Review series

The therapeutic promise of the cancer stem cell concept

Natasha Y. Frank,1,2 Tobias Schatton,3 and Markus H. Frank3,4
1Division

of Genetics, Brigham and Womens Hospital, Boston, Massachusetts, USA. 2Department of Medicine, VA Boston Healthcare System, West Roxbury, Massachusetts, USA. 3Transplantation Research Center, Childrens Hospital Boston, and Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Dermatology, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that selectively possess tumor initiation and self-renewal capacity and the ability to give rise to bulk populations of nontumorigenic cancer cell progeny through differentiation. As we discuss here, they have been prospectively identified in several human malignancies, and their relative abundance in clinical cancer specimens has been correlated with malignant disease progression in human patients. Furthermore, recent findings suggest that clinical cancer progression driven by CSCs may contribute to the failure of existing therapies to consistently eradicate malignant tumors. Therefore, CSC-directed therapeutic approaches might represent translationally relevant strategies to improve clinical cancer therapy, in particular for those malignancies that are currently refractory to conventional anticancer agents directed predominantly at tumor bulk populations.
Cancer stem cells: definition and experimental identification Physiologicaltissuesarehierarchicallyorganized,thatis,they arecomposedofcellpopulationswithdiverseself-renewaland proliferativecapacities.Relativelyrare,uncommitted,quiescent tissue-specificstemcellsarefoundattheapicesofthesecellular hierarchiesandaredefinedby2distinctproperties:thecapacity forprolongedself-renewalandthepotentialtogiverisetomore mature,transientlyamplifyingcellprogeniesthatinturngiverise tospecializedcellsofparticulartissuesthroughdifferentiation (1).Inaddition,suchstemcellspossessthecapacitytoproliferate extensively(1),forexample,inresponsetoinjuryandduringdevelopment(2).Bidirectionalinteractionsbetweenthesestemcellsand thecellularconstituentsoftheirindividualnichesinvolvedistinct developmentalsignalingnetworks,solublemediators,and/orcellmatrixprocesses.Theseinteractionsareessentialfortheestablishmentofastemcellpermissivemicroenvironmentandprovidea crucialregulatorybalancebetweenself-renewalanddifferentiation andbetweenquiescenceandproliferation(3). Somemalignanttumorscanalsobecomposedofmorphologicallyandphenotypicallyheterogeneouscellpopulations(4,5) withvaryingself-renewalcapacities,degreesofdifferentiation, andclonogenicandtumorigenicpotentials(610).Moreover, manyofthesignalingcascadesandinteractionswithstromal elementsthatorchestratephysiologicalstemcellbehavior,and consequentlynormaldevelopment,havealsobeenfoundtoplay importantrolesintheinitiationandprogressionoftumors(11). Takentogether,theseobservationshaveledtothedevelopmentof thecancerstemcell(CSC)theory,whichpositsthatneoplasms, likephysiologictissues,canbehierarchicallyorganized,andthat CSCs,whicharefoundattheapexofthiscellularhierarchyand seemtocompriseonlyasubpopulationoftumorcells,areessentialforitspropagation(1).Accordingtoaconsensusdefinition (12),aCSCisacellwithinatumorthatpossessesthecapacityto self-renewandtogeneratetheheterogeneouslineagesofcancer cellsthatcomprisethetumor.Therefore,CSCscanonlybedefined experimentallybytheirabilitytorecapitulatethegenerationofa
Conflict of interest:Theauthorshavedeclaredthatnoconflictofinterestexists. Citation for this article:J. Clin. Invest.120:4150(2010).doi:10.1172/JCI41004.

continuouslygrowingtumor(12).Consensusalsoexiststhatthe goldstandardassaythatfulfillsthesecriteriaisserialtransplantationinanimalmodels,which,althoughimperfect,isregardedas thebestfunctionalassayforthe2criticalcriteriaoftheconsensus CSCdefinition(12).Clearly,asdiscussedpreviously(13),tumorigenicityinhuman-to-mousexenotransplantationmodels,andas aresultcalculatedCSCfrequencyestimates,mightvarywiththe appliedexperimentalconditions,suchasthetissuesiteofxenotransplantationandthepresenceorabsenceofimmuneeffector mechanismsinrecipientimmunodeficientmice.Thedependence oftumorigenicpotentialontheimmunestatusofthetumorhost hasbeenconfirmedinhumanmalignantmelanomaxenograft models(14).However,thisstudydidnotdirectlyaddressCSCspecificfunctionssuchasself-renewalanddifferentiationcapacityinmarker-trackedserialxenotransplantationexperiments(14). Microenvironmentalfactorscanalsomarkedlyinfluencecancer celltumorigenicityinxenotransplantationmodels(15),asshown forhumanmelanoma,inwhichexogenouslyaddedECMfactors normallyproducedbytumorcellsmarkedlyenhancedtumorigenic potential(14),andforhumanbreastcancer,inwhichcografted cancer-derivedfibroblastssubstantiallyenhancedtumorgrowth inimmunodeficientnudemiceduetoincreasedproductionof stromal-derivedfactor1andresultantparacrinestimulationof breastcancerCXCR4(16). Thediversityandcomplexityofcurrentlyavailableexperimental tumorxenotransplantationmodels,andthedistinctresultsthat havebeengeneratedineachparticularassay,suggestthatthere mightnotexistasingleidealorbest-suitedmodelforthestudy ofCSCs,butratherthatcumulativeknowledgegeneratedinthe aggregateofexistingandpotentialfuturemodelswillyieldincreasinglyimportantanddefinitiveinsightsintoCSCbiology. AlthoughCSCshavebeenthoughttocomprisearelativelyrare subpopulationoftumorcellsinseveralmalignancies,relativerarityisnotadefiningcriterionaccordingtotheconsensusCSCdefinition(12)andisnotnecessarilyadefiningfeatureinallcancers inwhichCSCshavebeenidentified(1315,17,18).Inaddition, itisnoteworthythattheCSCconceptdoesnotmakeanyspecific assumptionsaboutthemultipotenttransdifferentiationplasticity oftheCSCsubset(12,18)oraboutthecellfromwhichthecancer arose(12),thatis,itdoesnotaddresswhetherthecancerarose
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Figure 1
CSCs, carcinogenesis, tumorigenesis, and tumor resistance. (A) Tumors can arise from somatic cells through genetic mutations of cancer-critical genes. In addition, dysregulation of microenvironmental factors can contribute to the carcinogenic process. Such events might predominantly affect long-lived somatic stem cells, which can represent the cancer cell of origin, for example in mouse models of colorectal cancer (48). However, the CSC definition does not imply a specific relationship between CSCs and physiological stem cells. Findings in other disease models support progenitors or terminally differentiated somatic cell types as the source of malignant transformation. (B) CSCs are posited to be exclusively capable of driving tumorigenesis through 3 defining features: (i) their ability for long-term self-renewal, (ii) their capacity to differentiate into tumor bulk populations devoid of CSC characteristics, and (iii) their unlimited potential for proliferation and tumorigenic growth. Furthermore, CSCs in certain malignancies possess the capacity to drive tumor angiogenic responses and/or to engage in vasculogenic mimicry, potential means of promoting tumor growth. In addition, immunoevasive features of CSCs might contribute to tumorigenesis and ultimately to tumor progression. (C) CSCs can exhibit increased resistance to chemotherapeutic agents and/or ionizing radiation. CSCs might also possess a preferential capacity to withstand immune-mediated rejection. If CSCs indeed represent the pool of resistant cells in human cancer patients, they likely also drive neoplastic progression, tumor recurrence, and metastasis. Although this hypothesis requires further validation, clinical tumor progression has already been correlated with CSC frequency in human melanoma patients.

fromastemcell,acommittedprogenitor,orevenaterminallydifferentiatedcell(12,1922)(Figure1). TumorigenicpopulationsfulfillingthedefinitionofCSCshave beenidentifiedinanumberofhumancancers,includingleukemias(2328),bladdercancer(29),breastcancer(30),CNScancers (31),coloncarcinoma(3234),headandneckcancer(35),ovarian cancer(36),pancreaticcancer(37,38),malignantmelanoma(13), livercancer(39),andEwingsarcoma(40).Itiscurrentlynotknown whetherallcancerscontainsubpopulationsofCSCs.ThemolecularphenotypesthathavebeenusedforCSCidentificationpurposesintheaforementionedhumanmalignanciesaresummarizedin Table1.Itshouldbenotedthatinsomeinstances,thesefindings havegivenrisetocontroversywithregardtothemarkersormarker combinationsusedandtheirrelevancetoclinicaldiseaseinthe respectivemalignancies.Forexample,withregardtobreastcancer,onestudythatexamined136clinicalbreastcancerspecimens foundthattheprevalenceofCD44+CD24tumorcellsshowedno correlationwithclinicaloutcomeandsurvival(41),potentially questioningthetranslationalrelevanceofthefindingofAl-Hajj etal.thatthisphenotypeidentifiescellswithtumorigenicpotentialinhumanbreastcancersamples(30).Nevertheless,thestudy didindicatethattheprevalenceofCD44+CD24tumorcellsmight favormetastaticdisease(41).AseparatestudythatalsoquestionedthetranslationalrelevanceofthefindingsofAl-Hajjetal. describedmolecularandphenotypicanalysesofCD24+andCD44+ breastcancercellsandfoundnocorrelationbetweenCD24and/or
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CD44expressioninprimaryinvasivebreastcarcinomaswithany specifictumorcharacteristics(42).Thisstudyalsosuggestedthat geneticdifferencesbetweenCD24+andCD44+cellswithinagiven tumorindicateindependentclonalevolutionasacauseoftumor heterogeneity.However,webelievethatfindingsofclonalevolutiondonotnecessarilycontradicttheexistenceofCSCsinagiven malignancy(15,43),includinginbreastcancer(30).Withregard toCD133asamarkerofbraincancerCSCs(31),Beieretal.found thatCD133gliomacellsalsocontainsubpopulationscapableof initiatingtumors,termedCD133gliomaCSCs(44).However,the self-renewalpotentialofCD133CSCscouldnotberigorously examinedinthisstudyinserialxenotransplantationexperiments duetoalackofsurfacemarkersthatwouldhaveallowedspecific purificationofCD133CSCsfromthelargerpoolofallCD133 gliomacells.Inaddition,aseparatestudyindicatedthepossibility ofinterconversionofCD133andCD133+gliomacellphenotypes (45),butitdidnotdemonstratethatCD133gliomacellsrepresentCSCsaccordingtotheconsensusdefinition(12).Withregard tocoloncancer,onestudy(46)raisedquestionsregardingCD133 (33,34)asauniversalmarkerofcolonCSCs,basedonfindings thatCD133canbewidelyexpressedbyhumanprimarycolon cancerepithelialcellsandthatCD133expressiondidnotidentify theentirepopulationofepithelialandtumor-initiatingcellsin humanmetastaticcoloncancer(46).Inthisstudy,bothCD133+ andCD133metastatictumorsubpopulationswerecapableof long-termtumorigenesisinaNOD/SCIDserialxenotransplanta-

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Table 1 CSCs in human malignancies
Malignancy AML Bladder Breast CNS Colon Colon Colon Ewing Head and neck Liver Melanoma Ovarian Pancreatic Pancreatic
CK5, cytokeratin 5.

Molecular phenotype CD34+CD38 LinCD44+CK5+CK20 CD44+CD24/loLinEPCAM+ CD133+ CD133+ CD133+ EPCAMhiCD44+Lin(CD166+) CD133+ CD44+Lin CD90+ ABCB5+ CD44+CD117+ CD44+CD24+EPCAM+ CD133+

Reference 23 29 30 31 33 34 32 40 35 39 13 36 38 37

interactionswiththemicroenvironmentandantitumorimmune responseofthetumorhostmightbesusceptibletotherapeutic interventionandthereforerepresentemergingfociofinvestigationalinterestandtranslationalsignificance.Furthermore,wediscussdirectCSCtargetingapproaches,forwhichtherapeuticproof ofprinciplehasrecentlybeenestablished(13). CSC functions in tumorigenesis CSC-associated angiogenesis and vasculogenic mimicry.In1971,Judah Folkmanadvancedthehypothesisofcancerangiogenesis,the notionthattumorsarecriticallydependentupontumor-related bloodvesselgrowthanddevelopment(51).Initiallycontroversial, thisconceptisnowbroadlyacceptedforitsbiologicandtherapeuticsignificance.Theprecisepathwaysbywhichcancersstimulate angiogenesis(i.e.,formationofnewvesselsfrompreexistingvessels;refs.52,53)intheperitumoralstroma,andthemechanisms responsiblefortheangiogenicresponsetotumorcells,havesince beensubjectsofintensestudy,andaspecificroleforCSCsinangiogenesishasbeenidentifiedrecently(54).Furthermore,although itisthoughtthatthetumorvasculatureismostlycomposedof nonmalignantendothelialcellpopulationsoriginatingfromhost angiogenesis(52,53),thereisevidencethattumorcellswithhigh degreesofdifferentiationplasticitymightcontributetothetumor vasculatureviaaprocesstermedvasculogenicmimicry(55),which seemstomimicvasculogenesis(i.e.,denovovesselformationthat occursindependentlyofangiogenesisthroughthedenovoproductionofendothelialcells).Inthecaseofhumanmelanoma,CSCs havebeenfoundtoberesponsibleforvasculogenicmimicry(13). Withregardtoangiogenesis,CSCscanreciprocallymodulate theirmicroenvironmentthrougheitherthesecretionofparacrine factorsordirectcell-cellcontact.Forexample,inhumanbrain cancer,CD133+Nestin+CSCsinmedulloblastomas,ependymomas,oligodendrogliomas,andglioblastomashavebeenfoundto residewithinaperivascularniche,wheretheyinteractcloselywith endothelialcells(56).Moreover,cograftingendothelialcellsin orthotopichumanmedulloblastomatumorxenografts,induced inadose-dependentfashionexpansionoftheself-renewing CSCfractionandacceleratedcancerinitiationandgrowth(56). Inanotherstudy(54),aspecificmechanisticrelationshipbetween CSCs and angiogenesis was established in glioma, in which glioma-initiatingcells,enrichedfromclinicalspecimensusing thecellsurfacemarkerCD133,wereshowntospecificallypromotetumorangiogenesis,andtherebytumorxenograftgrowth, throughthesecretionofhigherlevelsofVEGFcomparedwith bulktumorpopulations.Importantly,VEGF-specificneutralizingmAbtreatmentsuppressedthegrowthoftheCD133+human gliomacellderivedxenografts(54),whichindicatesthatthisCSC functionmayrepresentapotentialtherapeutictarget.Inaddition,Calabreseetal.havedemonstratedthatdepletionofvascular endothelialcells,whichwerefoundtobecapableofenhancingthe invivoself-renewalcapacityofCD133+brainCSCsthroughinhibitionofeitherERBB2orVEGFsignaling,reducedCSCabundance andsubstantiallyinhibitedtumorxenograftgrowth(56).Further studiesrevealedthatgliomaCSCsrespondeddifferentlytoangiogenesis-promotinghypoxia,withdistinctHIFresponsepatterns, thandidnon-stemtumorcellsandnormalneuralprogenitors (57).Specifically,HIF2andmultipleHIF-regulatedgeneswere preferentiallyexpressedingliomaCSCs.Inaddition,theintegrity oftheHIFresponsewasfunctionallyrequiredforgliomaCSCselfrenewal,proliferation,andsurvivalinvitroandtumorinitiation
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tionmodel.TheseresultsindicatethatCD133maynotuniformly identifycolonCSCsinalltumortypesandpatients. InadditiontocellsortingbasedCSCidentificationefforts, marker-specificgeneticlineagetrackingofsubpopulationsofcancercellsincompetitivetumordevelopmentmodelshasrecently beenusedtofurtherestablishinvivoevidencefortheexistence oftumorhierarchiesdrivenbymolecularlydefinedCSCs(13). Thisapproachhasprovidedtheadditionaladvantageofdetectingpotentialinteractionsbetween CSCsand bulk tumor cell populationsthatmayoccurinclinicalcancersandwasdesigned andusedbyourlaboratorytoidentifyCSCsinhumanmalignant melanoma(13).Basedonthedemonstratedutilityandfeasibility ofthegeneticlineagetrackingapproachforCSCidentificationin humanmalignantmelanomaandthesuccessofsimilarcellfate trackingtechniquesaspartofCSCidentificationstrategiesin humanbreastandbraincancer(47)aswellasinmodelsofcolon carcinomatumorigenesis(48,49),webelievethatinvivogenetic lineagetrackingshouldberoutinelyincludedintheexperimentalrepertoiretoassayandvalidateCSCidentityandfunction inhumanmalignancies.Moreover,webelieveittobeextremely importantinlightofthepotentiallimitationsofCSCassaysthat relyexclusivelyonsorted,untrackedcancercellsubpopulations,as arecentstudyhasshownthatexpressionof1putativeCSCmarker changesasvariousmalignantsubpopulationscycle(50).Invivo geneticlineagetrackingassaysprovidetheadditionaladvantageof facilitatingpotentialnicheinteractionsbetweenCSCsandtumor bulkpopulationsthatmaybeoperativeinnaturallyoccurring cancersbutmaytypicallyevadedetectionwhenpurified,isolated subpopulationsofcellsarestudied(13). The ability to prospectively identify CSCs has permitted researcherstobegincharacterizingspecificmolecularandcellular phenotypesandmechanismspreferentiallyassociatedwithCSCs thatmaycontributetotumorinitiation,growth,andprogression,inadditiontothoseassociatedwiththeirdefiningfeatures ofunlimitedself-renewalandproliferativecapacities(Figure1). AswediscussinthisReview,amongtherecentlyuncoveredCSC characteristicslikelytoinfluencetumorinitiation,growth,and progressionarefunctionsassociatedwithangiogenesisandvasculogenicmimicryaswellastumorimmuneevasion.TheseCSC

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potentialinvivo.Importantly,HIF2expressionwasalsofoundto correlatewithpoorsurvivalinhumanpatientswithaglioma(57), whichindicatesthatHIF2mightrepresentapromisingtargetfor antiglioblastomatherapies. Withregardtovasculogenicmimicry,MaryHendrixandcoworkersdescribedin1999aphenomenonwherebyhumanmelanomasdevelopedpatternednetworksofbasementmembranethat stainedwithperiodicacid-Schiffandperfusablechannelslinedby tumorcellsthatexpressedsome,butnotall,endothelialprotein andgenomicmarkers(58).Theyusedthetermvasculogenicmimicrytoemphasizethatthechannelsthatformedwerenotlinedby trueendothelialcells,asisthecaseinvasculogenesis.Itremains unknownwhethervasculogenicmimicryrepresentsamechanism oftumorperfusionwherebyaggressiveandmetabolicallyactive tumorsobtainthenutrientsrequisiteforcriticalstagesofgrowth andevolutionandwhetheritisrelatedto,orindependentof, angiogenesis.However,ithasbeenestablishedthatvasculogenic mimicrycharacterizedbyexpressionoftyrosinekinasewithIg-like andEGF-likedomains1(TIE-1)andCD144relatestomelanoma aggressiveness(59)andthatvasculogenicmimicrynetworksthat alsoexpresslamininrepresent,inthecaseofhumanmelanomas,a biomarkerassociatedwithincreasedclinicalmortality(60).Indeed, vasculogenicmimicryhasbeenproposedtoprovideapotential targetfortherapeuticintervention(59).Inadditiontodetection inuveal,cutaneous,andmucousmembranemelanomas,vasculogenicmimicryhasalsobeenidentifiedininflammatoryandductal breastcarcinomas,ovariancarcinomas,prostatecarcinomas,and softtissuesarcomas,includingsynovialsarcoma,rhabdomyosarcoma,osteosarcoma,andpheochromocytoma(61). Interestingly,additionofinhibitorsofangiogenesistomelanomacellculturesinvitrohasbeenshownnottoaffectthetubular network formation typically associated with vasculogenic mimicry,whereasangiogenesisbyHUVECswasabrogatedunder similarconditions(62).Thesefindingssuggestthatvasculogenicmimicrymightrepresentanimportantsurvivalmechanism thatcouldexplainthefailureofcurrentlyavailableinhibitorsof angiogenesistofullyeffecttumoreradication(63).Importantly, inthecaseofmelanoma,tumorcelldrivenvasculogenicmimicry hasbeenlinkedtobonemorphogeneticproteins(BMPs;ref.64), secretedmorphogenmembersoftheTGF-superfamilythatplay criticalrolesinearlyembryonicvasculardevelopment(65).Indeed, Rothhammeretal.showedthatimpairedBMPactivityresulted inreducedtumorgrowthinvivoinmurinemodelsofmelanoma (64).Inaddition,othershaveshownthatBMP4signalingviabone morphogeneticproteinreceptor,typeIA(BMPR1A)couldregulatethesizeoftheCSCpopulationinhumanglioblastomas(66). However,thepossibilitythatCSCsareselectivelyresponsiblefor vasculogenicmimicryhadnotbeenconsidereduntilrecently. WerecentlyfoundthathumanmelanomacellsexpressingABC, subfamilyB(MDR/TAP),member5(ABCB5)representasubpopulationofcellsenrichedforCSCs(13).Furthermore,ourresultsdemonstratedthatinbothclinicalpatienttumorsandexperimentaltumors inserialxenotransplantationexperiments,ABCB5+CSCspreferentiallyexpressedthevasculogenicmarkersTIE-1andCD144aswellas BMPR1A(13).ThesefindingssuggestedthattheCSCcompartment withinatumorcouldberesponsibleforvasculogenicmimicryand thatthisfunctionofCSCsmightrepresentonerolebywhichCSCs candrivetumorformation,growth,andprogression(67). FurtherinvestigationoftherelationshipbetweenCSCsand tumorangiogenesisandvasculogenicmimicry,anddissection
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ofthecellularandmolecularmechanismsinvolved,couldprovideopportunitiesforthedevelopmentofnovelCSC-targeted antiangiogenictherapieswithadvantagesovercurrentlyavailableconventionalantiangiogenictherapies.Indeed,despitegreat promise, conventional VEGF-targeted therapies have shown anunexpectedlylimitedsurvivalbenefitintheclinic(68),and thishadledtoadditionalstudiesaimedatfurtherdissecting theeffectsofVEGFinhibitiononprimarytumorgrowthand tumormetastaticpotential.Inthisregard,Ebosetal.foundin amousemodelofbreastcancerthat,despiteinhibitoryeffects oftheVEGFRtyrosinekinaseinhibitorsunitinibonestablished primarytumorgrowth,administrationofsunitinibpriortoor shortlyaftertumorcellinoculationresultedinacceleratedtumor metastasisanddecreasedsurvival(69).SimilareffectsofVEGF inhibitionhavebeenreportedinamodelofpancreaticcancer, inwhichprolongedtreatmentoftumor-bearingmicewithantiangiogenicdrugsresultedinincreasedlocaltumorcellinvasion andenhanceddistantmetastasis(70).Althoughtheexactmechanismsunderlyingthesefindingsremaintobeelucidated,recent studieshaveidentifiedimportantdifferencesbetweeninhibition of myeloid cellderived VEGF compared with tumor-derived VEGFontumorgrowth(71,72).Specifically,targetingofmyeloid cellderivedVEGFsignalingleadstodecreasedphosphorylationofVEGFR2andacceleratedtumorprogressionasaresult oftumorvasculaturenormalization(71),whereastargetingof bothmyeloidcellandtumor-derivedVEGFleadstocollapseof tumorgrowth(72).BecauseCSCscanrepresentthemajorsource oftumor-derivedVEGF(54),specifictargetingofCSCsorCSCderivedVEGFproductionmightrepresentamorepromisingform ofantiangiogenictherapycomparedwithcurrentmodalities. Immune evasion and modulation.Inadditiontotumor-promotingrolesinangiogenesisandvasculogenicmimicry,CSCsmight preferentiallyinitiateandsustainneoplasticgrowthanddisease progressionthroughimmunoevasiveandimmunomodulatory functions.Thispossibilityissupportedbyfindingsthatsuggest negativecorrelationsbetweentheimmunecompetenceofthe tumorhostandratesoftumorformation.Forexample,markedlyincreasedcancerriskshavebeendescribedinimmunocompromisedhumanpatients(73).Similarly,inexperimentalmodel systems,immunocompromisedanimalssuchasSCIDandRag/ mice,whicheachlackTandBcells,exhibitsubstantiallyincreased ratesofspontaneousmalignancies(74).Furthermore,fewertumor cellsarerequiredtoinitiatetumorgrowthinprofoundlyimmunocompromisedxenograftrecipients(14,23)thaninlessseverely immunocompromisedhosts(13,75).Specifically,inoculaofat least2105CD34+CD38humanacutemyeloidleukemia(AML) cellswererequiredtoinitiateleukemicdiseaseinSCIDmice(75), whereasonly5103cellswereneededtocauseleukemiainmore severelyimmunocompromisedNOD/SCIDhosts(23).Inaddition,CD34+CD38AMLcellscouldbeseriallypassagedtoform secondaryneoplasmsinNOD/SCIDmice(23),butnotinthe lessseverelyimmunocompromisedSCIDhosts(75),pointingto interdependenceofCSCphenotypeandfunctionandrecipient immunestatusinthismalignancy.However,patient-dependent differencesinAMLCSCfrequenciesalonemightaccountforthe observeddifferencesinthesestudies,becausenodirectcomparisonofAMLxenograftsfromthesameindividualstoNOD/SCID andSCIDrecipientswasperformedinthesestudies(23,75).In contrast,tumorigenicityclearlydependsonhostimmunocompetenceinhumanmalignantmelanoma(13,14).CSCfrequencies

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inhumantoNOD/SCIDmousexenotransplantationexperiments havebeenestimatedtobeapproximately1in106tumorcellswhen tumorformationwasassessed8weeksafterxenotransplantation (13,14).Ontheotherhand,higherfrequenciesofcellscapableof tumorinitiationhavebeenobservedinside-by-sidecomparative studiesinmoreseverelyimmunocompromisedxenograftrecipients,NOD/SCIDmicelackingthecommoncytokinereceptor -chain(c;alsoknownasIL-2R;ref.14).Theseresultsdemonstrateheterogeneityamongcancercellswithregardtoevasion ofthehostantitumorimmuneresponseandshowthatIL-2R dependenthostimmuneeffectorresponseseliminatesome,but notall,melanomacellscapableoftumorinitiation(13,14),therebyindicatingtheexistenceofanimmunoevasivesubpopulation ofCSCs(17).Moreover,theseresultsindicatethatCSCinteractionswithhostimmunitymightcontributetothevariabilityof experimentallydeterminedCSCfrequenciesinhumantomouse xenotransplantationmodelswithvaryingdegreesofhostimmunocompetence(1315,17,18),andthatCSCimmunomodulatory functionsmightleadtooverestimationofCSCfrequencieswhen clinicallyhighlyimmunogeniccancers,suchashumanmalignant melanoma(67,76),arestudiedinprofoundlyimmunocompromisedexperimentalhosts,suchasIl2rg/NOD/SCIDmice(17, 18).Thepossibilitythatnon-CSCpopulations,whichdonotnormallyinitiatetumors,mightcauseexperimentaltumorgrowthin suchmodelsissuggestedbyclinicalfindingsofdetectablecirculatingtumorcellpopulationsthatfrequentlydonotinitiatetumors inhumanmelanomapatientswithrelativelyintactimmunity(77). Thisisconsistentwiththeviewthatitispossibledependingon howdifferentthetumorenvironmentiswithinpatientscompared withmousemodelsthatdifferentcancercellsformtumorsin mousemodelsthaninhumanpatients(14).Furthermore,itis possiblethat,despiteobservationsofahighpercentageofmelanomacellswiththepotentialtoproliferateextensivelyandform newtumorsinparticularmodels,anevengreater,oramuchsmaller,fractionofmelanomacellsactuallycontributestodiseaseprogressioninpatients(14).Thus,whileitisconceivablethatmodel systemscouldbedesignedthatallowmostcancercellstoforman experimentaltumor,itislikelythatthosemodelsthataccountfor, butdonotmask,CSC-specificfunctionsrelatedtohostmicroenvironmentalandimmuneinteractionsmightbemorerelevantto thestudyofclinicallyandtranslationallyimportantCSCpopulations.Suchmodelsmightconsist,forexample,ofchimericmurine xenograftrecipients:micethatareorthotopicallyxenograftedwith humancancercellsintosyngeneichumantissueofcancerorigin, alreadyxenograftedfromthesamepatient,inthepresenceofan adoptivelytransferredhematopoieticsystemoriginatingfromthe samedonorthatiscapableofsyngeneicantitumorimmunity.This mightbefeasible,particularlyinthecaseofhumanmelanomas, forwhichchimericorthotopicxenotransplantationmodelshave previouslybeenestablished(78). Thefindingsofhigherratesofcancerdevelopmentinimmunocompromisedpatientsandanimalmodelsimplythathostimmunosurveillancemightservetoeliminatemalignantcellsatearly stagesoftumorigenesis,potentiallyexplainingtherelativelylow frequencyoftumordevelopmentinhealthyindividuals(79).The mechanismsbywhichCSCsmightattenuateantitumorimmune responses(17,80,81)arecurrentlyunknown,butcouldinclude immunoregulatorymechanismsknowntobeoperativeinphysiologicstemcells(17,8285),forexample,contact-dependent mechanismsrequiringengagementoftheinhibitorymoleculepro

grammedcelldeath1andsecretionorinductionofsolubleimmunomodulatoryfactorsthatarerequiredforimmunosuppression, suchasTGF-1andIL-10.Initialevidenceforanimmunoevasive phenotypeofCSCshasbeenprovidedbyrecentfindingsinhuman malignantmelanoma(17).ThisstudyshowedthatamongA375 humanmelanomacells,which,despiteclonalorigin,areheterogeneousforABCB5expression,theABCB5+CSCsubpopulation selectivelylackstheimmunogenicmelanoma-associatedantigen recognizedbyTcells(MART-1;ref.17).Thus,ABCB5+CSCscan possessaphenotypeofrelativeimmuneprivilegethatsuggests resistancetoMART-1directedimmunotherapeutictreatment strategies.Accordingly,thepossibilityofCSC-driventumorescape fromimmune-mediatedrejectionhasimportantimplicationsfor currentcancerimmunotherapyandmightrepresentaresistance mechanismsusceptibletotherapeuticintervention. Translational relevance of CSCs as targets for therapy Conventionalanticancerapproachesaredirectedpredominantly atbulktumorpopulations.Suchstrategiesoftenhavelimited efficacybecauseofintrinsicoracquireddrugresistanceand/or resistancetoionizingradiation(86).Mechanismsoftherapeutic resistanceincludeincreasedrecognitionandrepairofDNAdamagedbythedrugorionizingradiation,alteredcellcyclecheckpointcontrol,impairedfunctioningofapoptoticpathways,and reduceddrugaccumulationasaresultofincreasedexpressionof ABCtransportersthateffluxdrug(86,87).Evidencehasemerged thatCSCsrepresentasubpopulationofcellswithincancersthatis characterizedbyincreasedresistancetochemo-andradiotherapy, indicatingthatconventionalanticancerapproachesmightfrequentlyfailtoeradicatethecellsubsetthatinitiatesandperpetuatestumorigenesis(Figure1).Forexample,CSCchemoresistance hasbeenreportedinhumanleukemias(27,80,8892),inmalignantmelanoma(13,93),andinbrain(94),breast(95,96),pancreatic(37),andcolorectal(97)cancers.Furthermore,CSCradioresistancehasbeenidentifiedinbrain(98)andbreast(99,100)cancers. IfCSCsareindeedthemajorculpritsoftumordevelopmentand responsiblefortherapeuticresistanceandmalignantprogression inhumanpatients,treatmentapproachesthattargetCSCscould potentiallyincreasetheefficacyofcurrentlyavailabletreatment regimensandreducetheriskoftumorrelapseandmetastasis. ProofofprincipleforthepotentialtherapeuticefficacyofCSC targetingrequiresevaluationoftheinvivotumor-inhibitoryeffects oftherapiesdirectedatCSCsmolecularlydefinedbyaprospective markernotexpressedbynontumorigenicbulkcancerpopulations (13).Suchapproachesarewarrantedbecauseantitumoreffectsof therapiesdirectedatbothCSCsandbulkcancerpopulationscannotbeascribedwithcertaintytoaneffectoneithercellpopulation alone.Accordingly,provisionofthisproofofprincipleiscurrently notexperimentallyfeasibleincircumstancesinwhichcandidate CSCpopulationshavebeenidentifiedthroughmarkercombinationsthatincludepositivityformoleculesalsoexpressedbymost ofthetumorcellsandnegativityformoleculesexpressedbybulk cancerpopulations,forexample,positivityforCD34andnegativityforCD38ofCSCsinAML(Table1andref.23).Incontrast,itis feasibleinthosemalignanciesinwhichCSCshavebeenreported tobeenrichedamongcellpopulationsidentifiedbyasingleprospectivemolecularmarkernotsharedbythebulkcancerpopulations,forexample,theCSCsinbraincancer(31),coloncancer(33, 34),pancreaticcancer(37),andEwingsarcoma(40),allofwhich havebeenidentifiedbyexpressionofCD133;theCSCsinlivercan45

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resistancetoanticancertherapeutics,andneoplasticprogression inhumanmalignantmelanoma.Wethereforeexaminedwhether targetedablationofaprospectivelyidentifiedCSCcompartment inhibitstumorgrowth(13).Interestingly,administrationofan anti-ABCB5mAbtonudemouserecipientsofhumanmelanoma xenograftssubstantiallyinhibitedtumorformationandgrowth whenstartedpriortoxenotransplantation.Furthermore,theantiABCB5mAblimitedthegrowthofestablishedtumorscompared withcontrolxenograftrecipients,throughCSC-specificAb-dependentcell-mediatedcytotoxicity(13).Thesefindingshaveprovided initialproofofprinciplethattargetedablationofCSCsusingan Figure 2 agentdirectedataCSC-definingmolecularmarkerissufficientto The therapeutic promise of CSC-directed targeting strategies. A num- substantiallyinhibittumorigenesisandtumorgrowth(13).Also ber of therapeutic strategies directed at CSCs are beginning to be inhumanmalignantmelanoma,shRNA-mediatedknockdownof experimentally validated. These approaches could potentially enhance CD133whichisexpressedonsubsetsofhumanmelanomacells responsiveness to current anticancer treatment regimens and might (93,104,105),includingasubsetofABCB5+CSCs(93)resultreduce the risk of relapse and dissemination. The approaches include ablation using antitumor agents that target prospective markers of edinreducedmelanomaclonogenicityandmotilityinvitroand CSCs (e.g., monoclonal antibodies and activated immune cells); rever- ininhibitionofmelanomametastaticpotentialinexperimental sal of chemo- or radioresistance mechanisms operative in CSC; CSC modelsystemsinvivo(106). pathway interference; differentiation therapy; disruption of protumoriDirect CSC killing by targeting a CSC-expressed molecular genic CSC-microenvironment interactions; antiangiogenic or antivas- markerhasalsobeenreportedinexperimentalmodelsofhepaculogenic therapy; and disruption of immunoevasion pathways. tocellularcarcinoma(39).Inthismalignancy,Yangetal.identifiedapopulationofCSCsexpressingCD90thatshowedan increasedcapacitytoinitiateexperimentaltumorscomparedwith ceridentifiedbyexpressionofCD90(39);andtheCSCsinhuman CD90cancercellsinahumantonudemousexenotransplantamalignantmelanoma,identifiedbyexpressionofABCB5(Table1 tionmodel(39).AmongthisCSCpopulation,asubpopulation andref.13).Indeed,initialproofofprincipleforthepotential ofCD90+CD44+cellsdemonstratedaphenotypemoreaggressive therapeuticutilityoftheCSCconcepthasrecentlybeenprovided thanthatofCD90+CD44cells,characterizedbyformationof inhumanmelanomabythedemonstrationthatselectivekilling metastaticlesionsinthelungsofimmunodeficientmice.SystemoftheCSCsubpopulationidentifiedbyABCB5expressionissuf- icadministrationofahumanCD44specificmAbatthetimeof ficienttoinhibitexperimentaltumorgrowth(13).Thesefindings subcutaneousCD90+CD44+carcinomacellinoculationmarkedly supportthetherapeuticpromiseofCSC-targetedapproachesin inhibitedtumorinitiationandgrowthcomparedwithcontrols. humanmalignantmelanoma.Furthermore,theyprovidearatio- QuantificationofCD90+CD44+tumorcellapoptosisafterinvitro nale for the development of additional therapeutic strategies treatmentwiththehumanCD44specificmAbsuggestedmAbdirectedattargetingfunctionallyrelevantmolecularpathwaysin inducedCSCapoptosisasapossiblemechanismunderlyingthe CSCsinothercancers,regardlessofwhethersuchpathwaysmight observedinhibitionoftumorxenograftformation(39). beexclusivelyassociatedwithCSCsubsetsorwhethertheyare Inhumangliomas,shRNA-mediatedknockdownoftheneuoperativeinbothCSCsandbulkcancerpopulations.Indeed,those ralcelladhesionmoleculeL1celladhesionmolecule(L1CAM), therapeuticsthattargetbothCSCsandcancerbulkpopulations whichispreferentiallyexpressedonCD133+gliomaCSCs,submightprovemosteffectivefortumoreradication. stantiallydecreasedtheabilityoftheseCSCstoformneuroSeveralCSC-targetedapproachesharborpromiseforincreasing spheres(anonadherentinvitrogrowthpatternassociatedwith cancertherapeuticefficacy(Figure2).Inadditiontotheaforemen- the CSC phenotype) and induced apoptosis of CD133 +, but tionedpotentialindirectstrategiesrelatedtotheangiogenic/vas- notCD133,gliomacellsinvitro(107).Invivo,L1CAMknockculogenicfunctionsandtheimmunoevasivepropertiesofCSCs, downinCD133+gliomacellspriortoxenotransplantationinto therearedirectstrategies,suchasCSCablationusingagentsthat immunodeficientmicemarkedlyinhibitedinvivotumorigentargettheirmolecularmarkers,reversalofresistancemechanisms esisandprolongedsurvivalofthetumorxenograftrecipients. operativeinCSCs,anddifferentiationtherapy.Itisimportantto Furthermore,intracranialadministrationoflentiviralpreparecognizeinthisregardthattheCSCphenotypecandisplayboth rations expressing L1CAM shRNA to immunocompromised intra-andinterindividualvariabilityinparticularmalignancies mice5daysafterCD133+gliomacellxenotransplantationalso andthatCSC-directedtherapiesmightthereforehavelimitations substantiallysuppressedtumorgrowthandprolongedsurvival withregardtotargetingeveryCSCinallpatients. ofthetumor-bearingmice(107).Inaddition,Vlashietal.have CSC ablation using agents that target their molecular markers and signal- reportedthat,amonghumangliomacellsandhumanbreast ing pathways.AbundanceoftheCSCsubsetidentifiedbyexpression cancercells,malignantsubpopulationsthatexhibitCSCcharofthechemoresistancemediatorABCB5correlatespositivelywith acteristicsandpreferentialresistancetoionizingradiationcan neoplasticprogressioninhumanmelanomapatients(13).Consis- beidentifiedbyreduced26Sproteasomeactivitycomparedwith tentwiththis,theABCB5geneisalsopreferentiallyexpressedby bulktumorpopulations(47).Targetedkillingofthesecellsviaa melanomaswithhighinvivotumorigeniccapacityinhumanto proteasome-dependentthymidinekinasesuicidegeneresulted murinexenotransplantationmodels(101,102)andbymelanomas inexperimentaltumorregression(47),whichsuggeststhatdifofmetastatictumororigin,butnotthosethatareprimarytumors ferencesinproteasomeactivityrepresentpotentialtargetsfor (103).Thus,ABCB5providesadirectanduniquelinkamongCSCs, CSC-directedcancertherapy.
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In human bladder cancer, CSCs were recently identified as LinCD44+CK5+CK20(29).ThisstudyalsorevealedthatCD47,a proteinthatprovidesaninhibitorysignalformacrophagephagocytosis,ismorehighlyexpressedbybladderCSCsthantherest ofthetumorcells.BlockadeofCD47usingamAbresultedin engulfmentoftheCSCsbymacrophagesandsubsequentbladder cancercellkillinginvitro(29),whichpointstoapotentialtherapeuticvalueofthisCSC-targetedapproachforinvasivebladder cancerifsimilareffectsofCD47-specificmAbarefoundininvivo preclinicalstudies(29). Inhumanleukemias,inhibitionofNF-Binducedapoptosisof CD34+CD38CSCsinvitroandinhibitedtumorgrowthinexperimentalanimalmodelsinvivo,whilesparingthephysiologicHSC compartment(108).Furthermore,alsoinhumanleukemia,expressionofthesubunitoftheIL-3receptor(CD123)wasdetectedat higherlevelsonbothAMLCD34+CD38stemcellsandbulkAML populationscomparedwithnormalhematopoieticcells(109). TreatmentofhumanAMLcellswithaCD123-specificneutralizing mAbresultedinimpairedcancercellengraftmentandproliferationinmurineNOD/SCIDxenografthostsandimprovedlongtermsurvivalthroughtargetingbothleukemicstemcellsandbulk populations(109).LeukemicCSCshavealsobeentargetedthrough modulationofthePI3K/phosphataseandtensinhomolog/mammaliantargetofrapamycin(PI3K/PTEN/mTOR)pathwayina mousemodelofleukemia(110).ConditionaldeletionofthePten tumorsuppressorgeneinadulthematopoieticcellsinducedtransplantableleukemiasinwhichleukemia-initiatingcellscouldbe depletedbyrapamycinthroughinhibitionofmTOR(110). Inaggregate,theseresultsdemonstratethefeasibilityandtherapeutic promise of CSC ablation through targeting molecular markersspecificallyexpressedbyCSCsorexpressedbybothCSCs andbulkcancercellpopulations.MAb-andshRNA-basedstrategiesmayprovetobeespeciallyusefulinthisregardbecauseof theirhighdegreesoftargetspecificity. Reversal of resistance mechanisms operative in CSCs.Experimental evidencehasbeengeneratedinseveralhumanmalignanciesthat inhibitionofthemechanismsresponsibleforCSCresistanceto chemo-andradiotherapyrepresentsapromisingCSC-directed therapeuticstrategy.ReversingchemoresistanceinCSCpopulationscanbeachievedthroughspecificblockadeofmultidrug resistanceABCtransporters,asshowninhumanmelanoma(93). TheCSCmarkerABCB5mediatesmelanomaresistancetothe chemotherapeuticagentdoxorubicin(93,111),andthiseffectis reversiblebybothmAb-mediatedinhibitionofABCB5-dependent drugefflux(93)andsiRNA-mediatedABCB5genesilencing(111). Inaddition,ABCB5genesilencingincreasessubstantiallythesensitivityofhumanmelanomacellstotheanticancerchemotherapeutics5-fluorouracil(5-FU)andcamptothecin(112).Apotential broaderroleforABCB5inanticancerchemotherapeuticresistance issuggestedbytheobservationthatacrossapanelofhumancancercelllinesusedbytheNationalCancerInstitutefordrugscreening,ABCB5geneexpressionlevelscorrelatewithchemoresistance to45of119anticanceragents(93),andthefindingthathuman chronicmyeloidleukemiacellsresistanttotheanticancerchemotherapeuticvincristineexhibitABCB5geneamplificationand enhancedexpression(113,114).Theseresultswarrantfurtherin vivoexaminationtodeterminewhetherABCB5-targetedsensitizationtoclinicallyrelevanttherapeuticagentsrepresentsafeasible andeffectivestrategytoeradicatechemoresistantCSCs.Recent findingsincoloncancerfurthersupportthepotentialtherapeutic

utilityofCSCchemosensitizingagents(115).Inthisstudy,the researchersdemonstratedthatpretreatmentofCD133+colon CSCswithanIL-4specificneutralizingAbenhancedapoptosis mediatedby5-FUandoxiplatininvitroandinxenotransplanted immunodeficientmice(115). CSC-targetedtherapeuticapproachesmightalsoincludestrategiesdirectedatreversalofradioresistance.Inrecentyears,several studieshavereportedthatCSCshaveincreasedradioresistance comparedwithbulkcancerpopulationsandproposedthatreversalofCSCradioresistancemightbecriticalforcuringsolidcancersforwhichradiationremainsthefirst-linetreatment(116). Baoetal.reportedthat,inhumangliomas,CD133+gliomaCSCs contributedtotumorradioresistancethroughpreferentialactivationoftheDNAdamagecheckpointresponseandthrough increasedDNArepaircapacity(98).Inthisstudy,inhibitingthe Chk1andChk2checkpointkinasesreversedtheradioresistance ofCD133+gliomaCSCs(98).Additionalmechanismsofincreased radioresistanceingliomashavebeendescribedbyChangetal. (117),whofoundthatCD133+gliomaCSCsexpressedhigherlevelsofsirtuin1(SIRT1)comparedwithCD133bulkcancerpopulationsandthatspecificSIRT1inhibitionresultedinmarkedly enhancedradiosensitivityofCD133+gliomaCSCs.Theseresults suggestedthatSIRT1isapotentialtargetforreversingglioma CSCresistancetoradiotherapy(117).Inbreastcancer,increased radioresistanceofCD24/loCD44+CSCshasbeenattributedto decreasedproductionofROSinresponsetoradiationasaresult ofhighexpressionlevelsoffree-radicalscavengers(99,100)and activationoftheNotchsignalingcascade(100).Consistentwith theformer,pharmacologicdepletionofROSscavengersresulted inradiosensitization(99).Inaggregate,thesestudiesindicatethat targetingmechanismsofradioresistanceoperativeinCSCsrepresentsapromisingapproachtoultimatelyfacilitatetumoreradicationandpreventcancerrecurrence. Differentiation therapy.Thepossibilitythatdifferentiationofmore primitivecellswithinamalignancymayleadtotumordegenerationandincreasedsusceptibilitytoconventionalcytotoxicanticancertherapieshasbeenrecognizedforsometime(118).Therefore,differentiationtherapyholdspromiseasanapproachto targetCSCs.PotentialstrategiesthatinducequiescentCSCsto differentiateintomorematuretumorcellsincludeactivationof distinctsignalingpathways,suchasmorphogen-drivensignaling cascades(66);alterationofgeneexpressionprofilesusingmicroRNAs(miRNAs;ref.96);andepigeneticdifferentiationtherapy (119,120).Piccirilloetal.haveharnessedBMPsignalingtodifferentiateCSCsinexperimentalmodelsofhumanglioblastoma(66). AdministrationofBMP4tohumancancerbearingmiceinduced glioblastomadifferentiationandmarkedlyattenuatedCD133+ CSCfrequency(66).Inaddition,implantationofBMP4-treated glioblastomaxenograftstomurinerecipientsresultedinsmaller tumorlesionscomparedwithuntreatedcontrolsandsubstantially prolongedhostsurvival(66).PreferentialexpressionofBMPR1A onABCB5+CSCsinhumanmelanomas(13)suggeststhatsimilar BMP4-dependentdifferentiationstrategiesmightalsoholdtherapeuticpromiseinthismalignancy.Inmedulloblastoma,modulationofCSCsignalingpathwayshasalsobeenshowntoinduceCSC differentiation(121).Specifically,administrationofNotchpathwayinhibitorsresultedindepletionofmedulloblastomastem-like cells.SmallnoncodingmiRNAshavealsobeenshowntobecapableofregulatingCSCdifferentiationandfunction.Forexample, inbreastcancer,enforcedexpressionofthelet-7miRNAinduced
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differentiationofCD44+CD24/loCSCsandinhibitedtheirability toformtumorsinmice(96).Mostrecently,alsoinhumanbreast cancer,Guptaetal.usedahigh-throughputscreeningapproach todeterminetheanticanceractivityofapproximately16,000compoundsandidentifiedapotassiumionophore,salinomycin,that selectivelytargetsCD24loCD44hiCSCs(122).Treatmentofmice withthisagentinducedepithelialdifferentiationoftumorcells andresultedininhibitionoftumorgrowth,pointingtoapromisingroleforthisapproachinbreastcancertherapy. InhumanAML,Jinetal.(123)reportedatherapeuticapproach usinganactivatingmAbdirectedtotheadhesionmoleculeCD44. InvivoadministrationofthisAbtoNOD/SCIDmicetransplantedwithhumanAMLmarkedlyreducedleukemicrepopulation. Absenceofleukemiainseriallytransplantedmicedemonstrated thatAMLCSCsweredirectlytargeted.Mechanistically,CD44-specificAbtreatmentwasshowntoinducedifferentiationtomore maturecancercellprogenythatwereunabletoestablishrobust leukemiauponxenotransplantation(123).Interferencewithcancercelltransporttostemcellsupportivemicroenvironmental nicheswasfoundtorepresentanadditionalmechanismunderlyingCD44-specificAbmediatedAMLCSCeradication,indicatinga roleforthemicroenvironmentinregulatingCSCfunction(123). EpigenetictherapycouldpotentiallyalsobeusedtoinduceCSC differentiation,therebyrenderingtheseaggressivecellsmoresusceptibletoconventionalcytotoxictreatment.Indeed,inhibitorsof DNAmethyltransferasesand/orhistonedeacetylaseshavebeen showntoinduceCSCdifferentiationthatbypassescancer-associatedgeneticabnormalities(119).Inrecentclinicalfindingsin patientswithlocallyadvancedbreastcancer,atherapeuticregimen consistingofthedemethylatingagenthydralazineandthehistone deacetylaseinhibitormagnesiumvalproatefollowedbydoxorubicinandcyclophosphamidetherapyresultedinatrendtoward improvedclinicaloutcomecomparedwithdoxorubicinandcyclophosphamidetherapyalone(124).ThedoxorubicinchemoresistancemediatorABCB5(93,111)isexpressedinhumanbreast cancerandwassubstantiallydownregulatedafterdifferentiation therapyinthesestudies(124),raisingthepossibilitythatlossof more primitive and doxorubicin-resistant ABCB5-expressing breastcancercellsmighthavecontributedtotheobservedresults. Inaggregate,theresultsregardingdifferentiationtherapysuggest thattheseCSC-targetedapproachesholdpromiseforimproving currentlyavailableformsofcancertherapy.
1.Reya T, Morrison SJ, Clarke MF, Weissman IL. Stemcells,cancer,andcancerstemcells.Nature. 2001;414(6859):105111. 2.MorrisonSJ,KimbleJ.Asymmetricandsymmetricstem-celldivisionsindevelopmentandcancer. Nature.2006;441(7097):10681074. 3.ScaddenDT.Thestem-cellnicheasanentityof action.Nature.2006;441(7097):10751079. 4.Fidler IJ, Hart IR. Biological diversity in metastaticneoplasms:originsandimplications.Science. 1982;217(4564):9981003. 5.HanahanD,WeinbergRA.Thehallmarksofcancer.Cell.2000;100(1):5770. 6.GriffinJD,LowenbergB.Clonogeniccellsinacute myeloblasticleukemia.Blood.1986;68(6):11851195. 7.SabbathKD,BallED,LarcomP,DavisRB,Griffin JD.Heterogeneityofclonogeniccellsinacutemyeloblasticleukemia.J Clin Invest.1985;75(2):746753. 8.BruceWR,VanDerGaagH.Aquantitativeassay forthenumberofmurinelymphomacellscapable ofproliferationinvivo.Nature.1963;199:7980. 9.Hamburger AW, Salmon SE. Primary bioas48

Conclusion CSC-specificphenotypesandmechanismsthatrelatetofunctions intumorigenicity,cancerprogression,andtherapeuticresistance havebeenidentified.TheseresultsindicatethatCSCsmaycontributetothefailureofexistingtherapiestoconsistentlyeradicate malignanttumors.Therefore,CSCsrepresentnovelandtranslationallyrelevanttargetsforclinicalcancertherapy.Importantly, proof-of-principleexperimentshavestrengthenedtherationale fordevelopingCSC-targetedtherapeuticmodalitiesthatmight complementmoreconventionalcancertherapies.Indeed,CSCtargetedapproacheshaveshownpromiseinpreclinicalmodels. Theseapproachesincludedirectstrategies,suchasablationby targetingmolecularmarkersofCSCsorCSC-specificpathways, reversalofresistancemechanisms,anddifferentiationtherapy, andindirectstrategies,suchasantiangiogenictherapy,immunotherapeuticapproaches,anddisruptionofprotumorigenic interactionsbetweenCSCsandtheirmicroenvironment.Insome instances,mAb-basedstrategiesthattargetmoleculesexpressed byCSCs,forexample,epithelialcelladhesionmolecule(EPCAM) onbreastandcolonCSCs(Table1andrefs.30,32),arealready beingtranslatedtotheclinic(125).Thesedevelopmentsunderline thetherapeuticpromiseoftheCSCconcept.Ultimately,patient cureswillrequireeradicationofallcellswithinacancer;therefore, combinationtherapiesthattargetbothCSCsandbulkcancer populationsarelikelytoemergeasparticularlyeffectiveclinical strategies,especiallyinthosemalignanciescurrentlyrefractory toconventionalanticanceragentsdirectedpredominantlyatthe bulktumorcellpopulations. Acknowledgments ThisworkwassupportedbyNationalCancerInstitute,NIH,grants 1RO1CA113796,1R01CA138231,and2P50CA093683-06A20006 (toM.H.Frank)andbyNationalInstituteofNeurologicalDisordersandStroke,NIH,grant1KO8NS051349(toN.Y.Frank). T.SchattonistherecipientofaPostdoctoralFellowshipAward fromtheAmericanHeartAssociationFoundersAffiliate. Addresscorrespondenceto:MarkusH.Frank,Transplantation ResearchCenter,ChildrensHospitalBostonandBrighamand WomensHospital,HarvardMedicalSchool,300LongwoodAvenue,Boston,Massachusetts02115,USA.Phone:(617)919-2993, Fax:(617)730-0129,E-mail:markus.frank@childrens.harvard.edu.
15(9):10101012. 16.OrimoA,etal.Stromalfibroblastspresentininvasive human breast carcinomas promote tumor growthandangiogenesisthroughelevatedSDF-1/ CXCL12secretion.Cell.2005;121(3):335348. 17.Schatton T, Frank MH. Antitumor immunity and cancer stem cells. Ann N Y Acad Sci. 2009; 1176:154169. 18.SchattonT,FrankNY,FrankMH.Identification andtargetingofcancerstemcells.Bioessays.2009; 31(10):10381049. 19.JamiesonCH,etal.Granulocyte-macrophageprogenitorsascandidateleukemicstemcellsinblastcrisisCML.N Engl J Med.2004;351(7):657667. 20.HuntlyBJ,etal.MOZ-TIF2,butnotBCR-ABL, conferspropertiesofleukemicstemcellstocommittedmurinehematopoieticprogenitors.Cancer Cell.2004;6(6):587596. 21.KrivtsovAV,etal.Transformationfromcommittedprogenitortoleukaemiastemcellinitiatedby MLL-AF9.Nature.2006;442(7104):818822. 22.SunB,ChenM,HawksCL,Pereira-SmithOM,

say of human tumor stem cells. Science. 1977; 197(4302):461463. 10.Park CH, Bergsagel DE, McCulloch EA. Mouse myelomatumorstemcells:aprimarycellculture assay.J Natl Cancer Inst.1971;46(2):411422. 11.SchattonT,FrankNY,FrankMH.Solidtumor stemcellsimplicationsforcancertherapy.In: RajasekharVK,VemuriMC,eds.Regulatory Networks in Stem Cells.NewYork,NY:HumanaPress; 2009:527544. 12.ClarkeMF,etal.Cancerstemcellsperspectives on current status and future directions: AACR Workshoponcancerstemcells.Cancer Res.2006; 66(19):93399344. 13.SchattonT,etal.Identificationofcellsinitiating humanmelanomas.Nature.2008;451(7176):345349. 14.QuintanaE,ShackletonM,SabelMS,FullenDR, JohnsonTM,MorrisonSJ.Efficienttumourformationbysinglehumanmelanomacells.Nature. 2008;456(7222):593598. 15.Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med. 2009;

The Journal of Clinical Investigation http://www.jci.org Volume120 Number1 January2010

review series
HornsbyPJ.Theminimalsetofgeneticalterations requiredforconversionofprimaryhumanfibroblaststocancercellsinthesubrenalcapsuleassay. Neoplasia.2005;7(6):585593. 23.Bonnet D, Dick JE. Human acute myeloid leukemiaisorganizedasahierarchythatoriginates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730737. 24.CastorA,etal.Distinctpatternsofhematopoietic stemcellinvolvementinacutelymphoblasticleukemia.Nat Med.2005;11(6):630637. 25.Cox CV, Evely RS, Oakhill A, Pamphilon DH, Goulden NJ, Blair A. Characterization of acute lymphoblasticleukemiaprogenitorcells. Blood. 2004;104(9):29192925. 26.CoxCV,MartinHM,KearnsPR,VirgoP,EvelyRS, BlairA.CharacterizationofaprogenitorcellpopulationinchildhoodT-cellacutelymphoblasticleukemia.Blood.2007;109(2):674682. 27.IshikawaF,etal.Chemotherapy-resistanthuman AMLstemcellshometoandengraftwithinthe bone-marrowendostealregion.Nat Biotechnol.2007; 25(11):13151321. 28.MatsuiW,etal.Characterizationofclonogenicmultiplemyelomacells.Blood.2004;103(6):23322336. 29.ChanKS,etal.Identification,molecularcharacterization,clinicalprognosis,andtherapeutictargetingofhumanbladdertumor-initiatingcells.Proc Natl Acad Sci U S A.2009;106(33):1401614021. 30.Al-HajjM,WichaMS,Benito-HernandezA,MorrisonSJ,ClarkeMF.Prospectiveidentificationof tumorigenicbreastcancercells.Proc Natl Acad Sci U S A.2003;100(7):39833988. 31.SinghSK,etal.Identificationofhumanbraintumour initiatingcells.Nature.2004;432(7015):396401. 32.DalerbaP,etal.Phenotypiccharacterizationof humancolorectalcancerstemcells.Proc Natl Acad Sci U S A.2007;104(24):1015810163. 33.OBrien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumourgrowthinimmunodeficientmice.Nature. 2007;445(7123):106110. 34.Ricci-VitianiL,etal.Identificationandexpansionof humancolon-cancer-initiatingcells.Nature.2007; 445(7123):111115. 35.PrinceME,etal.Identificationofasubpopulation ofcellswithcancerstemcellpropertiesinheadand necksquamouscellcarcinoma.Proc Natl Acad Sci U S A.2007;104(3):973978. 36.ZhangS,etal.Identificationandcharacterizationof ovariancancer-initiatingcellsfromprimaryhuman tumors.Cancer Res.2008;68(11):43114320. 37.HermannPC,etal.Distinctpopulationsofcancer stemcellsdeterminetumorgrowthandmetastatic activityinhumanpancreaticcancer.Cell Stem Cell. 2007;1(3):313323. 38.LiC,etal.Identificationofpancreaticcancerstem cells.Cancer Res.2007;67(3):10301037. 39.Yang ZF, et al. Significance of CD90(+) cancer stemcellsinhumanlivercancer.Cancer Cell.2008; 13(2):153166. 40.SuvaML,etal.Identificationofcancerstemcellsin Ewingssarcoma.Cancer Res.2009;69(5):17761781. 41.AbrahamBK,FritzP,McClellanM,Hauptvogel P,AthelogouM,BrauchH.PrevalenceofCD44+/ CD24-/lowcellsinbreastcancermaynotbeassociatedwithclinicaloutcomebutmayfavordistant metastasis.Clin Cancer Res.2005;11(3):11541159. 42.ShipitsinM,etal.Moleculardefinitionofbreasttumor heterogeneity.Cancer Cell.2007;11(3):259273. 43.ShackletonM,QuintanaE,FearonER,Morrison SJ.Heterogeneityincancer:cancerstemcellsversus clonalevolution.Cell.2009;138(5):822829. 44.BeierD,etal.CD133(+)andCD133(-)glioblastoma-derivedcancerstemcellsshowdifferential growth characteristics and molecular profiles. Cancer Res.2007;67(9):40104015. 45.WangJ,etal.CD133negativegliomacellsform tumorsinnuderatsandgiverisetoCD133positivecells.Int J Cancer.2008;122(4):761768. 46.ShmelkovSV,etal.CD133expressionisnotrestrictedtostemcells,andbothCD133+andCD133 metastaticcoloncancercellsinitiatetumors.J Clin Invest.2008;118(6):21112120. 47.VlashiE,etal.Invivoimaging,tracking,andtargetingofcancerstemcells.J Natl Cancer Inst.2009; 101(5):350359. 48.BarkerN,etal.Cryptstemcellsasthecells-of-originof intestinalcancer.Nature.2009;457(7229):608611. 49.ZhuL,etal.Prominin1marksintestinalstemcells thataresusceptibletoneoplastictransformation. Nature.2009;457(7229):603607. 50.JakschM,MuneraJ,BajpaiR,TerskikhA,OshimaRG. Cellcycle-dependentvariationofaCD133epitopein humanembryonicstemcell,coloncancer,andmelanomacelllines.Cancer Res.2008;68(19):78827886. 51.FolkmanJ.Tumorangiogenesis:therapeuticimplications.N Engl J Med.1971;285(21):11821186. 52.FolkmanJ.SeminarsinMedicineoftheBethIsrael Hospital,Boston.Clinicalapplicationsofresearchon angiogenesis.N Engl J Med.1995;333(26):17571763. 53.Risau W. Mechanisms of angiogenesis. Nature. 1997;386(6626):671674. 54.BaoS,etal.Stemcell-likegliomacellspromote tumorangiogenesisthroughvascularendothelial growthfactor.Cancer Res.2006;66(16):78437848. 55.HendrixMJ,SeftorEA,HessAR,SeftorRE.Vasculogenicmimicryandtumour-cellplasticity:lessons frommelanoma.Nat Rev Cancer.2003;3(6):411421. 56.CalabreseC,etal.Aperivascularnicheforbrain tumorstemcells.Cancer Cell.2007;11(1):6982. 57.LiZ,etal.Hypoxia-induciblefactorsregulatetumorigenic capacity of glioma stem cells. Cancer Cell. 2009;15(6):501513. 58.ManiotisAJ,etal.Vascularchannelformationby humanmelanomacellsinvivoandinvitro:vasculogenicmimicry.Am J Pathol.1999;155(3):739752. 59.FolbergR,HendrixMJ,ManiotisAJ.Vasculogenic mimicry and tumor angiogenesis. Am J Pathol. 2000;156(2):361381. 60.LinAY,etal.Distinguishingfibrovascularsepta fromvasculogenicmimicrypatterns.Arch Pathol Lab Med.2005;129(7):884892. 61.Folberg R, Maniotis AJ. Vasculogenic mimicry. APMIS.2004;112(78):508525. 62.van der Schaft DW, et al. Effects of angiogenesisinhibitorsonvascularnetworkformationby humanendothelialandmelanomacells.J Natl Cancer Inst.2004;96(19):14731477. 63.Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007;6(4):273286. 64.RothhammerT,BatailleF,SprussT,EissnerG, BosserhoffAK.FunctionalimplicationofBMP4 expression on angiogenesis in malignant melanoma.Oncogene.2007;26(28):41584170. 65.LiuW,etal.Bmp4signalingisrequiredforoutflowtractseptationandbranchial-archarteryremodeling. Proc Natl Acad Sci U S A.2004;101(13):44894494. 66.Piccirillo SG, et al. Bone morphogenetic proteinsinhibitthetumorigenicpotentialofhuman brain tumour-initiating cells. Nature. 2006; 444(7120):761765. 67.Schatton T, Frank MH. Cancer stem cells and humanmalignantmelanoma.Pigment Cell Melanoma Res.2008;21(1):3955. 68.Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008;358(19):20392049. 69.EbosJM,LeeCR,Cruz-MunozW,BjarnasonGA, ChristensenJG,KerbelRS.Acceleratedmetastasis aftershort-termtreatmentwithapotentinhibitorof tumorangiogenesis.Cancer Cell.2009;15(3):232239. 70.Paez-RibesM,etal.Antiangiogenictherapyelicitsmalignantprogressionoftumorstoincreased localinvasionanddistantmetastasis.Cancer Cell. 2009;15(3):220231. 71.GreenbergJI,etal.AroleforVEGFasanegative regulatorofpericytefunctionandvesselmaturation.Nature.2008;456(7223):809813. 72.StockmannC,etal.Deletionofvascularendothelial growthfactorinmyeloidcellsacceleratestumorigenesis.Nature.2008;456(7223):814818. 73.GrulichAE,vanLeeuwenMT,FalsterMO,Vajdic CM.IncidenceofcancersinpeoplewithHIV/AIDS comparedwithimmunosuppressedtransplantrecipients:ameta-analysis.Lancet.2007;370(9581):5967. 74.Shankaran V, et al. IFNgamma and lymphocytespreventprimarytumourdevelopmentand shape tumour immunogenicity. Nature. 2001; 410(6832):11071111. 75.Lapidot T, et al. A cell initiating human acute myeloidleukaemiaaftertransplantationintoSCID mice.Nature.1994;367(6464):645648. 76.RosenbergSA,RestifoNP,YangJC,MorganRA, DudleyME.Adoptivecelltransfer:aclinicalpath toeffectivecancerimmunotherapy.Nat Rev Cancer. 2008;8(4):299308. 77.MedicS,PearceRL,HeenanPJ,ZimanM.Molecularmarkersofcirculatingmelanomacells.Pigment Cell Res.2007;20(2):8091. 78.Juhasz I, et al. Growth and invasion of human melanomasinhumanskingraftedtoimmunodeficientmice.Am J Pathol.1993;143(2):528537. 79.MaparaMY,SykesM.Toleranceandcancer:mechanismsoftumorevasionandstrategiesforbreakingtolerance.J Clin Oncol.2004;22(6):11361151. 80.CostelloRT,etal.Humanacutemyeloidleukemia CD34+/CD38progenitorcellshavedecreasedsensitivitytochemotherapyandFas-inducedapoptosis,reducedimmunogenicity,andimpaireddendriticcelltransformationcapacities.Cancer Res.2000; 60(16):44034411. 81.KawasakiBT,FarrarWL.Cancerstemcells,CD200 and immunoevasion. Trends Immunol. 2008; 29(10):464468. 82.Frank MH, Sayegh MH. Immunomodulatory functionsofmesenchymalstemcells.Lancet.2004; 363(9419):14111412. 83.LeBlancK,etal.Treatmentofsevereacutegraftversus-host disease with third party haploidentical mesenchymal stem cells. Lancet. 2004; 363(9419):14391441. 84.LeBlancK,RingdenO.Immunomodulationby mesenchymalstemcellsandclinicalexperience. J Intern Med.2007;262(5):509525. 85.Uccelli A, Moretta L, Pistoia V. Mesenchymal stemcellsinhealthanddisease.Nat Rev Immunol. 2008;8(9):726736. 86.GottesmanMM,FojoT,BatesSE.Multidrugresistanceincancer:roleofATP-dependenttransporters.Nat Rev Cancer.2002;2(1):4858. 87.DeanM,FojoT,BatesS.Tumourstemcellsand drugresistance.Nat Rev Cancer.2005;5(4):275284. 88.deGrouwEP,etal.Preferentialexpressionofa highnumberofATPbindingcassettetransporters inbothnormalandleukemicCD34+CD38-cells. Leukemia.2006;20(4):750754. 89.Ito K, et al. PML targeting eradicates quiescent leukaemia-initiating cells. Nature. 2008; 453(7198):10721078. 90.MichorF,etal.Dynamicsofchronicmyeloidleukaemia.Nature.2005;435(7046):12671270. 91.Viale A, et al. Cell-cycle restriction limits DNA damageandmaintainsself-renewalofleukaemia stemcells.Nature.2009;457(7225):5156. 92.WulfGG,etal.Aleukemicstemcellwithintrinsic drugeffluxcapacityinacutemyeloidleukemia. Blood.2001;98(4):11661173. 93.FrankNY,etal.ABCB5-mediateddoxorubicintransportandchemoresistanceinhumanmalignantmelanoma.Cancer Res.2005;65(10):43204333. 94.Eramo A, et al. Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ. 2006; 13(7):12381241.

The Journal of Clinical Investigation http://www.jci.org Volume120 Number1 January2010

49

review series
95.LiX,etal.Intrinsicresistanceoftumorigenicbreast cancercells tochemotherapy.J Natl Cancer Inst. 2008;100(9):672679. 96.Yu F, et al. let-7 regulates self renewal and tumorigenicityofbreastcancercells.Cell.2007; 131(6):11091123. 97.Dylla SJ, et al. Colorectal cancer stem cells are enrichedinxenogeneictumorsfollowingchemotherapy.PLoS One.2008;3(6):e2428. 98.BaoS,etal.GliomastemcellspromoteradioresistancebypreferentialactivationoftheDNAdamageresponse.Nature.2006;444(7120):756760. 99.DiehnM,etal.Associationofreactiveoxygenspecieslevelsandradioresistanceincancerstemcells. Nature.2009;458(7239):780783. 1 00.PhillipsTM,McBrideWH,PajonkF.Theresponseof CD24(/low)/CD44+breastcancer-initiatingcellsto radiation.J Natl Cancer Inst.2006;98(24):17771785. 1 01.HoekKS,etal.Invivoswitchingofhumanmelanomacellsbetweenproliferativeandinvasivestates. Cancer Res.2008;68(3):650656. 1 02.HoekKS,EichhoffOM,WidmerD,DummerR. Stemmingtheflood.Pigment Cell Melanoma Res. 2009;22(1):67. 1 03.SousaJF,EspreaficoEM.Suppressionsubtractive hybridizationprofilesofradialgrowthphaseand metastaticmelanomacelllinesrevealnovelpotentialtargets.BMC Cancer.2008;8:19. 1 04.KleinWM,WuBP,ZhaoS,WuH,Klein-Szanto AJ,TahanSR.Increasedexpressionofstemcell markersinmalignantmelanoma.Mod Pathol.2007; 20(1):102107. 1 05.MonzaniE,etal.MelanomacontainsCD133and ABCG2positivecellswithenhancedtumourigenic potential.Eur J Cancer.2007;43(5):935946. 1 06.RappaG,FodstadO,LoricoA.Thestemcell-associatedantigenCD133(Prominin-1)isamolecular therapeutictargetformetastaticmelanoma.Stem Cells.2008;26(12):30083017. 1 07.BaoS,etal.Targetingcancerstemcellsthrough L1CAM suppresses glioma growth. Cancer Res. 2008;68(15):60436048. 1 08.GuzmanML,etal.Anorallybioavailableparthenolideanalogselectivelyeradicatesacutemyelogenousleukemiastemandprogenitorcells.Blood. 2007;110(13):44274435. 1 09.JinL,etal.Monoclonalantibody-mediatedtargetingofCD123,IL-3receptoralphachain,eliminates humanacutemyeloidleukemicstemcells.Cell Stem Cell.2009;5(1):3142. 1 10.YilmazOH,etal.Ptendependencedistinguishes haematopoieticstemcellsfromleukaemia-initiatingcells.Nature.2006;441(7092):475482. 1 11.ElliottAM,Al-HajjMA.ABCB8mediatesdoxorubicinresistanceinmelanomacellsbyprotecting themitochondrialgenome.Mol Cancer Res.2009; 7(1):7987. 1 12.HuangY,etal.Membranetransportersandchannels:roleofthetransportomeincancerchemosensitivityandchemoresistance.Cancer Res.2004; 64(12):42944301. 1 F 13. rank NY, Frank MH. ABCB5 gene amplification in human leukemia cells. Leuk Res. 2009; 33(10):13031305. 1 14.LehneG,etal.Upregulationofstemcellgenesin multidrugresistantK562leukemiacells.Leuk Res. 2009;33(10):13791385. 1 15.TodaroM,etal.Coloncancerstemcellsdictate tumorgrowthandresistcelldeathbyproduction ofinterleukin-4.Cell Stem Cell.2007;1(4):389402. 1 16.V lashi E, McBride WH, Pajonk F. Radiation responsesofcancerstemcells.J Cell Biochem.2009; 108(2):339342. 1 17.ChangCJ,etal.Enhancedradiosensitivityandradiation-inducedapoptosisingliomaCD133-positive cellsbyknockdownofSirT1expression.Biochem Biophys Res Commun.2009;380(2):236242. 1 18.PierceGB.Thecancercellanditscontrolbythe embryo.Rous-WhippleAwardlecture.Am J Pathol. 1983;113(1):117124. 1 19.LotemJ,SachsL.Epigeneticsandtheplasticityof differentiationinnormalandcancerstemcells. Oncogene.2006;25(59):76637672. 1 20.JonesPA,BaylinSB.Theepigenomicsofcancer. Cell.2007;128(4):683692. 1 21.Fan X, et al. Notch pathway inhibition depletes stem-likecellsandblocksengraftmentinembryonal braintumors.Cancer Res.2006;66(15):74457452. 1 22.GuptaPB,etal.Identificationofselectiveinhibitors of cancer stem cells by high-throughput screening.Cell.2009;138(4):645659. 1 23.JinL,HopeKJ,ZhaiQ,Smadja-JoffeF,DickJE.TargetingofCD44eradicateshumanacutemyeloidleukemicstemcells.Nat Med.2006;12(10):11671174. 1 24.ArceC,etal.Aproof-of-principlestudyofepigenetic therapyadded toneoadjuvant doxorubicin cyclophosphamideforlocallyadvancedbreastcancer.PLoS One.2006;1:e98. 1 25.ZhouBB,ZhangH,DamelinM,GelesKG,GrindleyJC,DirksPB.Tumour-initiatingcells:challengesandopportunitiesforanticancerdrugdiscovery. Nat Rev Drug Discov.2009;8(10):806823.

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