Exosomes released by hepatocarcinoma cells endow adipocytes with tumorpromoting properties | Journal of Hematology & Oncology | Full Text

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AbstractBackground

Theinitiationandprogressionofhepatocellularcarcinoma(HCC)arelargelydependentonitslocalmicroenvironment.Adipocytesareanimportantcomponentofhepaticmicroenvironmentinnonalcoholicfattyliverdisease(NAFLD),whichisasignificantriskfactorforHCC.GiventheglobalprevalenceofNAFLD,abetterunderstandingoftheinterplaybetweenHCCcellsandadipocytesisurgentlyneeded.Exosomes,releasedbymalignantcells,representanovelwayofcell-cellinteractionandhavebeenshowntoplayanimportantroleincancercellcommunicationwiththeirmicroenvironment.Here,weexploretheroleofHCC-derivedexosomesinthecellularandmolecularconversionofadipocytesintotumor-promotingcells.

Methods

ExosomeswereisolatedfromHCCcelllineHepG2andaddedtoadipocytes.Transcriptomicalterationsofexosome-stimulatedadipocyteswereanalyzedusinggeneexpressionprofiling,andsecretionofinflammation-associatedcytokineswasdetectedbyRT-PCRandELISA.Invivomousexenograftmodelwasusedtoevaluatethegrowth-promotingandangiogenesis-enhancingeffectsofexosome-treatedadipocytes.Proteincontentoftumorexosomeswasanalyzedbymassspectrometry.Activatedphospho-kinasesinvolvedinexosome-treatedadipocytesweredetectedbyphospho-kinaseantibodyarrayandWesternblot.

Results

OurresultsdemonstratedthatHCCcellHepG2-derivedexosomescouldbeactivelyinternalizedbyadipocytesandcausedsignificanttranscriptomicalterationsandinparticularinducedaninflammatoryphenotypeinadipocytes.Thetumorexosome-treatedadipocytes,namedexo-adipocytes,promotedtumorgrowth,enhancedangiogenesis,andrecruitedmoremacrophagesinmousexenograftmodel.Invitro,conditionedmediumfromexo-adipocytespromotedHepG2cellmigrationandincreasedtubeformationofhumanumbilicalveinendothelialcells(HUVECs).Mechanistically,wefoundHepG2exosomesactivatedseveralphopho-kinasesandNF-魏Bsignalingpathwayinexo-adipocytes.Additionally,atotalof1428proteinswereidentifiedinHepG2exosomesbymassspectrometry.

Conclusions

Ourresultsprovidenewinsightsintotheconceptthattumorcell-derivedexosomescaneducatesurroundingadipocytestocreateafavorablemicroenvironmentfortumorprogression.

Background

Hepatocellularcarcinoma(HCC)nowrepresentsthefifthmostcommoncancerworldwideandthethirdleadingcauseofcancer-relatedmortality[1,2].AlthoughbothdiagnosticandtherapeuticstrategiesforHCChaveimprovedoverthepastdecades,the5-yearsurvivalrateonlyisaround10%,andHCCcontinuestobeaglobalhealthissue,especiallyinAsiancountries[3,4].Emergingevidencesuggestedthatnonalcoholicfattyliverdisease(NAFLD),acommondisorderinobesepeople,isasignificantriskfactorforHCC[5,6].Giventheglobalprevalenceofobesity,thereistheloomingthreatofarapidlyrisingoccurrenceofNAFLD-relatedHCC.Therefore,itisurgentandparamounttounderstandthemechanismsbywhichNAFLDcontributestoHCCdevelopment.

Tumorbehaviorisdeterminedbynotonlythemalignantpotentialoftumorcellitselfbutalsothesignalsfromitsmicroenvironment.Thus,itisclearthatthecrosstalkbetweentumorcellsandtheirsurroundingmicroenvironmentiscrucialforHCCdevelopment.InNAFLD,thehepaticmicroenvironmentcomprisesmultiplecelllineagesincludingendothelialcells,hepaticsatellitecells,immunecells,andadipocytes[7,8].PreviousstudieshavefocusedintensivelyontheinteractionsbetweenHCCcellsandawidevarietyofimmunecellssuchasKupffercells,NKcells,Tcells,andseveralantigen-presentingcells.Forexample,necroticdebrisofHCCcellscaninducepotentIL-1尾releasebymacrophageswhichsubsequentlypromoteHCCmetastasisinmousemodels[9].TheworkdonebyWolfetal.showedthathepaticNKTcellspromotedNAFLDbysecretingLIGHTandactivatedNF-魏Bsignalinginhepatocytestoenhancemalignanttransformation[10].However,theinterplaybetweentheHCCcellsandadjacentadipocytesremainspoorlyunderstoodsofar.

Currently,howcancercellscommunicatewiththeirlocalanddistantmicroenvironmentisundergoingare-evaluationwiththediscoveryofanovelwayofcell-cellinteractionexosomes[11,12].Inadditiontodiffusiblefactors,suchasgrowthfactors,cytokines,andextracellularbioactivemolecules,exosomesaresmallmembranevesiclesthatarereleasedbymanydifferentcelltypes,includingcancercells.Increasingevidencesuggeststhattumor-derivedexosomessupporttumordevelopmentandprogressionbygeneratingafavorablemilieuthroughimmunesuppression,angiogenesisenhancement,extracellularmatrixremodeling,andstromalcellconversion[13,14,15].Exosome-mediatedtransferofproteins,DNA,noncodingRNAs,andmRNAscouldinducephenotypicchangesintargetcells[16].Inmelanoma,thetumor-derivedexosomeseducatedbonemarrowprogenitorstowardapro-metastaticphenotypethroughthereceptortyrosinekinaseMET[17].Inpancreaticcancer,thesecretedexosomesinducedlipolysisinsubcutaneousadiposetissue[18].InHCC,exosomesderivedfrommetastaticHCCcelllinessignificantlyenhancedthemigratoryandinvasiveabilitiesofnonmotilehepatocytes[19].However,toourknowledge,nostudyhasreportedontheeffectsoftumor-derivedexosomesonadipocytes,whichrepresentanabundantcelltypewithintumormicroenvironmentinoverweightpatients.

Inthisstudy,weexploredtheroleofHCC-derivedexosomesinthecellularandmolecularconversionofadipocytesintotumor-promotingcells.OurresultsdemonstratedthatHCCcelllineHepG2-derivedexosomescouldbeactivelyinternalizedbyadipocytesdifferentiatedfrommesenchymalstemcells(MSCs)andcausedsignificanttranscriptomicalterations,andinparticular,inducedaninflammatoryphenotypeinadipocytes.Thetumorexosome-treatedadipocytes,namedexo-adipocytes,promotedtumorgrowth,enhancedangiogenesis,andrecruitedmoremacrophagesinmousexenograftmodel.Invitro,conditionedmediumfromexo-adipocytespromotedHepG2cellmigrationandincreasedtubeformationofhumanumbilicalveinendothelialcells(HUVECs).Mechanistically,wefoundHepG2exosomesactivatedseveralkinasesandNF-魏Bsignalingpathwayinexo-adipocytes.OurfindingsshowedforthefirsttimethatHCC-derivedexosomescouldconvertadipocytesintotumor-promotingcells,whichmayprovidenewinsightsintounderstandingtheinteractionsbetweentumorcellsandsurroundingmicroenvironment.

MethodsCellculture

HumanadiposetissuesandumbilicalcordswereobtainedaccordingtotheproceduresapprovedbytheEthicsCommitteeattheChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege.MSCswereisolatedandculture-expandedfromhealthyvolunteersaspreviouslyreported[20].Passage3MSCswereusedforfollowingexperiments.Toobtainadipocytes,MSCswereinducedunderadipogenicdifferentiationmedium,whichishighglucoseofDulbecco鈥檚modifiedEagle鈥檚medium(H-DMEM)supplementedwith10%FBS,1聽渭Mdexamethasone,0.5聽mM3-isobutyl-1-methylxanthine,and5聽渭g/mL0.1聽mMl-ascorbicacid.AdipocyteswerecharacterizedbyOilRedOstainingaccordingtothemanufacture鈥檚(BeyotimeBiotechnology)instructions.HUVECswereisolatedandculturedasroutinelydescribed[21].HCCcelllineHepG2waspurchasedfromcellbankattheChineseAcademyofMedicalSciencesandculturedinDF12containing10%FBS,penicillin(100聽U/mL),andstreptomycin(100lg/mL)at37聽掳Cwith5%CO2.

Exosomesisolation

Exosomeextractionwasperformedaspreviouslydescribed[22].Briefly,HepG2cellswereculturedinserum-freeDF12mediumfor24聽h.Then,theculturemediumwascollectedandcentrifugedat800gfor5聽minandadditional2000gfor10聽mintoremoveliftedcells.Thesupernatantwassubjectedtofiltrationona0.1-mm-porepolyethersulfonemembranefilter(Corning)toremovecelldebrisandlargevesicles,followedbyconcentrationbya100,000-Mwcutoffmembrane(CentriPlus-70,Millipore).Thevolumeofsupernatantwasreducedfromapproximately250鈥?00聽mLtolessthan5聽mL.Thesupernatantwasthenultracentrifugedat100,000gfor1聽hat4聽掳Cusing70TiRotor(BeckmanCoulter).Theresultingpelletswereresuspendedin6聽mLPBSandultracentrifugedat100,000gfor1聽hat4聽掳Cusing100TiRotor(BeckmanCoulter).IntheexperimentsinvolvingHepG2exosomes,weusePBSasanegativecontrol.

Transmissionelectronmicroscopy

Purifiedexosomeswerefixedwith1%glutaraldehydeinPBS(pH7.4).Afterrinsing,a20-uLdropofthesuspensionwasloadedontoaformvar/carbon-coatedgrid,negativelystainedwith3%(w/v)aqueousphosphotungsticacidfor1聽min,andobservedbytransmissionelectronmicroscope.

Quantitativereal-timepolymerasechainreaction

TotalRNAwasextractedusingTRIzol(Invitrogen)accordingtothemanufacturer鈥檚instruction,andcDNAwasprepared.Real-timePCRamplificationwasperformedintriplicatesaccordingtotheproceduresreportedpreviously[23].RelativeexpressionofmRNAwasevaluatedbythe2-螖螖CtmethodandnormalizedtotheexpressionofGAPDH.

Westernblotting

Proteinswereextractedwithradioimmunoprecipitation(RIPA)lysisbufferwithPMSF,quantifiedbyBCAProteinAssayKit(Beyotime).Westernblotwasperformedintriplicatesaccordingtotheproceduresreportedpreviously[24].GAPDHwasusedasaninternalcontrol.Weusedthefollowingantibodies:p-AKT(1:2000;rabbitIgG,CST,4060T),p-ERK1/2(1:5000;rabbitIgG,Abcam,ab76299),p-STAT5伪(1:1000;rabbitIgG,Abcam,ab30648),p-GSK(1:5000;rabbitIgG,Abcam,ab75814),AKT(1:1000;mouseIgG,proteintech,60203-2-Ig),ERK1/2(1:1000;rabbitIgG,proteintech,16443-1-AP),STAT5伪(1:1000;rabbitIgG,Abcam,ab32043),GSK3尾(1:1000,rabbitIgG,proteintech,22104-1-AP),CD63(1:500;rabbitIgG,proteintech,25682-1-AP),TSG101(1:500;rabbitIgG,Abcam,ab83),HSP70(1:100;rabbitIgG,SBI,EXOAB-KIT-1),calnexin(1:2000;rabbitIgG,CST,2433s),GAPDH(1:10000;rabbitIgG,proteintech,10494-1-AP)(1:10000;mouseIgG,proteintech,60004-1-Ig),HRP-conjugatedanti-rabbit-IgG(NeoBioscience),HRP-conjugatedanti-goat-IgG(NeoBioscience),andHRP-conjugatedanti-mouse-IgG(NeoBioscience).

Cytokineanalysis

Culturemediumwascollected24聽hafterthetreatmentwithorwithoutexosomes.TheconcentrationsofallcellcytokinesinsupernatantsweremeasuredusingELISAkits(BDTechnologies).

Immunofluorescencestaining

Theculturedcellswerefixedat4聽掳Cinice-coldmethanolfor10聽min,washedthreetimesinphosphate-bufferedsaline(PBS),andthenpermeabilizedin0.1%TritonX-100/PBSfor10聽minatroomtemperature.Nonspecificbindingwasblockedwith0.5%Tween-20/PBScontaining1%bovineserumalbumin(BSA)for30聽min.Theprimaryantibodieswereincubatedat4聽掳Covernight.Thesecondaryantibodieswereincubatedfor1聽hatroomtemperature.TheincubatedcellswerewashedinPBS,andHoechst33342(Sigma-Aldrich)wasusedtovisualizenuclei.p65antibody(10745-1-AP)waspurchasedfromProteintech.

Mousexenograftexperiments

NudemicewerepurchasedfromtheLaboratoryAnimalCenteroftheChineseAcademyofMedicalSciences(Beijing,China).AnimaluseandexperimentalprocedureswereapprovedbytheAnimalCareandUseCommitteeoftheChineseAcademyofMedicalSciences.Micewererandomlydividedintothreegroups,onegroupreceivedasubcutaneousinjectionof2鈥壝椻€?05exo-adipocytesand2鈥壝椻€?06HepG2cells,onegroupreceived2鈥壝椻€?05adipocytesand2鈥壝椻€?06HepG2cells,andthelastonereceived2鈥壝椻€?06HepG2cells.Thetumorweightwasmeasuredafter4聽weeks.Thetumortissueswerefixedwith10%PFA.EachgroupwastreatedwithHE,IL-6,Ki67,CD31,andF4/80staining.

TubeformationassayinMatrigel

InvitrocapillarynetworkformationwasdeterminedbyperformingatubeformationassayinMatrigel(BDBiosciences).1鈥壝椻€?04HUVECswereplatedonagrowthfactor-reducedMatrigel(BD)-coated96-wellplateintriplicateswith100聽uLserum-freemedium(control),exo-adipocyte-conditionedmedium,oradipocyte-conditionedmedium.After8聽hofincubation,tubeformationwasexaminedbymicroscopy(Olympus,Tokyo,Japan),andthebranchdensityandtubelengthwerequantifiedbyrandomlyselectingthreefieldsperwell.

Statisticalanalysis

Dataarepresentedasmean鈥壜扁€塖D.ComparisonsbetweengroupswereanalyzedviaStudent鈥檚ttest.Differenceswereconsideredstatisticallysignificantat*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,and***P鈥?lt;鈥?.001.

ResultsHepG2exosomesareactivelyinternalizedbyadipocytes

ToinvestigatewhetherexosomesaresecretedbyhepatocarcinomacellHepG2andplayaroleintheircommunicationwithadipocytes,weuseddifferentialcentrifugationtopurifyexosomesfromthesupernatantofHepG2cells.Isolatedparticleswereobservedundertransmissionelectronmicroscopyandfoundtopresentcharacteristicsofexosomes,withtypicalappearanceanddiameterrangingfrom30to200聽nm(Fig.聽1a).ThenanoparticlesizedistributionfortheHepG2exosomeswasfurtherobtainedbyNTA,andthepeaksofparticlesizewere~鈥?00聽nm,withintheexpectedsizeofexosomes(Fig.聽1b).EnrichmentforexosomeMarkerCD63,TSG101,andtheabsenceofthecell-specificmarkercalnexinwasdemonstratedbyWesternblot(Fig.聽1c).Adipocytesweredifferentiatedfromhumanmesenchymalstemcells(MSCs)underadipogenicinductionconditions.Figure聽1dcharacterizestheMSC-derivedadipocytesbymorphology,OilRedOstaining,BODIPYstaining,andexpressionofspecificadipogenicmarkergenesduringadipogenicdifferentiationofMSCs.ToexamineifadipocytesmightbetargetsofHepG2-derivedexosomes,alipid-associatingfluorescentdye,PKH67,wasusedtolabelexosomepreparationsandthenincubatedwithadipocytes.Exosomeincorporationwasobserved0.5聽haftertreatment,andexosomesaccumulatedinadipocytesovertime(Fig.聽1e).Collectively,weshowthatHepG2cellssecreteexosomes,whichareactivelyincorporatedinvitrobyadipocytes.

Fig.1

HepG2cell-secretedexosomesareactivelyincorporatedbyadipocytesinculture.aArepresentativeelectronmicroscopyimageofpurifiedHepG2exosomesshowedmorphology.Scalebar,200聽nm.bThenanoparticlesizedistributionforHepG2exosomeswasobtainedbyNTA.Theparticlesizeisbetween0and300,withapeakaround100聽nm.cWesternblotanalysisofexosomemarkerCD63,TSG101,andcell-specificmarkercalnexin.PositivecontrolwasHepG2celllysate,andnegativecontrolwasculturemedium.dCharacterizationofadipocytesdifferentiatedfromMSCsbymorphology,OilRedOstaining,BODIPYstaining,andexpressionofspecificadipogenicmarkergenesCEBP蓱,PPAR嗓,andLPL.eAdipocyteswereincubatedwith70聽ng/mLPKH67-labeledHepG2exosomesfortheindicatedtimes,anduptakeofexosomeswasdeterminedbyfluorescenceconfocalmicroscopy

FullsizeimageHepG2exosomesinduceaninflammatoryphenotypeinadipocytes

TodeterminetheeffectsofHepG2exosomesonadipocytes,weexposedadipocytesto25聽渭g/mLHepG2exosomesfor24聽handevaluatedthetranscriptomicalterationsusinggeneexpressionprofiling.Sevenhundredtwenty-fiveupregulatedand648downregulatedgeneswereidentified(Fig.聽2a),andthetop10up-anddownregulatedgenesinHepG2exosome-treatedadipocytes(exo-adipocytes)comparedtocontrolareshowninFig.聽2b.Interestingly,unsupervisedclusteringidentifiedexpressionchangesingenesignaturesrelatedtoinflammationinexosome-treatedadipocytes(Fig.聽2c).Consideringthatcancerisoftenassociatedwithaninflammatorymilieu,webegantoexplorewhetherHepG2exosomescouldchangeinflammatoryresponseinadipocytes.Inourpreviousreport,wefoundthatlungtumorexosomestriggerthereleaseofcytokinesincludingIL-6,IL-8,andMCP-1inMSC[24].Therefore,weextendedthisanalysisinadipocytesandconfirmedtheenhancedreleaseofIL-6,IL-8,andMCP-1byqRT-PCR(Fig.聽2dleft)andELISA(Fig.聽2dright).Todeterminewhetherincreasedexpressionofcytokinesinexosome-treatedadipocyteswasconcentration-dependent,wetreatedadipocyteswithdifferentconcentrationsofHepG2exosomesfor24聽handfoundapartialdose-dependenteffectofexosomesonthesecretionofIL-6,IL-8,andMCP-1(Fig.聽2e).

Fig.2

HepG2exosomeschangethetranscriptomeofadipocytesandinduceproductionofinflammatorycytokines.aThescatterplotassessedthegeneexpressionvariationsbetweenexosome-treatedadipocytesandcontrol.Thereddotsindicateupregulatedgenesandgreendotsdownregulatedgenes.bThetop10up-anddownregulatedgenesinHepG2exosome-treatedadipocytescomparedtocontrol(Erepresentsexosome-treatedadipocytes,Crepresentscontrol).cUnsupervisedhierarchicalclusteringbasedontheexpressionofgenesassociatedwithinflammation.dEnhancedreleaseofIL-6,IL-8,andMCP-1wasdetectedbyqRT-PCR(left)andELISA(right)(*P聽鈥?.05,**P聽鈥?.01,***P聽鈥?.001).eApartialdose-dependenteffectofexosomesonthesecretionofIL-6,IL-8,andMCP-1(*P聽鈥?.05,**P聽鈥?.01,***P聽鈥?.001)

Fullsizeimageexo-adipocytespromotetumorgrowthinvivo

Consideringtheimportantroleofinflammatorycytokinesintumordevelopment,wenextanalyzedwhetherexo-adipocytesaffectedHepG2cellsandprovidedabenefitfortumorgrowthbyusinganudemousexenografttumormodel.HepG2cellsweresubcutaneouslyco-implantedwithexo-adipocytes,adipocytes,orPBSataratioof10:1.Coinjectionwithexo-adipocytesresultedinanincreasedtumorweightcomparedwithtumorcellsinjectedwithadipocytesorPBS(Fig.聽3a).Todeterminetheeffectofexo-adipocytesonangiogenesisandproliferationoftumorcellsinvivo,weperformedIHCstainingtodetectCD31andKi67.Coinjectedexo-adipocytescouldenhancethevasculardensityasdemonstratedbytheincreasedexpressionofCD31(Fig.聽3b,c).Figure聽3d,erevealedthatthenumbersofKi67-positivecellswereincreasedsignificantlyinthepresenceofexo-adipocytes.Whenthetumorswereexcisedforassessmentofimmunecellinfiltration,weobservedthatthenumberofF4/80macrophageswashigherintumorsreceivingexo-adipocytesthanthosereceivingcontroladipocytes(Fig.聽3f,g).PreviousstudiessuggestIL-6tobeamajorregulatoroftumor-stromainteractionincancermicroenvironment[25].Here,weexaminedIL-6expressionlevelsintumorsectionsandfoundincreasedIL-6proteinlevels(Fig.聽3h,i),consistentwiththeupregulationofIL-6genesshowninFig.聽2e.Inaddition,weobservedtheappearanceofadipocytesamongcancercellsinthetumorsections,suggestingthatadipocyteswerenotconsumedbyneighboringcancercellsduringthe4-weektumorigenesisprocess(Fig.聽3j).Takentogether,exo-adipocyteswereendowedwithacapabilitybytumorexosomestopromotetumorgrowth,enhanceangiogenesis,andrecruitmacrophagesinvivo.

Fig.3

exo-adipocytespromotetumorgrowth,enhanceangiogenesis,andrecruitedmacrophagesinvivo.aRepresentativephotographsofHepG2tumorsgeneratedfromnudemiceinjectedwithtumorcellsaloneorco-injectedwithexo-adipocytesorcontroladipocytesataratioof10:1.bIHCstainingofbloodvesseldensityintumorsectionsfromxenograftsbystainingwithanti-CD31antibody.cCD31-positivecellswerequantified,andthedatarepresentsthemeannumberofCD31鈥?鈥塩ellsper200脳field(threefieldspergroup).dDetectionofproliferatingcellsintumorswithIHCstainingusingtheanti-Ki67antibody.RepresentativesofKi67stainingfromeachgroupareshown(magnification脳鈥?00).eKi67staining-positivecellswerequantified,andthedatarepresentthemeannumberper200脳field(threefieldspergroup).fThemacrophageinfiltrationwasexaminedbydetectingthenumberofF4/80-positivecellsusingimmunohistochemicalstaininginthetumortissuesharvested.RepresentativesofF4/80stainingfromeachgroupareshown(magnification脳鈥?00).gF4/80鈥?鈥塵acrophageswerequantified,andthedatarepresentthemeannumberofF4/80+macrophagesper200脳field(threefieldspergroup).hDetectionofIL-6expressionintumors.iIL-6expressionwasquantified,andthedatarepresentthemeannumberofIL-6+cellsper200脳field(threefieldspergroup).jArepresentativeHEstainingoftumorsectionsdemonstratedpresenceofadipocytesinmiceinjectedwithexo-adipocytesoradipocytes

Fullsizeimageexo-adipocyte-conditionedmediumischemotaxicandpromotesHepG2migration

TheincreasednumberofF4/80macrophagesintumorsreceivingexo-adipocytespromptedustoinvestigatetheeffectofexo-adipocyte-conditionedmediumonTHP-1cellsinvitroasTHP-1cellsareoneofthemostwidelyusedcelllinestoinvestigatethefunctionandregulationofmacrophages[26,27].Wefoundthatexo-adipocyte-conditionedmediumwasmorechemotacticthanadipocyte-conditionedmediumforTHP-1cells(Fig.聽4a).Additionally,asimilareffectwasobservedforHepG2tumorcells(Fig.聽4b).Wethenexaminedwhetherexo-adipocyte-conditionedmediumcouldaffecttumorcellmigration.Asexpected,comparedwiththeadipocyte-conditionedmedium,exo-adipocyte-conditionedmediumincreasedthemigrationcapacityofHepG2(Fig.聽4c).

Fig.4

exo-adipocyte-conditionedmediumischemotacticandpromotesHepG2migration.aTranswellmigrationassaysshowedthatTHP-1cellsweremorechemotactictowardexo-adipocyte-conditionedmediumthanadipocyte-conditionedmedium.Leftisarepresentativemicroscopicimageofcrystalvioletstaining;rightshowsthestatisticalresults.bTranswellmigrationassaysshowedthatHepG2cellsweremorechemotactictowardexo-adipocyte-conditionedmediumthanadipocyte-conditionedmedium.Leftisarepresentativemicroscopicimageofcrystalvioletstaining;rightshowsthestatisticalresults.cMigratoryabilitiesofHepG2co-culturedwithexo-adipocyte-conditionedmediumoradipocyte-conditionedmediumweredeterminedbywoundhealingassay

Fullsizeimageexo-adipocyte-conditionedmediumenhancedtubeformationofHUVECs

Toconfirmourinvivofindingsthatexo-adipocytespromoteangiogenesisintumors,weexaminedtheeffectofexo-adipocyte-conditionedmediumonHUVECs.Forty-eighthoursafterexposuretoexo-adipocyte-conditionedmedium,HUVECsexhibitedupregulatedexpressionofpro-angiogenicgenesAng1andFlk1aswellasdownregulatedtheexpressionofanti-angiogenicgenesVash1andTSP1(Fig.聽5a).Wefurtherevaluatedtheeffectsofexo-adipocyte-conditionedmediumonHUVECstubeformation.Asexpected,tubeformationofHUVECswassignificantlyincreasedinthepresenceofexo-adipocyte-conditionedmediumasdemonstratedbytheincreaseintubelengthsandareas(Fig.聽5b).Collectively,ourresultssuggestthatexo-adipocyte-conditionedmediumpromotedangiogenesisofendothelialcellsbothinvitroandinvivo.

Fig.5

exo-adipocyte-conditionedmediumenhancestubeformationofHUVECs.aHUVECswereincubatedwithexo-adipocyte-conditionedmediumoradipocyte-conditionedmediumfor48聽h.ThemRNAlevelsofAng1,Flk1,Vash1,andTSP1wereevaluatedbyqRT-PCR.Resultsaremean鈥壜扁€塖D(n鈥?鈥?foreachgroup).bComparedwithadipocyte-conditionedmedium,exo-adipocyte-conditionedmediumincreasedHUVECtubeformationinvitro.Scalebar,100聽渭m.Right鈥攔epresentativephotographoftubeformation.Left鈥攃alculationsinthreerandomlyselectedfields.Resultsaremean鈥壜扁€塖D(Student鈥檚ttest)

FullsizeimageHepG2exosomesactivatevariouskinasesandNF-魏Bsignalingpathwayinadipocytes

ToidentifywhichsignalingpathwayswereactivatedbyHepG2exosomes,weperformedphospho-kinaseantibodyarrayinadipocytestreatedwithorwithoutHepG2exosomesfor1聽h.AsshowninFig.聽6a,ofthe43kinasesexamined,15wasdetectedtohaveanincreaseofphosphorylationinexo-adipocytes.Thetop5increasedkinaseswereAKT,STAT5伪,GSK3alpha/beta,p38alpha,andERK1/2.UsingWesternblot,weconfirmedthestrongandrapidactivationofAKT,STAT5伪,ERK1/2,andGSK3尾(Fig.聽6b).SinceseveralkinasesactivatedinadipocytessuchasAKT,ERK1/2,andGSK3尾arecloselyassociatedwithNF-魏Bsignalingpathway,weinvestigatedthepossibleactivationofNF-魏BafterHepG2exosometreatment.Figure聽6cshowedthetranslocationofactivep65fromthecytoplasmtothenucleus.

Fig.6

HepG2exosomesactivateseveralkinasesandNF-魏Binadipocytes.aPhospho-kinaseantibodyarraywasperformedonproteinlysatesfromadipocytestreatedwithorwithoutHepG2exosomes.Data(right)arereportedaspercentageofincrease.Thepercentagewascalculatedas(exosome鈥夆垝鈥塩ontrol)/exosome鈥壝椻€?00%,andpercentageover20%isconsideredstatisticallysignificant.Thetop5kinaseswithanincreasedphosphorylationwerehighlightedbyredboxesintheleftpanel.bPhosphorylationofAKT,ERK1/2,STAT5伪,andGSK3尾wasconfirmedbyWesternblot.GAPDHwasusedasloadingcontrol.cRepresentativeimmunofluorescencestainingimagesofnucleartranslocationofp65inHepG2exosome-treatedadipocytes.Red(anti-p65antibody),blue(Hochest).dRelativemRNAexpressionofIL-6,IL-8,andMCP-1inadipocytestreatedwithexosomeinthepresenceorabsenceofNF-魏Binhibitor(*P聽鈥?.05,**P聽鈥?.01)

Fullsizeimage

Moreover,whenNF魏BinhibitorPDTCwasadded,theenhancedexpressionofIL-6,IL-8,andMCP-1inducedbyHepG2exosomesinadipocyteswasreduced(Fig.聽6d).Takentogether,theseresultsdemonstratedthatHepG2exosomesareabletoactivatevariouskinasesandNF-魏Bsignalingpathwayinadipocytes.

ProteomicanalysisofHepG2exosomes

Finally,weusedmassspectrometrytocharacterizeproteinscontainedwithinHepG2exosomes.Onethousandfourhundredtwenty-eightproteinsweredetectedinexosomes,whichwereclassifiedbyGOannotationaccordingtobiologicalprocess,cellularcomponent,andmolecularfunction.Theresultsshowedahighprevalenceofproteinsinvolvedinimmuneresponses(鈥渂iologicalprocess,鈥?Fig.聽7a),proteinswithbindingactivity(鈥渕olecularfunction,鈥?Fig.聽7b),andahighproportionofproteinsassociatedwithvesicleandgranule(鈥渃ellularcompartment,鈥?Fig.聽7c).Weselected32proteinswithknownfunctionsaccordingtoExocarta(http://www.exocarta.org/).AsdemonstratedinFig.聽7d,theseselectedproteinsincludedcommonexosomalmarkers,structureorsurfaceproteins,exosomalformationorsecretion-relatedproteins,andoncogenicproteins.Futurestudiesarerequiredtoidentifywhichproteinsareinvolvedinthemodificationofadipocytesintotumor-promotingcells.

Fig.7

ProteomicanalysisofproteinsrecoveredfromHepG2exosomes.Theidentifiedproteinswereclassifiedaccordingtobiologicalprocess(a),molecularfunction(b),andcellularcomponent(c).dSelectedproteinswithknownfunctionsinHepG2exosomes

FullsizeimageDiscussion

Tumorinitiationandprogressionrelyonthedynamicinteractionsbetweenmalignanttumorcellsandmultiplenormalcelltypeswithinitsmicroenvironmentsuchasfibroblasts,variousimmunecells,endothelialcells,andadipocytes.Ofthesecelltypes,adipocytesareprobablytheleastwellstudied,althoughtheyrepresentasignificantpartofthetissuesurroundingatumor[28].Emergingevidencesuggeststhatadipocytesshouldnotbeconsideredsimplyasanenergy-storagedepot.Instead,adiposetissuecanplayacentralroleinbothendocrineandmetabolicprocessesbyproducingabatteryoffactorsincludinggrowthfactorsandadipokines[29].Thus,understandinghowobesityandadiposetissue-relatedfactorsareconnectedtotumordevelopmentisparamount.In2010,Dirat鈥檚groupcoinedtheterm鈥渃ancer-associatedadipocytes(CAA)鈥?todemonstratethebidirectionalcrosstalkbetweenbreastcancercellsandtumor-surroundingadipocytesandthatCAAareakeyplayerintumorprogression[30].Subsequently,severalstudiesalsoshowedtheexistenceoftheputativeCAAinthevicinityofcancercells[31,32].Here,wechoseadipocytesasacellularmodelwhicharedifferentiatedbyculturinghumanMSCsunderadipogenicconditionsandarefullycharacterizedbymorphology,staining,andmarkergeneexpression.WedemonstratedthatHCCcelllineHepG2-derivedexosomescouldbeactivelyincorporatedbyadipocytesandconvertadipocytesintotumor-promotingcells(exo-adipocytes).Inthemousexenograftmodel,wefoundthatexo-adipocytespromotedtumorgrowthandenhancedangiogenesis.Fujisakietal.reportedthatinthepresenceofbreastcancercelllinesMCF7andMDA-MB-231,adipocytesrevertedtoanimmatureandproliferativephenotypeofCAAthatcouldpromotecancercellmigration[33].Leeetal.foundthatwhenindirectlyco-culturedwithbreastcancercells,adipocyteswouldbetransitedintoCAA,resultinginproliferation-enhancingeffectinER-positivebreastcancercellssuchasMCF7andZR-75-1butnotinER-negativecells[34].Thus,wepostulatethattheexo-adipocytesinourstudyareakindofCAAastheyexhibittumor-promotingcapacityandhigherexpressionofpro-inflammatoryfactorssuchasIL-6,IL-8,andMCP-1whosehigherexpressioninCAAhasbeenreported[33,34].IL-6playsdiverseregulatoryrolesincancerpathogenesisincludingremodelingthetumormicroenvironment,activationofEMTprocess,andpromotingdrugresistance[35,36].IL-8isknowntobeastimulatoryfactorfortumorangiogenesis[37],andMCP-1promotestherecruitmentofmacrophagesintotumors[38].Thesecytokinesmaybeatleastpartiallyresponsibleforthetumor-promotingandangiogenesis-enhancingeffectsofexo-adipocytes.

TheregulatorymechanismsoftheCAAtransitionarenotclearlyunderstood.Inthisstudy,weexploredtheroleofHCC-derivedexosomesonthecellularandmolecularchangesofexo-adipocytes,whichfurtherconfirmedthattumorcellscoulduseexosomesasanovelwayofcell-cellcommunication.Ourstudyisconsistentwithpreviousfindingsthattumorexosomesfromvariouscancertypescan鈥渆ducate鈥?neighboringcellssuchasMSCs[39],endothelialcells[40],monocytes[41],anddendriticcells[42].Forexample,exosomesfromovarianandbreastcancercellscanconvertadipose-derivedMSCs(AMSC)intomyofibroblast-likecells[43,44]whileprostatecancercell-derivedexosomestriggerbonemarrowMSCs(BMSC)todifferentiateintopro-angiogenicandpro-invasivemyofibroblasts[45].Ourresultssupportthepostulationthattheelementsofadiposetissuecanalsobemodifiedbycancercellsandparticipateinahighlycomplexviciouscycletoformatumor-favorablemicroenvironment.

Howexosomescausesignificantcellularandmolecularchangesintargetcellsremainsanareaofintensiveresearch.Usingmicroarray,Fangetal.foundthatHCCexosomescoulddelivermiR-1247-3pintofibroblastsandconvertedthemintocancer-associatedfibroblasttofosterlungmetastasis[46].Usingproteomicanalysis,Heetal.revealedthatexosomesderivedfrommetastaticHCCcelllinescarriedalargenumberofprotumorigenicproteins,suchasMETprotooncogene,S100familymembers,andthecaveolins[19].Here,wealsodetectedcommonexosomalmarkers,structureorsurfaceproteins,exosomalformationorsecretion-relatedproteins,andoncogenicproteinsinHepG2exosomes.UpontakingupHCCexosomes,725upregulatedand648downregulatedgeneswereidentified,andseveralcellsignalingpathwayswereactivated.Inourpreviousstudy[24],wefoundthatlungtumorexosomescouldactivateNF魏BsignalingpathwaythroughHSP70/TLR2.Here,wealsodetectedtheactivationoftheNF魏Bsignalingpathway.However,severalquestionsremainforfutureinvestigation,includingwhichreceptorsonthesurfaceofadipocytesparticipatedinHCCexosomeinternalizationandhowtheinternalizedexosomecargosactivatedthedownstreamsignalingpathways.

Conclusions

Collectively,ourdataindicatedthat(i)HCCtumor-derivedexosomeswereactivelyincorporatedintoadipocytesanddramaticallychangedadipocytestranscripomeandcytokinesecretion;(ii)exo-adipocytesstronglysupportedtumorgrowth,enhancedangiogenesis,andrecruitedmoremacrophages;and(iii)severalkinasesandNF-魏Bsignalingpathwaywereactivatedinexo-adipocytes(Fig.聽8).Ourresultsprovidenewinsightsintotheconceptthattumorcellcaneducatesurroundingadipocytestocreateafavorablemicroenvironmentfortumorprogressionandthatthiseffectmightbeamplifiedinoverweightpatients.

Fig.8

AschematicillustrationdemonstratesthatHCC-derivedexosomescanconvertadipocytesintotumor-promotingcellsthatcouldpromotetumorgrowth,enhanceangiogenesis,andrecruitmacrophages

FullsizeimageAbbreviationsEDTA:

Ethylenediaminetetraaceticacid

ELISA:

Enzyme-linkedimmunosorbentassay

exo-adipocytes:

Exosome-treatedadipocytes

FBS:

Fetalbovineserum

H-DMEM:

HighglucoseofDulbecco鈥檚modifiedEagle鈥檚medium

Hematoxylin-eosin

MCP-1:

Monocytechemotacticprotein1

MSCs:

Mesenchymalstemcells

NF-魏B:

Nuclearfactorkappa-light-chain-enhancerofactivatedBcells

Interleukin

qRT-PCR:

Quantitativereal-timePCR

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Funding

ThisstudywassupportedbyCAMSInnovationFundforMedicalSciences(2017-I2M-3-007),TheNationalKeyResearchandDevelopmentProgramofChina(2016YFA0101000,2016YFA0101003),BeijingKeyLaboratoryofNewDrugDevelopmentandClinicalTrialofStemCellTherapy(BZ0381),andNationalNaturalScienceFoundationofChina(81473450).

Availabilityofdataandmaterials

Thedatasetsusedand/oranalyzedduringthecurrentstudyareavailablefromthecorrespondingauthoronreasonablerequest.

AuthorinformationAuthornotes

ShihuaWangandMeiqianXucontributedequallytothiswork.

AffiliationsCenterofExcellenceinTissueEngineering,InstituteofBasicMedicalSciences,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciences,SchoolofBasicMedicinePekingUnionMedicalCollege,Beijing,100005,ChinaShihuaWang,聽MeiqianXu,聽XiaodongSu,聽XianXiao聽聽RobertChunhuaZhaoDepartmentofGeneticsandCellBiology,BasicMedicalCollege,QingdaoUniversity,308NingxiaRoad,Qingdao,266071,ChinaXiaoxiaLiCellTherapyTranslationalResearchLaboratory,PrincessMargaretCancerCentre,Toronto,Ontario,M5G2M9,CanadaArmandKeatingInstituteofBiomaterialsandBiomedicalEngineering,UniversityofToronto,Toronto,Ontario,M5G2M9,CanadaArmandKeatingInstituteofMedicalScience,UniversityofToronto,Toronto,Ontario,M5G2M9,CanadaArmandKeatingAuthorsShihuaWangViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarMeiqianXuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarXiaoxiaLiViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarXiaodongSuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarXianXiaoViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarArmandKeatingViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarRobertChunhuaZhaoViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarContributions

SHWdesignedandanalyzedtheexperimentsandwrotethemanuscript.MQXperformedandanalyzedtheexperimentsandpreparedthefigures.XXL,XDS,andXXperformedtheexperiments.AKandRCHZdesignedtheexperiment.Allauthorshavereadandapprovedthefinalmanuscript.

Correspondingauthors

CorrespondencetoArmandKeatingorRobertChunhuaZhao.

EthicsdeclarationsEthicsapproval

AllexperimentswereperformedundertheapprovaloftheEthicsCommitteeattheChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege.