Chapter 19

Markers of Stem Cells in Gliomas

P. Dell’Albani, R. Pellitteri, E.M. Tricarichi, S. D’Antoni, A. Berretta, and M.V. Catania

Abstract Gliomas are the most common neoplasmsof the Central Nervous System (CNS) and a fre-quent cause of mental impairment and death. Despitethe improved responsiveness to primary therapy, sur-vival of glioma patients is still very low. Therapiesof malignant gliomas are often palliative because oftheir infiltrating nature and high recurrence. Duringthe last decade, the concept that gliomas may arisefrom cancer stem cells (CSCs) has emerged. CSCsshare with neural stem cells (NSCs) the capacity ofcell renewal, multipotency and the expression of spe-cific proteins, such as CD133 and nestin. This chapterdescribes similarities and differences between NSCsand CSCs, and summarizes the emerging knowledgeon the possible role of stem cell markers as markers ingliomas, particularly in their tumoral grading. In addi-tion, the importance of specific niches in maintainingpools of CSCs is considered. The involvement of signaltransduction pathways, such as Notch, PDGF/PDGFR,Hedgehog-Gli1, and Bone morphogenetic protein andtheir implications in the control of CSCs functionin gliomas are analyzed. Furthermore, certain pro-teins expressed in tumor migrating cells and possiblyinvolved in recidive are evaluated.

Keywords Gliomas · Markers of cancer stem cells ·Neural stem cells (NSCs) · Cancer stem cells (CSCs) ·Side population cells (SP cells) · Signal transductionpathways

P. Dell’Albani (�)Department of Medicine, Italian National Research Council,Institute of Neurological Sciences, Section of Catania, GaifamiP., 18, 95126, Catania, Italye-mail: p.dellalbani@isn.cnr.it

Introduction

Gliomas represent the most frequent primary tumorsof the Central Nervous System (CNS) and animportant cause of mental impairment and death.Gliomas are histologically classified according totheir hypothesized line of differentiation (e.g., astro-cytes, oligodendrocytes, or ependymal cells), and aregrouped into four clinical World Health Organization(WHO) grades according to their degree of malig-nancy. Gliomas of astrocytic origin (astrocytomas)are classified into pilocytic astrocytomas (Grade I),astrocytoma (Grade II), anaplastic astrocytoma (GradeIII) and glioblastoma multiforme (GBM) (GradeIV). Tumors arising from oligodendrocytes includeoligodendrogliomas (Grade II) and oligoastrocytomas(Grade III). Grade I tumors are biologically “benign”,while grade II tumors are low-grade malignancies withlong clinical courses. Grade III and IV are malignantgliomas and are lethal within a few years and 9–12months, respectively. Furthermore, more than 50% ofgrade II gliomas transform into grade III and IV tumorswithin 5–10 years of diagnosis. Different therapeu-tic approaches are needed in each case. Even though,with the exception of grade I tumors, which are sur-gically curable if resectable at the time of diagnosis,all other grade gliomas are not curable with surgerybecause of their tendency to affect the cerebral hemi-spheres in a diffuse manner. Malignant gliomas arehighly recurrent tumors even after surgery, chemother-apy, radiation, and immunotherapy. Ionizing radiation(IR) represents the most effective therapy for glioblas-toma but radiotherapy remains only palliative becauseof radioresistance. The treatment strategies for gliomashave not changed appreciably for many years and mostare based on a limited knowledge of the biology of thedisease.

175M.A. Hayat (ed.), Tumors of the Central Nervous System, Volume 1,DOI 10.1007/978-94-007-0344-5_19, © Springer Science+Business Media B.V. 2011

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In the past most of the research on human braintumors has been directed on the molecular and cellu-lar analysis of the bulk tumor mass until during thelast few years when it became clear that the tumorkey cell is able to self-renew, proliferate, differen-tiate, and maintain the tumor mass. Essentially, inbrain tumor two main cellular populations could beobserved: the bulk of malignant cells, which are “celldeath committed” cells, and a rare fraction of selfrenewing, multipotent and tumor initiating cells calledcancer stem cells (CSCs). In 2003 Singh et al. pub-lished an article showing the identification of CSCs inhuman brain tumors, followed by another one relativeto the identification of human brain tumor initiatingcells (Singh et al., 2004). During the last 10 yearsnumerous publications on CSCs appeared and thistopic became the most fascinating subject of researchin the tumor field. The majority of studies of brainCSCs have attempted to characterize and define thebehaviour and the molecular mechanisms adopted bythese cells to survive, proliferate, and repair any dam-age they can suffer escaping from apoptotic cell death.Brain tumor stem cells (BTSCs) resemble neural stemcells (NSCs) in terms of phenotype, signalling, andbehaviour in vitro. Normal and CSCs share the expres-sion of several markers, the ability for self-renewaland differentiation, and signalling pathways involvedin the regulation of cellular survival and proliferation.Recently, a small population of stem cells, termed SidePopulation (SP), has been identified in several normaltissues and tumors. To develop anti-cancer and morespecifically anti-CSCs therapies it will be importantto analyze each piece of the puzzle “tumor”. For thispurpose the principal goal is the definition of specificprotein markers that identify uniquely the CSCs versusNSCs to create a tumor targeted therapy. This chap-ter will discuss neural and cancer stem cell similaritiesand differences, the most accredited mechanisms ofcancerogenesis and the emerging knowledge on mark-ers of stem cells eligible as markers in gliomas, whichin some instances could be correlated to the tumoralgrading. The known strategies or methods to identifyCSCs will be briefly mentioned. The importance ofspecific niches in maintaining pools of CSCs will alsobe discussed. The role of signalling pathways suchas Notch, PDGF/PDGFR, Hedgehog-Gli1, and Bonemorphogenetic protein and their implications in thecontrol of CD133+ CSCs function in gliomas, will bediscussed. Furthermore, certain proteins expressed in

tumor migrating cells and possibly involved in recidivewill also be evaluated.

Neural and Cancer Stem Cells

Neural stem cells (NSCs) are the CNS tissue-specificstem cells. NSCs have self-renewal, proliferativecapacities and multi-potentiality. In fact, NSCs areable to maintain the number of quiescent stem cellsin a given brain region or to increase it in particu-lar situations, and they are able to generate neurons,astrocytes, and oligodendrocytes (Gritti et al., 2002).During an early embryonic phase of development,NSCs divide symmetrically maintaining stemness andexpanding the cellular population. In a second neu-rogenic phase, NSCs undergo asymmetric division,giving rise to new stem cells and to proliferating pre-cursors belonging to the neuronal lineage. After thisphase a glial progeny will develop with the progressivedecline of stem cells, even though a small number ofstem cells persists in specific regions of the adult brain,primarily in the subventricular zone (SVZ) of the fore-brain lateral ventricles and in the dentate gyrus of thehippocampus.

In the past, tumor cells in the brain were hypoth-esized to derive mostly from the transformation ofmature neural cells such as astrocytes, oligodendro-cytes or neuronal precursors. Recently, this theoryhas changed because the concept of CSCs has beenextended to brain tumors. To date, two models havebeen proposed to explain cellular cancer proliferation.In the classic stocastic model all the cells in a tumorhave similar tumorigenic potential that is activatedasynchronously and at a low frequency. In contrast,the hierarchical model proposes that only a rare subsetof cells within the tumor have significant prolifera-tion capacity and the ability to generate new tumorsresembling the primary tumor, while the other tumorcells are terminally differentiated and committed tocell death. The hierarchical hypothesis correlates withthe cancer-stem-cell theory now supported by accu-mulating experimental data showing that cancers, likenormal organs, may be maintained by a hierarchicalorganization that includes stem cells, transient ampli-fying cells (precursor cells), and differentiated cells(Reya et al., 2001). Malignant gliomas contain bothproliferating and differentiating cells, which express

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either neuronal or glial markers, and can be gener-ated from both NSCs and glial lineage cells, such asoligodendrocyte precursor cells or astrocytes, whichcan behave as NSCs under appropriate conditions.These observations raise the possibility that they maycontain multipotent neural-stem-cell (NSC)-like cells(Ignatova et al., 2002). Stem cells can be identified bythe expression of specific markers, although they donot appear to be organ-specific. Normal stem cells andCSCs share the expression of several markers, the abil-ity for self-renewal and differentiation, and signallingpathways involved in the regulation of cellular survivaland proliferation. Furthermore, both show telomeraseactivity, resistance to apoptosis and increased mem-brane transporter activity.

Side Population Cells

Recently, a small population of stem cells, termed SidePopulation (SP), has been identified in several nor-mal tissues and tumors on the basis of the abilityto extrude fluorescent dyes, by the “flow cytometry-based side population technique” (Sing et al., 2003,2004; Yuan et al., 2004). SP-stem cells have severalfundamental properties: (1) they are generally veryrare (~0.01–5% of total number of cells from tumors),(2) they rarely divide, even though they have an ele-vated proliferative potential, (3) they can self-renew.SP cells are capable of sustained expansion ex vivoand are able to generate, through asymmetric divi-sion, both SP and non-SP progeny (Hirschmann-Jaxet al., 2004). SP cancer stem cells obtained frombrain tumors form neurospheres, which have the capac-ity for self-renewal and are able to differentiate intophenotypically diverse populations including neuronal,astrocytic, and oligodendroglial cells when dissociatedin single cell suspension. Recently, the presence ofstem cell – enriched SP in long-term cultured humanand rat glioma cell lines has been shown. SP stemcells demonstrate elevated chemoresistance. A recentreport suggests that SP cells are heterogeneous withrespect to the expression of drug transporter proteinsbelonging to adenosine triphosphate-binding cassette(ABC) superfamily, one of which is ABCG2 that isexpressed in proliferating cells preferentially. SP cellsalso express high levels of the ABCA3 transporter geneand have greater capacity to expel cytotoxic drugs,

such as mitoxantrone, resulting in better survival thannon SP-cells. Thus, SP phenotype defines a classof CSCs with high resistance to chemotherapeuticagents that should be targeted during the treatmentof malignant disease. These cells can undergo toasymmetrical division giving rise to ABCG2 positive(+) and negative (–) cancer cells. ABCG2+ cells havebeen identified as tumor progenitor cells, while amongthe ABCG2- cells both primitive cancer stem cells,which show high self-renewal, proliferative potentialand high expression levels of “stemness” genes suchas Notch-1, and differentiated tumor cells, which arepartially or fully differentiated cells that constitute thebulk of tumor mass have been endowed.

Strategies for Cancer Stem CellsIdentification

Since the identification and isolation of CSCs, the needto obtain a good number of these cells to study themboth at cellular and molecular levels has becomingevident. Thus, it was important to find out specificmethodologies to enrich and amplify CSCs from pri-mary tumor tissue or even from tumor cell lines.

Four methods are available for the identification ofCSC:

1. Isolation of CSCs by Fluorescence Activated CellSorting (FACS) and Magnetic Cell Sorting (MACS)which use the presence of specific surface markers.This method was used by Bonnet and Dick (1997)to isolate, from a patient affected by acute myeloidleukemia (AML), cells that were able to startleukaemia when transplanted in immuno-deficientmice. Now this method is adopted to isolate CSCsfrom various tumors such as breast cancer, malig-nant glioma, lung cancer, and others.

2. Isolation of “Side Population” (SP cells) enrichingCSCs.This technique is based on the capability of somecells to extrude the fluorescent dye Hoechst 33342,which depends on the expression of an ATP –binding cassette (ABC) transporter on the surfaceof these cells. This technique is one of the mostused method to select and identify CSCs fromhuman cancers (such as glioma, neuroblastoma,

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lung cancer). The SP method has a few disadvan-tages such as the high sensitivity to staining con-ditions (Hoechst concentration, cell concentration,staining time and staining temperature); further-more, when the staining process is complete, thecells should be maintained at 4◦C in order to inhibitfurther dye efflux. Another disadvantage is dueto harvesting relatively small number of SP cells(0.01–0.05%). Moreover, Hoechst 33342 is cyto-toxic because of its association with DNA, whichdetermines the inhibition of DNA topoisomeraseI causing DNA strand breaks; some researchersthink that differences in clonogenicity and tumoro-genicity between SP and non-SP cancer cells mightdepend on the Hoechst 33342 staining itself.

3. Isolation of CSCs by using a serum-free mediumthat allows the formation of cellular aggregatescalled “tumorsferes”In 1992 Reynolds and Weiss published a methodthat allows the in vitro enrichment of NSCs inbrain tissues. In this method, tissue is disrupted intosingle cells and cultured in the presence of epi-dermal growth factor (EGF) and basic fibroblastgrowth factor (bFGF) until the non-adherent cellsform three-dimensional spheres that are enrichedfor NSCs. The same method has also been used forthe isolation of CSCs from brain tumors and othermalignancies.

4. Isolation of CSCs by using their functional charac-teristics.Yu and Bian (2009) published an intriguing newmethod to isolate CSCs, which does not use anysorting based on stem cell markers, nor the Hoechst33342 that could be toxic during SP-populationselection. Four steps can be recognized, the firstis based on the property that CSCs have to firmlyadhere to extracellular matrix (ECM) while the bulkof committed tumor cells can be removed roughly;the second step is based on the higher chemotac-tic activity of CSCs versus tumor differentiatedcells, the third step is based on the CSCs prop-erty of destructing and remodelling the ECM bywhich these cells can move earlier than commit-ted cells; and the last step is to verify all the threeimportant characteristics of CSCs: self-renewal,multipotency, and tumorigenicity. This method hasrecently appeared in the literature and needs to befurther confirmed by other investigations. However,in addition to these methods, new protocols for the

isolation and enrichment of CSCs based on theirmolecular functional characteristics are needed tostudy and understand their ability to escape phar-macological or ionizing radiation therapies, theirinfiltrating nature, and the possible role they havein angiogenesis and tumoral vascularisation.

Stem Cell Markers in Brain Tumors

As mentioned above in brain tumors two main cellularpopulations could be observed: (1) the bulk of malig-nant cells, which are “cell death committed” cells and(2) a rare fraction of self renewing multipotent andtumor initiating cells, the CSCs. The identification ofprotein markers that identify uniquely the CSCs versusNSCs is of paramount importance to create a tumor tar-geted therapy. Moreover, since from all published datathe concept is emerging that a significant heterogene-ity is evident within specific subtypes of solid tumorssuch as GBMs, it will be rational to develop specificmolecular targeted drugs which could be personalizedfor individual tumors.

During this decade new important progress has beenmade in the recognition of possible markers of CSCs.Most important markers are: CD133, Nestin, Musashi,Sox-2 and CXCR4 as indicated in the summary pre-sented in Fig. 19.1.

CD133

CD133 (prominin-1) was the first identified memberof the prominin family of pentaspan membrane gly-coproteins. It was identified in both human and miceand was originally classified as a marker of primitivehaematopoietic stem cells and NSCs. CD133 is con-sidered to be a marker for embryonic NSCs. In theearly postnatal stage, CD133 is expressed in interme-diate radial glial/ependymal cell types, while in theadult CNS it is present in a subset of NSCs (ependymalcells) and is down-regulated in normal differentiatedcells (Kania et al., 2005). In spite of growing datapresent in literature, CD133 function is still not com-pletely understood. Its restricted localization in mem-brane protrusions of epithelial and other cell types,in association with membrane cholesterol, suggests aninvolvement in the dynamic organization of membraneprotrusions and in mechanisms such as migration

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Fig. 19.1 Scheme representing the differential expression ofCSC markers in glioma grading. (a) Diagram showing gliomainitiation and growth with a correlation among cell proliferationand tumor grading. (b) Summary of CSC markers expressed inthe different gliomas grade related to prominent cellular func-tions altered in gliomas. The different proteins are grouped in

cluster, which are related to specific cellular functions, but pro-teins position in a cluster is not stringent. (–) the negative sign isrelated to the absence or very low level expression of the protein;(+/–) is related to low levels of the protein; (+) the positive signis related to the presence of the protein, while ↑ or ↑↑ is relatedto high or very high expression levels of the proteins

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and interaction of stem cells with neighbouring cellsand/or ECM.

CD133 also represents a marker of tumor – initiat-ing cells. CD133 expression was investigated both atprotein and mRNA levels in non-neoplastic brain tis-sue. No CD133 protein expression was observed innormal brain, while RT-PCR analysis displayed posi-tivity in tumor samples. From these observations it waspossible to hypothesize that there could be a poten-tial post-translational down regulation of CD133 pro-tein accompanying cell differentiation, while CD133mRNA is constitutively transcribed even at very lowlevels in individual cells. CD133 expression patternhas been correlated with CSCs obtained from glioblas-tomas, but there are also experimental evidences ofCD133+ cells in low grade tumors. Moreover, thenumber of CD133 cells quantitatively correlates withtumor grade indicating an involvement of these cellsin glioma progression. CD133 cells have also the ten-dency to accumulate in dense clusters most of whichhave been found in highly vascularised regions of highgrade gliomas; in parallel, NSCs have the tendency toaccumulate in areas called “niches” within the SVZduring early cortical development. Because vascular-isation is important to support tumor cell migration,it could be speculated that CD133 cells are present instrategic positions for tumor cell invasion, with singleCD133 cells sporadically present within glioma tissueas the reminiscence of the invasive pathways.

In 2006, Liu et al. isolated two populations ofCD133 positive (+) and negative (–) cells from pri-mary culture of adult glioblastoma by FACS analysis.CD133+ cells exhibited stem cell properties in vitroand were able to initiate and drive tumor progres-sion in vivo, strongly suggesting that CD133+ cellsmight be the brain tumor initiating cells. This notionhas been challenged by studies demonstrating thatglioblastoma CD133– cells have also properties ofstem cells, even though, with lower proliferation index,and are tumorigenic when engrafted intra-cerebrallyinto nude mice (Beier et al., 2007). These data wereconfirmed by Wang et al. (2008), who observed thatCD133– cells implanted in brain of nude mice gaverise to brain tumor demonstrating that CD133+ cellswere not important for brain tumor initiation, but fortumor progression associated with increased angio-genesis and shorter survival. Thus, CD133 expressionseems to be related to angiogenesis. CD133+ cells arestrongly resistant to drugs and toxins because they

express several ABC transporters and have active DNArepair capacity and resistance to apoptosis. Bao et al.(2006) demonstrated that CD133+ tumor cells iso-lated from both human glioma xenografts and primarypatient glioblastoma specimens preferentially activatethe DNA damage checkpoint in response to radia-tion, and repair radiation-induced DNA damage moreeffectively than CD133– tumor cells. Furthermore,these authors could reverse CD133+ glioma stem cellsradioresistance by using a specific inhibitor of theChk1 and Chk2 checkpoint kinases, and consequentlyhypothesized that CD133+ tumor cells represent thecellular population that confers glioma radioresistanceand may be the source of tumor recurrence afterradiation.

Other characteristics of CD133+ cells are derivedfrom a gene expression analysis that revealed a higherexpression of drug transporters BCRP1/ABCG2 andMGMT versus CD133– (Liu et al., 2006), but alsohigher levels of apoptosis suppressors such as Bcl-2, FLIP, BCL-XL and several inhibitor of apoptosisproteins (IAPs), such as (XIAP, cIAP1, cIAP2, NAIPand survivin) (Schimmer, 2004). IAPs bind and inhibitcaspases 3, 7, and 9, preventing apoptosis, modulat-ing cell division, cell cycle progression, and signaltransduction pathways. Interestingly, CD133 expres-sion is different in primary GBM and in recurrentGBM being significantly higher in recurrent GBMtissue obtained from patients as compared to theirrespective newly diagnosed tumors. In addition, ithas been observed that CD133+ cells isolated fromglioblastoma biopsies highly express the cell surfacechemokine receptor CXCR4 (see below). From thesedata it appears that CD133+ CSCs may have impor-tance not only in tumoral recurrence after chemo-and radio-therapies, but also in brain invasion. Whilethis chapter was in preparation Lottaz et al. (2010)published an article that gives new insights into thepossible sub-classification of GBM. These authorswere able to distinguish two sub-groups of GBM byusing a newly derived 24-gene signature: the type ICSC lines that display “proneural” signature genesand resemble fetal neural stem cell (fNSC) lines andtype II CSC lines that show “mesenchymal” transcrip-tional profiles resembling adult NSC (aNSC) lines.The phenotype analysis of type I CSC lines evidencedthat they express CD133 and grow as neurospheres,while type II CSC cells lack CD133 and display a(semi-) adherent growth. From these data and from an

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accurate molecular analysis of seventeen GBM CSClines, authors hypothesize that GBM may derive fromcells that have retained or acquired properties of eitherfNSC or aNSC, but that have lost the differentiationpotential (Lottaz et al., 2010).

NESTIN

Nestin is a protein belonging to class VI of intermedi-ate filaments (IFs), that is produced in stem/progenitorcells in the mammalian CNS during development(Zimmerman et al., 1994) and is a marker of pro-liferating and migrating cells. IFs are highly diverseintra-cytoplasmic proteins which show cell type speci-ficity of expression. IFs are cytoskeleton constituentsinvolved in various cellular functions such as the con-trol of cell morphology, adhesion, proliferation, andcellular migration. Changes in the expression of IF pro-teins regulate remodelling of cell cytoskeleton duringdevelopment. This is particularly evident in the CNSwhere IFs have sequential expression. Embryos in pre-implantation phase express cytokeratins (classes I andII). During embryogenesis, a developmental periodcharacterized by tremendous cell migration, nestin(class VI) is coexpressed with vimentin (class III) inneuroephithelial and radial glial cells of the ventricularand the pial surfaces of the neural tube. When differen-tiation starts, cells leaving the cell cycle down-regulatenestin and subsequently up-regulate alternative IFssuch as neurofilaments (NFs) (class IV) in committedneurons and glial fibrillary acid protein (GFAP) (classIII) in glial precursors. In adult CNS, nestin expressionpersists in small populations of stem/progenitor cellsof the SVZ and to a lesser extent in the choroid plexuseven though several morphological types of nestin-positive cells (neuron-like, astrocyte-like, cells withsmaller cell bodies and fewer processes) are detectablein different areas of forebrain of normal human adultbrain. Moreover, nestin may be reexpressed in theadult organism under certain pathological conditionssuch as brain injury, ischemia, inflammation, andneoplastic transformation (Holmin et al., 1997). IFsubtypes have been linked to enhanced motility andinvasion in a number of different cancer subtypes.Nestin has been detected in brain tumors both oflow grade, pilocytic astrocytomas, and of high grade,malignant gliomas including glioblastoma multiforme.Dahlstrand et al. (1992) showed high nestin expression

in high malignant tumors, such as glioblastoma multi-forme, when compared to less anaplastic glial tumors.Nestin expression in tumor cells may be related to theirdedifferentiated status, enhanced cell motility, inva-sive potential, and increased malignancy. In addition,nestin has also been identified in the cell nucleus oftumor cell lines obtained from glioblastoma patients,suggesting that nuclear nestin may have a role in chro-matin organization or may act as specific regulatorof gene expression (Veselska et al., 2006). Furtherstudies should be addressed regarding the relationshipbetween nestin re-expression and tumor malignancy.

MUSASHI

Musashi is a conserved family of RNA-binding pro-teins. Its expression has been evolutionary conservedfrom nematodes to vertebrates. In mammals theMUSASHI family controls neural stem cell home-ostasis, differentiation, and tumorigenesis by repress-ing translation of particular mRNAs. Musashi-1 isa member of the MUSASHI family in vertebrates,which is preferentially expressed in neural progeni-tor cells, including NSCs of the periventricular areaand it is down-regulated during neurogenesis. Lowlevels of musashi are observed in periventricular neu-ral stem/progenitor cells (ependymal cells and SVZastrocytes) in the adult brain. Musashi-2 is a sec-ond gene belonging to the MUSASHI family, whichwas discovered by Sakakibara et al. (2001) throughhomology analysis. Musashi-1 and -2 cooperate toactivate Notch signalling through repression of trans-lation of the mRNA of the intracellular Notch signalrepressor m-Numb, and to maintain the self-renewingability of NSCs (Fig. 19.2). Elevated expression ofmusashi-1 (high mRNA and protein expression levels)has been reported in astrocytomas of different WHOgrades and more specifically in grade IV, while in othertumors including melanomas and prostate cancer lowerexpression has been observed.

SOX-2

SOX (SRY-like HMG box) genes represent a familyof transcriptional cofactors implicated in the controlof diverse developmental processes. To date, more

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Fig. 19.2 Notch signalling is a ligand-receptor initiated path-way. The interaction between a ligand (DLL-1, -3,-4 andJAG-1, -2) (1) and Notch receptors triggers two successivecleavages (2), the first mediated by TACE (tumor necrosisfactor-α-converting enzyme) and the second by γ-secretase,originating an intra-cytoplasmic fragment of Notch (NICD).NICD translocates to the nucleus (3) where it binds to the

transcription factor CBF1/Su(H)/LAG1 (CSL). This interac-tion results in the displacement of the co-repressor (CoR)(4) and recruitment of the co-activator (CoA) (5) leadingto transcriptional activation of target genes (6). Maintenanceof activated signalling depends on positive or negative reg-ulation through the action of Musashi (a) and Numb (b)respectively

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than 20 SOX genes have been described in mam-mals and are divided into six distinct groups accordingto their HMG-box homology (Schepers et al., 2002).SOX genes exhibit highly dynamic expression pat-terns during embryogenesis. In early stages of mouseembryonic development SOX1-3 are highly expressedwithin the CNS and are down-regulated when neuralcells are in the cell cycle and start to differentiate.SOX-2 has a role in growth and self-renewal of sev-eral stem cell types, both embryonic and adult. Infact, its activity is integrated in a complex network oftranscription factors that influence both the pluripo-tency and differentiation of embryonic stem cell. Innormal mouse brain SOX-2 is expressed in NSCs,early precursors and a few mature neurones. Recently,different research groups have shown the selectiveover-expression of SOX-2 in malignant gliomas bothat mRNA and protein levels, whereas SOX-2 was notdetectable in normal cortex (Gangemi et al., 2009).

CXCR4

Cancer cells obtained from glioblastoma biopsieshighly express the cell surface chemokine recep-tor CXCR4. This chemokine in NSCs may have asignificant role in directing NSC migration duringCNS development. Interestingly, an over-expressionof CXCR4 has been related to an highly inva-sive potential of gliomas (Ehtesham et al., 2006).Recent studies have analyzed the involvement of theCXCR4/CXCL12 signaling pathway to the prolifera-tion, survival, and motility of a human GBM cell line(do Carmo et al., 2010). These authors demonstratedthat CXCR4/CXCL12 axis determined an increase incell proliferation and motility. Moreover, the blockageof CXCR4 induced a significant increase of apopto-sis. These observations indicate that CXCR4/CXCL12signalling pathway may contribute to the growth andinvasive characteristic of GBM development. Thesedata add new possible targets for improved gliomatherapies.

Prognostic Roles of Cancer Stem CellMarkers in Glioma Grading

Numerous studies attempt to shed light on possibleprognostic markers of CSCs in gliomas. Nestin could

be considered as a potential prognostic marker forGBM patients as highlighted by numerous experimen-tal evidence. Immunohistochemical detection of nestinprotein expression could be used as indicator of ded-ifferentiation and progression in astrocytomas. In thisregard, it has been observed that low-grade astrocy-tomas contain low levels of nestin, while most high-grade gliomas express high levels of nestin with nestin-positive cells more abundant at the transition zone ofthe tumor. In addition to nestin, musashi expressionwas also investigated in low and high grade gliomas.Musashi-1 positive cells have been observed in a patch-like pattern in close proximity of tumor vessels; addi-tionally, some musashi positive cells could be widelydistributed within tumor parenchyma occasionally sur-rounding necrotic areas. Even though the expressionpattern of musashi correlate with malignancy beinghigh in glioblastoma, it is weaker than nestin in tumorcells of high grade gliomas, suggesting that these twoproteins follow a differential pattern of expression. Infact, musashi expression, that is a marker of asym-metric cellular division may be stopped early, whilenestin may be stopped late in the brain tumor stemcell dedifferentiation versus glioblastoma progression.Moreover, high level of nestin, but not musashi inbrain cancer cells, indicates significantly shorter sur-vival of glioma patients (Strojnik et al., 2007). Similarto nestin also CD133+ cells show a quantitative cor-relation with gliomas grade (low and high grade) andtissue distribution. In fact, CD133+ cells are presentin cluster in surrounding tumor parenchyma and arefrequently associated with tumor vessels. Nestin andCD133 expression may be considered an importantfeature of human gliomas. Low expression of these twomarkers significantly correlate with long survival ofglioma patients. In addition to Nestin, CD133, musashiand SOX-2 have been shown to increase with increas-ing WHO grade. In conclusion, nestin and CD133 andother up-regulated markers may be potential indicatorsof biological aggressiveness of gliomas. Moreover, thepresence of Ki67, a marker of cellular proliferation,together with CD133 could be considered independentprognostic factors of tumor recurrence and short sur-vival. The ABCG2 drug transporter may also be usedas a predictor for the outcome of glioma treatment.Enhanced expression of ABCG2 has been detected inhuman glioma tissue while low levels were found innormal brain. Furthermore, high-grade gliomas havethe tendency to express higher levels of ABCG2 thanlow grade ones.

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Cancer Stem Cell Markers Associated toGlioma Chemo- and Radio-Resistance

Despite the effort of biomedical research in the set-up of glioma therapies, the current treatments remaininsufficient. From data present in literature it is knownthat the following two mechanisms are the mainmechanisms by which cancer cells are able to sur-vive and proliferate infiltrating surrounding tissue. Thefirst mechanism is dependent on a multidrug resistancethat is the result of over-expression of membrane pro-teins that pump-out from the tumoral cell anticanceragents decreasing the intracellular drug concentration;the second mechanism is the resistance to apoptosis.

Multidrug resistance (MDR) refers to the ability thatcancer cells have to resist to a broad spectrum of struc-turally unrelated cytotoxic drugs that have differentmodes of action. MDR may be either intrinsic resis-tance (it could be established since the beginning ofthe chemotherapy) or acquired resistance (it could beacquired after the treatment period and could be deter-mined by genetic mutations). MDR cancer phenotypecould depend on the expression of drug transporterssuch as the over-expression of adenosine triphosphate-binding cassette (ABC) superfamily. This family con-sists of 48 human ABC genes that give rise to proteinsknown as ATP-dependent efflux pumps, which are ableto translocate a range of substrate molecules, such aslipids and xenobiotic compounds across the cellularmembranes using energy of ATP hydrolysis or formingmembrane channels. ABC genes have been organizedinto seven subfamilies ranging from ABCA-ABCG,depending on their amino acid sequence and domainstructure. ABCG2, also known as breast cancer resis-tance protein (BCRP), belongs to the ABC superfam-ily, and is a drug transporter that has been found to playan important role in conferring the MDR phenotype.ABCG2 is able to efflux > 20 different cytotoxic drugssuch as mitoxantrone, daunorubicin, doxorubicin andtopotecan (Doyle and Ross, 2003). ABCG2 has beendetected in microvessel endothelial human brain andglioma cells, implicating an important role in both nor-mal brain functions and glioma treatment. SP stemcells demonstrate elevated chemoresistance. MalignantSP cells export readily many cytotoxic drugs becauseof the high expression levels of drug-transporters suchas MDR-1 (i.e., ABCB1 or P-glycoprotein), MRP-1(ABCC1), ABCA2, ABCA3, and ABCG2. Moreover,

the expression of the MRP-1 protein specifically hasbeen verified in multiple human brain tumor typesincluding astrocytomas, glioblastomas, meningiomas,neuroblastomas, and oligodendrogliomas. In additionto efflux pumps other defence mechanisms could beused by CSCs. Alkylating agents such as carmustine(BCNU) and temozolomide (TMZ) are drugs used foradjuvant glioma therapy. These and similar drugs acton cancer cells alkylating their DNA on specific basesand damaging surviving mechanisms.

CSCs appear to posses enhanced DNA repaircapacity compared to other cells within the tumors.This capacity is dependent on the activity of theO6-methylguanine-DNA-methyltransferase (MGMT)gene. MGMT gene encodes for an important DNArepair protein, which acts by removing alkylating prod-ucts from the O6 position on guanine. The result-ing alkylated MGMT, also called “suicide enzyme”,is then marked for degradation by ubiquitinization.Physiological functioning of MGMT is importantfor maintaining cell integrity. Moreover, epigeneticsilencing of the MGMT gene by methylation of theCpG islands of its promoter region has been demon-strated to determine loss of gene transcription andprotein expression. Loss of MGMT expression willresult in decreased DNA repair and retention of alky-lated groups. The analysis of the MGMT expressionlevel with immunohistochemistry, and the methyla-tion status of its promoter could be used as valu-able predictors for clinical response to alkylatingagents. In fact, glioma patients lacking tumor MGMTexpression have benefited from O6-alkylating agent-based chemotherapy, meaning a prolonged survivalcompared to patients with MGMT-proficient tumors.Recently, Rivera et al. (2010) demonstrated that inGBM patients who received radiotherapy alone fol-lowing resection, the methylation of MGMT promotercorrelated with an improved response to radiother-apy, while unmethylated tumors demonstrated dou-ble progression during radiation treatment. Even themedian time of interval between resection and tumorprogression was approximately half that of methy-lated tumors. From these data authors suggest thatMGMT promoter methylation could be a predic-tive biomarker of radiation response. Furthermore,because this biomarker has also been demonstratedto be predictive of response to alkylating agents, itcould represent a general favourable prognostic factorin GBM.

19 Markers of Stem Cells in Gliomas 185

Resistance to apoptosis is the main mechanismthat is not mediated by efflux pumps, and it isknown to depend on the activation of cellular anti-apoptotic defence. Recent data show that XIAP (X-linked inhibitor of apoptosis protein) is the mostpotent member of IAP gene family acting on cas-pase inhibition and apoptosis suppression. Moreover,XIAP is over-expressed in chemoresistant cancer cells(Zhang et al., 2008). It appears that chemo-andradio-resistance depends on diverse mechanisms thatcould act in concert or have a prominent role depend-ing on the insult from which cancer cells would defend.New therapeutic approaches should target all sensitiveproteins playing key roles in mechanisms of insults,repair and maintenance of CSCs survival and pro-liferation in order to obtain not only the reductionof tumor mass but the arrest of tumor growth andsurvival.

Brain Tumor Cell Niches

Recent data have provided evidence that stem cellsof various tissues are present in protective niches.Stem cell niches are specialized microenvironments fornormal stem cells. In adult mammalian brain, neuroge-nesis persists in two germinal areas, the subventricularzone (SVZ) of the forebrain lateral ventricles and thedentate gyrus of the hippocampus, where continuouspostnatal neuronal production seems to be supportedby NSCs. The central structural element of the neu-ral stem cell niche is provided by capillaries withinthese areas. Stem cells reside in proximity of endothe-lial or other vascular cells facilitating communica-tions among these cell types. Niches are not a simplyrepositories but also complex dynamic “places” thatactively regulate the stem cell functions. Componentsand organization of the stem cell niches are differ-ent in various organisms and among tissues of thesame organism and might include heterogeneous celltypes, matrix glicoproteins, proteins secreted locallyor distally that drive specific signals and specific-localmetabolic conditions. Important functions of stem cellniches are the regulation of stem cell self-renewal andcell fate. Constitutive cells of niches provide a cellcontact and secrete factors that essentially maintainstem cells in a quiescent status. The exact mix ofsecreted factors, that regulate NSCs, have not yet been

determined, even though it is possible to indicate fewpossible factors or proteins such as the brain-derivedneurotrophic factor (BDNF), the vascular endothelialgrowth factor C (VEGF-C) (Le Bras et al., 2006),the bone morphogenic proteins (BMPs) (Piccirillo andVescovi, 2007) and the Pigment Epithelium-DerivedFactor (PEDF) (Ramirez-Castillejo et al., 2006). Itis important to note that in niches a bidirectionalcommunication does exist among cells that consti-tute the niche and the stem cells. There is in factin vitro evidence, showing the effects of NSCs onbrain-derived endothelial cells which survive and formvascular tubes due to the secretion of VEGF and BDNFfrom NSCs. Thus, it is possible to hypothesize thatdiseased stem cell niches might contribute to tumorige-nesis. In fact, recently published data have shown aber-rant vascular niches in glioblastomas. A disorganizedand excessive blood vessel formation is characteris-tic of glioblastoma. This feature could be explainedby the fact that CSCs residing in diseased stem cellniches need a huge trophic support due to the rapidgrowth of tumoral cells. This observation is supportedby reports that correlate the number of capillariesof glioblastoma with patient prognosis. Furthermore,there is evidence that CD133+ cells obtained fromglioblastomas are able to form highly vascularizedtumors in the brain of immuno-compromised mice.Like NSCs, glioblastoma stem cells appear to possespotent angiogenic activity and recruit vessels dur-ing tumorigenesis. Using multi-photon laser scanningmicroscopy it has been shown that CD133+/nestin+cells within sections of human glioblastomas, oligo-dendrogliomas, medulloblastomas, and ependymomasare situated in close proximity of tumor capillaries.Moreover, a specific role has been assigned to endothe-lial cells present in neural niches; in fact, endothelial-derived factors have been shown to accelerate theinitiation and growth of brain tumors. Despite theconspicuous number of studies in this field, numer-ous questions remain unanswered such as: Does thediseased stem cell niche drives the tumor develop-ment? Or do CSCs recruit other CSCs? How do cancerstem cells and their niches subvert the tight regula-tory conditions that feature normal stem cell niches?Answering these questions will be of relevant interest,because with the disclosure of the pathologic strategiesadopted by cancer stem cell niches, new therapeuticapproaches could be adopted to stop aberrant tumoralgrowth.

186 P. Dell’Albani et al.

Cancer Stem Cells and Invasiveness

In addition to the evaluation of niches, another impor-tant feature of tumor growth is the invasiveness.Despite the progress in early diagnosis of gliomas,surgery and radiation protocols, only 15% of gliomaspatients who underwent radical surgery are stillalive 2 years after diagnosis. This dismal prognosisis essentially due to the highly invasive nature ofglioblastoma cells. Tumor cells that migrate into thesurrounding brain parenchyma escape surgical resec-tion and are the putative source of recurrent tumors.The invasion is triggered by several cell surfacereceptors including receptor tyrosine kinases (RTKs),G protein-coupled receptors (GPCRs), TGF- recep-tors, integrins, immunoglobulins, tumor necrosis factor(TNF) family, cytokine receptors, and protein tyrosinephosphatase receptors.

Local cell invasion involves the detachment fromthe original site, the attachment to extracellular matrix(ECM), the degradation of the ECM, and migra-tion. In each step numerous proteins are involved.In detachment the down regulation of cell adhesionmolecules is necessary, while the attachment to theextracellular matrix (ECM) is sustained by integrins.Furthermore, ECM should be remodelled through theaction of several classes of proteases including matrixmetalloproteinases (MMPs, e.g., MMP-2, MMP-9 andmembrane-type MMP, MT-MMP), ADAMs (a disin-tegrin and metalloproteinase), plasmin, and cathep-sins. Finally, last step of invasion is determined bycytoskeletal rearrangements and formation of lamel-lipodia and filopodia (Teodorczyk and Martin-Villaba,2010). CSCs isolated from gliomas over-express arepertoire of molecules involved in infiltrating pro-cess such as CXC chemochine receptor 4 (CXCR4),CD44, vascular cell adhesion molecule-1 (VCAM-1)and integrins (ανβ5, ανβ3, α5β51). Furthermore,there are experimental evidences that glioma stemcell over-express some proteins involved in destruc-tion and remodelling of ECM such as matrixmetalloproteinase -2 (MMP-2) and MMP-9. Analysisof spatial distribution of glioma cells under laser con-focal microscopy performed by Yuan et al. (2004)showed an elevated proportion of CD133+ and nestin+cells in the margin of the tumor. Moreover, numerousnestin+/Sox2+ glioma cells were observed to infiltrateinto the surrounding normal brain tissue.

Notch, PDGF, Hedgehog-Gli1, and BoneMorphogenetic Protein SignallingPathways Implications in Control ofCD133+ CSC Function in Gliomas

Some of the signalling pathways that are involvedin differentiation and proliferation of glial progen-itors are altered in gliomas. The Notch family oftransmembrane receptors comprise Notch-1, -2, -3 and-4. Mature Notch receptors are heterodimers derivedfrom the cleavage of Notch pre-proteins into anextracellular and a trans-membrane subunits includ-ing the intracellular region. Mammalian Notch genesare widely expressed during embryonic development.Notch signalling is a ligand-receptor initiated pathway(Fig. 19.2). Maintenance of an activated Notch path-way is influenced by at least two factors, the negativeregulator Numb, and the positive regulator Musashi-1.Numb negatively regulates Notch by promoting itsubiquitination and by interfering with nuclear translo-cation. The RNA-binding protein Musashi-1 increasesNotch activity by inhibiting Numb translation by bind-ing to its cis-acting repressor sequences in the 3′-untranslated region (Fig. 19.2).

Notch signalling activates a diverse repertoire ofgenes regulating different cellular functions, and it hasbeen referred to as a gatekeeper against differentia-tion. In fact, Notch signalling blocks differentiationtowards a primary differentiation fate in a cell, andinstead directs the cell to a second alternative differ-entiation program or forces the cell to remain in anundifferentiated state. In the CNS, Notch is essentialfor the maintenance of the neural stem cell. Recently,Andreu-Agullò et al. (2009) have reported that treat-ment of NSCs with the Pigment Epithelium-DerivedFactor (PEDF) enhances Notch-dependent transcrip-tion of the downstream effectors Hes1 and 5 of theHES family of basic helix-loop-helix transcription fac-tors. Hes transcription factors negatively regulate theexpression of pro-neurogenic genes, therefore Notchsignalling enhances self-renewal by antagonizing dif-ferentiation, even though it can also regulate prolifer-ation and survival. These authors suggest that PEDFmaintains/induces the activity of CBF1 transcriptionfactor in dividing NSCs, determining increased self-renewal and multi-differentiation potentials. In addi-tion, the observation that Egfr promoter is a targetof CBF1 has highlighted the involvement of Notch

19 Markers of Stem Cells in Gliomas 187

activation on proliferation. Moreover, because Hes1and Hes5 block the execution of neurogenic programs,Notch signalling may act allowing the reversibilityfrom the quiescent state of NSCs. Therefore, a coor-dinated expression of EGFR and Hes1 may determinestemness by promoting mitogenic response in a highlyregulated quiescent population.

Studies performed in transgenic Notch activity-reporter (TNR) mice have demonstrated that highCBF1 activity discriminates stem cell from committedprogenitors, while loss-of-function experiments haveshown that down-regulation of Notch activity is nec-essary for the transition from radial glia/astroglia-likecells to committed progenitors (Mizutani et al., 2007).Furthermore, Notch promotes differentiation of vari-ous glial cell types (astrocytes, Müller glial cells, andradial glial cells). In addition to these functions, ithas been shown that Notch activity prevents nestindegradation during stem cell differentiation (Mellodewet al., 2004). Recently, it has been observed thatNotch-1 and its ligands are over-expressed in manyglioma cell lines and primary human gliomas. In addi-tion, the experimental knockdown of Notch-1 andits ligands determined apoptosis and inhibited pro-liferation of cultured glioma cell lines prolongingthe survival in murine orthotopic brain tumor model(Purow et al., 2005). This evidence suggests thatNotch-1 signalling might be critical for tumorigenesisand might represent an important target in the treat-ment of gliomas. Moreover, recent data suggest thatNotch signalling promotes the formation of CSCs ingliomas.

Notch signalling can directly up-regulate nestinexpression in gliomas and cooperate with KRASto generate peri-ventricular lesions characterized bycontinued proliferation of stem cells in the SVZ.In addition, it has been observed that a constitu-tive activation of Notch signalling in glioma celllines promotes growth and increases the formation ofneurosphere-like colonies in the presence of growthfactors. Moreover, it has been observed that in medul-loblastoma cell cultures a blockade of Notch sig-nalling through inhibition of γ-secretase drasticallyreduces the number of CD133+ cells, totally abol-ishes the SP cells, and inhibits the ability of formingtumors in vivo. These data suggest that the loss oftumor forming capacity could be due to the deple-tion of stem-like cells (Fan et al., 2006). Accordingly,Hes1 mRNA, a marker of Notch pathway activity,

is substantially up-regulated in the CD133-enrichedfraction of medulloblastoma cell line cultures. Thisconfirms that Notch signalling is especially activein stem-like cancer cells and supports the possibil-ity that Notch pathway inhibition may target thispopulation.

PDGF was originally identified in platelets andin serum as a mitogen for fibroblasts, smooth mus-cle cells (SMC), and glial cells in culture. To date,four PDGF ligands PDGFA-D are known. The fourPDGF polypeptide chains form five dimeric PDGF iso-forms: PDGF-AA, -AB, -BB, -CC, and -DD. PDGFisoforms exert their cellular effects through tyrosinekinase α- (PDGFR-α) and β- (PDGFR-β) receptors.During embryogenesis, glial and neuronal progenitorsexpress the PDGFR-α, whereas neurons and astrocytesexpress PDGF. The PDGFR-α is constantly expressedduring differentiation of neural stem cells, but is phos-phorylated only after PDGF-AA treatment, while thePDGFR-β is very low or not detectable in uncommittedcells, but its expression increases with differentiation.During the post-natal period, as glial progenitors dif-ferentiate into oligodendrocytes, PDGFR-α expressionis down-regulated. In adult brain, PDGFR-α is presentin the ventricular and sub-ventricular zone of the lat-eral ventricles possibly restricted to neural stem cells,whereas PDGF is widely expressed by neurons andastrocytes.

Ablation of the PDGFR-α in a subpopulation ofpost-natal neural stem cells shows that this receptor isrequired for oligodendrogenesis, but not for neurogen-esis. Interestingly, the infusion of PDGF-AA alone intomice SVZ arrests neuroblast production and inducesSVZ B cell proliferation contributing to the generationof large hyperplasias with some features of gliomas(Jackson et al., 2006). Thus, activation of PDGF sig-nalling in SVZ B stem cells might represent an eventcontributing to initiate tumorigenesis. Numerous stud-ies have demonstrated coexpression of the PDGF-A,PDGF-B, and of the PDGFRs in glioblastomas, sug-gesting that both autocrine and paracrine stimulationcould play an important role in glial tumorigenesis.Lokker et al. (2002) observed a decrease in cellu-lar survival and proliferation of glioma cell lines byblocking the PDGF autocrine signalling, providingevidence for a critical role of the autocrine loop inmaintaining cell transformation. Furthermore, ampli-fication of the PDGFR-α gene has been observed inlow grade and in a subset of high-grade gliomas. In

188 P. Dell’Albani et al.

neural progenitors and in more mature astrocytes ofnewborn mice the overexpression of the PDGFR-βdetermines the formation of oligodendrogliomas andoligoastrogliomas, respectively. Data present in liter-ature on PDGF/PDGFRs expression in gliomas showhow important it is to understand the diverse molecularevents that play a role in PDGF/PDGFRs expres-sion, signalling activation, and cellular responses ingliomagenesis.

HedgeHog (HH)-GLI signaling also has a rolein gliomas. SONIC HH (SHH) activates a signaltransduction cascade with the involvement of mem-brane proteins such as PATCHED1 (PTCH1) andSMOOTHENED (SMOH), leading to the action ofGLI transcription factors. Recently, Clement et al.(2007) demonstrated that human gliomas and theirCSCs require HH-GLI pathway activity for prolifera-tion, survival, self-renewal, and tumorigenicity. Theseauthors showed that in human gliomas (grade III) HH-GLI signaling regulated the expression of stemnessgenes, such as NANOG, OCT4, SOX, and BMI1 and theself-renewal of CD133+ glioma CSCs. The stemnesssignature observed in grade III could be extended tograde IV (GBM) and grade II tumors, even though withlower expression levels. The high stemness signatureobserved in grade III gliomas has been related to abun-dance of CSCs. The decreased levels in GBMs couldbe due to the increased number of more differentiatedtumor-derived cells, to increased participation of non-tumor cells and to increased angiogenesis. Moreover,in GBMs with expressing SHH new vessels, angio-genesis provides not only the nutrients to the tumor,but also determines its growth and CSCs self-renewal.The involvement of HH-GLI signalling in the reg-ulation of proliferation of normal brain and cancerstem cells has also been proved by the experimen-tal use of cyclopamine, a specific SMOH inhibitor.Adherent primary cultures of gliomas treated withcyclopamine showed decreased cell proliferation. Thecyclopamine treatment resulted in a decreased GLI1expression and complete cell death after 10 days oftreatment. Interestingly, when the drug treatment wasinterrupted a culture recovery was observed, indicat-ing temporally distinct cytostatic and cytotoxic effects.The authors have hypothesized that cyclopamine treat-ment could spare normal quiescent stem cells in theirniches, and thus would allow the regeneration of nor-mal adult tissues after cessation of treatment, offeringnew therapeutic prospectives.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transform-ing growth factor beta (TGF-beta) superfamily. Theroles of BMPs in embryonic development and cel-lular functions in postnatal and adult animals havebeen extensively studied in recent years. Signal trans-duction studies have revealed that the canonical pro-teins, Smad1, 5 and 8 are the immediate downstreammolecules of the BMP receptors, playing a centralrole in BMP signal transduction (Chen and Panchision,2007). Studies from transgenic and knockout mice forBMPs and related genes have shown that BMP signal-ing plays a critical role in heart, neural, and cartilagedevelopment. BMPs are well characterized inducers ofCNS stem cell differentiation, astroglial fate, mitoticarrest, and apoptosis. In adult brain, BMPs play aninstructive role in the stem cell niche, where the inter-action of these proteins and their inhibitor Nogginregulates the acquisition of an astroglial phenotype inthe stem cell progeny. BMP activities are regulated atdifferent molecular levels. In addition to their phys-iologic roles, it has been observed that some BMPsare implicated in the development of several cancers.Recent data show that BMP4, a protein belongingto the BMPs family, strongly activates BMP recep-tors (BMPRs), triggering Smad signalling cascade incells isolated from human GBMs. BMP4 treatmentdetermined a decreased proliferation and an increasedexpression of differentiated neural markers, with noeffect on cell viability. BMP4 treatment resulted in areduced clonogenic ability (>70%), both in the sizeof the CD133+ side population (~50%) and in thegrowth kinetics of GBM cells, indicating that it isable to gain a reduction of the CSC pool. Moreover,human GBM cells transiently exposed to BMP4 wereno more able to establish intracerebral GBMs whenimplanted in cerebral hemispheres of nude mice. Basedon these data it has been hypotesized that the tran-sient exposure to BMP4 depletes the brain tumorinitiating cancer population and produces a signifi-cant decrease in the in vivo tumor-initiating abilityof GBM cells (Piccirillo and Vescovi, 2007). Theseobservations prompted the authors to hypothesize thatBMP4, induces differentiation of the tumor-initiatingcells rather than killing them; it could be used aftersurgery to block new GBM development. However,more experiments need to be done to better under-stand this mechanism and try to set a therapeuticapproach.

19 Markers of Stem Cells in Gliomas 189

Conclusion

In the past, most of the research on human braintumors has been directed to the bulk of tumor mass,while only recently the attention of researchers isfocused on tumor key cells, the CSCs. CSCs repre-sent a small subset of cells within the tumor massable to self-renew and proliferate, which are respon-sible of both the initiation of primary disease and ofits recurrence. Considering that cancer initiation andprogression is a multi-step process involving numerouscellular mechanisms, including altered gene expres-sion, signal transduction pathway activation and/orinhibition and secretion of growth factors, new ther-apeutic protocols should take into account all thesevariables. The definition of a panel of CSC markershas a relevant interest for several reasons: CSC mark-ers may serve as prognostic or predictive tools; theyare fundamental both to select CSCs and attempt theirdepletion through specific therapeutic approaches. Infact, the definition of a specific GBM profile and theidentification of markers predictive of survival willenable clinicians to use individualized therapies.

Because gliomas, as other tumors, are very het-erogeneous in their cellular composition, molecularexpression, and clinical outcomes, the design of indi-vidualized therapies will be one of the most ambitiousgoals for the biomedical research.

Acknowledgments We thank Mr. Francesco Marino for hishelpful work for figure editing.

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