Frontiers | Apoptotic Tumor Cell-Derived Extracellular Vesicles ...

Cells undergoing apoptosis produce heterogeneous populations of membrane ... Cell (2000) 100:57–70. doi:10.1016/S0092-8674(00)81683-9. ThisarticleispartoftheResearchTopic TheImmunomodulatoryPropertiesofExtracellularVesiclesfromPathogens,ImmuneCellsandNon-ImmuneCells Viewall 12 Articles Articles ShoheiHori TheUniversityofTokyo,Japan NaohiroSeo MieUniversity,Japan AiKotani TokaiUniversityIseharaHospital,Japan Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction:Apoptosis,andtheOnco-RegenerativeNiche(ORN) Apo-EVsandApoptoticBodies Apo-EVProductionMechanisms CargoesandFunctionalActivities ConclusionandFuturePerspectives AuthorContributions ConflictofInterestStatement Funding References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData PERSPECTIVEarticle Front.Immunol.,23May2018 |https://doi.org/10.3389/fimmu.2018.01111 ApoptoticTumorCell-DerivedExtracellularVesiclesasImportantRegulatorsoftheOnco-RegenerativeNiche ChristopherD.Gregory*and IanDransfield MedicalResearchCouncilCentreforInflammationResearchattheUniversityofEdinburgh,TheQueen’sMedicalResearchInstitute,Edinburgh,UnitedKingdom Cellsundergoingapoptosisproduceheterogeneouspopulationsofmembranedelimitedextracellularvesicles(Apo-EVs)whichvarynotonlyinsize—fromtensofnanometerstoseveralmicrons—butalsoinmolecularcompositionandcargo.Apo-EVscarryavarietyofpotentiallybiologicallyactivecomponents,includingsmallmolecules,proteins,andnucleicacids.LargerformsofApo-EVs,commonlytermed“apoptoticbodies,”cancarryorganelles,suchasmitochondriaandnuclearfragments.Moleculesdisplayedonthesurfaceofextracellularvesicles(EVs)cancontributesubstantiallytotheirsize,aswellastheirfunctions.Thusfar,relativelylittleisknownofthefunctionalsignificanceofApo-EVsapartfromtheirrolesinfragmentationofdyingcellsandindicatedimmunomodulatoryactivities.Here,wediscussEVproductionbydyingtumorcellsandconsiderthepossiblerolesofApo-EVsinacelldeath-drivensectorofthetumormicroenvironmentknownastheonco-regenerativeniche(ORN).Weproposethattumor-derivedApo-EVsaresignificantvehiclesoftheORN,functioningascriticalintercellularcommunicatorsthatactivateoncogenictissuerepairandregenerationpathways.WehighlightimportantoutstandingquestionsandsuggestthatApo-EVsmayharbornoveltherapeutictargets. Introduction:Apoptosis,andtheOnco-RegenerativeNiche(ORN) Inadditiontoitsactivitiesindevelopmentalsculptingandadulttissueinvolution,apoptosisisrenownedforitscapacitytoregulatetissueturnoverandhomeostasisinwhich,simplistically,theexpansionofcellpopulationsisbalancedbyregulatedcelldeath(andviceversa).Incancer,thisbalancebetweencellgainandcelllossbecomesdysregulated,resultinginaccumulationoftumorcellsandnetgrowthofneoplastictissues(Figure1).Byeffectingcontrolledcelldeletion,apoptosisimposesabrakeononcogenesis,alogicalconceptthathaslongbeenprovenandiswidelyaccepted.Indeed,inhibitionofthetumorsuppressorfunctionofapoptosisledtothecategorizationofanewclassofoncogenes—BCL2beingtheprototypicmember—thatcouldpromotecellsurvivalthroughsuppressionofapoptosisandtherebyimposeanoncogenicimbalanceonthecellbirth/celldeathequation(1).Furthermore,theapoptosiseffectorprotease,caspase-8,ismutatedinmultiplecancertypesandthesurvivalpathwayPI3K/Akt/mTORisdysregulatedfrequentlyintumors(2).Bycontrast,pro-apoptoticregulatorssuchasp53andBIM(3)amongothershavefirmlyestablishedtumor-suppressiverolesforapoptosis.Fortheseandotherreasons,thecapacitytoevadeapoptosishasbecomeawell-acceptedhallmarkofcancer(4). FIGURE1 Figure1.Left:oncogenicextracellularvesicle(EV)networksintheonco-regenerativeniche(ORN).SchematicrepresentationoftheORNillustratingthepotentialrolesofApo-EVsfromdyingtumorcells(Apo)inprovidingoncogenicsignalstoneighboringcellsintheniche,exemplifiedbymacrophages(M),viabletumorcells(Tu),andendothelialcells(E).WeproposethatApo-EVstargetsuchcellsandmodulatecellularfunctions,includingmacrophagepolarizationtowardareparativephenotype(M2-likeactivationstate),promotionoftumorcellsurvivalandproliferation,andangiogenesis.EVsfromviablecells(Via-EVs)oftumorandstromalcelloriginalsoseemlikelytoparticipateintheseprocesses.Right:mechanismsofcelltargetingandmodulationby(Apo)-EVs.ExamplesofpossiblemodesofinteractionofApo-EVswithtargetcellsthatmayleadtomodulationofcellularfunctionswithorwithouttransferofintactEVcargoes(biologicallyactivecargoesrepresentedbysmallredcircles).1.Membranefusion(receptor-dependentor-independent)permitstransferofEVcargoestocytosoliclocations.2.DockingofEVsthroughreceptor–ligandinteractionmayactivateintracellularsignalingpathwaysintheabsenceofcargotransfer.Ligandssuchasphosphatidylserine(PtdSer)(green)exposedatEVsurfacesmayinteractdirectlyorindirectlywithtargetcellreceptors(examplesinthecaseofPtdSerincludingBAI1,TIM-4,Stabilin2,Axl,Mer,aswellasintegrinsαvβ3andαvβ5).3.Endocyticpathways(includingphagocytosis)resultinginlysosomaldegradationofcargoesarealsolikelytomodulatecellularfunctionssuchasthroughmetabolitesupplyandviareceptorligation.4.PutativeendocyticuptakeofEVswithoutlysosomaldegradation.WeproposethatApo-EVcargoesaretransferredintacttomultipleintracellularcompartmentsviathistypeofpathway. However,setoppositeitstumorsuppressorfunctions,theapoptosismachinerycanendowdyingcellswiththeabilitytostimulateproliferationofneighboringcells,eitheraspartofdevelopmentalprogrammesorintissuerepairandregenerationinadulttissues(5–9).Highlevelsofapoptosisarecommonlyassociatedwithpoorprognosisinmultiplecancertypes(10–17)andexpressionofpro-apoptoticeffectormoleculessuchasactiveCaspase-3andBaxcancorrelatewithaggressivedisease(18,19).Furthermore,low-levelactivationoftheapoptosisprogrammecanpromotegenomicinstabilityandoncogenictransformation(20). Emergingevidencesuggestsstronglythatbothconstitutiveandtherapy-inducedapoptosiscanengenderpro-oncogenicresponsesthatenhancetumorgrowthandcausepost-therapeuticrelapse(21–24).Inthisscenario,tumor-cellapoptosisitselfpromotesimbalanceinthecellbirth/celldeathequationthatultimatelyfavorsnettumorgrowth.SuchregenerativeeffectsofapoptosisinthecontextofthetumormicroenvironmentledoneofustoproposerecentlytheconceptoftheORN:atumor-promotingnetworkoftumorcells,stromalcells,andimmunecellswhich,togetherwithassociatedextracellularcomponents,includingEVs,solublefactorsandmatrixmolecules,isorchestratedbytumor-cellapoptosis(Figure1)(25,26).Wespeculatethatpervasiveapoptotictumorcell-derivedsignalsintheORNprovideimportantpathwaysfortumorgrowth,metastasisandtopost-therapeuticrelapse.Here,weconsiderthepotentialrolesofapoptotictumorcell-derivedEVsinprovidingsuchsignals. Apo-EVsandApoptoticBodies ItisbecomingincreasinglyclearthatEVsareimportantintercellularcommunicationvehiclesinthetumormicroenvironment,shuttlinganarrayofbiologicallyactivemoleculesreciprocallybetweentumorandnon-tumorcells,modulatingthedevelopmentofprimarytumorsandmetastases.WeproposethatApo-EVs—aswellasEVsgeneratedinviablecellsrespondingtotheirapoptoticneighbors—areimportantelementsoftheORN(Figure1).EVproductionisawell-establishedhallmarkofapoptosis,asissurfaceblebbing(zeiosis)oftheplasmamembraneincellsrespondingtoapoptosisstimuli.BlebsmaybeimportantprecursorstoApo-EVs,butitremainsuncleartowhatextenttheprocessofsurfaceblebbingisrelatedmechanisticallytotheproductionofApo-EVs.Here,weusetheterm“Apo-EV”toencompassallclassesofsubcellularvesiclesproducedasaconsequenceofapoptosis.Theseincludesmall(~50–1,000nm)aswellaslargervesicles(1toseveralmicronsindiameter),oftenreferredtoas“apoptoticbodies,”whichharborcaspase-modifiedautoantigens,nuclearremnantscontainingcondensedchromatin,andwell-definedorganelles,suchasmitochondriaandendoplasmicreticulum.AmongthesmallervesiclesarelikelytobeexosomesofendosomalpathwayoriginandbuddingplasmamembraneEVsalsoknownasmicrovesiclesorectosomes(27).AswithallEVs,thesizeofApo-EVsmatters—notonlyinrelationtowhatmolecularandorganellecargoescanbecarriedbutalsowithrespecttothespecificcontributionofmolecularcargoes,suchascellsurfaceproteins,tooverallEVsize(Figure2).WhileApo-EVsareundoubtedlyheterogeneousbothinsizeandcontent(28),theunderlyingcausesofthisheterogeneityremainobscure. FIGURE2 Figure2.Considerationsofvesicularcargoesinrelationtoextracellularvesicle(EV)sizes.SchematicrepresentationsofApo-EVsof~500nm(left)and50nm(right)areshownwithdelimitinglipidbilayer(green),asmallmitochondrion(brown),ribosomes(gray),andexamplesofsurfaceproteinsalldrawnapproximatelytoscale.Notethesignificantpotentialimpactofsurfacemoleculesizeontotalvesiclesizeinthecaseofsmallvesiclesdisplayingrelativelylargesurfacemolecules[measurementandrepresentationofcellsurfaceproteinsadaptedfromRef.(29)]. Apo-EVProductionMechanisms Duringapoptosis,caspase-dependentcleavage,andactivationofRho-activatedkinase,ROCKIaltersactomyosincontractility,resultinginmembraneblebbing(30).BlebbingoccursindependentlyofalteredK+andCl−channelactivitythatresultsinincreasedK+permeabilityandthereductionofcellvolumethataccompaniesapoptosis.Instead,blebformationoccursasaconsequenceofdetachmentoftheplasmamembranefromtheactincortexandincreasedhydrostaticpressuregeneratedbytheactomyosinsystem(31).Asaconsequence,thereisrapidlocalinfluxofcytosolicmaterialand“ballooning”ofthemembrane,resultinginblebenlargement.Althoughcytoskeletalproteinsarelackinginnewlyformedblebs,asblebbingprogresses,thereisreassemblyofthecorticalcytoskeletonunderneaththemembrane.TreatmentofcellswiththeROCKinhibitorY-27632reducesboththeformationofapoptoticbodiesandthecapacityforapoptotic-cellclearance(32).FormationofsmallApo-EVsmayalsoberegulatedthroughsimilarROCK-dependentmechanisms(33),asareEVsgeneratedbyavarietyoftumorcelltypes(34). Ithasbeenwidelyassumedthattheplasmamembraneofapoptoticcells,apoptoticblebs,andApo-EVsaremolecularlyhomogeneous,displayingbroadlysimilarchanges,suchasphosphatidylserine(PtdSer)exposure.However,thereisevidencethatapoptoticbodiesexhibitlossofmembraneintegritythatallowslimitedmolecularexchange(35),whichmayallowselectivereleaseofmoleculesthatareabletomodulateinnateinflammatorymechanisms.Proteinreleasefromapoptoticbodies,particularlyofnucleosomalhistones,wasreducedfollowinginhibitionofactivityofeitherROCKormyosinATPase(35).Lossofmembranepermeabilitymaybephasedasapoptosisprogresses,priortothecatastrophiclossofmembraneintegrityduringsecondarynecrosis.FormationofApo-EVsandgraduallossofmembranepermeabilitycouldrepresentamechanismtoprovidetransientprotectionofproteinsfromlocalproteolyticdegradationand/orclearance,potentiallyallowingsignalsrelatingtocellulardemisetobedisseminateddistally,forexampletootherpartsofthetumormicroenvironmentandtometastaticsites. Studiesoftherecognitionandphagocytosisofapoptoticcellshaverevealedpotentiallycontrastingrolesformembraneblebbingandtheformationofapoptoticbodies.Formationofapoptoticblebsmaypromotephagocytosisofapoptoticcells.Compromisedapoptoticcelluptakefollowinginhibitionofblebbing(36)couldbepartiallyreversedbyadditionofthePtdSeropsonin,MFG-E8.OtherphospholipidbindingproteinssuchasC1qhavebeendemonstratedtobindavidlytoapoptoticblebsandC1qbindstoneuronalblebs,actingtoaugmentphagocytosisbymicroglia(37).ROCK-dependenthigh-densityopsonizationofapoptoticblebscouldgenerateatopologythatpromotesphagocyterecognition,providinganexplanationforwhylow-levelPtdSerexposureisnotsufficienttosignalphagocytosisofviablecells.Thus,membraneblebbinglikelyfacilitatesmaintenanceofself-toleranceandsuppressionofantitumorimmunitythroughdirecteffectsonapoptoticcellclearance.Otherphenomenarelatedtovesiculationduringapoptosishavealsobeennotedrecently.Followingthedescriptionofapoptopodia—fineprotuberancesfromapoptoticcellsthatappeartobeinvolvedinthereleaseoflargervarietiesofApo-EVs(>1µm)(38)—EVproductionfromcertainapoptoticcelltypeshasbeenobservedtoinvolvefragmentationofmembraneprotrusionsresemblingbeadsonastring(39).Whilethesignificanceoftheseobservationshasnotbeenfullyelucidated,theyprovidecluesastothemoleculareventsunderlyingtheproductionofApo-EVsandtheircargoloadingpreferences.Intriguingly,Apo-EVsproducedfrombeadedapoptopodiawerefoundtobedepletedofnuclearcomponentsincludinghistonesandnuclearDNA(39)thatarewell-knownconstituentsofapoptoticbodies(40). CargoesandFunctionalActivities WhileEVsostensiblyofnon-apoptoticcelloriginshavebeenthesubjectofintenseresearchincancerbiologyinrecentyears,thebiologyofApo-EVsremainslessclear.FollowingonfromseminalworkshowingthatglioblastomaEVscarryRNAandproteincargoeshavingtumorgrowth-promotingpropertiesandutilityasdiagnosticbiomarkers(41),awealthofevidencenowimplicatesEVsinregulatingtumorgrowthandmetastaticspreadthroughcontrolofangiogenesis,drugresistance,andantitumorimmunity.Furthermore,therolesofEVsinintercellularcommunicationinthetumormicroenvironmentarebecomingbetterdefined.Takingsomerecentexamples,inmurinemelanoma,tumorcell-derivedexosomeshavebeenreportedtopromotetheaccumulationofpro-tumormacrophagesviatheirabilitytoeducatemesenchymalstromalcellswhich,liketumor-associatedmacrophages,areabletopromotemalignantdiseaseviamultiplemodes,includinggrowthfactorproduction,suppressionofantitumorimmunityandangiogenesis(42).EVsfromcirculatingtumorcellsarealsogeneratedunderconditionsofshearflow.TheseEVsmayplayimportantrolesinestablishingthemetastaticnicheinthelungthroughinteractionwiththelungvasculatureandrapidlyaccumulatingmyeloidcells(whichphagocytosethem)(43).ItisnoteworthythatEVsprovideanintercellularsignalingmechanismtotransferdrugresistancetosusceptiblecells.Forexample,transferofresistancetothemulti-receptortyrosinekinaseinhibitordrug,Sunitinibcanbeachievedbyalong,non-codingRNA(lncARSA)whichactsbycompetingforbindingtomir-34andmir-449topromoteAXLandc-METexpressioninrenalcellcarcinomacellsbycarriageinexosomesandtransfertosusceptiblecells,therebypropagatingresistance(44).Intriguingly,EVsisolatedfromcancer-associatedfibroblastsareabletoalterthemetabolicprofileofpancreatictumorcellsthatinteractwith,andinternalizethem(45).MetabolicreprogrammingbyEVsinvolvedinhibitionofoxidativephosphorylationbymitochondriaresultinginpromotionofglycolysisandglutamine-dependentreductivecarboxylation(46)intherecipienttumorcells.Furthermore,EVswerefoundtobecapableoftransferringmultiplemetabolicconstituentsincludingaminoacids,lipids,citrate,andpyruvateamongothers,totumorcellsendowingthemwiththecapacitytogrowinnutrient-deficientmediainvitro(45).TheseresultsstronglysupportthenotionthatEVsinthetumormicroenvironmentprovidetumorcellswithcriticalmetabolicsignalsandconstituentswhichpermitgrowthoftumorclonesunderconditionsofstresssuchashypoxiaandnutrientdeprivation. TheextenttowhichApo-EVs—includingthelargervariety,apoptoticbodies—canperformsimilarlydiversefunctionstotheirnon-apoptoticcounterpartsawaitsdetailedclarification.However,severalstudieswouldtendtosuggestthatApo-EVsrepresentfarmorethanbiological“wastedisposal”units.WesupportthedefinitionofApo-EVsasthoseEVs,regardlessofsizeorcargo,thatareproducedasaconsequenceofactivationoftheapoptosiseffectormachinery(suchasexecutionercaspaseactivation)andthatultimatelyresultsincelldeath.Thus,activeApo-EVproductionpresagescelldeathandamajorchallengefortheallocationoffunctionalpropertiestoApo-EVsspecificallywillbetheirdiscriminationfromEVsproducedbycellsactivatedbyother(forexample,stress)pathways,includingthoseenroutetoapoptosis.LikeallEVs,Apo-EVsareovertlyheterogeneousasillustratedbytheirsizeprofilealone,which,rangesfromaround30nmtoseveralmicrons(47,48).TowhatextentsizeofApo-EVsrelatestofunctionalpropertiesislargelyunknown,althoughsmallEVs(30–100nm,whichtheauthorstermed“exosome-like”)producedbyvascularendothelialcellsdownstreamofcaspase-3activationwerefoundtobedistinctfromtheirlargercounterparts(microvesiclesandapoptoticbodies)bothincargocompositionandbiologicalfunction(48).Vascularendothelialcell-derivedapoptoticbodiescarryhistonesandothernuclearproteinsaswellasabundantmarkersoforganellesincludingmitochondria,endoplasmicreticulum,andribosomes(48),confirmingobservationsofapoptoticbodycargoesinothersystems.Bycontrast,theexosome-likeEVswerefoundtobeenrichedinlysosomal,basementmembraneandextracellularmatrixproteins(48).Intriguingly,certainhallmarkproteinsofexosomes,includingTSG101,CD9,andCD81,weremissingfromtheexosome-likeEVswhereasothers,notablyfibronectin,synteninandtranslationallycontrolledtumorprotein(TCTP)werepresent.Critically,exosome-likeEVswerefoundtobeimmunogenic,incontrasttoapoptoticbodies(48),confirmingthepresumptionthatthelatter,asmajorremnantsofapoptoticcells,aregenerallytolerogenic. TheserecentstudiesextendearlierinvestigationsdemonstratingthesegregationofnuclearcomponentsintogranularandvesicularstructuresandextrusionfromthecellinEV-likestructuresandapoptoticbodies(49–54).Strikingly,DNAandRNAfromapoptoticcellshavebeendescribedassegregatingintonon-overlappingvesicularentities,addingtothecomplexityofApo-EVheterogeneity.Itiswellestablishedthattheblebsofapoptoticcellsurfacesharborantigensofcommonsignificanceinautoimmunedisease,includingtheribonucleoproteinsLaandRoandnucleosomalDNA(55).Theimmunogenicityofexosome-likeEVsfromapoptoticendothelialcellsaddsafurtherdimensiontothisphenomenon.Thus,theC-terminalfragment(LG3)ofthebasementmembranecomponentPerlecancarriedbytheexosome-likeEVsishighlyimmunogenicandmayberesponsiblefortheproductionofautoantibodiesthatcanseverelycompromisesuccessfulrenaltransplantation(48).Substantialfurtherinvestigationsarewarrantedinordertoclarifythedifferentialcapacityofapoptoticcellsandtheirderivedvesiclestomodulatetoleranceandimmunity. Besidesimmuno-regulatoryproperties,Apo-EVshaveadditionalfunctionalattributesbased,likeotherEVclasses,ontheirabilitytotransferbioactivemoleculesto“target”cells.Forexample,apoptoticbodies(1–4µm)derivedfrommatureendothelialcellshavebeenshowntostimulatetheproliferationanddifferentiationofcirculatingendothelialprogenitorcells(56).Indeed,Apo-EVsofendothelialcellorigincarryavarietyofbiologicallyactivecomponentsinadditiontotheaforementionedimmunogenicPerlecanLG3,includingTCTP,whichcaninhibitapoptosisinvascularsmoothmusclecells(57).Apo-EVsmayalsoallowthetransferofintactorganellesbetweencells.Inthiscontext,itisnoteworthythatmitochondrialtransferviaEVsmayrepresentanimportantresponsetostressfulconditionsasexemplifiedbythetransferofintactmitochondriafromastrocytestoneuronsinordertoprovidesurvivalsignalsduringtheischemicconditionsofstroke(58).OneofthemostintriguingcargoesofApo-EVsisgenomicDNAsinceithasbeenshownthatapoptoticbodiesareabletomediatethehorizontaltransferofDNAbetweensomaticcells.WhilethedetailsofthemodesoftransferandfundamentalrolesofApo-EVs(versustheremnantsofapoptoticcells)havenotbeenstudied,DNAfromapoptoticcellscanundoubtedlybetransferredtoneighboringcellsincludingtumorcells,endothelialcells,fibroblasts,andmacrophagesleadingtoapoptoticcell-derivedgeneexpressionintherecipientcells.Innormalphysiology,cellsareprotectedbyaDNAdamageresponserequiringDNAseII,Chk2,andp53/p21,anddeficiencyinp53andp21canultimatelyrendermurineembryonicfibroblastsoncogenicfollowingtransferofDNAfromapoptoticcellsharboringc-mycorH-Rasoncogenesincombinationwithadrugresistancegene(59–61).TheseresultshavesignificantimplicationsnotonlyforgenomicstabilityandheterogeneityoftumorcellsbutalsofortheacquisitionofaberrantDNAbynon-tumorcellcomponentsoftheORN,notablyendothelialcells,macrophagesandfibroblasts,allofwhichhaveknowncapacitytoengulfapoptoticcellsandbodies.SuchgeneticchangesintheORNcouldprovideimportantpro-oncogenicsignalseveniftheresultant“exogenous”geneexpressionistransient. ConclusionandFuturePerspectives Whileitisclearthatthebreakdownofapoptoticcellsintomembrane-boundedfragmentsofbroadsizerangesvariesbetweendifferentcelltypes,thefullextentofthefunctionalpropertiesofApo-EVsremainsunknown.Ithasbeenreportedthatformationof“bite-sized”apoptoticbodiescanaidinthephagocyticclearanceofdyingcells(36).Thismaybeimportantfortheapoptotic-cellclearanceprocessesofso-callednon-professional(i.e.,non-macrophage)phagocytes.However,macrophagesandotherphagocyteshaveovertcapacitytoengulfwholeapoptoticcellsrapidly(62).WeproposethatthemostimportantfunctionofApo-EVsinthecontextofcanceristhepropagationofintercellularsignalsoffundamentalimportancetotheORN.Understandingtheirmodesofinteractionwithrecipientcells,theirmechanismsofinternalizationandintracellularprocessingwillbecrucialtounderstandingfullythephysiologicalandpathologicalattributesofApo-EVs.Todate,virtuallynothingisknownoftheseprocesses,althoughitmaybeexpectedthatsomeclearance/engulfmentmechanismsofapoptoticcellsandApo-EVswillprovetosharemolecularcomponents(Figure1).ItisnoteworthyinthiscontextthatPtdSerexposedonEVsisinvolvedintheiruptakebytargetcellsexpressingPtdSerreceptorssuchasTIM-4,knownforphagocytosisofapoptoticcells(63).AcriticalquestioniswhetherendocytosedorphagocytosedApo-EVcargoisnecessarilydegradedbylysosomes,asisgenerallyassumed.Thus,thetargetingmechanismsofApo-EVsalongwiththedestiniesoftheircargoesrequiredetailedclarification. Pro-inflammatoryextracellularvesicles(EVs)areproducedbymacrophagesrespondingtoATPviaP2X7receptors.IthasbeenreportedrecentlythatthisresultsinNLPR3inflammasomeactivationinhumanmacrophages,whichconsequentlyundergovesicle-mediatedunconventionalsecretionofIL-1β(64).Conversely,alveolarmacrophage-derivedEVshavebeenshowntosuppressairwayinflammation(65).Thus,thevesicularintercommunicationthatresultsfromtissuedamageislikelytoinvolveavariedmixofvesiclepopulations,includingpro-andanti-inflammatory,derivednotonlyfromdyingcellsbutalsofromtheirresponsiveneighborsorrecruitedphagocytes(Figure1).SincetheORNrepresentsasectorofthetumormicroenvironmentengagedindysregulated,celldeath-driventissuerepairandregeneration,itseemslikelythattheintercellularcommunicationssoachievedbyEVsoftheORNwillprovetooverlapwiththoseinhealingorchronicwounds.Futureworkaimedatidentifyingtheunderlyingmechanismsmayyieldnovelmoleculartargetsforbothcancerandwoundtreatments. 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