84
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper

      research-article
      a , b , c , d , e , f , g , h , g , i , j , k , l , m , n , o , p , q , r , s , t , r , s , u , v , w , x , y , z , e , aa , ab , ac , ad , ae , af , ag , j , ah , j , *
      Journal of Extracellular Vesicles
      Taylor & Francis
      Extracellular vesicles, exosomes, non-coding RNA, mRNA, function, sorting, RNA binding proteins, quantification

      Read this article at

      Bookmark

          Summary

          The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data. 

          ABSTRACT

          The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

          Related collections

          Most cited references162

          • Record: found
          • Abstract: found
          • Article: not found

          Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder.

          Increasing evidence indicates that metabolic disorders in offspring can result from the father's diet, but the mechanism remains unclear. In a paternal mouse model given a high-fat diet (HFD), we showed that a subset of sperm transfer RNA-derived small RNAs (tsRNAs), mainly from 5' transfer RNA halves and ranging in size from 30 to 34 nucleotides, exhibited changes in expression profiles and RNA modifications. Injection of sperm tsRNA fractions from HFD males into normal zygotes generated metabolic disorders in the F1 offspring and altered gene expression of metabolic pathways in early embryos and islets of F1 offspring, which was unrelated to DNA methylation at CpG-enriched regions. Hence, sperm tsRNAs represent a paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorders.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells.

            Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior

              Summary Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.
                Bookmark

                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2017
                7 March 2017
                : 6
                : 1
                : 1286095
                Affiliations
                [ a ]Department of Biology, Swiss Federal Institute of Technology Zurich (ETH Zürich) , Zurich, Switzerland
                [ b ]Department of Biology, Massachusetts Institute of Technology , Cambridge, MA, USA
                [ c ]Department of Nephrology and Hypertension, UMC Utrecht , Utrecht, the Netherlands
                [ d ]Experimental Asthma Research, Priority Area Asthma & Allergy, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL) , Borstel, Germany
                [ e ]Department of Science and Technology, CSIR-Indian Institute of Chemical Biology , Kolkata, India
                [ f ]Department of Genetics, Cell- and Immunobiology, Semmelweis University , Budapest, Hungary
                [ g ]Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh , Edinburgh, UK
                [ h ]IRCCS MultiMedica , Milan, Italy
                [ i ]Cardiovascular Research Institute, Massachusetts General Hospital , Boston, MA, USA
                [ j ]Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University , Utrecht, the Netherlands
                [ k ]Immunology Service, Hospital La Princesa , Madrid, Spain
                [ l ]Department of Molecular Genetics, German Cancer Research Center (DKFZ) , Heidelberg, Germany
                [ m ]Department of Medicine, Helen Diller Family Comprehensive Cancer Center, UC San Francisco , San Francisco, CA, USA
                [ n ]Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora, Australia
                [ o ]Molecular Immunogenetics & Vaccine Research Section, Vaccine Branch, CCR, NCI , Bethesda, MD, USA
                [ p ]Neurogenomics Division, TGen , Phoenix, AZ, USA
                [ q ]Institute of Biomedical Engineering, National Tsing Hua University , Hsinchu, Taiwan
                [ r ]Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School , Boston, MA, USA
                [ s ]Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Sahlgrenska Academy, University of Gothenburg , Gothenburg, Sweden
                [ t ]Department of Experimental Biomedicine and Clinical Neurosciences (BIONEC), University of Palermo , Palermo, Italy
                [ u ]Center for Molecular Medicine, University Medical Center Utrecht & Regenerative Medicine Center , Utrecht, the Netherlands
                [ v ]Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School , Boston, MA, USA
                [ w ]Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK
                [ x ]Institute of Technology, University of Tartu , Tartu, Estonia
                [ y ]Instituto de Investigación del Hospital 12 de Octubre , Madrid, Spain
                [ z ]Institute of Neurology (Edinger Institute), Frankfurt University Medical School , Frankfurt am Main, Germany
                [ aa ]Department of Pathology and Forensic Medicine, Ribeirão Preto School of Medicine, University of Sao Paulo , Sao Paulo, Brazil
                [ ab ]Department of Pathology, Exosomes Research Group, VU University Medical Center , Amsterdam, the Netherlands
                [ ac ]Animal Physiology and Immunology, School of Life Sciences, Technical University of Munich (TUM) Weihenstephan , Freising, Germany
                [ ad ]Laboratory Clinical Chemistry & Haematology, University Medical Center Utrecht , Utrecht, the Netherlands
                [ ae ]Department of Cellular and Molecular Biology, Institute of Biomedical & Health Sciences, Hiroshima University , Hiroshima, Japan
                [ af ]Institut Curie, PSL Research University, INSERM U932 , Paris, France
                [ ag ]Functional Genomics Unit, Institut Pasteur de Montevideo, Nuclear Research Center, Faculty of Science, Universidad de la República , Montevideo, Uruguay
                [ ah ]Department of Molecular and Comparative Pathobiology and Department of Neurology, The Johns Hopkins University School of Medicine , Baltimore, MD, USA
                Author notes
                CONTACT Esther N. M. Nolte-‘t Hoen e.n.m.nolte@ 123456uu.nl Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 2, 3584CM Utrecht, the Netherlands
                [* ]

                These authors contributed equally to this work Except for Bogdan Mateescu, Emma Kowal, and Esther Nolte-‘t Hoen, authors are sorted alphabetically based on their last names

                Author information
                http://orcid.org/0000-0002-8992-7608
                http://orcid.org/0000-0001-5581-2354
                http://orcid.org/0000-0002-9488-7719
                http://orcid.org/0000-0003-1664-4233
                Article
                1286095
                10.1080/20013078.2017.1286095
                5345583
                28326170
                da2e58d6-0185-4188-b50a-7c148978b251
                © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 October 2016
                : 25 December 2016
                Page count
                Figures: 4, Tables: 6, References: 249, Pages: 34
                Funding
                Funded by: National Institutes of Health 10.13039/100000002
                Award ID: Extracellular RNA Communication Consortium
                Funded by: U. S. National Cancer Institute - Center for Cancer Research
                BM is supported by grant 1U19CA179513-01 from the NIH Common Fund. ENMN-TH receives funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement number 337581. MN received funding from FAPESP (Sao Paulo Research Foundation, Proc. No. 12/24574‑3) and CAPES (Coordination for the Improvement of Higher Education Personnel, Proc No. BEX 7057/15-6).
                Categories
                Article
                Original Research Article

                Cell biology,Biochemistry,Molecular biology,Bioinformatics & Computational biology,Biotechnology,Genetics
                mRNA,extracellular vesicles,exosomes,quantification,RNA binding proteins,exosome sorting,function,non-coding RNA,Exosomes packaging,microRNA

                Comments

                Comment on this article