Brain Tumor Stem Cells Bringing Order to the Chaos of Brain Cancer_2008

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Brain Tumor Stem Cells: Bringing Order to the Chaos of Brain CancerPeter B. Dirks

From the Arthur and Sonia Labatt BrainTumor Research Center, Program inDevelopmental and Stem Cell Biology,Hospital for Sick Children, University ofToronto, Toronto, Ontario, Canada.

Submitted April 14, 2008; acceptedApril 15, 2008.

Supported by the Canadian CancerSociety/National Cancer Institute ofCanada, Canadian Institutes for HealthResearch, Genome Canada, Stem Cell

Network of Canada, Jessica’s FootprintFoundation, and the Hospital for SickChildren Foundation.

Author’s disclosures of potential con-icts of interest and author contribu-tions are found at the end of thisarticle.

Corresponding author: Peter B. Dirks,MD, PhD, Arthur and Sonia Labatt BrainTumor Research Center, Program inDevelopmental and Stem Cell Biology,Hospital for Sick Children, University ofToronto, 555 University Ave, Toronto,Ontario, Canada, M5G 1X5; e-mail:[email protected].

© 2008 by American Society of ClinicalOncology

0732-183X/08/2617-2916/$20.00

DOI: 10.1200/JCO.2008.17.6792

A B S T R A C T

Brain tumors are generally incurable cancers. Work from a number of laboratories stronglysuggests that they are organized as a hierarchy based on a subset of cancer cells that havestem-cell properties. These cells have now been shown to be resistant to conventional therapyand responsive to differentiation therapy. New in vitro and in vivo models for interrogating braintumor cells in stem-cell conditions have been developed that provide important new opportunitiesfor elucidating the key pathways responsible for driving the proliferation of these cells. Continuedapplication of the principles of stem-cell biology to the study of brain cancers is likely to continueto bring further important insight into these aggressive cancers, bringing new treatments andunderstanding of the origins.

J Clin Oncol 26:2916-2924. © 2008 by American Society of Clinical Oncology

INTRODUCTION

Brain tumors are diverse group of neoplasms thatremain a formidable cancer problem that has seenfew therapeutic advances during the last 40 years.Glioblastoma multiforme, the most common pri-marytumorofadults,hasamediansurvivalofabout12 months, now modestly increased to 15 months

with the up front use of temozolomide chemother-apy.1 Chemotherapy has improved the survival of childhood medulloblastomas,withsurvival rates re-ported up to 65% even for disseminated disease;however, many children who survive suffer long-term sequelae of chemotherapy and radiation ther-apy.2,3 Brain tumors now approach leukemia as thenumber onecancer killer of children.

Advances inbrain tumor therapy, aswith many other human cancers, lag behind the accumulatingknowledge of the molecular genetic alterations seenin human brain tumors. The genetic and cellularheterogeneity of the cancer tissues poses furtherchallenges to treatment. Will all of the cells in thetumor respond to the treatment? More specically,will the treatment directed at potential moleculartargets kill the key cells driving the growth of thetumor? We need to know that we are hitting theright target in the rightcell.

These questions bring tumor cellular heteroge-neity into focus, andhave led toa reconsideration of a functional tumor hierarchy for proliferation anddifferentiation as an explanation for tumor cellularheterogeneity. Are human brain tumors organizedas a stem-cell system, with mixtures of cells with

differing potentials in terms ofself-renewaland pro-liferation,and mostimportantly in terms of theabil-ity to propagate tumor growth and initiate newtumors? Evidence from a number of labs several years agostronglysuggeststhathumanbrain tumorscontain a minority population of cells that have po-tent tumorigenicity, a property not shared by thebulk of the tumor cells. 4-8 This tumorigenic sub-

population has many of the properties that can beascribed to a stem cell, namely the expression of neuralstem-cellmarkers,and moreimportantly, theproperties of self-renewal, extensive proliferation,and the ability to differentiate into more matureneural lineages.Humanbrain tumorsthereforehavebeenshowntotacancerstem-cellmodel;however,our current understanding remains only a begin-ning,andtheevidence ofcancerstemcells inhumanbrain tumors is stillbasedon thestudy ofa relatively smallnumber of human tumor samples.

In this review, the evidence that supports thecancer stem-cell model in human brain tumors willbe reviewed.Inaddition, becausethis eldis movingrapidly, I will review recent experimental insightsinto brain tumor resistance to therapy that havebeen found on the basis of the cancer stem-cellmodel, as well as exciting new approaches to braintumor treatment that may involve targeting braintumor stem cells. Because the data supporting theexistence of cancer stem cells in human brain tu-mors are as good as the experimental models thathave been used to generateexperimental evidence, Iwill review some of the strengths and limitations of the experimental systems that have been developed

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to study cancer stem cells in human brain tumors. The cancer stem-cell model has also led to a renewed interest in the cell of origin of brain tumors.

IDENTIFICATION OF BRAIN TUMOR STEM CELLS

Serum-Free Culture and Brain Tumor Stem-Cell Enrichment

Brain tumors, as a relatively uncommon subset of human can-cers, havereceived substantialattention in thecancer stem-celleldinpart because of the application and adaptation of the methodologiesthat were already developed for thestudy of normalneuralstem cells.In addition, application of the principles of purication and func-tionalanalysisof normal hematopoietic stem cells and leukemia stemcells hasbeencritical to theidenticationandcharacterization ofbraintumor stemcells. 9-12

Brain tumor cells withstem-cellproperties canbe enrichedusingseveralstrategies,namelyserum-freeculture,andcell sortingof freshly dissociated single-cell suspensions of tumor cells. The conditions forbrain tumorstem-cell cultureare adaptedfromthedenedconditionsoriginally developed forcultureof normal neural stemcells, as neuro-spheres.13 These conditions facilitate studying the cardinal propertiesof stem cells: self-renewal, proliferation, and differentiation in vitro

and after transplantation in vivo (Fig 1).In serum-freeculture,in thepresenceof epidermal growth factorandbroblast growth factor,humanbrain tumorcellscanbegrown assingle cell–derived colonies of cells that prominently express neuralpre-cursor markers, such as nestinand thecell-surfacemarker CD133. 4,6,8,14

Normalneurospheresand tumor spheres are heterogeneous, and alsocontain smaller numbersof cells that express markers of morematureCNSlineages, that likely represent lineagecommitted progenitors butnot fully differentiated progeny. Therefore, brain tumors grown as

A

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Tumor progenitorsRapid proliferation

lineage commitment

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Differentiated tumorcells (bulk)

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Framework ofStem Cell Biology

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Sort fresh cells

In vitro clonogenicassay

In vitro clonogenicassay

In vivo orthotopicxenotransplant

In vivo orthotopicxenotransplant

BulkEnrichment forstem cells

Then serial

Bulk

Fig 1. The brain tumor stem-cell hierarchyand principles of analysis. (A) Advances instem-cell knowledge and experimental tech-niques have led us to consider that thecellular heterogeneity of brain tumors maybe the result of a stem-cell hierarchy. Braintumors contain mixes of stem cells, progen-itor cells, and differentiated tumor cells, andthese cells have different functional proper-ties. (B) Analysis of single-cell suspensionsof brain tumor cells then allowsexperimentalanalysis of tumor subpopulations, in vitro,with serum-free culture, and in vivo withorthotopic intracerebral xenotransplantation.

Brain Tumor Stem Cells

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neurospheres contain mixtures of small numbers of stem cells, largernumbers of immature progenitors, and small numbers of differenti-ated cells. Nestin andCD133 markers areexpressed in both stem cellsand progenitor cells. Neurosphere cells are mostaccurately describedas cultures of neural precursors. The term “precursor” refers to a mix of stemandprogenitorcells.Progenitorsare distinctfromstemcellsbecausethey have limited self-renewal and an increased proliferative ability.

A subset of tumor cells grown in neurosphere conditions can beserially passaged in vitro in replating assays, which demonstrate thatsingle cells can generate secondary colonies, displaying self-renewalability. Only cells that can repeatedly regrow colonies are stem cells;progenitorcells inculturemay generate progeny that form a colony of cells, but the cells in these colonies do not have a capacity to generatenew colonies on replating (ie, they lack self-renewal). Therefore,sphere formation should not be equated with being derived from astem cell, but repeated sphere-forming ability should. Thefraction of tumor cells demonstrating sphere-forming activity typically increasesafter formation of primary spheres from fresh cells, suggesting thatthese serum-free culture conditions enrich for cells with stem-cellability.However, a true separation of stemcellsandprogenitor cells in

the neurosphere assay remains problematic, and we are awaiting fur-therpuricationand functional denition of thestemand progenitorcells inthis assay. In this system, the presenceofa stemcellis alsoonly retrospectively inferred; if one has spheres that replate multiple timesand that show differentiation capacity, they are thought to be derivedfrom a stem cell.

Brain tumor sphere cells can be induced to differentiate, mor-phologically becoming complex and inducing the expression of ma-ture neural lineages in response to growth-factor withdrawal with orwithout serum addition, together with plating cells on dishes coatedwith substrate. 4,6,8,14 The differentiation potential of the brain tumorsphere cells does not match that of normal neurospheres, but malig-nant brain tumors typically show ability to differentiate into multi-

ple lineages.The ability to grow human brain tumor cells as neurospheres is

variable. Glioblastoma cells aremosteasily grown in these conditions,but only a fraction of primary lines can be established long-term incultureas passageable lines in these sphere conditions. 8 Other typesof brain tumors can be grown for short periods in neurosphere condi-tions, such as medulloblastoma and ependymoma, but long-termlines remain difcult to establish. 4,6,8,15 Benign brain tumors show alow primary clonogenic sphere-forming activity from plating freshcells. These data suggests that they could contain rare populations of stem cells that have a more limited differentiation ability, but thesecells grow so slowly that demonstration of replating and self-renewalability, and, therefore, denitive stem-cell activity in vitro, has notbeen denitively shown. It will be important to understand whetherthese lessaggressivetumors (socalled “benign”brain tumors)arealsoorganized as a hierarchy, as many of these tumors remain extremely difcult to treat if they are not in surgically accessible areas.

Therefore, we still have not optimized the conditions for sup-porting the growth of these tumor precursor cells long-term in vitro.Nevertheless, applicationof serum-freegrowthconditions toglioblas-tomas in particular has enabled growth of lines that retain the samegenotypeof thepatient’s primarytumor,andwhich showstablestem-cell properties in vitro and more faithful generation of models of thedisease after xenotransplantation in vivo. 16 These methods clearly showthe advantages of serum-free–based culturemethods for study-

inghuman brain tumorcells, andsuggestthat serum-based lineshavelimited utility. Establishment of patient-specic glioblastoma stem-cell lines in multiple laboratories should afford new opportunities forunderstanding the signaling pathways involved in proliferation andself-renewal of brain tumor stem cells, and for testing novel genetictherapies or performing novel small-molecule screens.

Prospective Sorting and Brain Tumor Stem-Cell Enrichment

Prospective sorting of freshly dissociated tumor cells in suspen-sion remains thegold standard for enriching forcells that show stem-cell properties. CD133 was identied as a marker that was a goodcandidate for testing the clonogenic abilities of distinct brain tumorsubpopulations. CD133 (human prominin-1) is a 120-kDa, ve-transmembrane cell-surface protein originally shown to be expressedin mouse neuroepithelium 17 and in a subset of subset of CD34human hematopoietic stem cells (identied as AC133). 18,19 Subse-quently,Uchidaet al 20 developed hybridomas producing monoclonalantibodies raised against human fetal neurospheres. One antibody recognized thesame antigen as CD133,andsorting human fetalbrain

cells forCD133enrichedhighlyforstem-cellproperties in vitro andinvivo. Brain tumor spheres express other stem-cell markers, but, cell-surface markers are limited. 6

Fresh sortingof human brain tumors (glioblastomasandmedul-loblastomas)for theneuralprecursor marker CD133andcomparisonof colony-formation ability in neurosphere conditions demonstratedan enrichment for in vitro clonogenic cells in the CD133 fraction. 4

Allof thesphere-forming abilityandproliferative potential fromfreshhuman glioblastomas and medulloblastomas in vitro resided withinthe CD133 fraction. Typically, however, in culture, an increasingfraction of cells in glioblastoma spheres express CD133, so that inadditionto identifyingclonogenic cells in vitro (andtumorigeniccellsin vivo), CD133 also marks cells that do not have clonogenic or

tumorigenicpotential. CD133therefore marks brain tumorstemcells,butnotallCD133 cellsaretumorstemcells,andCD133becomeslesshelpfulas a marker of clonogenicity or tumorigenicitywith increasingtime in cell culture.

Gold Standard for Cancer Stem Cells: Tumorigenic Assays In Vivo

In vitro assays are surrogate assays for the denition of a cancerstem cell. An inability of a fresh brain tumor to generate sphereculturesin vitrodoesnotmeanthat thetumordoesnot contain cancerstemcells.“Cancer-initiating cell” isa moreprecise andpreferredtermcompared with “cancer stem cell” because it directly refers to theoperational denition in vivo. Cancer initiating cells can be denedonlyusingthegold standardcell transplantationassaysinvivo.Tumorcells that can be successfully cultured adapt to the culture conditionsand may not behave the same as freshly isolated tumor cells. So,although cultured stem cells may closely resemble their unculturedcounterparts, there may be important differences, particularly withtheir tumor-initiating ability in vivo. It has been shown that cell sur-face–sorted fresh hematopoietic cells have more potent engraftmentability than the cells sorted using the same markers after 24 hours inculture, indicating a dissociation of engraftment ability in vivo andmarker expression after cell culture. 21

The denitive identication of a brain tumor stem cells wasdemonstrated by prospective sorting of CD133 tumor cells from

Peter B. Dirks

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fresh (uncultured) human glioblastomas and medulloblastomas. 5

CD133 brain tumor cells are highly enriched for tumor-initiatingactivity in vivo. As few as 100 CD133 human brain tumor cellswere capable of initiating fatal inltrative tumors in nonobesediabetic/severe combined immunodeciency mice after ortho-topic transplantation. Injection of 100,000 CD133 – cells did notlead to tumor formation, although viablehuman tumor cells couldbe identiedin themousebrains many weeks after transplantation,suggesting that these cells were viable, but were no longer able toinitiate tumor formation. These studies denitively demonstratedcritical functional differences between distinct tumor subpopula-tions, dened by CD133.

As well, tumor formation by injection of glioblastoma sphereswas also shown at the same time. In particular, glioblastoma spherescultured from single cells were able to engraft and cause a serially transplantable glioblastoma tumor in immunodecient mice. 8 Im-portantly, orthotopic injection intothe brain microenvironment may be critical to reading out the tumorigenic fraction because these samecells showed decreased engraftment ability subcutaneously. 8 Subse-quently CD133 has been shown to enrich for in vitro clonogenic cells

fromhuman ependymomas.15

It is important to note that sorting forCD133 enriches for cancer stem cells, and does not denitively iden-tify them.

CD133 may not always be an informative marker because nowseveral studies have shown that CD133– glioblastoma cells demon-strate tumorigenic ability and an ability to initiate a serum-free cul-ture. 22,23 Studies of dissociated suspensions of fresh human cellsremain challenging; there are likely technical differences between dif-ferent laboratories in processing human samples, in culture of thecells, and also in sorting using CD133. Clearly larger numbers of human samplesmust beinterrogatedto determine whetherthecancerstem-cell model holds, as well as to determine the consistency of markers that identify them, foreach patient tumor within a particular

brain tumor type, and for the diverse types of tumors that exist.Additional markers of brain tumor stem cells are needed to furtherconrmtheir existence in a broader spectrumof human tumors.

BRAIN TUMOR STEM CELLS: IMPLICATIONS FOR TREATMENT

Tumor Resistance to Radiation Therapy The importance of identication of brain tumor–initiating cells

is that it offers new insight into mechanisms of resistance of cancertissues to current treatments, and it identies a newcellular target fortreatment.InworkbytheRichgroup, 24 CD133 humanglioblastomacells were shown to be resistant to radiation therapy, retaining aclonogenic and tumorigenicpotential. CD133 cells increaseinnum-ber after irradiation of glioblastomas cells in culture and in tumorsgrowing in vivo. The CD133 cells undergo similar DNA damage tothose of their CD133 – counterparts, but they show a better ability torepair strand breaks, through a more potent activation of DNA-damage checkpoint mechanisms. This repair mechanism has beenshown to be targetable in proof-of-principle experiments, throughpharmacologic inhibition of the checkpoint kinases Chk1 and Chk2,which renders the CD133 glioblastoma cells more radiosensitive.CD133 cells from the Daoy medulloblastoma cell line have alsobeen shown to be radiation resistant compared with their CD133counterparts. 25

Novel Therapies for Brain Tumors Based on aStem-Cell Framework

The similarities between normal neural stem cells and braintumor cells suggeststhat furthermechanisticsimilaritiesexistbetweennormal stem cells and cancer stem cells in terms of the pathwaysregulatingself-renewaland proliferation.Brain tumorscanbeconsid-eredas a disease of unregulatedself-renewalwhere genetic alterationsin normal stem cell self-renewal pathways cause abnormal prolifera-tion and cancer growth. Another important property of neural stemcells is multilineage differentiation, and brain tumor stem cells retainan abilityto respondtocues thatpromote their differentiation(at leastpartially) both in vitro andin vivo.Therefore, oneof themost impor-tant insights that thecancer stem-cell modelbrings to an understand-ing of brain tumorigenesis is a consideration that the brain tumorgrowth represents aberrant organogenesis based on stem cells. Thisideasuggestsnewstrategiesfor treatment forbrain tumors(Fig2).Thetumor tissue grows because cancer stem cells self-renew inappropri-ately and generate large numbers of progeny that retain some differ-entiation capacity. Pathways regulating neural stem cell proliferationand differentiation are therefore potentially exploited for brain tu-

mor treatment.

Differentiation Therapy of Brain Tumor Stem Cells Promotion of tumor stem-cell differentiation maybe an impor-

tant strategy for treatment of brain tumor stem cells. Vescovi et al 26

have shown that bone morphogenic proteins (BMPs), which nor-mally induceastrocyte differentiationfromnormalneural precursors,havebeenshown to promote glioblastoma cell differentiation in vitroandinvivo.Most importantly,BMPs inducethesuppression ofglioblas-tomatumorigenicity in vivo, possiblythoughpromotion of thediffer-entiation of restricted tumor progenitors or by direct action on themost primitive cell (the cancer stem cell) in the tumor. Work by theFine group 27 has further demonstrated that a subset of glioblastoma

stemcellsare blockedintheir differentiation becauseof impairmentinthe BMP signaling pathway resulting from epigenetic silencing of theBMPR1B receptor. These glioblastomacells seemto bestuck ina stateresemblingthat ofembryonicneuralstemcells. InductionofBMPR1Bby transfection or demethylation restores their ability to respond toBMPs and reduces their tumorigenicity.

Targeting Developmental Signaling Pathways Implicated in Driving Stem-Cell Proliferation

Many studies implicate several developmental signaling path-waysas positiveregulators of stem-cell self-renewaland proliferation.These pathways are therefore potentially important targets in cancerstem cells. Notch,28-32 Sonic Hedgehog (Shh), 33-40 and Wingless41-46

pathways have received the most attention. As well, homeobox tran-scription factors (such as HoxB4, 47,48 the PTEN phosphatase pro-tein, 49 and the polycomb group of transcriptional repressors have[Bmi-1] 50-53 been also shown to play a role in stem-cell self-renewal.Bmi-1 has been shown tobe a targetof Shh signaling and is expressedin human brain tumors. 54 In brain tumors, the Shh and Notch path-ways have received the most attention, and targeting these pathwayswill be reviewed here.

The importance of aberrant Shh signaling in human medullo-blastomas has been demonstrated by the discovery of mutations of multiple Shh pathway members, Patched1 (Ptc1), 55-57 Smooth-ened (Smo) 58,59 andSuppressor of Fused (SuFu) 60 in these tumors.







Mice with mutations in the Shh receptor Ptc1, develop medullo-

blastoma,whichis phenotypically identical to the humandisease.61

Medulloblastomas express Shh target genes, demonstrating highpathway signalingactivity, andaresensitive to thepathway blockercyclopamine.62,63 Therefore, inhibiting Shh activity in medullo-blastomas is a focus forintensive translational research in thebraintumor eld.

The role of Shh signaling in normal neural stem cells and indiverse types of human cancer 64 suggests thatpathwayactivity maybefoundinother typesof humanbraintumors.Humanbrain tumor celllines of diverse phenotype express the Gli transcription factors, theeffectors of Shh signaling; brain tumor cell lines of different pheno-types are also inhibited by cyclopamine.37,65 Interestingly, Gli1 wasinitially found to be amplied in a glioblastoma line, although thisgenetic alteration has not been seen in primary glioblastomas. 66 Arecent study shows that grade 2 and 3 astrocytomas, butnotglioblas-tomas, express high levels of Ptc1mRNA in the primary tissue, colo-calizing with neural precursor markers, suggesting that subsets of gliomasin particularmayhave proliferationdrivenbyShhsignaling. 67

Several studiesnowshowthatcyclopaminetreatment of glioblastomastem cells attenuates their tumorigenicity, when given to cells in cul-turebefore transplant, 68 or systemicallyafter intracranial transplant. 69

Notch signaling plays multiple and complex roles in nervoussystem development. 70 Notch receptors 1-3, and ligands Delta-like1(Dll-1),Delta-like3 (Dll-3), andJagged1areexpressed in thegerminalzones of the embryonic and postnatal brain and are expressed by

undifferentiated neurospheres. 71-75 Many studies implicate Notch in

control of normal neural stem-cell self-renewal and proliferation. Arole for Notch in human brain tumors is also emerging, with ndingNotch receptor and ligand transcripts in cultures of gliomas, 14 andparticularly inmedulloblastomas where Notch1orNotch2transcriptshave been shown to be increased in medulloblastoma specimens. 76,77

Overexpression of Notch2-intracellular form (activated Notch) in-creasesproliferationof medulloblastoma cell lines 76 and medulloblas-toma cell-line growth has been shown to be suppressed by inhibitionof Notch proteolytic processing by gamma-secretase inhibitor. 77

Targeting the Brain Tumor Stem-Cell Niche Another key insight is that brain tumor stem cells may be sup-

ported by a specic microenvironment or niche. The idea of a key supportive niche also suggests the importance of interrogating braintumor stem-cell function in the brain microenvironment, by use of orthotopic transplant models. The normal neural stem cell and thebrain tumor stem cell may have a niche provided by blood vessels.Another strategy for treatment, therefore, is to target the cells thatprovide the supportive environment for the cancer stem cell. 78

CD133 glioblastoma cells secrete increased levels of vascular endo-thelial growth factor comparedwith their CD133 – counterparts, witha further increase in response to hypoxia. 79 Tumor stem cells may,therefore, be responsible for tumor angiogenesis. CD133 medullo-blastoma and glioblastoma cells have been shown to be localized inclose proximity to blood vessels in tumors in situ; they expand with

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Treatment

Test in vivo

Block self-renewal Radiationsensitize

Forcedifferentiation

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Block niche

Cultures enriched fortumor stem cells

Geneticscreens

• Characterize transcriptome andproteome

• Characterize surface markers

Chemicalscreens

Works invitro?

Antibodytherapy

T

Fig 2. Brain tumor stem-cell therapeutics.(A) Applying stem-cell thinking to brain can-cer leads to new considerations for therapy.Ideally, all tumor cells, both stem cell andbulk, should be eliminated, and likely multi-modality treatment willbe needed. Targetingself-renewal of the brain tumor stem cell islikely to be critical for long-lasting tumortherapy; however, other recently suggestedstrategies are promotion of differentiationand targeting the stem cell’s niche. (B) Ide-ally, we will make cultures from everypatient’s brain tumor in serum-free condi-tions, creating basic patient individualizedlines, and then they will be extensivelycharacterized to identify cell-surface andmolecular targets, and then subject themto a variety of screens that will be veriedin vivo after xenotransplantation of thepatient tumor cells.

Peter B. Dirks






coculture with endothelial cells, and these cells initiate larger tumorswhen grafted together with endothelial cells. 80 Therapies that targetthevasculaturemay effectively pull outthesupportiveenvironmentof the brain tumor stemcells, attenuating their tumorigenicity.

Cell-Based Screens for Novel BrainTumor Therapeutics

Although a candidate-geneapproachhas revealedvital pathwaysin neuralstem cell and tumor stem-cell biology, to date, relatively fewpathways have been extensively studied. Because of the advances inmethodologies forculturingandanalyzing stem-cell function in vitro,applications of screens on these cells may reveal a wider spectrum of pathwaysthatfunctionto control stem-cell self-renewaland differen-tiation.Recently, a chemical(small-molecule) screen on normal neu-ral precursors in culture recovered a surprising number of smallmolecules thatattenuated precursor-cellproliferation. 81 Although themechanisms of action of these agents have not yet been extensively characterized, a number of these molecules affect neurotransmis-sion pathways that were previously thought to be functionalmainly in the mature CNS, on differentiated neurons. Further-

more, a number of these agents had potent inhibitory effects oncultures enriched for brain cancer stem cells, from both mousemedulloblastomas and human glioblastomas. These data revealthat drugs that are effective on normal neural stem cells may ndapplication for brain tumor stem cells. These results also suggestthat clinically approved drugs that have application for humanneurologic and psychiatric illness (that were contained in the drugscreen) could nd application for human brain tumors. A numberof these agentshadmore potenteffects on thecultures enriched for

cancer stem cells, suggesting a partial selectivity for the cancer cellsversus the normal cells. Obviously, it will be important to ndagents that show selective effects on brain tumor stem cells butspare normal neural stem cells. In addition, the effectiveness of these agents has not yet been shown in animal models of braintumors, butthis recent study suggests thevalue of cell-based small-molecule screens as an additional strategy for new drug discovery for brain tumors.

Cell of Origin: From Normal Neural Stem Cells? Thequestionof brain tumor cell of origin receives a great deal of

attentionin theliterature (Fig 3). One could argue that, after cancer isestablished, it does not matter which cell it arose from. It is thereforemost criticalto identify thecells that aredrivingthetumor,and thentocharacterizethemfor theaberrantmolecularpathwaysthat drive theirinappropriate growth. However, identifying the cell of origin may beimportant for several reasons. The particular cell in which an onco-gene is expressed may determine the subsequent phenotype and theresulting aggressiveness of the tumor. The cancer-causing genetic orepigenetic alterationsmaybe quite different in thedistinctcells of the

normal stem-cell hierarchy, suggesting that different treatments tar-geting different pathways could depend on the cell of origin. As well,knowing the cells of origin and how they are targeted at the earlieststages of cancer may also allow for protection of cells of origin, andtherefore prevention of cancer initiation. It must be made clear thatthe term “cancer stem cell” does not imply cell of origin from normalstem cells. Although the properties of normal neural stem cells sug-gests that they areattractive candidates for thecell of origin of humanbrain tumors,currently there isexperimental evidencein mouse brain

Brain tumor withmultiple differentiated

lineages

Brain tumor withsingle differentiated

lineage

Committed progenitoror differentiated cell

has restricteddifferentiation capacity

Transformedstem cell:

multilineagedifferentiation

Restricted to SVZLarger SVZ in Children

Stem Cells ProgenitorCells

Differentiated Cells

D e d i f

f e r e n

t i a t i o n

S t e m c e l l : l i m i t e d

d i f f e r e n t i a t i o n d u e t o

t r a n s f o r m a t i o n e v e n t

Fig 3. Cell of origin for brain tumors.Stem cells are attractive candidates for thecell of origin for human brain tumors, par-ticularly because they have self-renewalability. However, experimental evidenceso far suggests that both stem cells andmore differentiated cells give rise to braintumors. Transformed stem cells may giverise to tumors showing multilineage poten-tial, reecting an intrinsic property of stemcells. A transformation event, however,could restrict the differentiation capacity ofstem cells, leading to a tumor with more

restricted expression of lineage markers.More differentiated cells may reacquirestem-cell abilities (and undergo dedifferen-tiation or “reprogramming ”) because of thetransforming events, and result in tumorsexpressing multiple lineages, or these dif-ferentiated cells may be intrinsically limitedto one or few lineages resulting in a tumorwith more uniform phenotype. SVZ, sub-ventricular zone.







tumorsfor celloforigin fromstem cellsandprogenitorcells, as well asmore differentiated cells.

Clues to the cell of origin lie in the phenotype of thebrain tumor.Brain tumors are composed of morphologically heterogeneous cells,with varying numbers of less differentiated cells identied by theneural precursor marker nestin 82,83 mixed with cells of differentiatedneurallineages,84 suggestingthat thetransformedcell hasmultilineagedifferentiation potential. A more limited expression of lineage mark-ersmay suggest cellof origin from a cellwith more restricteddifferen-tiation potential (a progenitor or a differentiated cell) or a stem cellthat has become blocked in its full differentiation potential.

Other clues also lie in the site of origin of brain tumors in thehuman brain, particularly related to the subventricular zone(SVZ),85 which is where normalstem cells are thought to exist. 86-90

As well, experimental brain tumor models developed in mice,beginning in the 1960s and 1970s suggest that viral tumorinduction, 91-93 or chemical carcinogenesis,94,95-103 preferentially induce tumors adjacent to the cerebral ventricles, therefore possi-bly from stem cells or progenitor cells.

Morerecently, furtherexperimental models in mice alsosupport

thelocationoftheSVZsiteoforigin.Paradaetal104

developeda mousemodel decient in p53 and NF1 in which 100% of mice developedglioblastoma. All of the early lesions were seen close to the SVZ eventhough full-blown tumors were occupying the cerebral hemispheres.A proliferative expansion of thegranule-cell precursors in theexternalgranule layer of thecerebellumof Patched1mutantmice alsosuggeststhatmedulloblastomas arise in precursor cells. 105 However, medullo-blastomas may also arise from stem cells, as suggested by recent dataidentifying cerebellar stemcells puried by CD133/prominin1 whichis not expressed in the external granule layer. 106 Brain tumors of different phenotypes,indifferent locations,occurringatdifferentages,mayhave distinctcells of origin.

The Holland group 107,108 has shown that human brain tumor–

derived oncogenes injected in vivo drive brain tumorigenesis morepotently when expressed in more primitive neural compartments. Amodel developed byDePinho, 109 however,suggests thatdifferentiatedastrocytes and cultured neurosphere precursors transduced with on-cogenes areequally permissive to transformation if thecells have a key genetic alteration (p16Ink4a/p19Arf deciency). These alterations inmore differentiated cells may allow acquisition of a stem cell–likephenotype in the resulting tumor. 109,110 Further work by Bruggemanet al111 demonstrates that tumor initiation in cultured proliferatingastrocytes may lead to more aggressive tumors compared with thesame oncogenic alterations occurring in neural precursors grown asneurospheres. One could speculate that the neural stem cells, which

are thought to be relatively quiescent, retain this feature after specictransformation events. Theexperimental work so far suggestsa com-plex heterogeneity for cell of origin of brain tumors in experimentalmodels, but intriguingly suggest that tumor phenotype depends onthe normal cell that receives the transformation signals. So far, allcell-of-origin questions remain limited because of our poor under-standingof the normal neural stem-cell hierarchy,and the identica-tion of specic neural progenitor populations. We are unable toexperimentally test cancer-initiating events in pure populations of stem cells versus pure populations of progenitors or differentiatedcells.Further,moreclear insightwillundoubtedlycomefromongoingadvances in normal neural stem-cell biology and additional advancesin experimental approaches.

SUMMARY

Applying the experimental and conceptual framework of stem-cellbiology to the study of human brain tumors has yielded importantnew insight into understanding brain tumor behavior. Identicationof a subpopulation of cells in these tumors that drives tumor propa-

gation has led to a deeper understanding of why these tumors are soresistant to conventional therapy. It has also led to new strategies fortreatment,such aspromotion of differentiation. In addition, inknow-ing the key cells driving the tumor, we will now be able to determinewhether a putative novel or existing molecular target or activatedsignaling pathway plays a critical role in cells that are thought to lie atthe root of the cancer. Furthermore, the methodologies of stem-cellbiology as applied to cancer cell growth in vitro may give us modelsthat more faithfully represent the patient’s disease. By having braintumor cell cultures that areenriched instem cells that can also beusedto recapitulate thedisease in experimental animals,we may nowhavemore relevantcell populations for screeningnewdrugs andperform-inggenetic screens invitro,withan abilityto verify invivo. Muchworkremains in improving invitroand invivoassaysand inmore precisely sorting and purifying brain tumor stem cells. New insight will con-tinue to come fromadvances in researchon normal stem-cell biology.Aswell, it isnot yet clear whether all types ofbrain tumors or whetherall patients’ individual tumors will follow the cancer stem-cell model.There is no doubt that this area will continue to be a focus for intenseresearch activity in the years to come.

AUTHOR’S DISCLOSURES OF POTENTIAL CONFLICTSOF INTEREST

The author(s) indicated no potential conicts of interest.

REFERENCES

1. Stupp R, Mason WP, van den Bent MJ, et al:Radiotherapy plus concomitant and adjuvant temozolo-mide for glioblastoma. N Engl J Med 352:987-996, 2005

2. Gilbertson RJ: Medulloblastoma: Signalling achange in treatment. Lancet Oncol 5:209-218, 2004

3. Crawford JR, MacDonald TJ, Packer RJ:Medulloblastoma in childhood: New biological ad-vances. Lancet Neurol 6:1073-1085, 2007

4. Singh SK, Clarke ID, Terasaki M, et al: Iden-tication of a cancer stem cell in human braintumors. Cancer Res 63:5821-5828, 2003

5. Singh SK, Hawkins C, Clarke ID, et al: Iden-tication of human brain tumour initiating cells.Nature 432:396-401, 2004

6. Hemmati HD, Nakano I, Lazareff JA, et al:Cancerous stem cells can arise from pediatric braintumors. Proc Natl Acad Sci U S A 100:15178-15183,2003

7. Yuan X, Curtin J, Xiong Y, et al: Isolation ofcancer stem cells from adult glioblastoma multi-forme. Oncogene 23:9392-9400, 2004

8. Galli R, Binda E, Orfanelli U, et al: Isolationand characterization of tumorigenic, stem-like neuralprecursors from human glioblastoma. Cancer Res64:7011-7021, 2004

9. Huntly BJ, Gilliland DG: Leukaemia stemcells and the evolution of cancer-stem-cell research.Nat Rev Cancer 5:311-321, 2005

10. Warner JK, Wang JC, Hope KJ, et al: Con-cepts of human leukemic development. Oncogene23:7164-7177, 2004

11. Dick JE, Bhatia M, Gan O, et al: Assay ofhuman stem cells by repopulation of NOD/SCIDmice. Stem Cells 15:199-203; discussion 204-207,1997 (suppl)

12. Bonnet D, Dick JE: Human acute myeloidleukemia is organized as a hierarchy that originatesfrom a primitive hematopoietic cell. Nat Med 3:730-737, 1997

Peter B. Dirks






13. Reynolds BA, Weiss S: Generation of neu-rons and astrocytes from isolated cells of the adultmammalian central nervous system. Science 255:1707-1710, 1992

14. Ignatova TN, Kukekov VG, Laywell ED, et al:Human cortical glial tumors contain neural stem-likecells expressing astroglial and neuronal markers invitro. Glia 39:193-206, 2002

15. Taylor MD, Poppleton H, Fuller C, et al:Radial glia cells are candidate stem cells of ependy-moma. Cancer Cell 8:323-335, 2005

16. Lee J, Kotliarova S, Kotliarov Y, et al: Tumorstem cells derived from glioblastomas cultured inbFGF and EGF more closely mirror the phenotypeand genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391-403, 2006

17. Weigmann A, Corbeil D, Hellwig A, et al:Prominin, a novel microvilli-specic polytopic mem-brane protein of the apical surface of epithelial cells,is targeted to plasmalemmal protrusions of non-epithelial cells. Proc Natl Acad Sci U S A 94:12425-12430, 1997

18. Yin AH, Miraglia S, Zanjani ED, et al: AC133,a novel marker for human hematopoietic stem andprogenitor cells. Blood 90:5002-5012, 1997

19. Miraglia S, Godfrey W, Yin AH, et al: A novel

ve-transmembrane hematopoietic stem cell anti-gen: Isolation, characterization, and molecular clon-ing. Blood 90:5013-5021, 1997

20. Uchida N, Buck DW, He D, et al: Directisolation of human central nervous system stemcells. Proc Natl Acad Sci U S A 97:14720-14725,2000

21. Dorrell C, Gan OI, Pereira DS, et al: Expan-sion of human cord blood CD34( )CD38(-) cells inex vivo culture during retroviral transduction withouta corresponding increase in SCID repopulating cell(SRC) frequency: Dissociation of SRC phenotypeand function. Blood 95:102-110, 2000

22. Beier D, Hau P, Proescholdt M, et al:CD133( ) and CD133(-) glioblastoma-derived cancerstem cells show differential growth characteristics

and molecular proles. Cancer Res 67:4010-4015,200723. Wang J, Sakariassen PO, Tsinkalovsky O, et

al: CD133 negative glioma cells form tumors in nuderats and give rise to CD133 positive cells. Int JCancer 122:761-768, 2008

24. Bao S, Wu Q, McLendon RE, et al: Gliomastem cells promote radioresistance by preferentialactivation of the DNA damage response. Nature444:756-760, 2006

25. Blazek ER, Foutch JL, Maki G: Daoy medul-loblastoma cells that express CD133 are radioresis-tant relative to CD133- cells, and the CD133 sectoris enlarged by hypoxia. Int J Radiat Oncol Biol Phys67:1-5, 2007

26. Piccirillo SG, Reynolds BA, Zanetti N, et al:Bone morphogenetic proteins inhibit the tumori-genic potential of human brain tumour-initiatingcells. Nature 444:761-765, 2006

27. Lee J, Son MJ, Woolard K, et al: Epigenetic-mediated dysfunction of the bone morphogeneticprotein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cell 13:69-80, 2008

28. Calvi LM, Adams GB, Weibrecht KW, et al:Osteoblastic cells regulate the haematopoietic stemcell niche. Nature 425:841-846, 2003

29. Hitoshi S, Tropepe V, Ekker M, et al: Neuralstem cell lineages are regionally specied, but notcommitted, within distinct compartments of thedeveloping brain. Development 129:233-244, 2002

30. Chojnacki A, Shimazaki T, Gregg C, et al:Glycoprotein 130 signaling regulates Notch1 expres-

sion and activation in the self-renewal of mammalianforebrain neural stem cells. J Neurosci 23:1730-1741, 2003

31. Maillard I, Adler SH, Pear WS: Notch and theimmune system. Immunity 19:781-791, 2003

32. Radtke F, Raj K: The role of Notch in tumor-igenesis: Oncogene or tumour suppressor? Nat RevCancer 3:756-767, 2003

33. Lai K, Kaspar BK, Gage FH, et al: Sonichedgehog regulates adult neural progenitor prolifer-ation in vitro and in vivo. Nat Neurosci 6:21-27, 2003

34. Machold R, Hayashi S, Rutlin M, et al: Sonichedgehog is required for progenitor cell mainte-nance in telencephalic stem cell niches. Neuron39:937-950, 2003

35. Wechsler-Reya RJ, Scott MP: Control ofneuronal precursor proliferation in the cerebellum bysonic hedgehog. Neuron 22:103-114, 1999

36. Dahmane N, Ruiz-i-Altaba A: Sonic hedgehogregulates the growth and patterning of the cerebel-lum. Development 126:3089-3100, 1999

37. Dahmane N, Sanchez P, Gitton Y, et al: TheSonic Hedgehog-Gli pathway regulates dorsal braingrowth and tumorigenesis. Development 128:5201-5212, 2001

38. Rowitch DH, S-Jacques B, et al: Sonic

hedgehog regulates proliferation and inhibits differ-entiation of CNS precursor cells. J Neurosci 19:8954-8965, 1999

39. Watkins DN, Berman DM, Burkholder SG, etal: Hedgehog signalling within airway epithelial pro-genitors and in small-cell lung cancer. Nature 422:313-317, 2003

40. Palma V, Ruiz i Altaba A: Hedgehog-GLIsignaling regulates the behavior of cells with stemcell properties in the developing neocortex. Devel-opment 131:337-345, 2004

41. Megason SG, McMahon AP: A mitogen gra-dient of dorsal midline Wnts organizes growth in theCNS. Development 129:2087-2098, 2002

42. Chenn A, Walsh CA: Regulation of cerebralcortical size by control of cell cycle exit in neural

precursors. Science 297:365-369, 200243. Chenn A, Walsh CA: Increased neuronal pro-duction, enlarged forebrains and cytoarchitecturaldistortions in beta-catenin overexpressing trans-genic mice. Cereb Cortex 13:599-606, 2003

44. Willert K, Brown JD, Danenberg E, et al: Wntproteins are lipid-modied and can act as stem cellgrowth factors. Nature 423:448-452, 2003

45. Reya T, Duncan AW, Ailles L, et al: A role forWnt signalling in self-renewal of haematopoieticstem cells. Nature 423:409-414, 2003

46. Kuhnert F, Davis CR, Wang HT, et al: Essen-tial requirement for Wnt signaling in proliferation ofadult small intestine and colon revealed by adenovi-ral expression of Dickkopf-1. Proc Natl Acad Sci U SA 101:266-271, 2004

47. Sauvageau G, Thorsteinsdottir U, Eaves CJ,et al: Overexpression of HOXB4 in hematopoieticcells causes the selective expansion of more primi-tive populations in vitro and in vivo. Genes Dev9:1753-1765, 1995

48. Antonchuk J, Sauvageau G, Humphries RK:HOXB4 overexpression mediates very rapid stemcell regeneration and competitive hematopoietic re-population. Exp Hematol 29:1125-1134, 2001

49. Groszer M, Erickson R, Scripture-Adams DD,et al: Negative regulation of neural stem/progenitorcell proliferation by the Pten tumor suppressor genein vivo. Science 294:2186-2189, 2001

50. Lessard J, Sauvageau G: Bmi-1 determinesthe proliferative capacity of normal and leukaemicstem cells. Nature 423:255-260, 2003

51. Molofsky AV, Pardal R, Iwashita T, et al:Bmi-1 dependence distinguishes neural stem cellself-renewal from progenitor proliferation. Nature425:962-967, 2003

52. Park IK, Qian D, Kiel M, et al: Bmi-1 isrequired for maintenance of adult self-renewinghaematopoietic stem cells. Nature 423:302-305,2003

53. Raaphorst FM: Self-renewal of hematopoi-etic and leukemic stem cells: A central role for thePolycomb-group gene Bmi-1. Trends Immunol 24:522-524, 2003

54. Leung C, Lingbeek M, Shakhova O, et al:Bmi1 is essential for cerebellar development and isoverexpressed in human medulloblastomas. Nature428:337-341, 2004

55. Wolter M, Reifenberger J, Sommer C, et al:Mutations in the human homologue of the Drosoph-ila segment polarity gene patched (PTCH) in spo-radic basal cell carcinomas of the skin and primitiveneuroectodermal tumors of the central nervous sys-tem. Cancer Res 57:2581-2585, 1997

56. Raffel C, Jenkins RB, Frederick L, et al:Sporadic medulloblastomas contain PTCH muta-tions. Cancer Res 57:842-845, 1997

57. Pietsch T, Waha A, Koch A, et al: Medullo-blastomas of the desmoplastic variant carry muta-tions of the human homologue of Drosophilapatched. Cancer Res 57:2085-2088, 1997

58. Reifenberger J, Wolter M, Weber RG, et al:Missense mutations in SMOH in sporadic basal cellcarcinomas of the skin and primitive neuroectoder-mal tumors of the central nervous system. CancerRes 58:1798-1803, 1998

59. Lam CW, Xie J, To KF, et al: A frequentactivated smoothened mutation in sporadic basalcell carcinomas. Oncogene 18:833-836, 1999

60. Taylor MD, Liu L, Raffel C, et al: Mutations inSUFU predispose to medulloblastoma. Nat Genet31:306-310, 2002

61. Goodrich LV, Milenkovic L, Higgins KM, et al:Altered neural cell fates and medulloblastoma inmouse patched mutants. Science 277:1109-1113,1997

62. Romer J, Curran T: Targeting medulloblas-toma: Small-molecule inhibitors of the Sonic Hedge-hog pathway as potential cancer therapeutics.Cancer Res 65:4975-4978, 2005

63. Romer JT, Kimura H, Magdaleno S, et al:Suppression of the Shh pathway using a smallmolecule inhibitor eliminates medulloblastoma inPtc1( /-)p53(-/-) mice. Cancer Cell 6:229-240, 2004

64. Beachy PA, Karhadkar SS, Berman DM: Tis-sue repair and stem cell renewal in carcinogenesis.Nature 432:324-331, 2004

65. Ruiz i Altaba A, Sanchez P, Dahmane N: Gliand hedgehog in cancer: Tumours, embryos andstem cells. Nat Rev Cancer 2:361-372, 2002

66. Kinzler KW, Bigner SH, Bigner DD, et al:Identication of an amplied, highly expressed genein a human glioma. Science 236:70-73, 1987

67. Ehtesham M, Sarangi A, Valadez JG, et al:Ligand-dependent activation of the hedgehog path-way in glioma progenitor cells. Oncogene 26:5752-5761, 2007

68. Bar EE, Chaudhry A, Lin A, et al: Cyclopamine-mediated hedgehog pathway inhibition depletesstem-like cancer cells in glioblastoma. Stem Cells25:2524-2533, 2007

69. Clement V, Sanchez P, de Tribolet N, et al:HEDGEHOG-GLI1 signaling regulates human gliomagrowth, cancer stem cell self-renewal, and tumori-genicity. Curr Biol 17:165-172, 2007







70. Gaiano N, Fishell G: The role of notch inpromoting glial and neural stem cell fates. Annu RevNeurosci 25:471-490, 2002

71. Lindsell CE, Boulter J, diSibio G, et al: Ex-pression patterns of Jagged, Delta1, Notch1,Notch2, and Notch3 genes identify ligand-receptorpairs that may function in neural development. MolCell Neurosci 8:14-27, 1996

72. Irvin DK, Zurcher SD, Nguyen T, et al: Ex-pression patterns of Notch1, Notch2, and Notch3suggest multiple functional roles for the Notch-DSLsignaling system during brain development. J CompNeurol 436:167-181, 2001

73. Stump G, Durrer A, Klein AL, et al: Notch1and its ligands Delta-like and Jagged are expressedand active in distinct cell populations in the postnatalmouse brain. Mech Dev 114:153-159, 2002

74. Johansson CB, Momma S, Clarke DL, et al:Identication of a neural stem cell in the adultmammalian central nervous system. Cell 96:25-34,1999

75. Irvin DK, Nakano I, Paucar A, et al: Patternsof Jagged1, Jagged2, Delta-like 1 and Delta-like 3expression during late embryonic and postnatalbrain development suggest multiple functional rolesin progenitors and differentiated cells. J NeurosciRes 75:330-343, 2004

76. Fan X, Mikolaenko I, Elhassan I, et al: Notch1and notch2 have opposite effects on embryonalbrain tumor growth. Cancer Res 64:7787-7793,2004

77. Hallahan AR, Pritchard JI, Hansen S, et al:The SmoA1 mouse model reveals that notch signal-ing is critical for the growth and survival of sonichedgehog-induced medulloblastomas. Cancer Res64:7794-7800, 2004

78. Gilbertson RJ, Rich JN: Making a tumour’sbed: Glioblastoma stem cells and the vascular niche.Nat Rev Cancer 7:733-736, 2007

79. Bao S, Wu Q, Sathornsumetee S, et al: Stemcell-like glioma cells promote tumor angiogenesisthrough vascular endothelial growth factor. CancerRes 66:7843-7848, 2006

80. Calabrese C, Poppleton H, Kocak M, et al: Aperivascular niche for brain tumor stem cells. CancerCell 11:69-82, 2007

81. Diamandis P, Wildenhain J, Clarke ID, et al:Chemical genetics reveals a complex functionalground state of neural stem cells. Nat Chem Biol3:268-273, 2007

82. Dahlstrand J, Collins VP, Lendahl U: Expres-sion of the class VI intermediate lament nestin inhuman central nervous system tumors. Cancer Res52:5334-5341, 1992

83. Tohyama T, Lee VM, Rorke LB, et al: Nestinexpression in embryonic human neuroepithelium

and in human neuroepithelial tumor cells. Lab Invest66:303-313, 1992

84. Burger PC, Scheithauer BW, Vogel FS: Surgi-cal Pathology of the Nervous System and its Cover-ings (ed 3). New York, NY, Churchill Livingstone, 1991

85. Globus JH, Kuhlenbeck H: The subependy-mal cell plate. The subependymal cell plate (matrix)and its relationship to brain tumors of ependymaltype. J Neuropath Exp Neurol 3:1-35, 1944

86. Smart I: The subependymal layer of themouse brain and its cell production as shown byradioautography after thymidine-H3 injection.J Comp Neurol 116:325-347, 1961

87. Altman J: Autoradiographic investigation ofcell proliferation in the brains of rats and cats. AnatRec 145:573-591, 1963

88. Lewis PD: Mitotic activity in the primatesubependymal layer and the genesis of gliomas.Nature 217:974-975, 1968

89. Doetsch F, Garcia-Verdugo JM, Alvarez-BuyllaA: Cellular composition and three-dimensional organi-zation of the subventricular germinal zone in the adultmammalian brain. J Neurosci 17:5046-5061, 1997

90. Doetsch F, Caille I, Lim DA, et al: Subven-tricular zone astrocytes are neural stem cells in theadult mammalian brain. Cell 97:703-716, 1999

91. Copeland DD, Vogel FS, Bigner DD: Theinduction of intractranial neoplasms by the inocula-tion of avian sarcoma virus in perinatal and adultrats. J Neuropathol Exp Neurol 34:340-358, 1975

92. Copeland DD, Bigner DD: The role of thesubependymal plate in avian sarcoma virus braintumor induction: Comparison of incipient tumors inneonatal and adult rats. Acta Neuropathol (Berl)38:1-6, 1977

93. Vick NA, Lin MJ, Bigner DD: The role of thesubependymal plate in glial tumorigenesis. ActaNeuropathol (Berl) 40:63-71, 1977

94. Hopewell JW, Wright EA: The importance ofimplantation site in cerebral carcinogenesis in rats.Cancer Res 29:1927-1931, 1969

95. Koestner A, Swenberg JA, Wechsler W:

Transplacental production with ethylnitrosourea ofneoplasms of the nervous system in Sprague-Dawley rats. Am J Pathol 63:37-56, 1971

96. Druckrey H, Ivankovic S, Preussmann R:Teratogenic and carcinogenic effects in the off-spring after single injection of ethylnitrosourea topregnant rats. Nature 210:1378-1379, 1966

97. Mennel HD, Kosse N, Heverhagen JT, etal: Primary and transplanted ENU induced rattumors in neurooncology. Exp Toxicol Pathol 56:25-35, 2004

98. Koestner A: Characterization of N-nitrosourea-induced tumors of the nervous system; their prospec-tive value for studies of neurocarcinogenesis and braintumor therapy. Toxicol Pathol 18:186-192, 1990

99. Pilkington GJ, Lantos PL: The developmentof experimental brain tumours a sequential light andelectron microscope study of the subependymalplate, II: Microtumours. Acta Neuropathol (Berl) 45:177-185, 1979

100. Schiffer D, Giordana MT, Pezzotta S, et al:Cerebral tumors induced by transplacental ENU:Study of the different tumoral stages, particularly ofearly proliferations. Acta Neuropathol (Berl) 41:27-31, 1978

101. Oda H, Zhang S, Tsurutani N, et al: Loss ofp53 is an early event in induction of brain tumors inmice by transplacental carcinogen exposure. CancerRes 57:646-650, 1997

102. Leonard JR, D’Sa C, Klocke BJ, et al: Neuralprecursor cell apoptosis and glial tumorigenesisfollowing transplacental ethyl-nitrosourea exposure.Oncogene 20:8281-8286, 2001

103. Wilhelmsson U, Eliasson C, Bjerkvig R, et al:Loss of GFAP expression in high-grade astrocyto-mas does not contribute to tumor development orprogression. Oncogene 22:3407-3411, 2003

104. Zhu Y, Guignard F, Zhao D, et al: Earlyinactivation of p53 tumor suppressor gene cooper-ating with NF1 loss induces malignant astrocytoma.Cancer Cell 8:119-130, 2005

105. Oliver TG, Read TA, Kessler JD, et al: Loss ofpatched and disruption of granule cell developmentin a pre-neoplastic stage of medulloblastoma. Devel-opment 132:2425-2439, 2005

106. Lee A, Kessler JD, Read TA, et al: Isolation ofneural stem cells from the postnatal cerebellum. NatNeurosci 8:723-729, 2005

107. Holland EC, Hively WP, DePinho RA, et al: Aconstitutively active epidermal growth factor recep-tor cooperates with disruption of G1 cell-cycle arrestpathways to induce glioma-like lesions in mice.Genes Dev 12:3675-3685, 1998

108. Holland EC, Celestino J, Dai C, et al: Com-bined activation of Ras and Akt in neural progenitorsinduces glioblastoma formation in mice. Nat Genet25:55-57, 2000

109. Bachoo RM, Maher EA, Ligon KL, et al:Epidermal growth factor receptor and Ink4a/Arf:Convergent mechanisms governing terminal dif-ferentiation and transformation along the neuralstem cell to astrocyte axis. Cancer Cell 1:269-277,2002

110. Passegue E, Jamieson CH, Ailles LE, et al:Normal and leukemic hematopoiesis: Are leukemiasa stem cell disorder or a reacquisition of stem cellcharacteristics? Proc Natl Acad Sci U S A 100 Suppl1:11842-11849, 2003

111. Bruggeman SWM, Hulsman D, Tanger E, etal: Bmi1 controls tumor development in an Ink4a/ Arf-independent manner in a mouse model for gli-oma. Cancer Cell 12:328-341, 2007

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