Frontiers | Early Dissemination of Circulating Tumor Cells

Circulating tumor cells (CTCs) play a causal role in the development of metastasis, the major cause of cancer-associated mortality worldwide ... ThisarticleispartoftheResearchTopic RevisitingSeedandSoil:ANewApproachtoTargetHibernatingDormantTumorCells Viewall 10 Articles Articles AnnF.Chambers WesternUniversity(Canada),Canada RobertoPiñeiro HealthResearchInstituteofSantiagodeCompostela(IDIS),Spain EVILIANIDOU NationalandKapodistrianUniversityofAthens,Greece Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. AbstractIntroductionIdentificationandCharacterizationofCTCsCTCsasPrecursorsofMetastasisCTCs,DTCsandTumorDormancyClinicalImplicationofEarlyDisseminationConclusionsAuthorContributionsFundingConflictofInterestReferences SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData MINIREVIEWarticle Front.Oncol.,07May2021 |https://doi.org/10.3389/fonc.2021.672195 EarlyDisseminationofCirculatingTumorCells:BiologicalandClinicalInsightsFrancescaChemi*,SumitraMohan,TatianaGuevara,AlexandraClipson,DominicG.RothwellandCarolineDive*CancerResearchUKManchesterInstituteCancerBiomarkerCentre,UniversityofManchester,Macclesfield,United KingdomCirculatingtumorcells(CTCs)playacausalroleinthedevelopmentofmetastasis,themajorcauseofcancer-associatedmortalityworldwide.Inthepastdecade,thedevelopmentofpowerfulcellularandmoleculartechnologieshasledtoabetterunderstandingofthemolecularcharacteristicsandtimingofdisseminationofCTCsduringcancerprogression.Forinstance,genotypicandphenotypiccharacterizationofCTCs,atthesinglecelllevel,hasshownthatCTCsareheterogenous,disseminateearlyandcouldrepresentonlyaminorsubpopulationoftheprimarytumorresponsiblefordiseaserelapse.WhiletheimpactofmolecularprofilingofCTCshasnotyetbeentranslatedtotheclinic,CTCenumerationhasbeenwidelyusedasaprognosticbiomarkertomonitortreatmentresponseandtopredictdiseaserelapse.However,previousstudieshaverevealedamajorchallenge:thelowabundanceofCTCsinthebloodstreamofpatientswithcancer,especiallyinearlystagediseasewheretheidentificationandcharacterizationofsubsequently“lethal”cellshaspotentiallythegreatestclinicalrelevance.TheCTCfieldisrapidlyevolvingwithdevelopmentofnewtechnologiestoimprovethesensitivityofCTCdetection,enumeration,isolation,andmolecularprofiling.HereweexaminethetechnicalandanalyticalvalidityofCTCtechnologies,wesummarizecurrentdataonthebiologyofCTCsthatdisseminateearlyandreviewCTC-basedclinicalapplications.IntroductionThemajorcauseofcancerrelatedmortalityismetastasis(1,2)whichisattributedtodisseminationofcancercells,referredtoascirculatingtumorcells(CTCs),fromtheprimarysiteviathebloodstreamorthelymphaticsystemtosubsequentlyformsecondarytumorsindistantsites.TheburdenofCTCsisstronglyassociatedwithcancerprognosisinseveralcancertypes(3).Metastasiswaslongthoughttooccurinthelaterstagesofcancerprogressioninpatientswithadvanceddisease.However,agrowingbodyofevidencereportsthepresenceofCTCsatearlierstagesoftumorgrowth,evenbeforethedetectionofprimarytumor(4,5).CTCsmayseedactivemetastatictumorsorremaininalatentstatecalledtumorcelldormancythat,atsomepointintimeandviamechanismsincompletelydefined,exitdormancytoformmetastases(6,7).ComprehensiveanalysisofCTCsiscentraltounderstandingmechanism(s)ofcancermetastasis.MolecularprofilingofCTCs,andparticularlyearlydisseminatingCTCscouldalsoleadtodiscoveryofnewprognosticandpredictivebiomarkerstoinformpatientmanagement.ThemajorchallengeforCTCdetectionandanalysisistheirrarityinatypicallysampledbloodvolume(10-50ml),andthisisobservedinmostpatientswithadvancedcancerwithsomenotableexceptionssuchasSmallCellLungCancer(8,9).ThischallengeoflowCTCprevalenceisfurthermagnifiedinearlydiseasesettings.Inadditiontotheirrarity,CTCsarealsogeneticallyandphenotypicallyheterogeneous(includingthespectrumofepithelialtomesenchymalphenotypes).TechnologiesthatcanaccommodateCTCheterogeneityarecriticallyneeded.Thisminireviewwillfocusonearlydisseminatingtumorcells;inparticular,wewillsummarizemethodsforidentificationandcharacterizationofCTCsandgiveanoverviewofcurrentknowledgeonthebiologyandclinicalrelevanceofCTCearlydissemination.IdentificationandCharacterizationofCTCsCTCenumerationisawell-establishedbiomarkerforcancerdiagnosis,prognosis,diseaseprogressionandpredictionoftherapeuticresponse(10).CTCscanbeseparatedand/orenrichedfromotherbloodcellsbyusingdifferenttechnologiesthatexploiteithertheirphysicalproperties(e.g.,size,weight,density,deformability,electrical),ordifferentialexpressionofmolecularmarkers(commonlyintracellularandsurfaceproteinexpression)oracombinationofboth(11–15).CTCenrichmenttechniquesemployingaffinity-basedcapturemethodsuseantibodiesbindingtocellsurfacemarkersandarebasedontwostrategies;1)thenegativeenrichmentapproachthatemploysupstreamimmunomagneticdepletiontoremoveCD45-positiveWhiteBloodCells(WBCs),thoughthisisrarelyachievedtocompletion,and2)thepositiveenrichmentapproachthatcapturesCTCsandthensubsequentlyremovesWBCs(14,16).WhilepositiveenrichmentfailstocapturecellswithlowornegativeexpressionoftheCTCmarkers,negativeenrichmentstrategiestypicallyhavealowerstringencycomparedtopositiveenrichment.ThecommercialRosetteSep™CTCEnrichmentCocktail(StemCellTechnologies)integratesnegativeimmunoaffinity-basedenrichmentwithdensitycentrifugation.Thetechnologyutilizestetramericantibodycomplexesagainstcellsurfaceantigensfoundonhumanhematopoieticcells(CD2,CD16,CD19,CD36,CD38,CD45,andCD66b)andglycophorinAthatenablestheremovalofwhiteandredbloodcellsfromwholeblood,therebyenrichingfortheremainingCTCs(17).Amongthepositiveenrichmentapproaches,CellSearch®technologyisbasedonimmunomagneticenrichmentwhichusesanepithelialcelladhesionmolecule(EpCAM)coatedonferromagneticparticles,withsubsequentimmunomagneticseparation(18).AlternativeCTCisolationtechniquesarerequiredtocapturemesenchymalCTCsorCTCsinthedynamicprocessofEMT.AdditionalCTCplatformsusingenrichmenttechnologyincludethosebasedonimmunomagneticseparationsuchasamagneticcellseparationsystem(MACS)(19)andCTC-ChipwhichstillemploysanEpCAM-basedenrichmentapproachcombinedwithamicrofluidicdevice(16,20).ThemostcommonlyusedantibodiestoidentifyepithelialcellsareEpCAMandcytokeratins(CK-19).However,CTCsundergoingepithelial–mesenchymal-transition(EMT)cangraduallylosetheirepithelialcharacteristics,havingnoorverylowexpressionofEpCAMandconsequentiallyevadecaptureandincreasinglyCTCplatformsarecombiningepithelialmarkerswithothersmarkers,suchasmesenchymalmarkers(e.g.,N-cadherin,vimentinorTWIST1),stemcellmarkers(CD133,CD44,CD34,ALDH1)(21)earlyapoptosismarkers(M30,Bcl-2)orcancerspecificmarkers(e.g.,HER2,PSMA)(22,23).AnotherplatformforCTCdetectionisAdnaTest(AdnagenAG)thatenrichesCTCsusingacocktailofantibodies(e.g.,EpCAM,MUC-1,AR,Her2)specifictothecancertype(e.g.,breast,lung,prostate,ovarian)followedbyasubsequentanalysisoftumorassociatedgeneexpressionbyRT-qPCR(24).Morerecently,anapproachusinganinvivopositiveenrichmenttechnologynamedGILUPICellCollector®(GILUPINanomedizin)hasbeendescribed.ThistechnologyallowscaptureofCTCsdirectlyfromthecubitalveinofthepatientbyusingantibodiesagainstEpCAMwiththeadvantageofusingthetotalvolumeofbloodandincreasingthechanceofCTCsisolation(18).CTCscanbeenrichedwithoutlabellingbasedontheirphysicalproperties.Tumorcellsarenormally(butnotalways)largerthanmostbloodcellsandthischaracteristichasbeenexploitedtocaptureCTCsbysize-basedfiltrationusingofmicrofluidicdevice/cartridgesormicrochipstoseparatecells.Oneoptionfordensity-basedCTCenrichmentistheAccuCyteassaywhichusesadensity-gradientseparationtechnologythatintegratesaseparationtubeandacollectordevice(25).AnotherexampleofCTCenrichmenttechnologybasedonphysicalpropertiesistheParsortix™systemthattrapscellsbasedonbothdeformabilityandsizeindisposablecassetteswithchannelsthatgraduallydecreasesinsizetoapproximately<10μm(26).Acombinatorystrategyusingamicrofluidicplatformsandananotechnology-assistedseparationhasbeenalsodevelopedforCTCisolation(27).Alternativemethodsusingmicrofiltration-basedenrichmentwhichisolatetumorcellsbysizeincludeISET®(RarecellsDiagnostics)(17)andScreenCell®(28).TheelectricalpropertiesofCTCscanalsobeusedfortumor-cellisolationbyapplyinganon-uniformelectricfieldthroughthephenomenonofdielectrophoresis(DEP).Here,apositive(pDEP)ornegative(nDEP)forceisappliedtoacell,movingittowardsorawayfromtheelectricfieldsource,respectively.SomesystemshavebeendescribedwhichemployDEPenrichmenttechnologiesincludingApoStream®(29)andDEParray™automatedsystem.ThelattertrapsstainedcellsinDEPcagesandiscombinedwithahigh-resolutionimagingdevice.Singlecells,selectedviamarkerexpressioncanbemovedwithinthechipbyelectricalforcesandphysicallyisolatedforfurthergenomicanalysis(30).GiventhelikelylossofCTCswithanyenrichmentstep,CTCplatformshavebeendesignedtocaptureallnucleatedcellsinthebloodstream.Thehigh-definitionsingle-cellassay(HD-SCA)developedinPeterKuhn’slaboratory(31,32)andcommerciallyavailablethroughEpicSciences,isbasedonsucha‘nocellleftbehind’approach,wheretheentirepopulationofcellsinaliquidsampleareplatedasabiologicalmonolayerontoglassslidesandimmune-stainedforHD-CTCidentification.EachslidesubsequentlyundergoessophisticatedimageprocessingtodetectrarecellsthatcanbephysicallypickedforgenomicanalysisorsubjectedtosinglecellCYTOFtoassessupto40proteinsperCTC(12,31,33).ForsingleCTCimageanalysis,differenttoolshavebeendevelopedforautomatedprocessesofclassification,sorting,anddetectionofCTCsforsubsequentgeneticanalysis.ArecentstudyintroducedananalysisprogramcalledACCEPT,whichclassifiedfluorescentimagesofsinglecellsfromCellSearch®platformasCTCsornotCTCswithanaccuracyof96%(34).Incontrast,anautomatictoolforlabel-freeCTCdetectionisalsopossiblewherebyCTCsandWBCsareidentifieddirectlyfrombrightfieldmicroscopyimages(35).WecannotdescribeallthecurrentlyavailableCTCplatforms,butreferthereadertoacomprehensiverecentreview(36).NotallcellsdetectedthatareclassifiedphenotypicallyasCTCscarrygenomicaberrations(30,37,38)andincreasinglyphenotypicidentificationofCTCsisfollowedbymolecularprofilingtoconfirmwhethercirculatingcells,howeverenrichedandisolated,aretumorcells.MolecularprofilingofCTCscouldalsoprovideunprecedentedwindowsontothemetastaticprocess,underlyingtumorheterogeneityandinformationontreatmentresponseandresistance(17,29,39–44).Withtheevolvingfieldofsingle-celltechnologies,evaluationofDNA,RNAandproteinalterationsatthesinglecelllevelisnowfeasibleandisbeingappliedtoCTCs(Table1)andanalysisofpairedprimarytumorandCTCshasthepotentialtoshedlightontotumorevolution.Astudyperformedon23patientsshowedthatsheddingofCTCsfromtheprimarytumorisnotrandom;instead,acquisitionofcopynumberaberrations(CNA)isdrivenbyaconvergentprocessacrosstumortypesthatultimatelyleadstothereleaseofCTCswithcomplexgenomicrearrangements(56).Inanotherstudyinbreastcancer,CTCsresembledCNAofprimarytumorsandcontainedalterationsassociatedwithbrainmetastasiswithhighclonality,suggestingthatbrainmetastasiscompetentcellshadundergoneclonalselection(57).Singlecellanalysis,althoughexcitingcanbelimitedbyfailuresinthetechnicallychallengingstepswithintheworkflows.Forthisreason,expandingCTCsin2Dor3DculturesorviainvivomodelscouldovercomethetechnicallimitationsofsingleCTCanalysisandfacilitatefunctionalstudies.PrimaryculturesfromCTCshavebeensuccessfullyestablishedinpatientswithadvancedstagecancer(58)whichmaintainedmolecularandphenotypicpropertiesoftheunculturedprimaryCTCs,matchedgeneticalterationsofthecorrespondingprimarytumorandcouldbeusedtoassessmolecularchangesovertimewithserialblooddraws(59).Incontrast,lowsuccessofCTCcultureshasbeenreportedforpatientswithearlystagecancers,mostlikelyduetothelowerabundanceofCTCscomparedtopatientswithadvancedstagecancer.OptimizationofcultureconditionsanddevelopmentofeventuallyCTCcelllinesisthusaworthygoalthatwillimproveourunderstandingofthebiologicalpropertiesofearlydisseminatingtumorcells.TABLE1Table1SummaryofstudiesthatperformedCTCmolecularprofiling.CTCsasPrecursorsofMetastasisMetastasisisacomplex,multi-stepprocessviawhichcancercellsleavetheprimarytumor,intravasateandsurviveinthebloodstream,extravasate,invadeandcolonizeasecondaryorgansitebeforegrowingintoamacroscopicmetastaticlesion(Figure1)(60).Forepithelialtumors,anearlystepofthemetastaticcascadeisproposedtooccurviaadedifferentiationprogramknownasepithelial-to-mesenchymaltransition(EMT).DuringEMT,tumorcellsdownregulateepithelialmarkerssuchasE-cadherin,detachfromneighboringcellsandacquireamoreinvasivemesenchymalphenotype(61).EMTprogramcanbestimulatedbymultiplefactorsincludinganactivatedtumorassociatedstromaorunderhypoxicconditions(62).Inaddition,theinvasivetumorcellsupregulatemetalloproteinaseactivityleadingtodegradationofextracellularmatrixandenablingtumorcellmigrationtoreachthevasculature(63).However,recentstudiesinmousemodelshaveshownthatinvasionandmetastasiscanoccurindependentlyofEMT(64–66).Inparticular,E-cadherinmayenhancesurvivalduringtumorcelldetachment,disseminationandmetastaticseedingbylimitingreactiveoxygen-mediatedapoptosis(66).ThesefindingsmayatleastinpartexplaintheprognosticroleofepithelialCTCsdetectedbyCellSearch®technologyinseveralcancertypes(14)andthepresenceofhybridphenotypes(epithelial/mesenchymal)inpatientswithcancer(67,68).FIGURE1Figure1Overviewofthemetastaticcascade.Themetastaticprocessincludesinvasion,intravasation,circulation,extravasationandcolonization.CTCsdetachingfromtheprimarytumorcantravelaloneorasclusters.Enumeration,molecularprofilingandexpansionofCTCsinnon-metastatictumorscouldprovideabetterunderstandingonthesignificanceofCTCearlydissemination.FigurecreatedinBioRender.com.Aggressivetumorcellscanalsotransitiontowardsavascularphenotypebyexpressingendothelialmarkersandformingbloodvessels,aphenomenoncalledvasculogenicmimicry(VM)(69).AlthoughVMhasbeendescribedinbreast,ovary,lung,prostate,andbladdercancerandhasbeenassociatedwithdisseminationandmetastasis,itremainsacontroversialissue,withconcernsincludingalackofrobustdiscriminationbetweenVMandendothelialbloodvessels(70).However,asubpopulationofsmallcelllungcancerCTCsthatco-expressedVE-cadherin(amarkerofVM)andepithelialmarkershadacopynumberprofileconfirmingtumororigin,implyingthatinthisaggressivelungcancer,VMmaybecausallyinvolvedinCTCdissemination(71).CTCscantravelassinglecellsorascellaggregatescalledCTCclustersorcirculatingtumormicroemboliwhichhavebeenreportedforseveralcancertypesincludingbreast,prostate,lung,andcoloncancers(72).Althoughtheyaredetectedatalowerfrequencyandhavesignificantlyshorterhalf-lifeinthebloodthansingleCTCs(73),CTCclustersaremorelikelytoformmetastasisinmousemodels(73).CTCclusterscanincludenon-tumorcelltypesincludingpericytes,immunecells,plateletsandcancer-associatedfibroblasts(74)whichmaysupportthesurvivaloftheclusteredCTCs.ArecentstudyidentifiedneutrophilsaccompanyingCTCsinpatientswithadvanced-stagebreastcancerwhereinteractionsbetweenneutrophilsandCTCsmediatedbyVCAM1,promotedcellcycleprogressionandmetastaticseeding,openingupnewtherapeuticvulnerabilitiestopreventbreastcancerspread(44).PlateletscanalsointeractwithCTCs,providingasurrounding‘shield’thatpreventsrecognitionbytheimmunesystemandprotectsagainstshearstressforceswithinthebloodstream(60).RNA-seqperformedonsingleCTCsrevealedthatplateletmarkerswerehighlyexpressedinasubsetofpancreaticCTCs,supportingtheinteractionofthesetwocelltypesinthecirculation(75).CTCsurvivalwithinthebloodstreammayalsobeachievedbyup-regulationofβ-globin(asubunitofhemoglobingenenormallyexpressedbyredbloodcells),asobservedinbreast,prostate,andlungcancers,withaconsequentreductionofoxidativestresswithinCTCs(76).OnlyaminorfractionofCTCsarethoughttocompleteallthestepsofthemetastaticcascade(77,78).CTCextravasationissuggestedtooccurinasimilarmannerasleucocyteextravasation,aprocessinvolvingnumerousligandsandreceptorsexpressedbybothtumorcellsandendothelialcellsincludingselectins,integrins,cadherins,CD44andimmunoglobulin(Ig)superfamilyreceptors(79).ThefirststepsofdistantorgancolonizationmaybepartiallydrivenbygeneticandepigeneticprogramspresentinasubpopulationoftumorcellsatanyprecedingstepofthemetastaticcascadebeforeCTCsseedmetastasisandmolecularprofilingofsampledCTCshasthepotentialtouncovertheirsubsequentcompetenciesandperhapsunveiltheirtissuetropism.Pertinenttothishypothesis,aninvivogenome-wideCRISPRscreeningperformedinbreastcancer-derivedCTCsidentifiedanupregulationofribosomalproteinsandregulatorsofthetranslationmachineryinasubsetofCTCsthatassociatedwithhighmetastaticburdeninmousemodels(80).Supportingthenotionofpredictingtissuetropism,comparisonoftranscriptomicprofilesbetweenbreastcancerCTCsassociatedwithbrainmetastasisandCTCsassociatedwithmetastasistootherorgansrevealedadistinctgenesignatureassociatedwithbrain-homingCTCs(81).Inaddition,CTCswithalterationsinmetabolicpathwaysshowedastrongerlivertropismincolorectalcancer(82)andproteinubiquitylationwasidentifiedasanimportantmechanismofbonemarrowmetastaticseedinginmelanoma(51).CTCs,DTCsandTumorDormancyOnceCTCshavesurvivedwithinthebloodstreamandextravasatedintoadistantsitetheycanresideinadormantstate[oftenreferredtoasdisseminatedtumorcells(DTCs)]foryearsbefore‘awakening’toproliferateandcauseovertmetastasis(83).SeveralstudieshaveshownthatDTCscanbefoundinthebonemarrowofpatientswithoutovertmetastases,indicatingthatthesecellsdisseminateearlyduringtumorprogression(84).Insupportofthishypothesis,geneticanalysisofbonemarrowDTCsfrombreast,prostate,andoesophagealcancerrevealedfewerchromosomalabnormalitiesinDTCsthaninmatchedprimarytumorcells,indicativeofaparallelprogressionmodelofmetastaticgrowth(85–87).IdentificationofDTCs,togetherwithanincreasedunderstandingandthentargetingofthe‘awakening’stimuliandmechanism(s)holdspotentialinimprovingpatients’clinicaloutcomesalthoughfinding,isolatingandanalyzingDTCsistechnicallychallengingandinvasive.NeverthelessknowledgeoftumordormancyandDTCshasimprovedinrecentyears.Extrinsicfactorsincludingalackofangiogenesis,immunesurveillance,andthebalancebetweenproliferationandapoptosishaveallbeenshowntodrivetumordormancy(88).MolecularprofilingofDTCshasenabledabetterunderstandingofthecell-intrinsicsignalsthatinducedormancy,suchasinhibitionofpathwaysinvolvedincell-cycleregulation,metabolicsignalsandautophagy(89).However,onlyafewstudieshaveinvestigatedwhetherCTCsfromperipheralbloodexpressmarkersoftumordormancy(90–92).Inabreastcancerstudy,CTCsubsetswereselectedforEpCAMnegativity,positivityforstemcellmarkers(CD44+/CD24−)andcombinatorialexpressionofuPAR/intβ1becausedownregulationofthesetwomarkershasbeendirectlyimplicatedinbreastcancerdormancy(90).TheuPAR+/intβ1+subgroupofCTCswerefoundtobemoreproliferativecomparedtouPAR-/intβ1-CTCsininvitroassays,suggestingthatthesetwomarkerscouldbeusedtodistinguishCTCsthatsubsequentlyproliferatevsbecomedormantatdistantsites(90).AlaterstudyfromthesamegroupidentifiedmTORsignalingasacriticaldeterminantinpromotingCTCseedingandmaintainedlong-termbonemarrow-residentbreastcancercelldormancy(91).Thebalancebetweenproliferationandapoptosishasbeenshowntobeassociatedwithtumordormancy(88,93).Inlinewiththisfinding,proliferationandapoptosismarkers(Ki67,M30)weremeasuredonCTCsderivedfrompatientswithbreastcancerwhoweredisease-freeforatleast5yearsorwhorelapsedmorethan5yearsaftersurgery.ThestudyfoundthatapoptoticCTCsweredetectedmorefrequentlyinpatientswhoremaineddisease-freecomparedtothosewhoexperiencedlaterelapse,suggestingthattheexpressionofthesetwomarkerscouldbepotentiallyusedtopredictescapefromdormancy(92).Inanotherstudy,36%ofpatientshaddetectableCTCs8to22yearsaftermastectomywithoutevidenceofprogressivetumorgrowth.Theauthorsofthisstudysuggestthatthiscouldbeassociatedwithafailuretocompletethefinalstagesofmetastasis,whichcouldbepotentiallybeingkeptincheckbyaprevailingapoptosis/proliferationbalancethatmaintainsadormantstateindistantsites(94).However,theclinicalapplicationofCTCdetectioninthetumordormancycontextstillremainsunclear.FutureresearchinthisfieldshouldfocusontheidentificationofCTCmolecularfeaturesthatcoulddistinguishbetweencancersthatarebehavingmoreaggressivelyfromthosethatwillenteradormantstate.ClinicalImplicationofEarlyDisseminationAnalysisofCTCshaveenhancedourunderstandingofcancerbiology(61)aswellasthepotentialvulnerabilitiesofthemetastaticcascade.TheapplicationofCTCbasedassaysinaclinicalsetting,especiallyinearlystagediseasehasbeenchallenging,primarilyduetothelowfrequencyofCTCs.QuestionsoftendebatedinthefieldofCTCresearcharewhetherthelownumberofsinglecellsanalyzed(typicallylessthan10)aresufficienttocapturetumorheterogeneityandifthisheterogeneityisbettercapturedinatumorbiopsy(95).However,mosttumorbiopsyproceduressampleasingleregionorlimitednumberofregionsofatumoranditisoftendifficulttoassesswhethertheaggressivetumorcloneshavebeencaptured(96).Incomparison,CTCsarecellsthathaveundergonetheselectionprocessandhavealreadyenteredthemetastaticcascade,thoughasmentionedpreviously,onlyaminorfractioncompleteit.WhethertheseCTCsareindeedabetterrepresentationoftheaggressivetumorclonesthantumorbiopsyisyettobedetermined,especiallyinearlystagecancers.Tothisend,inthepastdecadeseveralstudieshaveexploredthepotentialofCTCsinclinicalresearchandtheimplicationsofearlydisseminationofCTCsinpatientdiagnosisaswellasprognosis.Althoughtechnologiesforprofilingtheseraresinglecellshaveevolvedintherecentyears,singlecellmanipulationandanalysis(capture,enumeration,molecularprofiling,andbioinformaticworkflows)willlikelyneedtobesimplified,automatedandlessexpensivetobecomeroutinelyfeasibleandtakenupintheclinic.ThelowprevalenceofCTCsinearlydisease(97–100)clearlyhindersextensivestudies.Inpatientswithearlybreastcancer,theTREAT-CTCtrialwasthefirsttodemonstratetheclinicalutilityofCTCsusingtheCellSearch®platform.ThistrialaddressedtherequirementofadditionaltreatmenttoeliminateCTCspostadjuvantchemotherapyandCTCscreeningwasperformedattheendofadjuvantchemotherapyin1317patientswithHER2negativebreastcancer.Ofthe95CTCpositivepatients,63wererandomlyassignedtoobservationortrastuzumabadministration.ThetrialdemonstratedthefeasibilityofCTCbasedscreeninginanadjuvantsettingaswellasthehigherrateofrelapseamongstCTCpositivepatients(101).Morerecently,inacohortof75patients’withlimitedstageSCLC(LS-SCLC,definedastumorconfinedwithinonlyonelungand/orinthelymphnodesinthemediastinum)theCONVERTtrialdeterminedthat≥15CTCswasasanindependentprognosticmarkerwith60%ofpatientshaddetectableCTCsatpre-treatmentsampling(102).InlocalizedprostatecancertherewasadefinitetrendtowardsapositivecorrelationofCTCswithpathologicalstageaswellasatrendtowardsprognosticandpredictiveimpactofdetectingCTCswithseveralstudiesreportingcorrelationswithpatientsurvivaland/ordiseaserecurrenceposttreatment(103–107).Astudyin2014ofpatientswithchronicobstructivepulmonarydisease(COPD),founddetectableCTCsinsomepatients,withthesepatientsdevelopinglungnodules1–4yearslaterandwithfourpatientsdiagnosedwithinvasiveadenocarcinomaandafifthdiagnosedwithsquamouscellcarcinoma,demonstratingthepredictivevalueofCTCsinearlyNSCLC(4).However,thestudyalsoreportedfalsepositivesinthreepatientswhodidnotdevelopovertcancersuggestingtheneedforfurthervalidationusingbroaderCTCdetectionsystemsinlargenationwidescreeningprograms.Furthermore,instageI-IIINSCLCCTCscollectedatsurgerypriortotumorresectionfromthedrainingpulmonaryveinwerehigherincountcomparedtosamplingoftheperipheralblood(1–3,093vs.0–4CTCsintheperipheralblood)andalthoughalargerstudywillberequiredtovalidatethisfinding,CTCcountwasassociatedwithriskofrelapse(30,108,109).Strikingly,inacasestudywithinthiscohort,genomiccomparisonofindividualpulmonaryveinCTCstotheresectedprimarytumorandasecondarytumorwhichdeveloped10monthslater,revealedthatCTCshadmoregenomicvariantsincommonwiththemetastasisthantheprimarytumorimplicatingearlydisseminatingCTCsasresponsiblefordiseaserelapse(29).Furtherstudiesusingthisapproachtoconfirmthesefindingsarewarranted.AfurtherpotentialutilityofCTCs,giventhedataemergingonearlydissemination,isasbiomarkersofminimalresidualdisease(MRD)followingtreatmentwithcurativeintentwheretumorphenotypeandgenotypecanbeassessedasindicatorsof(aggressiveversusindolent)relapsetime-courseasacomplementaryapproachtoctDNAmonitoring(110).TheincompleteprimarytumoreradicationwithconsequentpersistenceofresidualcellsintheformofCTCsorDTCsremainsamajorchallengeintheclinicalmanagementofpatientswithcancer.ThedetectionofMRDafterprimarycurativetreatmenthasthepotentialtoidentifyhigh-riskpatientswhocanbenefitfromadditionaltreatmentsandmonitoring.TheroleofCTCsinMRDmonitoringhasbeeninvestigatedinseveralcancertypesincludingbreast,colorectal,lung,andprostatecancers(111).Inthesestudies,detectionofCTCsatafollow-uptimepoint(rangingfrom3monthsto5yearspostchemotherapy,accordinglytothetumortypes)wassignificantlyassociatedwithunfavorableoutcomes.Inparticular,studiesfromourgroupshowedthatthepresenceofCTCs(measuredbyCellSearch®)afteronecycleofchemotherapywasassociatedwithworseoverallsurvivalinbothpatientswithNSCLCandSCLC(9,112).GiventheprovenclinicalrelevanceofCTCsintheMRDsetting,ultrasensitiveassaysarenowrequiredinordertodetectsmallnumberofcellsandtocaptureabroadrangeofCTCphenotypes(epithelial,mesenchymalorboth).Morerecently,adistinguishingrolebetweenCTCsandDTCshasbeenreported:patientswithdetectableCTCsintheMRDsettingrelapsedearliercomparedtothosewithdetectableDTCsonly,whoshowedalaterrelapse(113,114).ConclusionsIntheclinic,analysisofCTCshasbeenusedforprognosticstratificationofmanysolidcancerssuchasbreast,smallcelllung,non-smallcelllung,colorectal,andprostatecancersaswellastomonitordiseaseprogression.However,CTCsasaliquidbiopsyhavenotyetfulfilledtheirundisputablepotentialtoinformofpersonalizedmanagementofpatientswithcancerwhichmayevenextendtothehighbarofearlierdetectionofcancers.ThelownumberofCTCsinthecirculationandthesensitivityoftheCTCassayscurrentlyinuseremainachallenge.Tothisend,intenseeffortshavebeenmadearoundtheworldtostandardizeCTCbasedassaystoovercomethetechnicalchallengesofenrichment,detection,enumeration,isolation,andNGSanalysesandtoincreaseassaysensitivity.DevelopmentsinthefieldofCTCenrichmentinstrumentsandNGSanalyseshaveelevatedCTCstudies,bringingexcitinginsightsintobiology,heterogeneityandevolutionoftumorsandbegintoilluminatethepathwaysthatunderlietumordisseminationandsubsequentstepsofthemetastaticcascade.ThesestudiesincludedataonCTCheterogeneity,interactionsinthebloodstreamwithothercelltypes,immuneevasion,metastaticpotentialandorgantropism.However,severalunansweredbiologicalquestionsremain,suchaswhatcausesthedisseminationofcellsintothecirculation,whatdeterminesthetropismoftheseCTCsatametastaticsiteandfurtherhowandwhichpathwaysneedtobetargetedtocurbthemetastaticpotentialofthesesinglecells.Theanswerstothesequestionscouldbeverydifferentdependingontheprimarytumorinquestionandmoreresearchmustbedonetoanswerthem.Furthermore,morestudiesinlargepatientcohortswillneedtobedesignedtoaddresstheclinicalutilityofCTCsbeyondsingleCTCandCTCclusterenumerationsothatCTCdatacanbeusedforindividualizedtestsfordrugsusceptibilityandinvestigatepredictivebiomarkersofresponsetotreatmentsaswellasforearlierdetectionofdiseaseprogression.Although,wehavecomealongwayinCTCresearchthequestionremainsifCTCsarereadyforprimetimeintheclinic.Inouropinion,thestandardizationofCTCassaysalongwiththecombiningoutputsfromotherliquidbiopsyreadoutssuchascellfreeDNA,cellfreeRNAandcirculatingproteinswillhelprealizetheirtruepotential.AuthorContributionsFC,SM,andTGdraftedthemanuscriptandAC,DR,andCDevolvedthemanuscripttothefinaldraft.Allauthorscontributedtothearticleandapprovedthesubmittedversion.FundingThisworkwassupportedthroughCorefundingtoCancerResearchUK(CRUK)ManchesterInstitute(A27412),theCRUKManchesterMajorCentreAward(A25254),theCRUKLungCancerCentreofExcellence(A20465)andaR01grantfromUSNationalCancerInstitute(R01CA197936).SupportwasreceivedfromtheManchesterNIHRBiomedicalResearchCentre,andtheManchesterExperimentalCancerMedicineCentre.ConflictofInterestTheauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest.References1.WeissL.MetastasisofCancer:AConceptualHistoryFromAntiquitytothe1990s.CancerMetastasisRev(2000)19(3-4):193–383.doi:10.1023/A:1010623111204CrossRefFullText|GoogleScholar2.WittekindC,NeidM.CancerInvasionandMetastasis.Oncology(2005)1:14–6.doi:10.1159/000086626CrossRefFullText|GoogleScholar3.VasseurA,KiavueN,BidardFC,PiergaJY,CabelL.ClinicalUtilityofCirculatingTumorCells:AnUpdate.MolOncol(2020).doi: 10.1002/1878-0261.12869PubMedAbstract|CrossRefFullText|GoogleScholar4.IlieM,HofmanV,Long-MiraE,SelvaE,VignaudJ-M,PadovaniB,etal.“Sentinel”CirculatingTumorCellsAllowEarlyDiagnosisofLungCancerinPatientsWithChronicObstructivePulmonaryDisease.PloSOne(2014)9(10):e111597.doi: 10.1371/journal.pone.0111597PubMedAbstract|CrossRefFullText|GoogleScholar5.RiedK,EngP,SaliA.ScreeningforCirculatingTumourCellsAllowsEarlyDetectionofCancerandMonitoringofTreatmentEffectiveness:AnObservationalStudy.AsianPacJCancerPrev(2017)18(8):2275–85.doi: 10.22034/apjcp.2017.18.8.2275PubMedAbstract|CrossRefFullText|GoogleScholar6.Recasens,A,MunozL.TargetingCancerCellDormancy.TrendsPharmacolSci(2019)40(2):128–41.doi: 10.1016/j.tips.2018.12.004PubMedAbstract|CrossRefFullText|GoogleScholar7.ValletteFM,OlivierC,LézotF,OliverL,CochonneauD,LalierL,etal.Dormant,Quiescent,TolerantandPersisterCells:FourSynonymsfortheSameTargetinCancer.BiochemPharmacol(2019)162:169–76.doi: 10.1016/j.bcp.2018.11.004PubMedAbstract|CrossRefFullText|GoogleScholar8.ChenL,BodeAM,DongZ.CirculatingTumorCells:MovingBiologicalInsightsIntoDetection.Theranostics(2017)7(10):2606–19.doi: 10.7150/thno.18588PubMedAbstract|CrossRefFullText|GoogleScholar9.HouJM,KrebsMG,LancashireL,SloaneR,BackenA,SwainRK,etal.ClinicalSignificanceandMolecularCharacteristicsofCirculatingTumorCellsandCirculatingTumorMicroemboliinPatientsWithSmall-CellLungCancer.JClinOncol(2012)30(5):525–32.doi:10.1200/JCO.2010.33.3716PubMedAbstract|CrossRefFullText|GoogleScholar10.ThieleJA,BethelK,KrálíčkováM,KuhnP.CirculatingTumorCells:FluidSurrogatesofSolidTumors.AnnuRevPathol:MechDis(2017)12:419–47.doi: 10.1146/annurev-pathol-052016-100256CrossRefFullText|GoogleScholar11.ShenZ,WuA,ChenX.CurrentDetectionTechnologiesforCirculatingTumorCells.ChemSocRev(2017)46(8):2038–56.doi: 10.1039/c6cs00803hPubMedAbstract|CrossRefFullText|GoogleScholar12.ThieleJA,PituleP,HicksJ,KuhnP.Single-CellAnalysisofCirculatingTumorCells.MethodsMolBiol(2019)1908:243–64.doi: 10.1007/978-1-4939-9004-7_17PubMedAbstract|CrossRefFullText|GoogleScholar13.VaidyanathanR,SoonRH,ZhangP,JiangK,LimCT.CancerDiagnosis:FromTumortoLiquidBiopsyandBeyond.LabChip(2019)19(1):11–34.doi: 10.1039/c8lc00684aCrossRefFullText|GoogleScholar14.MohanS,ChemiF,BradyG.ChallengesandUnansweredQuestionsfortheNextDecadeofCirculatingTumourCellResearchinLungCancer.TransLungCancerRes(2017)6(4):454–72.doi: 10.21037/tlcr.2017.06.04CrossRefFullText|GoogleScholar15.FerreiraMM,RamaniVC,JeffreySS.SciencedirectCirculatingTumorCellTechnologies5.MolOncol(2016)10(3):374–94.doi: 10.1016/j.molonc.2016.01.007PubMedAbstract|CrossRefFullText|GoogleScholar16.RawalS,YangY-P,.CoteR,AgarwalA.IdentificationandQuantitationofCirculatingTumorCells.AnnuRevAnalChem(2017)10(1):321–43.doi:10.1146/annurev-anchem-061516-045405CrossRefFull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emicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms.*Correspondence:FrancescaChemi,[email protected];CarolineDive,[email protected] 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