Concepts of metastasis in flux: the stromal progression model

Concepts of metastasis in flux: The stromal progression model

Jonathan P. Sleeman

, Gerhard Christofori

, Riccardo Fodde

, John G. Collard

, Geert Berx

, Charles Decraene

, Curzio Rüegg

aUniversitätsmedizinMannheim,UniversityofHeidelberg,CentreforBiomedicineandMedicalTechnologyMannheim(CBTM),TRIDOMUS-GebäudeHausC, Ludolf-Krehl-Str.13-17,D-68167Mannheim,Germany

bKITKarlsruheCampusNord,Hermann-von-Helmholtz-Platz1,76344Eggenstein-Leopoldshafen,Germany

cDepartmentofBiomedicine,InstituteofBiochemistryandGenetics,UniversityofBasel,Mattenstrasse28,CH-4058Basel,Switzerland

dDepartmentofPathology,JosephineNefkensInstitute,ErasmusMC,Rotterdam,TheNetherlands

eDivisionofCellBiology,TheNetherlandsCancerInstitute,Plesmanlaan121,1066CXAmsterdam,TheNetherlands

fUnitofMolecularandCellularOncology,DepartmentforMolecularBiomedicalResearch,VIB,9052Ghent,Belgium

gDepartmentofBiomedicalBiology,GhentUniversity,9052Ghent,Belgium

hTranslationalResearchDepartment,InstitutCurie,CentredeRecherche,Paris,France

iCNRS,UMR144,Paris,France

jPathology,DepartmentofMedicine,FacultyofScience,UniversityofFribourg,1RueAlbertGockel,CH-1700Fribourg,Switzerland

Theabilityoftumorcellstoleaveaprimarytumor,todisseminatethroughthebody,andtoultimately seednewsecondarytumorsisuniversallyagreedtobethebasisformetastasisformation.Anaccu- ratedescriptionofthecellularandmolecularmechanismsthatunderliethismultistepprocesswould greatlyfacilitatetherationaldevelopmentoftherapiesthateffectivelyallowmetastaticdiseasetobe controlledandtreated.Anumberofdisparateandsometimesconflictinghypothesesandmodelshave beensuggestedtoexplainvariousaspectsoftheprocess,andnosingleconceptexplainsthemechanism ofmetastasisinitsentiretyorencompassesallobservationsandexperimentalfindings.Theexciting progressmadeinmetastasisresearchinrecentyearshasrefinedexistingideas,aswellasgivingrise tonewones.Inthisreviewwesurveysomeofthemaintheoriesthatcurrentlyexistinthefield,and showthatsignificantconvergenceisemerging,allowingasynthesisofseveralmodelstogiveamore comprehensiveoverviewoftheprocessofmetastasis.Asaresultwepostulateastromalprogression modelofmetastasis.Inthismodel,progressivemodificationofthetumormicroenvironmentisequally asimportantasgeneticandepigeneticchangesintumorcellsduringprimarytumorprogression.Mutual regulatoryinteractionsbetweenstromaandtumorcellsmodifythestemnessofthecellsthatdrivetumor growth,inamannerthatinvolvesepithelial–mesenchymalandmesenchymal–epithelial-liketransitions.

Similarinteractionsneedtoberecapitulatedatsecondarysitesformetastasestogrow.Earlydisseminat- ingtumorcellscanprogressatthesecondarysiteinparalleltotheprimarytumor,bothintermsofgenetic changes,aswellasprogressivedevelopmentofametastaticstroma.Althoughthismodelbringstogether manyideasinthefield,thereremainneverthelessanumberofmajoropenquestions,underscoringthe needforfurtherresearchtofullyunderstandmetastasis,andtherebyidentifynewandeffectivewaysof treatingmetastaticdisease.

Abbreviations: BMDC,bonemarrow-derivedcell;CAF,carcinoma-associated fibroblast;CGH,comparativegenomichybridization;CSC,cancerstemcell;CTC,cir- culatingtumorcell;DTC,disseminatedtumorcells;ECM,extracellularmatrix;EMT, epithelial–mesenchymaltransition;FAK,focaladhesionkinase;mCSC,migrating cancerstemcell;MET,mesenchymal–epithelialtransition;TAM,tumor-associated macrophage.

∗Correspondingauthorat:UniversitätsmedizinMannheim,UniversityofHei- delberg, Centre for Biomedicine and Medical Technology Mannheim (CBTM), TRIDOMUS-Gebäude Haus C, Ludolf-Krehl-Str. 13-17, D-68167 Mannheim, Germany.Tel.:+496213839955;fax:+496213839961.

E-mailaddress:[email protected](J.P.Sleeman).

1. Introduction

Eversincemetastasishasbeeninvestigated,modelsandcon- ceptsabouthowthemetastaticdiseaseprocessworkshavebeen suggested [1]. These have provided a framework withinwhich to understand clinical observations and experimental findings, have servedasanimportanttoolfor directingfurtherresearch, and havesuggestedhow newtherapies thataddressmetastatic disease might be developed. Most early concepts were based onclinicalobservationsandautopsy findings.Theseincludethe

“seedandsoil”hypothesisthatenvisagestumorcellsasseedsthat requireaparticularorganmicroenvironmentor“soil”iftheyareto

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surviveoutsideoftheprimarytumorandgrowasmetastases[2], theanatomical/mechanicalmodelthatproposesthatpatternsof metastasiscanbeexplainedbythelocationoftheprimarytumor withrespecttothebloodandthelymphaticvasculature,whichin turndeterminestowhichorgansdisseminatingtumorcellswill betransportedandsubsequentlybecomemechanicallyentrapped [3,4],andtheoriesaboutmetastaticcascadesandgeneralizingsites thatholdthatmetastasesinoneorgancoulddisseminatetumor cellsandgiverisetometastasesinfurtherorgansinasequential manner[5,6].

The complexity of metastasis as a process determines that noneoftheseorindeedotherconceptscompletelyandaccurately describeshowtheprocessworks,nordotheyintegrateandencom- passallclinicalobservationsandexperimentalfindings.Thiscan havemajorconsequencesfortherapy.Forexample,Halstead’srad- icalmastectomyforthetreatmentofbreastcancerinwhichthe axillaanditslymphnodesareremovedinadditiontothebreast containingtheprimarytumorwasdevelopedonthebasisofthe metastaticcascadeconcept.Therationalwasthatiflymphnodes containingmetastatictumorcellswereleftinsitu,thentheselymph nodemetastasescouldthemselvesgiverisetometastasesinother organs.Removingalllymphnodesintheaxillashouldtherefore improvesurvivalrates.However,large-scalelong-termrandom- izedtrialshaveprovidedevidenceinrecentyearsthatforanumber oftypesofcancerremovingthelymphnodesthatdrainprimary tumorshasverylittleeffectonpatientsurvival[7].Furthermore, recentanalysisofthegrowthrateoftumorssuggeststhatwithin thelifetimeofacancerpatientthereisnotenoughtimefortheserial seedingofmetastasesfromametastasiselsewhere[8].Together, theseobservationsunderlinetheimportanceofanintegratedand accurateconceptofhowmetastasisworks,ifefficientandeffective therapiesaretobedeveloped.

Inthelastfew years,rapidprogresshasbeenmadeinmany areasofmetastasisresearch.Thesenewinsightsintotheprocess ofmetastasishavechallengedexistingacceptedparadigms,stim- ulatedthedevelopmentofnewconceptsandmodels,expanded ourunderstandingof hithertopoorlyunderstoodaspectsofthe process,andhavehighlightedtheneedtore-evaluateandinter- pretexistingdatainthelightofthesenewfindings.Inthisreview, wediscusslong-standingconceptsabouthowmetastasisdevel- opsinthecontextofsomeofthecontemporarytheoriesthathave arisenrecentlyasaconsequenceofthesenewobservations.We usetheconceptofthemetastatic“seed”andthe“soil”oftheorgan microenvironment–themostlong-lastingandinfluentialhypoth- esisinthefield–asaframeworkwithinwhichtodiscussthese ideas.

2. Howdoesthemetastaticseeddevelop?

2.1. Clones,heterogeneityandselection

Basedonaseriesofseminalobservationsinexperimentalani- mals[9,10],Fidlerandothersformulatedtheclonalselectionmodel to explain how tumor cells acquire the ability to metastasize.

Thismodelpostulatesthatduringtumorprogression,increasing genomicinstabilityintheprimarytumorresultsinthestochas- ticaccumulationofgeneticandepigeneticdefects,resultingina heterogeneouspopulationoftumorcellsthatdifferintheirgene expressionpatterns.Thegeneexpressionprofileofsomeofthese cellswillbesufficienttoendowthissubpopulationwiththeprop- ertiesrequiredforlocalinvasion,survivalinthecirculatorysystem, extravasationintosecondaryorgans,andgrowthasovertmetas- tasesatthesesites.Othersubpopulationsofcellsintheprimary tumorwillhavesomeofthepropertiesrequired,butwillnotsuc- cessfullycompleteallthenecessarysteps.Thustumorcellsthat

successfullyformmetastasesshouldbeconsideredas“decathlon winners”[10].

Inadditiontoexperimentalevidencefromanimalmodels,sup- portfortheclonalselectiontheorycomesfromhistologicaland geneticanalysisofhumantumorswhichprovidesevidenceforhet- erogeneouspatterns ofgene expression[11].A corollaryof the clonal selectiontheoryisthat organ-specificpatternsof metas- tasis may bedependent on tumor-intrinsic properties that are selectedfor as tumorcells disseminate.Initialevidence for the existenceofgenesdrivingorgan-specificmetastasiscamefromthe identificationofpoorprognosisgenesignaturethroughsupervised clustering ofcohorts ofprimary breast cancers [12–15].Subse- quently,geneexpressionsignaturesassociatedwithbreastcancer metastasistobone,lung andbrain weredefinedinexperimen- tal models and validated with human samples [16–18]. These experimentalstudies werebased onthe generationand analy- sisoforganotropicmetastaticlinesderivedfromaparentalline (mostlyMDA-MB-231)bymultipleroundsofinvivoselection.The brainandlungmetastasissignaturewerepartlyoverlappingand containedgenescontrollingvascularremodelingandpermeabil- ity,suchasCOX2,ANGPTL4, LTBP1andEGFRligands.Thebone metastasissignaturewasrather divergent,andcontainedgenes associatedwithboneosteolysisandcellsurvivalinthebonesuchas IL-11,PTHrPandOPN.Besidesallowingtheidentificationofindi- vidualgenes,thesestudiesprovedusefulfortheclassificationof metastasis-promotinggenesbasedontheirfunctionalcontribution to metastasis. Three categories were defined: (i) metastasis- initiating genes,comprising genesthatprovideanadvantagein tumorcellgrowth,escapeandinvasivenessattheprimarytumor site;(ii)metastasisvirulencegenes,givingsurvivaladvantagesto disseminatedtumor cells within thenewly colonized microen- vironment;(iii)genespromotingprogression,givingadvantages duringtheentiremetastaticprocessbyaffectinggeneralsteps,such as tumor angiogenesis, inflammation, epithelial–mesenchymal transition(EMT),or immune evasion.While thesestudieshave providedunprecedentedmoleculardetailsonthemechanismsof organ-specific metastasis,many questions that are relevant for thedevelopmentoftherapeuticstrategiesremainopen.Forexam- ple,theexperimentalmodelsarebasedontheuseofhumancell lines in immunosuppressed mice,thereby bypassinga possible role of the adaptive immune system in controlling metasta- sis. Also, cancer cells were injected directly into the vascular system in these models, thus mimicking only the final steps ofmetastasis.

Theclonalselectiontheorywouldnotseemconsistentwiththe observationthatprimarytumors areoftenphenotypically simi- lartothemetastases theygiveriseto[19],asaccordingtothis model,metastases shouldrepresentselectionof onlyasubpop- ulation in the primary tumor.Other observations, for example fromgene expressionprofiling of primarytumors, alsosuggest thattheclonalselectionmodelmayneedtobere-evaluated[20].

Thesestudieshavedefinedmolecularsignaturesinprimarytumors thatsuccessfullypredictpatientprognosis.Themajorityoftumor cells intheprimary tumormustexpressthesignaturefor it to bedetected, which doesnot seem toconform withthe notion that a small subpopulation of tumor cells develop metastastic properties,assuggestedbytheclonalselectionhypothesis.These dataratherindicatethat metastaticdevelopmentispre-defined by geneticchanges acquiredduring the initialstages of tumor development.Consistently,transcriptomeanalysissuggeststhat primarytumors arerathersimilartotheirmatchedmetastases, and are more similar with each other than with tumors from other individuals [21]. Nevertheless, a number of observations make it difficultto usetranscriptome analysistodraw conclu- sionsabouttheprovenanceofthetumorcellsthatseedmetastases withconfidence,asalthoughtranscriptomicallysimilar,primary

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tumorsandtheirmatchedmetastasesalsodisplayprofounddiffer- encesintheirgeneexpressionprofiles[8,22].Thedifferentgenetic backgroundsofindividualsmayaccountforthemoreextensivedif- ferencesbetweenindividualsthanbetweentheirmetastasesand theirprimarytumors.Moreover,recentstudiessuggestthatpri- marytumorsarecomposedofclonalareas,whichwouldnotbe detectedbystudiesthatsimplytaketotaltumormaterialforanal- ysis[23]. Furthermore,theexistenceofa predictive‘metastatic signature’inprimarytumorsmightnotbeinconsistentwiththe clonalselectiontheory,sincemetastatictumorcellsmayself-seed backtotheprimarytumorandtherefore‘contaminate’aprimary tumorsignaturewithametastaticsignature[24,25].Self-seeding oftheprimarytumorwithmetastasis-derivedcancercellsmight alsocomplicatetheinterpretationoftheestablishedrelationship betweenprimarytumorsizeandmetastaticpotential[26,27].

Variationsontheclonalselectionmodelhavebeenproposed thathelptoresolvesomeoftheseissues.Theclonaldominance modelsuggeststhatmetastaticallycompetentcells haveacom- petitiveadvantageandthereforeoutgrowothersubpopulationsin theprimarytumor[28].Thedynamic heterogeneitymodelsug- geststhattheacquisitionofmetastaticcompetencemayonlybe transient,andthatthefrequencywithwhichmetastaticallycom- petentcellsarisedeterminesthemetastaticpotentialofagiven tumor[29].

2.2. Isthedisseminationofthemetastaticseedanearlyorlate eventinthelifeofatumor?

Implicitintheclonal selectiontheoryand itsvariantsis the ideathatcancercellsneedtoaccumulateasufficientnumberof geneticandepigeneticalterationstoacquirefullmetastaticcapac- ity,requiringthatmetastasisisaratherlateeventduringtumor progression toallowthe accumulationof suchalterations [30].

Thisnotionisconsistentwiththegenerallyacceptedcorrelation betweenprimarytumorsizeandriskoflymphnodeanddistant metastasis [27], and the observation that metastatic genes are alreadyexpressedinprimarytumors[31].

Inthelastfewyearsasignificantbodyofevidencehasemerged whichindicatesthattumorcellsthatultimatelyformmetastases may disseminate very early aftertumorigenesis. This notionis basedonthegenomicanalysisofsingledisseminatedtumorcells (DTCs),aswellasmatchedprimarytumorsandtheirmetastases fromhumanpatients[8,22].Similarly,experimentalmanipulation ofanimalmodelsofmetastasissuggeststhatdisseminationmay occureven atpre-malignant stages oftumorigenesis[32].Con- sistently,circulatingtumorcells(CTCs)inthebloodandDTCsin thebone marrowcanbothbedetectedatearlystages oftumor developmentincancerpatients[30,33–37].Toaccommodatethese observations,analternative modelhasbeenproposedin which tumorcellsdisseminateearlyduringtumorprogression,andsub- sequentlyacquireadditionalgeneticchangesthatultimatelyallow themtogrowoutasmetastasesatthedistantsite.Inthismodel, primarytumorsandmetastasesprogressinparallelasindepen- dentlesions[8].Clonalselectioninprimarytumorsandmetastases would becompatiblewiththis model,but wouldnot beapiv- otaldeterminantof when disseminationof themetastaticseed occurs.

ThecomparativegenomicanalysisofDTCsfromlymphnode and bone and their corresponding primary tumors has been performed using comparative genomic hybridization (CGH) for a number of typesof cancer, and providessignificantevidence in support of a parallel progression model. For example, DTCs generally showfewer geneticabnormalities than theirprimary tumorsandthereisalsoextensivedisparitybetweenchromosomal gainsandlosseswhenDTCsandtheirprimarytumorsarecom- pared(reviewedin [8,22]).These studiesalsoprovideevidence

thatgeneticabnormalitiesinDTCswereacquiredindependently ofthose intheprimarytumor,andthatsubstantialnumbersof chromosomallosseswerefoundinprimarytumorsthatwerenot present in DTCs.Aslossof DNAis irreversibleand transmitted to progeny,these observations provideevidence for both early disseminationofmetastaticfoundercellsandparallelprogression.

However, studies on DTCs are potentially complicated by the useofepithelialmarkerstodetectthem.Tumorcellsundergoing EMT, for example(see below), maynot express thesemarkers andthereforewouldnotbeincludedintheanalysis,potentially skewingtheresults.Nonetheless,whenmatchedprimarybreast tumors and theirmetastases were alsocompared genomically, forexampleusingCGH,almosthalfofthepairedsamplesshowed morediscordancesthansharedchromosomalabnormalities,and a substantialnumberof chromosomallosses werefoundin the primary tumors that were not present in the metastases [38].

Similarfindingshavebeenmadeinotherstudies[39,40].

Inadditiontothisgenomicanalysis,otherevidencealsosup- portsthenotionofearlydisseminationandparallelprogression.

DTCsmayremaindormantover prolongedperiodsoftime,and arecentstudydemonstratedinvivoevolutionindormanttumor cells of theheritable abilityto escape dormancyand growout as metastases[41].Experimentally,when untransformedmam- maryepithelialcellscontaininginducibleoncogenesareinjected intravenously,theycanremainviableinlungtissueforprolonged periodsoftimebeforeassumingmalignantgrowthuponinduc- tionofoncogeneexpression[42],providingaproofofprinciple thatevennon-transformeddisseminatedcellshavethepotential toremaindormantandultimatelygrowastumors.Nevertheless, giventhatthedefinitionofmalignancyisthebreachingofthebase- mentmembrane, itiscurrentlydifficulttoenvisagehowtumor cellscouldphysicallydisseminateatapre-malignantstage,ashas beensuggested[32].However,recentstudiesshowthatinvasive- nessmayappearearlyduringtransformationincellsthatescape oncogene-inducedsenescence[43],providingamechanismfordis- seminationveryearlyduringtumorigenesis.

Genomicexonsequencingofcolorectal[44]andpancreaticpri- mary tumors and theirmatched metastases [23] revealed that themajorityofpoint mutationswerecommontobothprimary tumorsandtheirmetastases,andthatmetastaseshadacquireda fewadditionalmutations.Thismayargueagainstearlydissemina- tion.Indeed,thesedatawereusedtocalculatewhenthemetastastic foundercellsdeveloped,andconcludedthatfewifanyadditional mutationsarerequired formetastasticfounder cellstodevelop fromcarcinomas[44],andthatmetastaticdisseminationisalate event[23].However,therearesomeimportantcaveatsassociated withtheinterpretationofthesefindings.Exonanalysisofprotein- encoding genes was used, which by definition only addresses around 1%ofthegenome[45];analysisof thegenomes ofpri- marytumorsandtheirmatchedmetastasesonamoregloballevel comestodifferentconclusions(seeabove).Furthermore,theanal- ysisofpointmutationsinprotein-encodinggenesmayskewthe investigationtowardgeneticchangesthatunderliethetumorigenic properties ofthecancercells.Thus patternsofpoint mutations wouldbeexpectedtobesimilarbetweenprimarytumorsandtheir metastases,andthesedatasetsmaynotbeappropriateformaking robustconclusionsabouttheetiologyofmetastaticfoundercells.

Inaddition,disparityinpointmutationsbetweenprimarytumors andtheirmetastasesthatwerefoundinotherstudiessupportthe notionofparallelprogression[22].

2.3. Metastaticseedsandthepeckingorder:hierarchyandCSCs

Anotherconceptforhowmetastasisworksarisesasa corol- lary ofthecancerstem cell(CSC)hypothesis that predictsthat malignancies,likemanyhighturnovertissues,arecharacterized

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byahierarchicalorganization,withstem-likecellsendowedwith self-renewalandthecapacitytodifferentiate,butalsowithmore committedprogenitorcellsandfullydifferentiatedlineages[46].

AsbydefinitionCSCsarepredictedtobethecellsthatinitiateand drivesecondarytumorgrowth,theywouldbeexpectedtounderlie malignantbehaviorbyrespondingtoenvironmentalcuestodetach fromtheprimarytumoranddisseminatethroughoutthebodyas so-calledmigratingcancerstemcells(mCSCs)[19].ThusmCSCsare predictedtobethemetastaticseedsthatfoundsecondarytumors.

ExperimentalevidencetosupportthenotionthatCSCsplaya criticalroleinmetastasisremainsthinontheground.However, recentstudiespointtotheexistenceofspecificstem-likesubpopu- lationsofcancercellsendowedwithhighmigratoryandmetastatic capacity,andsuggestthatCSCsareheterogeneouspopulationsthat includeactivelycyclingCSCsthatdrivetumorgrowth,aswellas morequiescentstem-like cancercells. Thiscellularheterogene- itywithintheCSCcompartment withthedichotomyof cycling and quiescentCSCs wasfirststudied inpancreas cancerwhere theCSCpopulationisdefinedbyCD133expression.Thecombined expressionofCD133andCXCR4,achemokinereceptorimplicated incellularmigrationandhighmalignantandmetastaticpotential, earmarksCTCsdetectableintheportalveinwhicheventuallyform livermetastasis[47].Accordingly,depletionofthemigratingcancer stemcellsusingapharmacologicalinhibitoroftheCXCR4receptor abrogatedtheirmetastaticpotential[47].CXCR4expressioninCSCs islikelytomakethemresponsivetoachemotacticgradientestab- lishedbyitsspecificligand,stromalfactor1orSDF-1,expressedby severalorgansinwhichmetastasesdevelop.

AdditionalevidencefortheexistenceofdifferentCSCssubtypes responsiblefor metastasiscomesfromstudiesoncolon cancer, whereCSCscanbedetectedandprospectivelyenrichedwithavari- etyofcellsurfaceantigenmarkers[48–52].Threedistincttypesof CSCs(alsoreferredtoastumor-initiatingcells,TICs)arelikelyto existincoloncancer:extensiveself-renewinglong-term(LT-TICs), tumortransientamplifyingcells(T-TAC),anddelayedcontributing (DC-TICs)[53].Onlyself-renewingLT-TICswereshowntobeable tocontributetometastasisformation[53].Finally,amorespecific markerofmigratoryanddistantmetastasis-causingCSCsincolon cancerwasrecentlyidentified:asubpopulationofCD26⁺cellswas foundinbothprimaryandmetastatictumorsfromadvancedstage CRCpatientscapableofgivingrisetoCTCsintheportalveinandto distantmetastasis[54].

TheaboveexamplesofheterogeneityinCSCpopulations,aswell asseveralothers[55]arelikelytoreflectplasticityintheCSCpheno- type.Additionalplasticityisalsoreflectedinstudiesthatshowthat non-CSCscanacquireCSCproperties[56,57].Forexample,similar tonormalstemcells,amicroenvironmentalnichehasbeenshown toberequiredtomaintaingliomaandskincancerCSCs[58,59], andthis isprobablyalsothecase forothertumortypes[60].A perivascularlocationcanactuallybethedrivingforcethatleadsto theacquisitionofCSCpropertiesbynon-CSCsubpopulations[61].

Thusextrinsicmicroenvironmentalcuesareemergingasimportant determinantsoftheCSCpopulation.

2.4. Sleepingitoff:dormancy

Metastasescanoccurmanyyearsaftersurgicalremovalofthe primarytumor,whichhasgivenrisetotheconceptofdormancy.

These late-developing metastases are thought to develop from DTCsthathavebecomere-activatedafterremaininginastabledor- mantstateoveraprolongedperiod[62].Forexample,afterradical prostatectomyforprostatecancer,almosthalfofallpatientshave detectableDTCsintheirbonemarrowmorethan5yearsaftertheir surgery[63].Dormanttumorcellscanexistinaquiescentstate,or asmicrometastasesinwhichproliferationisbalancedbycelldeath throughapoptosis[7].Reactivationofthesedormantcellscanbe

duetochangesinthetumorcellsthemselves,forexampledueto lossofmetastasissuppressorgenesthatregulatedormancy[64], aswellastomodificationoftheirmicroenvironment,forexample extracellularmatrix(ECM)remodelingandrecruitmentofinflam- matorycells[65,66].Theactivationofthegrowthofindolenttumor cellsbybonemarrow-derivedcells(BMDC)recruitedinresponseto osteopontinproducedbyasecondremote“instigator”tumormay alsoreflectthere-animationofdormantcells[67].Duetotheir quiescenceorslowturnover,dormanttumorcellsareresistantto conventionalcytotoxictherapiesbecausetheirintrinsicquiescence makestheminsensitivetoDNA-damagingagentsthatspecifically targetcyclingcells[68].

Anelegantrecentstudythatlookedatthemechanismbehind there-activationofdormantbreastcancercellsinthebonemar- rowprovidesevidencethatintrinsicchangesingeneexpression intumorcellscanrelievedormancy[41].Metastasesgrowingout in the bone marrow after long latency periods were found to expressVCAM-1,incontrasttotheparentalclonethatwasorig- inallyinjectedintotheexperimentalanimals.Infurtherroundsof injectionintoanimals,theseVCAM-1-expressingcellswereable toformbonemetastaseswithoutenteringdormancy.Mechanisti- cally,VCAM-1allowsbreasttumorcellstorecruit␣4␤1-positive osteoclastprogenitors, therebyelevatingosteoclastactivitythat leads to bone destruction. These data nicely demonstrate that invivoevolutionoftumorcellscanleadtothelossofdormancy.

Thereareanumberofparallelsbetweendormanttumorcells and CSCs.Asmentioned above, CSCscan bequiescent,and are alsoresistanttochemotherapy.MechanismsthatCSCssharewith normalstemcellsunderlietheirinnateresistancetotherapy,for example multi-drug resistance due to up-regulation of cellular efflux pumps [69,70], activation of the DNA damage response [71],andlowerconcentrationsofreactiveoxygenspecies[72].A perivascularlocationregulatesCSCidentity(seeabove),andisalso requiredforthesurvivalofdormanttumorcellsthathavedissem- inatedtothebrain[73].

2.5. Re-evaluatingEMT:multiplefunctionsinmetastasis?

Aconceptthatcontinuestoattractattentionisthenotionthat themorphogenetic program of EMTbecomes activatedin can- cercellsastheyprogress,andthatthiscontributestometastasis formation.Duringthetransitionfrombenignadenomatomalig- nantcarcinomaandmetastasisformation,differentiatedepithelial tumor cells are thought to acquire a de-differentiated, migra- tory,and invasive phenotypethroughtheprocess of EMT[74].

This process of EMT is accompanied by dramatic changes in cellular morphology, the loss and remodeling of cell–cell and cell–matrixadhesion,andthegainofmigratoryandinvasivecapa- bilities[75,76].ThefunctionalcontributionofEMTtometastasis inpatientsisstilldebated,yetrecentprogressinthediscoveryof novelEMTmarkersprovidesincreasingevidencefortheoccurrence ofEMTinhumancancers[19,77,78].

ItisnowbecomingevidentthatEMTitselfisamultistagepro- cess,involvingdistinctgeneticandepigeneticalterationsandahigh degreeofcellularplasticity.Inthepastyears,alargenumberof geneshavebeenidentifiedthatseemtobecriticalforthis pro- cess[75].AmajormoleculareventduringEMTisthelossofthe epithelialcell–celladhesionmoleculeE-cadherin,whichbyitself cansufficetoinduceEMTandtumorprogression[79–81].Con- versely,cellsundergoingEMTacquireexpressionofmesenchymal markerssuchasvimentin.

A broad-spectrum oftranscriptionaland post-transcriptional regulatorsthathavebeenimplicatedinmalignantprogressionalso regulates EMT[82]. Many growth factorssuch astransforming growthfactor␤(TGF␤),andtheirassociatedsignaltransduction pathwaysinduceEMTbyactivatingoneorseveraltranscriptional

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repressors,suchasSnail1(Snail),Snail2(Slug),Zeb1(␦EF1),Zeb2 (Sip1),E47,andTwist,whichinturnrepressanumberofgenes, includingE-cadherin[75,83,84].Manyothertranscriptionfactors also play critical roles in EMT [75,85]. Moreover, a number of microRNAsthataredifferentiallyexpressedduringEMTarepiv- otalregulatorsofthecomplexcircuitsthatunderliethemultiple stagesofEMT[86–88].Furthermore,severalenzymaticactivities andfactorscriticalforepigeneticregulation,suchasDNAmethy- lation and histonemodifications, are themselves modulated in theirexpressionoractivitiesduringEMT[89,90].Together,these changesorchestratethedramaticreprogrammingofcellsthatchar- acterizesEMT.

CellpolarityisregulatedbytheScribble,thePartitioningdefec- tive(Par) and the Crumbs complexes [91]. Loss of apical-basal polarityasaresultofaberrantexpressionofpolarityproteinsis considereda prerequisitefor metastatictumorprogression and leadstoEMT.ThisiswellillustratedbytheParcomplexthatcon- sistsoftheproteinsPar3,Par6andtheatypicalproteinkinaseC [91].TGF␤downregulatesPar3expression,revealingamechanism bywhichTGF␤candisrupttightjunctionformation,mediateloss ofapical-basalcellpolarityandinduceEMT[92].Par6ofthePar complexpromotestumorinitiationandprogressionandinteracts withtheTGF␤receptor.BlockingtheTGF␤-dependentphosphory- lationofPar6inbreastcancermodelsreducesmetastasistothe lungsand highlights theimportanceof thelossof polaritysig- nalingforEMTand metastasis[93].Similarly, repressionof the Crumbspolaritycomplexinepithelialtumorsoccursconcomitantly withincreasedexpressionofvimentinandreducedexpressionof E-cadherin,anditsexpressionnegativelycorrelateswiththemigra- toryandmetastaticcapacityofcells.Importantly,theproteinsZEB1 andSnailmediaterepressionofCrumbs,linkingknownregulators ofEMTtopolarityproteinsignalingthroughtheCrumbsprotein [94].

EMTappears not tobe a unitary “blackand white” process thatleadsinvariablyandirreversiblyfromapurelyepithelialtoa purelymesenchymalphenotype;thereappeartobeshadesofgray inbetween[82,95].Ithassuggested,forexample,thatEMTshould be classified into three subtypes [95]. Furthermore, basal-like breast carcinomas often exhibit features associated with EMT, yetretainsomeepithelialcharacteristics[96].Suchintermediate stateshavebeenreferredtoasthemetastableEMTphenotype[97].

Moreover,thereisalsoconsiderableplasticityintheresponseto EMTinduction,andisoftenareversibleprocessbothphysiologi- callyandpathologically.Forexample,hypoxiainducesareversible EMTin breastcancercells [98]. Thereversibility ofEMTin the cancercontext hasbeenused tosuggest that EMTallows cells toinvadeand disseminate,andisthen reversedatdistantsites throughamesenchymal–epithelialtransition(MET)thatresultsin ametastasisthatphenotypicallyresemblestheoriginatingprimary tumor[19].Evidencefordynamicreversiblephenotypicchanges invivoduringdisseminationhasbeenobtainedformelanoma[99].

Autocrine motility factor [100]and expression of GATA3 [101]

havebeenshowntoreverseEMT.PartialEMThasbeenshownto decreasecelladhesionbutstillallowcollectivecellmigration[102], consistentwithobservationsthatthemesenchymal(single cell) andcollectivemodesofmigrationarereversiblyinterchangeable [103,104].Theseandothersuchdynamicreversiblechangeshave beensuggestedtobevitalfordissemination[105].Themultiple levelsatwhichEMTisregulated[82,106]providesaplatformfor thefine-tuningofmetastabletransitionalstatesbetweenpurely epithelialandpurelymesenchymalphenotypes.Thespatialand temporalexpressionandcombinationoftranscriptionalrepressors thatareinduced,forexample,caninfluencetheoutcomeofthe EMTprocess[107].ThusapictureemergesinwhichEMTdescribes aspectrumofphenotypesthatarereversiblyinterchangeableand subjecttodynamicregulationbythemicroenvironment.Dynamic

interchange in the “gray scale” between purely epithelial and purely mesenchymalphenotypes asevidenced bythe interplay between ZEB and miR-200 points to the importance of such transitionsintumorprogression[86].

Classically,theinductionofEMThasbeeninterpretedasbeing importantintheprocessofmetastasisbyendowingtumorcells withinvasive properties.However,recent findings suggestthat EMTprovidesmanymore propertiesof relevancetometastasis thanjustinvasiveness.Forexample,EMTservesasanescaperoute for tumor cells froma variety ofobstacles connectedwith cell transformationandrapidtumorgrowth,includingoncogeneaddic- tion,oncogene-inducedcellularsenescence,tumorhypoxia,and increasedapoptosis [43,108,109].Apparently, EMTensuresthat cancercellsnotonlygainmigratoryandinvasivecapabilitiesbut alsosurviveoncetheyhavelefttheiraccustomedprimarytumor environment.SignalingpathwayselicitedbytheEMTprocesspro- videavarietyofsurvivalsignalsthatovercomecellcyclearrestand celldeathbyapoptosisoranoikisthatotherwisewouldbetrig- geredbythecytokinestormoccurringwithintheprimarytumor environment,bytheinflammatoryresponseswithintheneighbor- ingtissueandbytheimmunedefensewithinthebloodcirculation.

Accordingly, the genetic program of EMT includes a variety of immunosuppressivefunctions.

Thecomplexchangesinthecytoskeletonassociatedwithmotil- ityandinvasivenessmaybeincompatiblewithcellproliferation [110].Accordingly,ithasbeenshownthatgrowtharrestcanbea featureofEMT,forexamplethroughincreasedlevelsofp16ink4a [111]andrepressionofcyclinDexpression[112,113].Consistently, persistent expression of Twisthas beenassociated withmain- tenance of dormancyand quiescence [107]. Conversely,METis associatedwithincreasedproliferation[86].

EMTalsoappearstoplayacriticalroleinthegenerationand maintenance of cancerstem cells,consistent withtheobserva- tionthatmanystemcellgenesareexpressedinmetastaticcancer cells [114,115]. Indeed,EMTincreases thestemness of cells, as aftercompletionofEMTcancercellsexpressmanystemnessmark- ers, they are able toform spheroids in culture, theyare more tumorigenicinxenografttransplantationexperimentsandtheyare moremetastatic[114–116].Abroadspectrumofsignalsfromthe tumormicroenvironmentmaytriggerEMTattheinvasivefront of epithelialmalignancies, wheretumor cellsare indirect con- tactwithstromalcomponentssuchasfibroblasts,myofibroblasts, granulocytes,macrophages,mesenchymalstemcells,andlympho- cytesthatareabletosecretediffusibleEMT-inducingsignals[117], therebyinducingEMT,stemnessproperties,andfacilitatingdetach- mentanddisseminationfromtheprimarysite[118,119].Moreover, quiescentstem-likecancercells areearmarkedbyexpressionof EMTmarkers[75].TheabilityofEMTtoinducebothcellcyclearrest andendowstemnesspropertiesoncellsmaythereforebyofrele- vancetothequiescentCSCsubpopulationsmentionedabove.The inductionofEMTmaycontributetotheplasticityintheCSCpheno- type,forexample,endowingnon-CSCswithstemnessproperties.

However,thedegreetowhichgeneticprogramsthatregulatestem- nessandEMToverlapremainstobeproperlyinvestigated.EMT hasalsobeensuggestedtogeneratemCSCsthatleavetheprimary tumoranddisseminatetodistantsites,subsequentlyundergoing METtoresumegrowthandformmetastasesthatarephenotyp- icallysimilartotheprimarytumorfromwhichtheyarederived [19,86].

Finally, cells that have undergone EMTare foundtoexhibit increasedresistanceagainstmany,butnotallchemotherapeutic agents[116].Interestingly,theconverseisalsotrue:chemicalenti- tieshavebeenfoundthateradicatewithhigherefficacycellsthat haveundergoneEMTascomparedtotheirepithelialcounterparts, raisingthepossibilityofdirectlytargetingcellsthathaveunder- goneEMT[120].

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3. Conceptsofthesoil

Thelastfewyearshaveseenadramaticincreaseinourknowl- edgeaboutkeyconstituentsofthemicroenvironmental“soil”that supportsthesurvivaland outgrowthofthemetastatic“seed”in distantorgans. It hasbecomeclear that themicroenvironment aroundDTCshasaprofoundinfluenceonwhethertheydie,remain dormantorgrowas metastases [7].Different tumortypesmay have differentmicroenvironmental requirementsfor metastatic outgrowth.Suchdifferencesmaycontributetodifferencesinintrin- sicmetastaticpotential,namelythetendencyforsometumortypes (e.g.melanomas)toformmetastasesevenwhentheprimarytumor isverysmall,whileothertumortypes(e.g.basalcellcarcinomas) rarelymetastasizeevenaftersizablegrowthoftheprimarytumor [6].Similarly,particularmicroenvironmentalrequirementsforthe survivalandgrowthofDTCsfromdifferenttypesofcancermay underlieorgan-specificpatternsofmetastasis.

3.1. Inflamingthesituation:anicheoccupation

AmicroenvironmentthatisconducivetothegrowthofDTCs has been termed a metastatic niche [121]. Recent years have seena numberofseminalstudiesthat haveidentifiedkeycon- stituentsofsuchniches.RemodelingoftheECMandrecruitmentof inflammatorycellsandotherBMDCplayacentralrole[122–125].

Growthfactor,cytokines,chemokinesandotherproteinsproduced bycellularcomponentsofthemetastaticnichearepivotalinthe formation of metastatic niches,for the attraction of CTCs, and forthesurvival and outgrowthof DTCs[122–124,126].Anum- berof observations alsosuggest that a perivascular location is a pre-requisitefor DTC survival and outgrowth [73], and there is increasing evidence that hypoxia plays animportant role in themetastasis-promotingfunctionofmetastaticniches[126–128].

Progressivechangesinthestromaofprimarytumorstakesplace duringtumorformationandprogression[129,130],andthereare alsomanysimilaritiesbetweenthesechangesandtheconstituents ofmetastaticniches.

Metastaticnichesmaybefoundendogenouslyinorganswhere metastasesform.Ahigherprevalenceofsuchnichesmayunder- liethepredilectionofDTCstogrowasmetastasesinorganssuch aslymphnodes,lungs,liver,brainandbone.Anumberofobser- vationssuggestthatbyoccupyingthenormalstemcellniche,for exampleinthebonemarrow,DTCsfindaprimednichethatsup- portstheirgrowth[131,132].Nevertheless,endogenousmetastatic nichesareprobablysparselydistributed,whichmayaccountinpart fortheinefficiencyofthemetastaticprocess.Forexample,injection oftensofthousandsoftumorcellsintravenouslyonlygenerates severalhundredmetastases,evenafterseveralroundsofselection fortheabilitytogrowasexperimentalmetastasesinthelungsafter intravenousinjectionwhichwouldbepredictedtohighlyenrichfor cellswithmetastasis-formingability[133].

Remodelingoftheorganmicroenvironmenthasbeendemon- strated in recent years to create metastatic niches that foster theoutgrowthofDTCs.Thesenichescanbeinducedbyprimary tumors prior tothesettling of DTCsin organs – so-called pre- metastatic niches – that can also attract CTCs through growth factors,cytokinesandotherchemoattractantsthatareproduced by niche components [122–124]. In experimentalmodels, pre- metastatic niche formation has been shown to be critical for theformationof fulminantmetastases [122–124].Formation of metastaticnichesafterremovaloftheprimarytumor,forexam- pledue toinflammatory processes, may beresponsible forthe re-activationofdormantDTCs,althoughexperimentalevidenceto supportthisnotionstillremainstobegarnered.

It is notable that many of the components of metastatic nichesandtheirformationarerelatedtoinflammatoryprocesses.

Pro-inflammatorymembersoftheS100familyandmembersof the Serum Amyloid A acute phase proteins have been identi- fied as having a pivotal role in the formation and function of metastaticniches,playingakeyrole,forexample,intherecruit- ment of CD11b+ myeloid cells to these sites [134]. Monocyte and macrophage-specific chemokines are also expressed [123].

The remodeling of the ECM (see below), such as the produc- tion and deposition of fibronectin and the activities of lysyl oxidasesandproteases,isa hallmarkofbothsitesofinflamma- tion and of pre-metastatic niches [135]. Hypoxia, an emerging nichefeaturethatalsoinducesexpressionoflysyloxidases,can alsopromoteinflammatoryresponses[136].Inadditiontoreleas- ing cells from dormancy in the bone [41], VCAM-1expression on tumorcells hasalso been recently shown to mediate their interactionwithmetastasis-associatedmacrophages,providinga survivaladvantage[137].Takentogether,theseobservationssug- gest that the formation of metastatic niches recapitulates the inflammatoryprocessesandtumor–stromainteractionsthatdrive primarytumorgrowth,andtherebyfostersmetastasisformation byDTCs.

3.2. Diggingoverthesoil

RemodelingoftheECMhasemergedasanimportanteventdur- ingtheestablishmentofmetastaticniches.MMP-9,producedfor examplebyVEGFR1+BMDC,isrequiredfortheformationofpre- metastaticnichesandtheoutgrowthofsecondarytumorsinthe lung [122,138].AdditionalECMcomponentssuchasfibronectin [122],periostin[139]andtenascin-C[140]areproducedinthese niches,andexistingECMcomponentsaremodified,forexample throughtheactivityoflysyloxidases,enzymesthatcross-linkcolla- genandelastin[126].Together,theseandothermechanismsserve tomodifytheECM,therebycreatingamicroenvironmentthatis permissiveforthegrowthofDTCs.

ECM remodeling may act in a number of ways to promote theoutgrowthofmetastases.Changesintheconstituentsofthe ECMcanofcourseservetomodifyepitopeswithwhichintegrins and other receptorsonthe surface of tumorcells can interact.

Integrin-mediatedactivationoffocaladhesionkinase(FAK)signal- ingpromotescellsurvivalandproliferation[141]andcanregulate CSCproperties[142].Remodelingof theECMcanalsobesuffi- cient tore-activatedormanttumorcells,for examplemediated byintegrin-FAKsignaling[65,143].Inductionofperiostinexpres- sionbyfibroblastsinmetastaticnichesisrequiredforrecruitment of Wntligandsand themaintenance of CSCproperties inDTCs [139].Evidence is alsoemerging thatan importantoutcome of matrix remodeling is an increase in the stiffness or rigidity of the microenvironment in a manner that can have a profound effectoncellbehavior.Forexample,matrix cross-linkingmedi- ated by the activity of lysyl oxidases increases focal adhesion formation and FAK activation, and promotes invasiveness and malignancy[144].Caveolin1expressiononcarcinoma-associated fibroblasts(CAFs)remodels andstiffenstheECM microenviron- ment, and consequently promotes metastasis formation [145].

MatrixstiffnessalsoregulatestheactivityoftheTAZtranscription co-activatorthatformspartoftheHippopathway[146],andTAZ activityconfersstemnesspropertiesonbreastcancercells[147].

Inhepatocellularcarcinomacells,stiffermatrices werefoundto promoteproliferationandchemoresistance,whilecellssurviving afterchemotherapyonsoftermatricesexhibitedareversibledor- mantphenotypeassociatedwithexpressionofCSCmarkers[148].

Finally,increasedmatrixstiffnessfavorsTGF␤-inducedEMTover apoptosis[149].Thusapictureemergesinwhichenhancedmatrix stiffnessmaintainsorendowsCSCspropertiesontumorcells,can regulatedormancy,anddeterminestheresponsetoEMT-inducing factors.

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3.3. Longrangefertilizersofthesoil

Aremarkablefindingthathasemergedfromthestudyofthe formationofpre-metastaticnichesisthelong-rangesignalingthat allows primarytumors toestablishmetastatic nichestructures.

FactorssuchasVEGF-AandPlGFproducedbyprimarytumorsact distantly onthebone marrowtomobilizeVEGFR1+BMDCthat contributetopre-metastaticnicheformation[122].Similarly,pri- marytumor-derivedVEGF-A,TNF␣andTGF␤induceexpressionof S100A8andS100A9indevelopingpre-metastaticniches,whichin turnrecruitsCD11b+myeloidcells[123].

Recent studies have implicated primary tumor-derived microvesiclesandexosomesinthelong-rangesignalinginvolved inpre-metastatic nicheformation[150].Microvesiclesand exo- somes contain membrane and cytoplasmic proteins, as wellas nucleicacidsderivedfromthecelloforigin.Theycanbetrans- portedviatheblood,andthecargotheycarrycaninteractwith targetcellsand modifytheirbehavior[151].Exosomes released fromratpancreaticadenocarcinomacells togetherwithCD44v6 in the soluble fractioncomplement each otherin generatinga nicheforefficienttumoroutgrowth[152].Microvesiclesreleased fromCD105-positiverenalcarcinomaCSCsstimulateangiogenesis, upregulateVEGF-A,MMP2andMMP9expressioninpre-metastatic sitesinthelung,andpromotelungmetastasis[153].Microvesicles havealsobeenshown tobeinvolvedinthebilateralcommuni- cationbetweentumorcells andfibroblasts,withtumor-derived microvesiclesactingtoupregulateMMP9expressioninfibroblasts [154].

Therequirementforlong-rangesignalsderivedfromprimary tumors that orchestrate theformation of pre-metastaticniches mayaccountfortheassociationbetweenelevatedriskofmetas- tasisdevelopment andincreasing primarytumor size.It would seemreasonabletoassumethatthetumor-derivedgrowthfactors andothersignalingmoleculesinvolvedwouldneedtoriseabove agivensystemicconcentrationthresholdbeforehavinganeffect in thebonemarrow orpotentialsitesof pre-metastatic niches.

Largertumorswouldbeexpectedtoproducemoreoftherequi- sitesignalingmolecules,andthereforetheconcentrationofthese moleculesinthecirculatorysystemshouldalsoriseconcomitantly.

ThusanicheenvironmentthatsupportstheoutgrowthofDTCsas metastasesmayonlydeveloponcetheprimarytumorreachesa sufficientsizetoproduceenoughsignalingmoleculestoactivate nicheformation.

4. Puttingitalltogether:conflictingorcompatible concepts?

As outlined above,a number of novel conceptshave arisen recentlyasaresultofnewgroundbreakingexperiments,andexist- ingconceptshavealsobeenmodifiedasaresult.Theseconcepts oftenonlyconsideroneparticularaspectofmetastasis,andnone ofthemcompletelyexplaintheprocess,noraccountforallexper- imentalfindings.Isitpossibletosynthesizeaconceptonthebasis ofthedatathathasbeengeneratedtodatethatunifiesthesedif- ferentconceptsandprovidesamorecomprehensiveoverviewof theprocessofmetastasis?Someoftheconceptsaboveareappar- entlyconflicting,forexampleregardingthequestionofwhether themetastaticdisseminationthatultimatelygivesrisetometas- tasisisanearlyeventaftertumorigenesisorratheroccurslatein tumordevelopment.Itispossiblethatnosingleconceptexplains theprocessofmetastasis,andthatthemechanismsdifferbetween cancertypesorevenbetweenindividualpatients.Nevertheless, theprocessofmetastasisiscomparableformanydifferenttypes ofcancer(localprogressionandinvasion,transportinthecircula- torysystem,extravasation,survivalandgrowthat(oftensimilar)

secondarysites),suggestingthatcommonmechanismsareprob- ably operative. Furthermore, there are considerable similarities betweenseveraloftheconceptsoutlinedabove,whichprovidea foundationforputtingtogetherthepiecesofthemetastasisconcept jigsawpuzzle.

Strikingareasofconvergencearethecommonalitiesthathave emergedbetweentheregulationofEMT,stemness,dormancyand therapyresistance.Manyofthesearepointedoutabove.Thesim- ilaritiesbetweenCSCsand cellsthathave undergoneEMThave beenrecentlyextensivelyreviewed[110,116].Afurtherexample isprovidedbyCXCR4.InadditiontomarkingCSCsthatwillform metastases,CXCR4anditsligandSDF-1havebeenimplicatedin regulatingEMTin breastcancer[155],oral SCC[156]and pan- creatic cancercells[157],and probablyactinconjunction with TGF␤[158,159].Similarly,CXCR4isassociatedwithchemoresis- tance[160]andreversibledormancy[148].

Itisalsostrikingthatmanyoftheconstituentsthathavebeen describedasbeingcrucialformetastaticnichefunctionserveto regulate EMT, stemness, dormancyand therapy resistance. For example, VEGF-Adrivesthe formationof pre-metastatic niches [122],createsaperivascularnichethatmaintainsthestemnessof skintumorCSCs[59]andsuppressesdormancy[73].EMTisinduced byinflammatoryregulatorsthatarepresentinmetastaticniches [161],asexemplifiedbyIL-1␤inheadandneckcancer[162].The ECMremodelingthattypicallyoccursininflammationandfibrosis isverysimilartothatfoundinmetastaticniches,andcontributes toEMT[95].Consistently,thelysyloxidaseLOXL2inducesEMT viaincreasingthestabilityandactivityofSnail1[163].MMPsare alsoactivatedinthemetastaticnicheandinduceEMT[164].The metastaticnicheconstituentperiostinregulatesCSCproperties,as wellasEMT[165].HypoxiapromotesCSCstemness,as wellas theformationofaCSCniche[166].Furthermore,hypoxiaisalso apotentandreversibleinducerofEMT[98],anda recentstudy implicatesitininducingdormancyinglioblastomaCSCs[167].

5. Thestromalprogressionmodel

Theaboveobservationsindicatethatthereisatightintercon- nectionbetweenEMT,stemness,dormancyandtherapyresistance, anditislikelythatthemetastaticnicheplaysacriticalroleinreg- ulatingtheseprocessesatsiteswheresecondarytumorsdevelop.

Theseandtheotherobservationsdescribedaboveallowustoten- tativelysuggestaconceptofmetastasisthatwehavecalledthe stromalprogressionmodel(Fig.1).Thetumorstromaiscomprised ofECM,non-malignantcellsandthesignalingmoleculestheypro- duce.Inthestromalprogressionmodel,progressiveco-evolution ofthetumorstromaandthegeneticmake-upoftumorcellsatboth theprimaryandsecondarysitesprovidetheplatformrequiredfor metastasisformation.Thismodelaccommodatesmanyaspectsof thedisparatemodelsandtheoriesthathavebeensuggestedtodate, andisoutlinedindetailinthefollowingtext.

Similar to clonal selection models, the stromal progression model suggeststhatserial acquisitionofgeneticmutationsand aberrations driven by increasing genomic instability occurs in tumorcellsduringprimarytumorprogression,togetherwithepige- neticchanges.However,stromalprogressionalsooccursinparallel, forexampletheprogressiveremodelingoftheECMinthetumor, activationandrecruitmentofstromalcellssuchasfibroblastsand BMDC, regional hypoxia,theinduction of angiogenesisand the developmentofaninflammatorymilieu.Breachofthebasement membraneandsubsequentinvasionfurtherexposestumorcells tonewmicroenvironments and furtherstimulates stromalpro- gression.Thusthedynamicstepwisemutualandinterdependent cross-regulation betweentumorandstromalcells leadstopro- gressionofthetumorasawhole.Intheabsenceofanappropriate

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Fig.1.Thestromalprogressionmodel.Duringthedevelopmentoftheprimarytumor,progressivegeneticandepigeneticchangestakeplaceintumorcells(upperpanel).

Inparallel,thestromaassociatedwiththetumorcellsalsobecomesprogressivelymodified,forexamplethroughextracellularmatrixremodelingandtheactivationand recruitmentofcellssuchascarcinoma-associatedfibroblasts(CAFs),tumor-associatedmacrophages(TAMs)andothercellsoftheimmunesystem.Forthesakeofclarity,only someofthesechangeshavebeendepicted.Interactionsbetweenthetumorstromaandtumorcellsmodifythestemnesspropertiesofthetumorscells,forexamplethrough epithelial–mesenchymalandmesenchymal–epithelial-liketransitions(EMTandMET),whichregulatesthecancerstemcell(CSC)subpopulation.Disseminationfromthe primarytumorbeginsearly.Uponenteringthesecondarysite,disseminatedtumorcells(DTCs)haveanumberofpossiblefates,someofwhicharedepictedinthelefthand sidebox,bottompanel.TheseinitialfatesaredeterminedbytheunderlyinggeneticandepigeneticstatusoftheDTC,andthroughinteractionswiththemicroenvironment atthesecondarysite.EMT-andMET-liketransitions,furthergeneticprogressioninthetumorcells,andstromalprogressioninthemicroenvironmentcansubsequently alterDTCfateandleadtothegrowthofovertmetastases.Continuingmutualinteractionsbetweentumorcellsandstromalcellsatthesecondarysitestimulatestromal progression,asintheprimarytumor.Furthermore,astheprimarytumorgrows,increasingamountsofmetastaticniche-inducingfactorsaccumulatesystemically,resulting ininductionofstromalprogressionatthesecondarysite,andmetastaticnicheformation.MetastaticnicheformationcanoccurpriortosettlingofDTCsatthesecondary site,asinthecaseofpre-metastaticnichesthatprimethemicroenvironmenttosupportoutgrowthofincomingDTCs.Self-seedingoftheprimarytumorormetastasis,or cross-seedingbetweentheprimarytumoranditsmetastasismaycontributetotheoverallmake-upofthetumorcellpopulationattheprimaryandsecondarysites.

stromalcompartment,thegeneticandepigeneticchangesintumor cellsareinsufficienttosupporttumorgrowthandsurvival.Tumor progressionisthereforebuiltonafoundationofgeneticandepi- geneticchangesintumorcells,butisalsoabsolutelydependent onstromal progression in parallel (Fig. 1).Animportant result oftheinterplaybetweentumorcellsandthestromaisthegen- erationof CSCs that drivetumorgrowth,whose properties are determinedbytheirunderlyinggeneticmakeup,butalsobythe microenvironment,ina processthatinvolvesdynamicEMTand METtransitionsthatmayonlybepartial.Thesetransitionsalsocon- tributetotumorcellsurvival,andregulatedormancy,invasiveness andtherapyresistance, andcanoccurinboth CSCandnon-CSC populations. This aspectof the stromal progression model has

parallelswiththedynamicheterogeneitymodelproposeddecades ago[29].

Cancercelldisseminationbeginsearly,forexampleafterescape fromoncogene-inducedsenescence[43],andcontinuesthrough- out tumorgrowth and progression. CTCs leaving thetumor no longerhave contactwiththesupportivestromal microenviron- ment they are accustomed to, and the genetic and epigenetic changestheycarryareusually insufficienttosupporttheirsur- vivalorgrowthasafulminantmetastasis.Anappropriatestromal compartmentthereforehastobere-establishedatsecondarysites ifDTCsaretosurviveandgrowoutasmetastases.DTCsthatdo notend upinanappropriate microenvironment(orwhichcan- notinitiateone)eitherdieorremaindormant,probablyeventually

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regressing.Ifthemicroenvironmentsupportsthesurvivalofthe DTCs,orismodifiedtosupporttheirsurvival,thentheDTCscan continue to acquire genetic mutations and aberrations at sec- ondarysites,andprogressgeneticallyinparalleltotumorscellsin theprimarytumor,asforeseenintheparallelprogressionmodel.

However,concurrentstromalprogressionalsoaccompaniesthese geneticchangesinthetumorcellsatthesecondarysite,similarto thecaseintheprimarytumor(Fig.1).

Stromalprogressionalsotakesplaceatsecondarysitestoform microenvironmentsthat supportoutgrowthofmetastases. Such microenvironmentsmaybeinitiatedanddevelopedinanumber ofconceivableways:(i)DTCsmaysettleinpre-existingmicroen- vironments that providestromal componentsthey need.These maybenormal stemcellniches,forexample,orpre-metastatic nichesinducedbytheprimarytumor.(ii)Factorsproducedbythe DTCsthemselvesmayactonthesurroundingstromaandinitiate orcontributetothestromalprogressionthatultimatelysupports secondarytumorgrowth.Thusgeneticchangesintumorcellscan promotestromalprogression.(iii)Tumor–stromainteractionsin theprimarytumorproduceincreasingquantitiesof solublefac- tors asthetumorsgrow, suchasgrowthfactors,cytokinesand chemokines. These begin to accumulate systemically and ulti- mately inducethe formation of metastatic niches as described above,eitherpre-metastaticallyorafterthedisseminationofDTCs.

Hencethesizeoftheprimarytumorcorrelateswiththeincidence of metastasis,assizeis proportionateto theamountoffactors produced.(iv)Onceaprimarytumorhasbeenremoved,parallel geneticprogressionintheDTCsand/orassociatedstromalprogres- sionmayeventuallyleadtooutgrowthofmetastases.Inaddition, otherpathologicaleventssuchastissuetraumaorchronicinflam- mationmayrelease sufficient systemic levelsofgrowthfactors and cytokinesthat inducemetastatic nicheformationsuchthat metastatic niche formation is kick-started and/or stromal pro- gressionis supported.Removalof theprimary tumormay also removecirculatingfactorsthatwererepressingdistantmetastatic outgrowth,suchasangiogenesisinhibitorsormatrixremodeling enzymes.

Inthestromalprogressionmodel,formationof(pre)metastatic niches can constitute an important component of the stromal remodelingrequiredatsecondarysitesfortheoutgrowthofmetas- tases(Fig.1).Asintheprimarytumor,theinteractionoftumorcells withthestromalmicroenvironmentatthesesitesplaysakeyrole inregulatingmetastableEMT–MET-liketransitionsthatdetermine stemnessproperties,controldormancy,providesurvivalfunctions andmodulateresistancetotherapy.ThusEMTcanendowCSCsin theprimarytumorwithmigratorypropertiesthatcanbereversed atsecondary sitesthroughMETin responsetoa newmicroen- vironment, ashas beensuggested [19]. Inthe absenceof MET, thesecellsmayremaindormantduetothequiescence-promoting effectsofEMT.Similarly,non-CSCDTCsthatsurvivemayeventu- allyacquirestemnessproperties,forexamplethroughepigenetic changesinresponsetoEMTinducedwhenanappropriatestromal environmentdevelops,and/orthroughgeneticchanges.Hencethe propertiesofthetumorcells,thenatureofthesurroundingstroma, theinteractionbetweenthetwocompartments,andthecontinu- inginterdependentprogressiveevolutionofthetumorcellsandthe tumorstromaacttogethertodeterminethestemnessproperties requiredfortheoutgrowthofmetastases,regulatethere-activation ofdormantcells and determinesensitivity totherapy.Like pri- marytumors,metastasesmaydisseminatecells,andcross-seeding betweenprimarytumorand theirmetastasesmaycontributeto thesimilaritiesbetweenthemthatareobservedhistologicallyand intranscriptomicstudies.

Thestromalprogressionmodelsuggeststhatthesparseexis- tenceofappropriateendogenousstromalmicroenvironmentsthat areabletosupporttumorgrowthcontributestothelowefficiency

of metastasisformationin experimentalmetastasisassays. This may alsobe a reason why largenumbersof cells arerequired togetanefficient“takerate”inexperimentalanimals,and why providingconstituentsofasupportivestroma,forexampleinthe formofMatrigel,increasestakerate.Themodelalsoprovidesan explanationforwhycontinuouspassagingoftumorcellsinexperi- mentalanimalsandselectionforgrowthinparticularorganswould giverisetotumorcellsthatmetastasizeefficientlytotheorgan inquestion.Here,tumorcellsareselectedthathavetheabilityto interactwithparticularstromalmicroenvironmentsoftheorgan concerned,toinducestromalprogressioninthosemicroenviron- ments,and/ortoundergogeneticorepigeneticchangesinresponse totheendogenousorinducedmicroenvironment.

6. Conceptsandclinicalperspectives

While the stromal progression model incorporates many theories, observations and experimental findings, several open questionsremain.Majorissuesincludethetimingofdissemina- tionofthemetastaticseed,thedegreetowhichtheregulationof stemnesspropertiesoverlapswiththepathwaysthatcontrolEMT, dormancyandtherapyresistance,keystagesinstromalprogres- sion,andthemutualinterdependencebetweengeneticchangesin cancercellsandchangesintheassociatedstroma.Understanding howmetastasisworksisofmorethanjustacademicinterest,asan accurateconceptualgraspoftheprocessisfundamentaltoeffec- tivetherapy.Forexample,ifthetumorcellsthatseedmetastases disseminatelate,awindowofopportunityopenstoremovethe primarytumorbeforemetastaticdepositshavetakenroot.Ifon theotherhand,earlydisseminationandparallelprogressionisthe overridingmodeofmetastaticseeding,thenatthetimeofcancer diagnosis,DTCswiththepotentialtodevelopintometastaseswill alreadybepresent,andthereforethetherapeuticstrategywillneed tobedifferent.Anotherimplicationofparallelprogressionisthat thechoiceoftargetedtherapiestotreatmetastasesshouldbebased onmolecularandbiologicalfeaturesobservedinmetastasesrather thaninprimarytumors[22].

ThedormancyofDTCsoverlongperiodsoftimeandtheirrel- ativestability,togetherwithrelapseoccurringmanyyears after diagnosis,surgeryandinitialtreatmentdemandsthatmoreeffort isplacedonunderstandingtheregulationofdormancy.Thismay provideanovelopportunitytopreventmetastaticoutgrowthand keepdisseminatedcancerasadormant,chronicbutmanageable disease.Keyissuesaretounderstandhowquiescent,disseminated cancercellsinteractwiththemicroenvironment,andtodefinethe criticalcuesthatawakecancercellsformdormancyandallowthem toprogresstofullmetastasis.

Understandingthenatureofthetumorcellsthatinitiatemetas- tasescouldbekeytosuccessfultherapy.Ifmetastasesareseeded byparticularCSCsubpopulations,thentargetingthemwouldbe expectedtoeffectivelysuppressmetastasisformation.Theexpres- siononCSCsofspecificmembersofthefamilyofCXCchemokines receptorshasrecentlyreceivedinterestinthisregard.Chemokines serve as chemoattractants for cells endowed with CXC recep- torssuchasCXCR4andCXCR1thathavebeenfoundtoearmark migratorysubpopulationsofCSCsinpancreaticandbreastcancer, respectively[47,168].SelectiveblockadeofCXCR1targetsbreast CSCsinhumanxenograftsslowdownprimarytumorgrowthand reduce metastasisformation [169]. Clinical trials withpharma- cological inhibitors and monoclonalantibodies directedagainst specificCXCRswillassesstheircapacitytoblockCSCsdissemina- tionandpreventmetastasisformationin cancerpatients.These and similarstudiesmayprovidenoveltherapeuticstrategies to selectivelytargetcancerCSCsafterdisseminationthroughoutthe bodyofthecancerpatientandpreventthemfromformingdistant metastases.

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