors.affiliation: Center for Immunology and Inflammatory Diseases and Department of Medicine, Massachusetts General Hospital, and Program in Immunology, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
authors.forename: Thorsten R
authors.initials: TR
authors.lastname: Mempel
authors.affiliation: Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA.
authors.forename: Bakhos A
authors.initials: BA
authors.lastname: Tannous
authors.affiliation: Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA.
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D000067128:Extracellular Vesicles / Q000502:physiology*; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D051379:Mice; D009374:Neoplasms, Experimental / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*
chemical_list: D019922:GAP-43 Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA