Received4March2019Accepted25July2019 DepartmentofMolecularBiomedicalSciencesandComparativeMedicineInstituteNorthCarolinaStateUniversityRaleighNC27607USAJointDepartmentofBiomedicalEngineeringUni ID: 953921
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RESEARCHARTICLECirculatingtumorcellsexitcirculationwhilemaintainingmulticellularity,augmentingmetastaticpotentialTylerA.Allen,DanaAsad,EmmanuelAmu,M.TaylorHensley,JhonCores1,2,AdamVandergriff1,2JunnanTang,Phuong-UyenDinh,DeliangShen,LiQiao,TengSu,ShiqiHu,HongxiaLiangHeatherShive,ErinHarrell,ConnorCampbell,XinxiaPeng1,4,5,JeffreyA.YoderandKeCheng1,2,ABSTRACTMetastasisaccountsforthemajorityofallcancerdeaths,yettheprocessremainspoorlyunderstood.Apivotalstepinthemetastasisprocessistheexitingoftumorcellsfromthecirculation,aprocessknownasextravasation.However,itisunclearhowtumorcellsextravasateandwhethermulticellularclustersoftumorcellspossessth
eabilitytoexitasawholeormustfirstdisassociate.Inthisstudy,weuseinvivozebrafishandmousemodelstoelucidatethemechanismtumorcellsusetoextravasate.Wefoundthatcirculatingtumorcellsexitthecirculationusingtherecentlyidentifiedextravasationmechanism, Received4March2019;Accepted25July2019 DepartmentofMolecularBiomedicalSciencesandComparativeMedicineInstitute,NorthCarolinaStateUniversity,Raleigh,NC27607,USA.JointDepartmentofBiomedicalEngineering,UniversityofNorthCarolinaatChapelHillandNorthCarolinaStateUniversity,ChapelHill,NC27607,USA.DepartmentofCardiology,TheFirstAffiliatedHospitalofZhengzhouUniversity,Zhengzhou,Henan450001,China.BioinformaticsRes
earchCenter,NorthCarolinaStateUniversity,Raleigh,NC27607,USA.BioinformaticsGraduateProgram,NorthCarolinaStateUniversity,Raleigh,NC27607,USA.*Authorforcorrespondence(kcheng3@ncsu.edu) T.A.A.,0000-0002-8729-6339;M.T.H.,0000-0002-7202-2116;D.S.,0000-0002-1826-5141;L.Q.,0000-0002-6372-7157;H.L.,0000-0002-3229-1426 ©2019.PublishedbyTheCompanyofBiologistsLtdJournalofCellScience(2019)132,jcs231563.doi:10.1242/jcs.231563 JournalofCellScience directlyintothecirculationoftg(fli1a:egfp)zebrafish,whichpossessfluorescentbloodvessels(Fig.1A)(Blumetal.,2008).Followinginfusion,HeLacellsweretrackedthroughintravitallightsheetmicroscopy,forupto24h,tovisualiz
etheextravasationprocessinreal-time.HeLacellsexitedbloodvesselsthroughboththerecentlyidentifiedangiopellosis(AP)mechanismofextravasation,andthecanonicaldiapedesismechanism(Fig.1B,C).TheAPmethodcharacteristicallyelicitedremodelingoftheendothelialcells(indicatedbytheyellowdottedline),andactivelyexpelledtheHeLacellsfromthelumenintothesurroundingtissue(Fig.1B;Movie1).Incontrast,whenHeLacellsunderwentdiapedesisthroughthecharacteristicpenetrationoftheendothelialwall,therewereonly Fig.1.HumancervicalcancercellspossesstheabilitytoextravasatebloodvesselsthroughbothAPanddiapedesis.(A)Illustrationoftheinjectionoffluorescenttumorcellsintothecirculatio
nofzebrafishembryos.Scalebar:200m.(B)RepresentativetimelapseimagesofaHeLacell(cyan)undergoingextravasationtoexitbloodvessels(red)throughAP;theyellowdottedlineindicatesvascularremodeling;thewhitedashedlineindicatestheborderoftheoutermostportionofthebloodvessel.Scalebar:20m.(C)RepresentativetimelapseimagesofaHeLacell(cyan)undergoingextravasationtoexitbloodvessels(red)throughdiapedesis.Scalebar:20m.Arrowheadshighlightthepositionoftheextravasatingtumorcell.(DF)Graphicalrepresentationofthevascularactivityofthevesselduringextravasation,thedurationoftheextravasationeventsandthenumberofcellswhichexitedduringeachextravasationevent;dataisrepresentat
iveofatleast=4injectedzebrafish,and=30fortumorcells(multipleextravasationeventsoccurineachzebrafish).(GI)Graphicalrepresentationofthecircularityoftumorcellsduringtheextravasationevents,thepercentageofcellsthatextravasatedthevessels,andthepercentageofcellswhichexitedthroughAPordiapedesis;dataisrepresentativeofatleast=4injectedzebrafish,and=30fortumorcells.*5;ns,notsignificant.A.U.,arbitraryunits. RESEARCHARTICLEJournalofCellScience(2019)132,jcs231563.doi:10.1242/jcs.231563 JournalofCellScience lowlevelsofvascularremodeling(Fig.1C;Movie2).Themovementofendothelialcellsduringthisprocesswastracked/measuredasvascularactivity,whichrevealedcellsun
dergoingAPempiricallyelicitedhigherratesofvascularremodeling(Fig.1D).Onaverage,thetumorcellsrequired136.5minlongertoundergoacompleteAPextravasationevent,comparedtothetumorcellsundergoingdiapedesis(0.01),withtheaveragedurationfortheAPextravasationeventsbeing263.5min,and127minfordiapedesis(Fig.1E).WeobservedthatmultipletumorcellswereabletoexitvesselssimultaneouslythroughAP,whileonlyindividualcellswereobservedtoextravasatethroughdiapedesis(Fig.1F).Interestingly,tumorcellswhichextravasatedthroughdiapedesiswereobservedtoexitexclusivelyasindividualcells,evenwhentravellingincirculationasclusters.Anotherdistinctionobservedbetweenthetwomethodswasth
echangeincellcircularityduringtheextravasationevents.Tumorcellsundergoingdiapedesisexhibitedadumbbellshapeandtheysqueezedthroughtheendothelialwall,whilecellsundergoingAPretainedtheircircularitywhileundergoingtheevent(Fig.1G;Movie5).Ofthe2030cellsinfused,49.66%extravasatedintothesurroundingtissue,withtheothersremaininginsidethelumenoverthecourseofthe24-hobservation(Fig.1H).Nearly15timesmoreHeLacellsexitedthroughAP(46.5%),thanthediapedesismethod(3.16%),suggestingAPastheleadingextravasationmechanisminthismodel(Fig.1I).APsupportscancerclusterextravasationToevaluatetheextravasationbehaviorofmulticellularCTCclusters,weinjectedhigh-densityamounts
oftumorcells(50tostimulatecellclusteringincirculation(preclusteredcellaggregatesaretoolargetoinject).Tumorcellclusterswereobservedtoexitwhilemaintainingmulticellularity(clusterphenotypewithoutdisassociation)throughAP,addingtothedifferencesobservedbetweenAPanddiapedesis(Fig.2A,B).Specifically,weobservedHeLacellsthatformedclustersinvivopossessedtheabilitytoexitbloodvesselsasclusters,throughAP,intothesurroundingtissueandextravascularcavities(Fig.2C;Movie3).ThisphenomenonwasalsoobservedinZF3primarycancercellsderivedfromatumorisolatedfromanadulttransgenictg(brca2zdf1)mutantzebrafish,and Fig.2.Humancervicalcancerandmelanomatumorcellclustersexita
smulticellularaggregatesthroughAP.(A)Illustrationofatumorcellcluster(cyan)undergoingextravasationofabloodvessel(red)thoughAP.(B)TablelistingkeydifferencesobservedintumorcellextravasationthroughAPdiapedesis.(C)RepresentativetimelapseimagesofaHeLacellcluster(cyan)undergoingextravasationtoexitbloodvessels(red)throughAP;theyellowdottedlineindicatesvascularremodeling;thewhitedashedlineindicatestheborderoftheextravascularcavitythetumorcellsextravasatedinto.Scalebar:20(D)RepresentativetimelapseimagesofaZF3zebrafishprimarytumorcellcluster(cyan)undergoingextravasationtoexitbloodvessels(red)throuAP;theyellowdottedlineindicatesvascularremodeling.Scal
ebar:20m.(E)RepresentativetimelapseimagesofaB16F10cellcluster(cyan)undergoingextravasationtoexitbloodvessels(red)throughAP;yellowdottedlineindicatesvascularremodeling.Numbersdenoteindividualcells.Arrowheadshighlightthepositionoftheextravasatingtumorcell.Scalebar:20m.(F,G)Graphicalrepresentationofvascularactivity(inarbitraryunits)oftumorcellclustersduringtheAPextravasation,andthepercentageofCTCswhichexitedassinglecellsormulticellularclusters;=8.(H)GraphicalrepresentationofthedurationoftheeventsofCTCclustersandsingleCTCs;=10.*N.S.,notsignificant. RESEARCHARTICLEJournalofCellScience(2019)132,jcs231563.doi:10.1242/jcs.231563 JournalofCellScien