BJ Goldie

H Peinado

H Valadi

J Ratajczak

J Skog

JD Arroyo

JL Hood

JR Chevillet

KC Vickers

L Vojtech

Muneesh Tewari

W Zhou

Y Zhang

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Tewari Genome Biology  (2015) 16:47 DOI 10.1186/s13059-015-0613-5Q AND A Open AccessA functional extracellular transcriptome in animals?implications for biology, disease and medicineMuneesh TewariAbstractMuneesh Tewari shares his views on possiblefunctions and applications of RNA transport byextracellular vesicles in animals.intercellular communication was stimulated by studiesIntroductionThe past couple of years have seen a flurry of papers onRNAs that are transported by extracellular vesicles(ECVs) in animals. These RNAs have been implicated inintercellular communication in diverse biological con-texts, from neuronal function to immunosuppressiveeffects of semen to cancer metastasis (see [1-3] for ex-amples). Muneesh Tewari is an Associate Professor atthe University of Michigan, where he studies extracellu-lar RNAs in cancer and other diseases. He is currentlyinvolved in the NIH Extracellular RNA CommunicationConsortium, which focuses on comprehensively charac-terizing the human extracellular transcriptome as well asinvestigating the potential functions and applications ofRNAs in ECVs. Here, he talks to Genome Biology aboutthis exciting area of research and the challenges thatneed to be addressed.What has stimulated the recent interest instudying RNAs transported by extracellularvesicles?The idea that there might be lateral transfer of nucleicacids between animal cells has been around for sometime. As early as the 1970s there were reports thatnaked, purified mRNA could be taken up by mammaliancells and translated, though interest apparently wanedover the next couple of decades and there was no satis-fying mechanism proposed or any independent valid-ation. Then, around 10 years ago, it became clear that inCorrespondence: mtewari@med.umich.eduDepartments of Internal Medicine and Bioengineering, Biointerfaces Instituteand Center for Computational Medicine and Bioinformatics, University ofMichigan, 109 Zina Pitcher Place, 4029 BSRB, SPC 2200, Ann Arbor, MI48109-2200, USA© 2015 Tewari; licensee BioMed Central. This iAttribution License (http://creativecommons.oreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.the nematode Caenorhabditis elegans short interferingRNAs are transported in between cells, and uptake ofRNA from the environment was reported in both C.elegans and several insect species.More recently, interest in mammalian RNA-basedfrom Mariusz Ratajczak's lab in 2006 [4] and from JanLötvall's lab in 2007 [5] that not only suggested thatRNA could be transferred between cells, but proposedas a mechanism that RNA is carried across in extracellu-lar vesicles. Extracellular vesicles themselves have beenknown to exist for decades, and a subclass of theseknown as exosomes has been studied for some time inthe context of being protein-presenting vehicles. But thefinding of RNA being associated with ECVs was a newdiscovery. Ratajczak's team's studies of microvesiclesfrom embryonic stem (ES) cells [4] suggested that thevesicles could transfer ES-cell-specific mRNAs intohematopoietic progenitor cells in a functional form.Lötvall's group [5] purified exosomes from a mast cellline and found that there were RNAs associated withthese exosomes, including both non-coding (nc)RNAs -specifically micro (mi)RNAs - and mRNAs. In cellculture experiments they provided evidence that themRNAs could be transferred to recipient cells in func-tional form, as evidenced by the production of corre-sponding proteins in the recipient cells. Soon after this,in 2008, Johan Skog and Xandra Breakefield [6] providedadditional evidence. They looked at glioblastoma-associated extracellular vesicles from cells transfectedwith green fluorescent protein (GFP)-encoding plasmidDNA. They found that GFP mRNA was associated withthese vesicles and, when added to recipient cells, thevesicles could deliver GFP mRNA to recipient cells inwhich it was then translated to protein.So I think the recent surge in excitement has comefrom the idea that these vesicles may provide a mechan-ism for RNA-mediated intercellular communication. Thebasic idea is that RNA gets packaged into the vesicles,which are then released from the cell and travel (eithers an Open Access article distributed under the terms of the Creative Commonsrg/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Tewari Genome Biology  (2015) 16:47 Page 2 of 4locally or systemically like a hormone) to other cells,where the vesicles are taken up and functional RNA is de-livered to the target cells in which it exerts its action.Once you have a possible mechanism there's a more di-rected path for investigation.Have technological advances been important?As interest in extracellular vesicles has increased, thetechnology has also started to develop. The basic tech-nologies were in place for studying small particles inother settings, far removed from extracellular vesicles inbiology, and these technologies are now starting to beadapted for studying vesicles. An example of this is theuse of nanoparticle tracking analysis technology to quan-tify and determine the size of exosomes, as well as theintroduction of nanopore-based methods for similar pur-poses. The ability to globally characterize RNAs usingprofiling techniques such as RNA-seq has also contrib-uted to progress in the field. Yet there are still manyquestions in the ECV field awaiting technology to an-swer them. I think we're going to see the developmentof more technologies for ECV analysis as the fieldmatures.What do you think is the extent of this transport?It's hard to say at this point. In experimental settingsthere are some very interesting data demonstrating suchRNA transport, where purified preparations of extracel-lular vesicles are added to cells and taken up by the re-cipient cells along with the associated RNAs. But it'shard to say yet whether this is a physiological mechan-ism that is relevant in diverse biological contexts. Is itsomething that's widespread and that we were com-pletely oblivious to before? Or is it going to turn out tobe a niche mechanism that operates in very specific con-texts or in experimental systems only?What kinds of ncRNAs are being found in thesevesicles?It's still very early in this field, but in the studies thathave come out so far very diverse classes of RNAs arebeing reported. Not all studies find the same kinds ofRNAs, though. So far sequences have been found thatmap to miRNAs, long non-coding (lnc)RNAs, otherncRNAs such as small nucleolar (sno)RNAs and tRNAs,as well as RNA reads mapping to mRNAs. So I thinkthere's a high degree of diversity. That said, the studiesare early.It's worth noting than the NIH has initiated a CommonFund program in this area. Common Fund programs areintended to fund transdisciplinary projects in up and com-ing fields, for developing new paradigms as well as devel-oping the new technologies needed to investigate them.The NIH has created a project called the ExtracellularRNA Communication Program (http://commonfund.nih.gov/Exrna/index) and as part of this they are fundingdozens of labs that will be taking up a range of questionsand challenges around extracellular RNAs, including thosefound in vesicles. One of these challenges is to bettercharacterize all extracellular RNAs: both RNAs that areassociated with vesicles, and extracellular RNAs that arenot associated with vesicles and that may be associatedwith proteins or other macromolecules [7,8]. My expect-ation is that in the coming years we will get a lot moredata on the breadth of RNA species that are associatedwith various kinds of extracellular vesicles.What do we know about how certain ncRNAs getinto vesicles for extracellular transport?We really don't know much yet. There are some specu-lations at least for miRNAs, with respect to the miRNAeffector machinery and where it localizes with respect tomembranes inside cells, but really it's too early to sayhow they get loaded.How much evidence is there that the transportedRNAs are functional at their destinations?There is strong evidence that exosomes and other typesof ECVs, when added to recipient cells, can mediatephenotypic effects. There's also evidence in experimentalsettings that some of these phenotypes can be mediatedby ECV-associated RNAs [9]. But the jury is still out onthe extent to which it is specifically RNAs in vesiclesthat mediate ECV-based modulation of target cells innatural physiologic settings.Might the RNA transport in ECVs have other roles?One hypothesis that has been proposed is that one ofthe functions might be to rid the cell of certain RNAs.It's hard to say why the normal RNA degradation ma-chinery in cells wouldn't be able to do this, but it wouldbe interesting to look at whether there are some RNAsthat are not degraded easily by the cell and whetherthese are preferentially enriched in vesicles. One couldspeculate that this might be especially the case for viralRNAs or other infectious RNAs that might escape thecellular RNA degradation machinery.What are the challenges in studying thisphenomenon?ECVs have been studied for some time but this intensefocus on them is recent. As a result one basic challengeis nomenclature. The reality is that there are multipletypes of ECVs and you can distinguish them by morph-ology and size using transmission electron microscopy.They also appear to have different mechanisms of bio-genesis; a lot of this is now being teased out. The morewe know about biogenesis the easier it will be to haveTewari Genome Biology  (2015) 16:47 Page 3 of 4clear and useful nomenclature and definitions for thedifferent types of ECVs. In the meantime the Inter-national Society for Extracellular Vesicles (http://www.isevmeeting.org/) has come up with working definitionsand nomenclature that they plan to refine on a regularbasis.Another challenge is analyzing the vesicles. There aretechnologies that can be used for this, as I mentioned,that in the past have been used for nanoparticlecharacterization, but they have their limitations. Theyare bulk technologies; for example, they can tell youhow many vesicles there are in a population and theirsize distribution, but it is difficult to isolate and analyzea single vesicle in molecular detail. You also have to becareful because protein aggregates might sometimesshow up as vesicles. Single vesicle analysis is very diffi-cult, especially for smaller vesicles like classical exo-somes that are smaller than the diffraction limit of lightand can't be visualized by light microscopy. Thesesmaller vesicles are the size of retroviral particles. Wehave lots of great technologies now to study single cells,such as flow cytometry, which can probably be used tostudy some of the larger vesicles, but single vesicle isola-tion and analysis is still a challenge.Tracking and imaging vesicles in vivo is another chal-lenge. We want to be able to see where they go andwhere they are taken up. Fluorescent labeling can behelpful for some of these studies but there is still morework to be done.There is an additional, critical challenge for testing thehypothesis that RNA transport in ECVs is functional.Ideally, we would like to deplete ECVs of a specific RNAand determine how that affects the function of the ECVsfor a given phenotype. We currently don't have any goodways of doing that. I think some of the CRISPR-CAS9technologies will get us closer to that, and I expect we'llstart seeing those studies in the near future.Can you speculate on how this transport mightchange our understanding of animal physiology?If it turns out that a robust, ECV-based intercellularRNA transport mechanism is broadly operative in naturalsettings, it would change our understanding dramatically,because it suggests that the boundaries between cells aremuch more fluid than we previously thought. The abso-lute number of vesicles is very high: there can be on theorder 1012 to 1013 of these particles per milliliter in thebloodstream. So although the vesicles are very small, nu-merically there are a lot of them around. If the hypothesisproves true, then it challenges the idea that organismshave compartmentalized cells. We know already that cellsexchange information and molecules, but the boundariesmight be much more fluid than we thought, between cells,tissues and organs.For example, in cancer biology there is accumulatingevidence that exosomes are secreted from a primarytumor and go to distant sites in the body, which theyprecondition for metastasis [10,11]. A key challenge is todetermine how much the phenotype is due specificallyto the RNA molecules associated with the vesicles versusother activities of exosomes - for example, through theproteins carried on the cell surface. In our own work wehave found that when analyzed quantitatively, the abso-lute number of miRNA molecules associated on averagewith an exosome is tiny [12]. More work is needed todissect the contribution of RNA associated with ECVs tothe functional effects associated with ECVs.What are the implications for biomedicine?Two main areas are being explored and both are thesubject of large projects in the NIH Extracellular RNACommunication Program (http://commonfund.nih.gov/Exrna/index). One is therapeutic, the idea being that youcan load ECVs with RNAs of therapeutic benefit and in-ject or otherwise deliver them for uptake by target cells.My own bias is that when we co-opt systems that havealready evolved for a purpose we are interested in, itturns out to be successful more of the time than whenwe try to engineer systems. Thus, if ECVs are a natural,physiologic mechanism of RNA delivery, then I wouldexpect that it could be very powerful therapeutically.There could also potentially be fewer safety concernswith this approach than with putting more artificial, syn-thetic materials into the body.An important question is how much RNA you canload into ECVs. As mentioned above, we did a study ofnaturally occurring exosomes and tried to quantify howmany RNA molecules on average are in each exosome,and we found that they were very scarce. However, thismay not be true when you load exosomes experimentally.A second area is diagnostics using RNA in vesicles.There are multiple companies now working on exosome-based diagnostics, trying to measure RNAs - not justncRNAs but also mutated sequences in mRNAs in pa-tients with cancer. Johan Skog and Xandra Breakefield infact provided initial evidence for this approach in theirpaper in 2008 on glioblastoma-associated vesicles [6]. Theidea is that cancer cells release vesicles into the circula-tion, so this is a way of sampling the tumor without havingto do a biopsy. Vesicles would serve as replacement for abiopsy to look at gene expression profiles as well as spe-cific mutant RNAs that are present. This could conceiv-ably be used to look over time at the evolution of thetumor. There are some theoretical advantages over usingcirculating tumor DNA in that gene expression profilesmeasured from ECV-associated RNAs could provide moreinformation about dynamic changes in tumor cell statesthan DNA, which tends to remain more static. But thereTewari Genome Biology  (2015) 16:47 Page 4 of 4are a lot of hurdles and unknowns. One challenge is isolat-ing the vesicles that come from the tumor from the rest ofthe vesicles in the bloodstream - this can be challengingbecause there are a lot of background vesicles!Competing interestsThe author reports that he is a paid scientific advisor to Miroculus, Inc.,which is developing a platform for microRNA detection, including ofextracellular microRNAs present in body fluids.AcknowledgementsThe author acknowledges support from the following sources, which hascontributed to the perspectives and ideas presented in this article: NationalInstitutes of Health (NIH) Transformative R01 Grant R01DK085714, NIHExtracellular RNA Communication Common Fund Grant U01 HL126499-01,Stand Up To Cancer Foundation Innovative Research Grant SU2C-AACR-IRG1109,Damon Runyon-Rachleff Innovation Award, United States Department of DefensePeer-Reviewed Cancer Research Program Award CA100606, and funding from theCanary Foundation and a Prostate Cancer Foundation Creativity Award.References1. Goldie BJ, Dun MD, Lin M, Smith ND, Verrills NM, Dayas CV, et al.Activity-associated miRNA are packaged in Map1b-enriched exosomesreleased from depolarized neurons. Nucleic Acids Res. 2014;42:9195–208.doi:10.1093/nar/gku594.2. Vojtech L, Woo S, Hughes S, Levy C, Ballweber L, Sauteraud RP, et al.Exosomes in human semen carry a distinctive repertoire of smallnon-coding RNAs with potential regulatory functions. Nucleic Acids Res.2014;42:7290–304. doi:10.1093/nar/gku347.3. Zhou W, Fong MY, Min Y, Somlo G, Liu L, Palomares MR, et al. Cancer-secretedmiR-105 destroys vascular endothelial barriers to promote metastasis. CancerCell. 2014;25:501–15. doi:10.1016/j.ccr.2014.03.007.4. Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, et al. Embryonicstem cell-derived microvesicles reprogram hematopoietic progenitors:evidence for horizontal transfer of mRNA and protein delivery. Leukemia.2006;20:847–56. doi:10.1038/sj.leu.2404132.5. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediatedtransfer of mRNAs and microRNAs is a novel mechanism of genetic exchangebetween cells. Nat Cell Biol. 2007;9:654–9. doi:10.1038/ncb1596.6. Skog J, Würdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, et al.Glioblastoma microvesicles transport RNA and proteins that promotetumour growth and provide diagnostic biomarkers. Nat Cell Biol.2008;10:1470–6. doi:10.1038/ncb1800.7. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al.Argonaute2 complexes carry a population of circulating microRNAsindependent of vesicles in human plasma. Proc Natl Acad Sci U S A.2011;108:5003–8. doi:10.1073/pnas.1019055108.8. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT.MicroRNAs are transported in plasma and delivered to recipient cells byhigh-density lipoproteins. Nat Cell Biol. 2011;13:423–33. doi:10.1038/ncb2210.9. Zhang Y, Liu D, Chen X, Li J, Li L, Bian Z, et al. Secreted monocytic miR-150enhances targeted endothelial cell migration. Mol Cell. 2010;39:133–44.doi:10.1016/j.molcel.2010.06.010.10. Hood JL, San RS, Wickline SA. Exosomes released by melanoma cellsprepare sentinel lymph nodes for tumor metastasis. Cancer Res.2011;71:3792–801. doi:10.1158/0008-5472.CAN-10-4455.11. Peinado H, Alečković M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-BuenoG, et al. Melanoma exosomes educate bone marrow progenitor cells towarda pro-metastatic phenotype through MET. Nat Med. 2012;18:883–91.doi:10.1038/nm.2753.12. Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, et al.Quantitative and stoichiometric analysis of the microRNA content ofexosomes. Proc Natl Acad Sci U S A. 2014;111:14888–93. doi:10.1073/pnas.1408301111.

A functional extracellular transcriptome in animals? implications for biology, disease and medicine

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A functional extracellular transcriptome in animals? implications for biology, disease and medicine

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