Journal of Neurocytology 31, 507–521 (2002)

NG2 precursor cells in neoplasia: Functional,histogenesis and therapeutic implications formalignant brain tumoursM . CHEKENYA 1,∗ a n d G. J . P I LKI NGTON 2

1The Faculty of Medicine, Department of Anatomy and Cell Biology, University of Bergen,◦

Arstadveien 19, N-5009 Bergen, Norway;2Experimental Neuro-oncology Group, Department of Neuropathology, Institute of Psychiatry King’s College, London SE5 8AF, UKMartha.Chekenya@pki.uib.no

Received January 2002; revised June 2002; accepted June 2002

Abstract

Diffusely infiltrating astrocytic tumours of the central nervous system (CNS) are the most frequent intracranial neoplasms andaccount for more than 60% of all primary brain tumours in man. Until recently, it was generally accepted that the glial componentof the mature CNS, consisted of differentiated astrocytes, ependymal cells, oligodendrocytes and the non-neuro-ectodermalmicroglial cells. There exists a recently recognised population of glial cells that express the NG2 proteoglycan (NG2 cells). NG2cells are dynamic and undergo rapid morphological changes in response to a variety of CNS pathologies. They are highly motilecells, which interact with various extracellular matrix (ECM) in association with the integrin receptors. During angiogenesisand response to tissue injury, NG2 precursor cells are recruited to sites where vessel growth and repair are occurring. NG2 isover-expressed by both tumour cells and pericytes on the blood vessels of malignant brain tumours. The function of NG2 cellsin the CNS, and the notion of them as a source of and/or lineage marker for some gliomas are discussed. In addition, theirpossible role in glioma angiogenesis, proliferation and invasion will be considered as will their value in provision of targets forclinical and pre-clinical therapeutic strategies in brain tumours.

Introduction

Diffusely infiltrating astrocytic tumours of the centralnervous system (CNS) are the most frequent intracra-nial neoplasms and account for more than 60% of allprimary brain tumours in man (Kleihues, 2000). Theincidence differs between regions but is usually citedas around 5–7 new cases per 100,000 capita (Kleihues,2000; Davis, 1998), the majority of these being of glialorigin. In children under the age of 15 years, CNS tu-mours account for 23% of all cancers and, in terms ofincidence of paediatric cancers, rank second only to theleukaemias.

Although gliomas may arise at any site in the CNS,in adults they preferentially originate in the cerebralhemispheres. The tumours express a great diversityin histopathological features and biological behaviourand they often exhibit extensive diffuse infiltrationof adjacent parenchyma and distant brain structures(Kleihues et al., 1993). Migration is not unique to malig-nant cells, since endothelial cells, reactive astrocytes,lymphocytes and microglial cells also invade native

∗To whom correspondence should be addressed.

tissue during neovascularisation (Folkman et al., 1989;Folkman, 1971). This process involves interactions be-tween the host and tumour cell-derived extracellu-lar matrix (ECM), which provides a substrate ontowhich cells adhere and migrate (Rutka et al., 1988;Giordana et al., 1985). The terms migration and inva-sion are frequently used interchangeably but, strictlyspeaking, migration is the active movement of cellsthrough a tissue while invasion implies migrationwith associated destruction of normal cellular ele-ments of the brain (Bolteus et al., 2001). Diffuse inva-sion in glioma invariably leads to recurrence, whichis often associated with the transition to a highergrade of malignancy and, ultimately, the acquisition ofhistological and biological characteristics of glioblas-toma. The majority of glioblastomas develop veryrapidly (the so-called de novo or primary glioblastomas),without clinical, radiological or morphological evi-dence of a less-malignant precursor lesion (Kleihues& Ohgaki, 2000). Conversely, secondary glioblastomas

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develop over years and progress from low-gradediffuse or anaplastic astrocytoma. These glioblastomasubtypes evolve through different genetic pathways,affect patients of different ages and are likely to differin their responses to therapy. One of the most remark-able advances in our understanding of cancer progres-sion is that solid tumour growth is dependent upon an-giogenesis (Humphries, 2000; Folkman, 1995; Folkmanet al., 1989). Indeed, one of the hallmarks of high-gradegliomas is the presence of microvascular proliferation(Kleihues, 2000). Data from transgenic mouse models oftumourigenesis indicates that angiogenesis, the sprout-ing of new vessels from pre-existing vasculature, oc-curs early during tumour development (Hanahan et al.,1996).

The non-neuronal component of the mature CNSparenchyma consists mainly of astrocytes, ependymalcells, oligodendrocytes and microglial cells. In addi-tion, another population has recently been describedthat express the transmembrane chondroitin sulphateproteoglycan, neuron-glial 2 (NG2). These cells arefound both in the developing and mature CNS andthey have been most often referred to as oligoden-drocyte progenitor cells (OPCs/NG2 cells) because oftheir ability to differentiate into oligodendrocytes fromoligodendrocyte/type-2 astrocyte (O-2A) progenitorcell cultures (Wolswijk & Noble, 1989; Raff et al., 1983).NG2 cells also display differentiated morphologies sug-gesting that they are not all oligodendrocyte progen-itor cells. This review discusses the role of the NG2cells in tumour cell biology. We will focus on three ma-jor areas of interest, (1) their function in the CNS andmore specifically in neural neoplasms, (2) their possi-ble significance as lineage markers and provision of thecellular source for some gliomas, and (3) NG2 expres-sion as a possible target for therapeutic intervention inmalignant gliomas.

OPC cells in the adult CNS

Adult OPCs develop from perinatal OPCs (Wren et al.,1992). They can be distinguished from their perinatal

Table 1. Distinguishing characteristics of perinatal and adult oligodendrocyte progenitor cells.

Perinatal oligodendrocyteAdult oligodendrocyte progenitor cells progenitor cells

Morphology Stellate Unipolar or bipolarPhenotypic antigens NG2+; PDGFα-R+; O4+; GFAP− ;

GalC− ;NG2+; PDGFα-R+; O4− ;

Migration rate Slow (4.3 ± 0.7 µm/hour) (Wolswijk& Noble, 1989; Wolswijk et al., 1990;Wren et al., 1992)

Rapid and extensive (21.4 ±1.6 µm/hour (Noble et al., 1988)

Cell cycle duration Long, 65 ± 18 hours Shorter, 18 ± 4 hoursDifferentiation time into

oligodendrocytes without mitogensSlow (50% differentiation within

5 days)Faster (50% differentiation within

2 days)

counterparts on account of their morphology, cell cycleduration, differentiation times, antigenic phenotype,and migration rates (Wolswijk et al., 1990; Wolswijk &Noble, 1989; Wren et al., 1992; Noble et al., 1988; Raffet al., 1983) (see Table 1). Until recently, it has been dif-ficult to identify OPCs within the adult CNS due tothe lack of reliable phenotypic markers (Dawson et al.,2000). Adult OPCs can be identified by expression of theNG2 proteoglycan and the platelet derived growth fac-tor α receptor (PDGFα-R) (Hall et al., 1996; Fulton et al.,1992; Levine et al., 1993; Reynolds & Hardy, 1997; Wilsonet al., 1981). NG2 cells also take up cobalt when stimu-lated with quisqualate (Fulton et al., 1992). During earlyembryogenesis, NG2 is expressed in the CNS on devel-oping capillaries, as well as on premature cells of neu-roepithelial origin (Grako & Stallcup, 1995). The relativeproportion of NG2 cells increases exponentially duringthe period of rapid expansion of the brain vasculature,but subsides during the second postnatal week uponterminal differentiation (Nishiyama et al., 1996a; Grako& Stallcup, 1995). It has been debated as to whether theactual numbers decrease during the postnatal stage, orwhether their density declines with the expansion ofbrain volume. An additional element that needs to bereconciled is the proportion of NG2 cells that gives riseto terminally differentiated oligodendrocytes. For ex-ample, if an NG2 precursor cell divided to give rise totwo daughter cells, one of which terminally differenti-ated into oligodendrocytes and the other remained asan NG2 precursor cell, there would be no decrease inNG2 cell numbers.

This reasoning might resolve the paradoxical findingthat NG2 cells persist in numbers that are comparableto mature oligodendrocytes in the gray matter, but arefewer in the spinal cord (Dawson et al., 2000). NG2 cellsdo not express S-100β, a calcium binding protein ex-pressed by differentiated astrocytes (Kligman & Hilt,1988), the intermediate filaments glial fibrillary acidicprotein (GFAP) or vimentin (Levine et al., 1993). More-over, they do not express other markers of mature cells,such as glutamine synthase for astrocytes (Butt et al.,1999; Reynolds & Hardy, 1997), or galactocerebrosidase

NG2 expression in malignant brain tumours 509

(GalC) for oligodendrocytes (Levine & Card, 1987), in-dicating that NG2 cells are distinct from both matureastrocytes and oligodendrocytes. Previous studies re-ported that NG2 cells in the cerebellum and forebraindid not express microglial OX-42 (Levine & Card, 1987;Leong & Ling, 1992; Reynolds & Hardy, 1997), GSAI-B4 lectin (Nishiyama et al., 1997; Streit & Kreutzberg,1987), or F4/80 (McKnight et al., 1996; Nishiyama et al.,1997). Conflicting reports however, demonstrate thatafter CNS injury, an influx of OX42 and ED1 positive mi-croglia/macrophage cells co-expressed NG2 (Bu et al.,2001). Indeed, some studies indicate that there exists apopulation of NG2 cells in the normal adult and foetalhuman brain that co-express the microglial lineagemarkers CD68, and CD11c (Pouly et al., 1999). Recently,a combination of morphological analyses and doubleimmuno-labelling for NG2 and the ionised calcium-binding adapter molecule-1 (IBA1) expressed by mi-croglia and macrophages (Ito et al., 1998) demonstratedthat macrophages constitute the primary source of NG2after spinal cord injury (Jones et al., 2002; Bu et al., 2001).Most importantly this study reconciles the apparentconflict in the literature regarding the issue of whethermicroglial cells express NG2. Jones et al. elegantly showthat resident ramified microglia from intact CNS tissue,although localised in close proximity to the lesion, didnot express NG2 (Jones et al., 2002; Bu et al., 2001).

The function of NG2 cells in the normaland neoplastic CNS

REACTIVE RESPONSE TO INJURY

Adult OPCs isolated from the adult optic nerve arecharacterised by a unipolar or bipolar morphologyin vitro and small cell bodies (10–15 µm in diam-eter) that give rise to multiple stellate branches invivo (Levine et al., 2001; Tanaka et al., 2001). The pro-cesses have a radial orientation in the gray matter,whereas they are more longitudinal in the white matter(Levine et al., 2001). NG2 cells undergo morphologi-cal changes in response to a variety of injuries, such asdemyelination or inflammation (Reynolds et al., 2001),spinal cord contusion (McTigue et al., 2001), hypoxic-ischaemia (Back et al., 2001), kainate induced neurotox-icity (Shee et al., 1998), cerebellar puncture lesions andherpes viral encephalitis (Levine et al., 1998; Levine,1994). These changes are in all cases characterised by(1) upregulation of NG2 expression within hours ofinjury (Jones et al., 2002) (2) an increased density ofprocesses that change to become shorter and thicker(Levine et al., 1998, 2001; Levine, 1994; Tanaka et al.,2001) and (3) fine filopodia appear on the cell bodyand processes of reactive NG2 cells (Levine et al., 1998).The onset is rapid and may persist for several weeks,suggesting that NG2 cells are a dynamic populationthat can be regulated by the CNS microenvironment

(Levine, 1994; Levine et al., 1998). Although, it is notclear whether the reactive NG2 cells are all endoge-nous, resident brain OPCs. There is evidence that aproportion of the reactive cells with large, round cellbodies and short processes are infiltrating mononuclearphagocytes (Jones et al., 2002; Bu et al., 2001). Moreover,the fact that IBA1+/NG2+ macrophages are recruitedonly acutely and sub-acutely into the lesion, suggeststhat NG2 expressing macrophages are derived fromthe blood and not from CNS microglia. The idea thatNG2 positive macrophages are derived from blood andmigrate into the degenerating tissue is supported byseveral studies that show that no cells that co-expressNG2 and OX42 were ever observed in hippocampalslice cultures treated with kainic acid, suggesting thatNG2+/OX42+ cells are not derived from endogenousresident brain cells (Jones et al., 2002; Bu et al., 2001).

CELL ADHESION, MIGRATION AND INVASION

Cell adhesion and migration on ECM components—integral features of the invasion process—are promotedby a number of membrane receptors, of which, integrinsrepresent a major class (Humphries, 2000). Several linesof evidence indicate that NG2 chondroitin sulfate pro-teoglycan is also capable of modulating both cell-ECMand cell-cell adhesion. For example, the NG2 core pro-tein is localised on microspikes in melanoma cells andparticipates in cellular adhesion (Garrigues et al., 1986).The human antigen shares 85% homology with rat NG2,and the human 9.2.27 monoclonal antibody (mAb) caninhibit melanoma migration on endothelial cells andECM proteins (Harper et al., 1984). NG2 chondroitinsulfate proteoglycan is deposited at the interface be-tween the substrata and the migrating cells (de Vrieset al., 1986).

Recent in vitro studies however, demonstrate thatalthough brain chondroitin sulfate proteoglycans canpromote transient adhesion of neuronal cells, they gen-erally inhibit stable cell adhesion and neurite out-growth (Gladson, 1999). NG2 negatively modulates ad-hesion of melanoma cells via cluster of differentiation44 (CD44), hyaluronic acid (HA), fibronectin, and α4β1integrin (Burg et al., 1998). CD44 is ubiquitously ex-pressed in several tumours (East et al., 1993), includ-ing gliomas (Kuppner et al., 1992), where it mediatestumour cell adhesion and migration (Merzak et al.,1994; Akiyama et al., 2001). NG2 may indirectly regulatecell—ECM interactions by serving as a co-receptor forα4β1 integrin during adhesion and migration (Iida et al.,1995). The NG2 core protein communicates with α4β1integrin by outside-in and inside-out signalling mech-anisms (Midwood & Salter, 2001; Iida et al., 1995). Sinceadhesion to fibronectin is mediated by α4β1 integrinsvia the same leucine/aspartic acid/valine (LDV) ‘hotspots’, present on the NG2 proteoglycan (Nishiyamaet al., 1991), competitive inhibition might explain the

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weak interactions between α4β1 and fibronectin in thepresence of NG2. It follows that blocking NG2 with the9.2.27 mAb would augment the α4β1 mediated bindingto fibronectin (Burg et al., 1998).

NG2 also interacts with the β1 integrin subunit,where it modulates adhesion to type II and VI colla-gen (Doane et al., 1998; Midwood & Salter, 2001). NG2is best characterised as a cell surface receptor for type VIcollagen (Tillet et al., 1997; Burg et al., 1996; Nishiyama& Stallcup, 1993; Stallcup et al., 1990), which is a majorcomponent of the basement membrane in some typesof vasculature (Kuo et al., 1997; Rand et al., 1993). Thenormal brain ECM is composed of very little collagen,laminin, and fibronectin but contains an abundance ofHA. However, during glioma invasion and migration,neoplastic cells can synthesise tenascin, HA and colla-gen de novo (Chintala et al., 1996; Paulus et al., 1988). Thefact that NG2 interacts with laminin, tenascin and cer-tain collagen types, (Burg et al., 1997) suggests that NG2may modulate glioma cell adhesion to various ECMcomponents. It is established that NG2 poses a barrierto cell migration and axonal growth on laminin sub-strates (Lemmon et al., 1989; Edgar et al., 1984; Fidleret al., 1999; Fawcett & Asher, 1999; Dou & Levine, 1994)through Gi protein receptor mediated signal transduc-tion (Dou & Levine, 1994). Indeed, we previously re-ported that NG2 expressing glioma cells failed to mi-grate on laminin, vitronectin and fibronectin substrates,and only migrated efficiently on collagen IV coated sub-strates in vitro (Chekenya et al., 1999).

Studies of glial progenitors revealed that while type1 astrocyte progenitors were relatively sedentary, cellson the type 2 astrocyte/oligodendrocyte (O-2A) lineagepathway were highly motile (Small et al., 1987). Sincethe major distinguishing characteristic between thesecells appeared to be the presence of gangliosides, in-cluding GD3, recognised by the A2B5 antibody, thepossible correlation between GD3/A2B5 gangliosideexpression and motility in glioma cells was assessed.On segregation of A2B5 positive and negative gliomasub-populations, the positive fractions showed a sig-nificantly higher migration rate (Gratsa et al., 1997).Addition of gangliosides such as GD3 to glioma celllines significantly reduced proliferation rates but in-creased migration (Merzak et al., 1995). Morover, usinga combination of immunocytochemistry and the “Tran-swell’’modified Boyden chamber invasion/motility as-say (Pilkington, 1994), migratory cells showed a highexpression of cell surface gangliosides while they werenot labelled by either of the cell cycle indicators, prolif-erating cell nuclear antigen (PCNA) or Bromodeoxyuri-dine (Pilkington, 1992). The apparent picture of neo-plastic glia transiently arresting from the cell cycle dur-ing the migratory phase of invasion (Pilkington, 1996)has been alluded to by several groups (Schiffer et al.,1998; Giese et al., 1996; Mariani et al., 2001) and hasbeen termed the “go or grow’’ hypothesis (Berens &

Giese, 1999). Since NG2 expression appears to be coin-cident with Ki67 labelling and increases in expressionwith grade of malignancy, the possibility of a mutualexclusivity of NG2 and GD3/A2B5 became an inter-esting concept. Recently, we compared the NG2 spatialdistribution in neoplastic tissue that originated fromthe tumour main mass with that obtained from the in-terface between the tumour and the infiltrated brain.The results indicated that the expression of NG2 wasconfined to the proliferative tumour main mass (withhigh Ki67 labelling) and was markedly reduced in theinfiltrative edge (with low Ki67 labelling) (Chekenyaet al., 2002a) (see Fig. 1), whereas the distribution ofGD3 was more widespread (Hedberg et al., 2001). Al-though NG2 and GD3 were both highly expressed inthe main tumour mass, the expression patterns of thesetwo antigens were mutually exclusive at the infiltrativeedge. Indeed, NG2 is expressed on perivascular andbasement membrane components of glial neoplasms,as is GD3 (Koochekpour & Pilkington, 1996; Ziche et al.,1992). We previously reported that laminin was not apermissive substrate for NG2 expressing glioma mi-gration in vitro (Chekenya et al., 1999). Since lamininis abundantly deposited in the border zones betweenthe tumour tissue and the brain parenchyma in vivo(Pedersen et al., 1993; Giordana et al., 1985), it is tempt-ing to speculate whether NG2 expressing cells may berestricted to the tumour main mass due to their inabil-ity to adhere and migrate on certain ECM components(Chekenya, 2002a).

OPCs are produced in restricted foci within the ger-minative neuroepithelium during embryogenesis andmigrate to distant sites where they function (Noble,2001). OPC migration in the optic nerve and chiasmais guided by repulsive sematophorins secreted fromthe optic chiasma. Netrin 1 guides a subtype of NG2cells with small nuclei, while Sema3a repels a subtypewith large nuclei (Sugimoto et al., 2001). The mech-anisms by which NG2 may be involved in tumourcell motility are also believed to involve the modifi-cation of cytoskeletal dynamics (Fang et al., 1999). Inboth the gray and white matter, NG2 localises to ra-dial actin spikes associated with filopodia that are po-sitioned at opposite poles from fascin containing lamel-lipodia in migrating cells. This indicates a role for NG2in the establishment of cellular protrusions, which areessential for cell polarity during morphogenesis or mi-gration through Rho-dependent mechanisms (Stallcup& Dahlin-Huppe, 2001; Lin et al., 1996; Fang et al.,1999). NG2 interacts with the ECM via its core protein(Nishiyama & Stallcup, 1993; Stallcup et al., 1990; Tilletet al., 1997). NG2-expressing glioma cells migrate mostefficiently on type VI collagen. This migration is specif-ically inhibited by NG2 blocking antibodies or by mu-tations in NG2’s collagen-binding domain, indicating arole for NG2 in cell signalling during migration (Burget al., 1997) (see Fig. 2).

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TUMOUR GROWTH AND CELL PROLIFERATION

Fibroblast growth factor 2 (FGF-2) and PDGF AA in-duce proliferation and alter the differentiation of cul-tured OPC lineage cells. PDGF, FGF-2 and their re-ceptors are present in the normal adult CNS, and areupregulated during pathological conditions (Redwineet al., 1997). Both NG2 and PDGFα receptor are co-expressed on OPCs throughout the late embryonic andpostnatal life (Nishiyama et al., 1996a), where they alsointeract on the surface of vascular smooth muscle cells(Nishiyama et al., 1996a, b; Grako et al., 1995; Goretzkiet al., 1999). NG2 binds and activates both PDGF-AAand FGF-2, modulating their pleiotropic effects on thesignalling receptors. Anti-PDGFα receptor antibodiesco-immunoprecipitate with NG2 from cell lysates ofO-2A progenitor cells (Nishiyama et al., 1996a) suggest-ing a physical link between the two molecules. More-over, antibodies against NG2 block the mitogenic ef-fects of PDGF-AA on both OPC and aortic smoothmuscle cells. The PDGFα receptor is unresponsive toPDGF-AA in aortic smooth muscle cells derived fromNG2 knockout mice (Paulus et al., 1988; Goretzki et al.,1999; Grako & Stallcup, 1995; Nishiyama et al., 1996b),indicating that NG2 is important for both the pro-liferative and migratory responses to the PDGF-AA/PDGF-α receptor pathway. Using Ki67 (MIB-1) im-munolabelling, which recognizes proliferation associ-ated nuclear proteins expressed during G1 to M phasesof the cell cycle, we showed that NG2 expressingglioblastoma cells xenografted into nude rat brainswere more proliferative than their NG2 negative coun-terparts (Chekenya, 2002b). NG2 expressing humanglioblastomas in adults exhibited high MIB-1 labellingindices in the tumour main mass areas compared tothe NG2 negative tumours (Chekenya, 2002a). Similarobservations have been reported for melanoma cells,where the expression of NG2 increased their prolifer-ation rates in vitro, tumorigenicity and their metastaticpotential in vivo (Burg et al., 1998). Using magnetic res-onance imaging (MRI) we showed that NG2 express-ing xenografts grew faster and exhibited greater spreadwithin the brain compared to NG2 negative controltumours (Chekenya, 2002b). NG2’s binding and acti-vation of the PDGF-AA/ PDGF-αR pathway is oneputative mechanism by which NG2 expression couldenhance the growth of neoplastic cells. The PDGF-αRgene on chromosome 4q11-p12 is amplified in someglioblastomas (Hermanson et al., 1996) and is over-expressed in most astrocytomas (Nister et al., 1988;Fleming et al., 1992) and this may lead to both au-tocrine and paracrine growth factor stimulation. In ad-dition, NG2-collagen interactions may also potentiategrowth factor driven proliferative and migratory re-sponses. Since the collagen types are capable of bindingPDGF, an intriguing possibility is that NG2 may bindthe collagen-PDGF complex and enhance the growth

factor’s interaction with the PDGF receptors on theglioma cell surface. Simple gangliosides such as GD3,have been widely reported to suppress proliferation butare consistent with increased invasive behaviour in ma-lignant gliomas (Merzak et al., 1995; Gratsa et al., 1997).Since PDGF is among those growth factors that induceGD3 expression (Pilkington et al., 1993), a dual role ofthis factor may be envisaged in NG2/GD3 mediatedprocesses of proliferation and invasion in glioma.

VASCULAR MORPHOGENESIS AND TUMOURANGIOGENESIS

NG2 is widely expressed by vascular cells during bothnormal and pathological angiogenesis (Humphries,2000; Ozerdem et al., 2001, 2002; Grako & Stallcup, 1995;Nishiyama et al., 1996a). Although it has been previ-ously reported that capillary endothelial cells expressNG2 in the CNS microvasculature (Pouly et al., 2001;Grako & Stallcup, 1995; Schrappe et al., 1991), emerg-ing evidence suggests that NG2 is invariably expressedby the mural cell component of the neovasculature(Ozerdem et al., 2001). NG2 is expressed by pericytes onimmature brain capillary vessels as early as embryonicday 10–12 (E10–12) in the rat and continues to be ex-pressed throughout the period of rapid expansion of thebrain vasculature (Miller et al., 1995; Nishiyama et al.,1996a; Ozerdem et al., 2002; Grako & Stallcup, 1995).Outside the CNS, the earliest and most prominent ex-pression of NG2 is on cardiomyocytes at E10 (Grakoand Stallcup, 1995; Ozerdem et al., 2001). In the dorsalaorta, smooth muscle cells express NG2 from E10 to E14(Ozerdem et al., 2001). During angiogenesis and in re-sponse to injury, NG2 expressing mural cells respond toenvironmental cues by migrating to sites where vesselgrowth and repair are occurring (Ross, 1993; Schwartzet al., 1990; Grako et al., 1999). There is substantial di-versity in the pericyte/endothelial cell ratios in thevarious micro/macro-vessels, suggesting that NG2 ex-pressing pericytes may have an important role in de-termining the structural and functional properties ofa given blood vessel (Balabanov & Dore-Duffy, 1998;Speiser et al., 1968; Sims et al., 1994; Tilton et al., 1979). Forexample, the seamless pericyte investment of CNS cap-illaries may contribute to the relatively impermeableblood-brain barrier (Balabanov & Dore-Duffy, 1998).

The close apposition of pericytes to endothelialcells also suggests a seminal role during angiogene-sis (Orlidge & D’Amore, 1987). Recent evidence indi-cates that the NG2 proteoglycan binds specifically andsaturably to plasminogen and its kringle domains con-sisting of angiostatin (K1–4) and miniplasminogen (K5)(Goretzki et al., 2000). In agreement with these find-ings, we have recently shown that over-expression ofNG2 in human glioblastoma cells directly increased tu-mour angiogenesis, which resulted in increased cellular

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NG2 expression in malignant brain tumours 513

proliferation, tumour initiation, and growth rates(Chekenya, 2002b) (see Fig. 3). Expression of NG2 wasalso indicative of a poorer survival outcome of theNG2 tumour bearing animals. The binding and seques-tration of angiostatin by NG2 might promote neovas-cularisation by neutralising the inhibitory effects ofangiostatin on endothelial cell proliferation and mi-gration (Chekenya, 2002b; Goretzki et al., 2000). As acell surface component of mural cells, NG2 is in posi-tion to sequester angiostatin, which otherwise wouldbe available to inhibit proliferation and migration ofendothelial cells (Chekenya, 2002b; Goretzki et al.,2000).

Implications of NG2 and PDGFα receptorexpression in the histogenesis of gliomas

NG2 has been implicated as a reliable marker for OPCs,particularly when used in combination with PDGFα-R(Hall et al., 1996; Dawson et al., 2000). The expressionof these antigens in neoplasia suggests that transfor-mation of neuroepithelial precursors may be the initialevent in the development of CNS tumours (Goldman,2000; Cushing, 1928). Paediatric brain tumours couldarise from transformed perinatal precursor cells, whichare capable of self-renewal by asymmetric cell divi-sion and do not persist into adulthood (Noble, 2001).Neoplastic transformation of this precursor may giverise to a variety of tumour types, including astrocy-tomas, oligodendrogliomas, mixed oligo-astrocytomasas well as secondary glioblastomas, representing differ-ent stages of differentiation at the time of oncogenesis.

Oncogenic transformation during adulthood onadult OPCs could lead to the development of primary

Fig. 1. NG2 is expressed in areas of high cell proliferation in the tumour main mass and is downregulated at the tumourinfiltrative areas of lower cellular proliferation. In order to examine the spatial distribution of NG2 in the tumour main massrelative to the brain parenchyma in whole brain sections, biopsy spheroids from human glioblastoma were xenografted intonude rat brains. (A) NG2 expression (green) within the high tumour burden areas. (B) Three dimensional reconstruction ofconfocal images from the immunostained sections seen in panel (A) showing NG2 expression (green) in the tumour main mass.(C) NG2 cells (green) are confined to the tumour main mass (T) and are reduced towards the infiltrative brain (B). (D) Thenumbers of Ki67 (MIB-1) positive cells (green) in the tumour main mass (T) attenuate towards the infiltrated brain (B). A, C–D:Scale bars = 60 µm, Magnification ×160. B: Scale bar = 250 µm, Magnification ×160. Propidium iodide (red) was used tocounterstain the nuclei in all immunostaining. Based on Chekenya et al. (2001a).

Fig. 2. Schematic representation of the signalling pathways which may be involved in NG2 mediated malignant progression.As a membrane-spanning protein, NG2 interacts with macromolecules on both sides of the plasma membrane. Intracellularly,NG2 interacts with actin and upon its engagemnet by the substratum, NG2 triggers cytoskeletal rearrangements that leadto cell migration. These cytoskeletal changes involve the Rho family of small GTPases rac and cdc42. NG2 activates rac andcdc42 by interacting via its C-terminus with the MUPP-1 PDZ-domain containing scaffolding protein. The signalling cascadewould trigger recruitment from blood and cell migration of NG2 expressing tumour cells, vascular pericytes, oligodendrocyteprecursor cells and mononuclear phagocytes into the tumour which may provide stimulatory signals for tumour growth andprotection from apoptosis. Extracellularly, NG2 interacts with a variety of ligands, including ECM components. For e.g. NG2interacts with type VI collagen to mediate cell migration and proliferation. Type VI collagen plays a role in tissue remodelling,vascular development and wound healing. NG2 binds kringle domain proteins and sequesters angiostatin, resulting in luxurianttumour angiogenesis due to the neutralisation of the inhibitory effect of angiostatin on endothelial proliferation. NG2 binds themitogens PDGF-AA and FGF-2 and acts as an auxillary receptor for these growth factors, potentiating their ability to interactwith and activate the receptor tyrosine kinases.

(de novo) glioblastomas. Several direct lines of evidencesupport this proposal for the transition from the normalto the pathological state. Totipotential stem cells do in-deed exist in the adult rodent, primate (Palmer et al.,1997; Gould et al., 1998, 1999; Gage, 1998) and humanbrain (Scolding et al., 1999; Eriksson et al., 1998; Frisenet al., 1998). Bromodeoxyurdine (BrdU) labelling and3H- thymidine incorporation techniques have demon-strated that adult OPC cells continue to replicate in vivo(Levison et al., 1999; Gould et al., 1998; Doetsch et al.,1999; Johansson et al., 1999). Furthermore, transforma-tion of an OPC cell rather than a mature glial cell mayexplain the existence of histologically “mixed’’ glial tu-mours. The manner in which gliomas diffusely infil-trate the adjacent brain parenchyma, as contrasted tothe well-circumscribed contours of peripheral tumoursand their metastases, suggests that CNS tumours retainthe ability of their cell of origin to migrate through theparenchyma (Silbergeld & Chicoine, 1997).

Nevertheless, experiments to test this hypothesishave been hampered by the difficulties in phenotypicidentification, isolation and manipulation of the pre-cursor cells in vivo. These studies have also been lim-ited by the lack of biological systems that representearly stages of the disease in adult animals (Kokkinakiset al., 2001). However, Barnett and co-workers demon-strated that transformation of an O-2A progenitor cellline with the c-myc and H-ras oncogenes generatedpathologies strikingly similar to human glioblastomaswhen implanted into the adult rat brain (Barnett et al.,1998). Recently, the cell types that undergo neoplastictransformation to give rise to gliomas after exposure toN-methylnitrosourea (MNU) (Rushing et al., 1998) havebeen characterised and it has been demonstrated that

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Fig. 3. NG2 expression in angiogenic vasculature from human glioblastoma biopsies and in human glioblastoma biopsiesvxenografted into nude rat brains. (A) T1-weighted MRI of a highly vascular glioblastoma in a patient, showing markedgadolinium ring enhancement, indicating neovascularisation and vascular permeability. The low signal area corresponds tocentral necrosis. (B) NG2 is expressed on pericytes lining vascular ‘tufts’, which are characteristic of microvascular proliferationsof human glioblastoma. (C) CD31 expression on endothelial cells within the tumour vasculature. (D) The proliferation markerKi67 (MIB-1) detected proliferating endothelial and tumour cells (insert, black arrow heads) within the regions of high NG2expression in the tumour. (E) In order to examine the spatial distribution of NG2 in the tumour main mass relative to the brainparenchyma in whole brain sections, biopsy spheroids from the same human glioblastoma were xenografted into nude rat brains.Histologically, the tumours derived showed a highly diffuse infiltration of single cells into the contiguous brain parenchyma.Moreover, T1-weighted MRI imaging of the rat brain revealed that these tumours were also very representative of the humanglioblastoma in situ. Panel (E) shows ‘ring’ contrast-enhancement of the humnan xenograft, a feature which is indicative ofneovascularisation and vascular permeability. The hypodense regions indicate a central necrosis. (F) NG2 expression withinthe tumour main mass was highly expressed on the tumour and pericyte cells of the associated neovasculature in the regionscorresponding to high signal intensity on T1-weighted MRI. (G) There was high CD31 labelling of vascular endothelial cellsin the same regions. (H) Ki67 labelling indicated that these regions of NG2 expression were proliferatively active, with higherlabelling indeces (insert) compared to the human biopsy. B, C, F, and G: Scale bars = 100 µm, Magnification ×200. D: Scale bar= 100 µm, Magnification ×100. Inserts in D and H: Scale bar = 50 µ, Magnification ×630.

the target cells for MNU were the OPCs (Kokkinakiset al., 2001).

Histopathological studies have asserted that the pres-ence of both NG2 and the PDGFα receptor in vari-ous glioma tissues might be indicative of their deriva-tion from OPCs (Shoshan et al., 1999; Nishiyama,2001). NG2 is upregulated on both the glioma cell sur-face and on microvascular proliferations of high-gradegliomas (Schlingemann et al., 1990; Schrappe et al.,1991; Chekenya, 2002a). NG2 and PDGFα receptor arerarely expressed on normal, myelin forming oligoden-drocytes (Sung et al., 1996), but they are both expressedby oligodendrogliomas, suggesting that the latter maybe derived from immature OPC cells (Shoshan et al.,1999; Nishiyama, 2001) NG2 expression is also upreg-ulated during other malignant conditions, such as onmelanoma cells (Bumol et al., 1984; Grako & Stallcup,1995; Reisfeld et al., 1984; Burg et al., 1998) and onpaediatric acute myeloid leukaemia (AML) monoblasts

(Mauvieux et al., 1999; Behm et al., 1996; Smith et al.,1996). Moreover, NG2 is a tumour specific antigen in thechemically induced rat chondrosarcoma HSN (Legeret al., 1994).

Given the great diversity of neoplastic tissues thatexpress NG2, and its location on tumour vasculature,these studies do not rule out the possibility that NG2cells might display extensive tropism for areas of in-jury or pathology in the adult brain. Gliomas pro-duce immunosuppressive signals, such as transforminggrowth factor β (TGFβ) (Bodmer et al., 1989), a chemoat-tractant for monocytes (Wahl et al., 1987), and otherhumoral factors, such as glioma derived chemotacticfactors (GDCF)-1 and 2, which stimulate the clonalexpansion of microglial cells. In turn, microglia se-crete cytokines such as interleukin-1 (IL-1), IL-6 andtumour necrosis factor α (TNF-α), which regulate theproliferation of astrocytes and OPCs (Barron, 1995; Ar-nett et al., 2001; Gehrmann et al., 1995). Furthermore,

NG2 expression in malignant brain tumours 515

growth factors such as PDGF, FGF-2, IGF-1, and EGFthat are released during tumour progression are mito-gens for vascular endothelial cells and NG2 expressingmural cells (Yamamoto & Yamamoto, 1994; Grako &Stallcup, 1995). Indeed, release of endogenous PDGFmediates the migration and proliferation of mural cellsafter balloon catheter-induced injury (Ferns et al., 1991).NG2 cells accumulate in the rat aortal intima follow-ing balloon angioplasty injury, and are abundant inartherosclerotic arteries obtained from human biopsies(Grako, et al., 1995).

While NG2 expression appears to be consistentwith increased proliferation rates and coincident withKi-67 labelling in adult glioma and levels increasedwith increasing histological grade of malignancy(Pilkington et al., 1993; Shoshan et al., 1999; Chekenyaet al., 1999), recent findings from our laboratories sug-gest that intrinsic brain tumours of childhood maynot share these relationships (Pilkington, unpublishedobservations). Indeed from a series of 65 paediatricintrinsic brain tumours (pilocytic astrocytomas, diffuseastrocytomas, ependymomas, dysembryoplastic neu-roepithelial tumours, supratentorial primitive neuroec-todermal tumours (PNETs) and medulloblastomas),there was no evidence of a consistent positive correla-tion between NG2 expression and proliferation as de-termined by Ki-67 labelling in paraffin embedded waxsections. Surprisingly, in the low grade malignancy pi-locytic astrocytomas, the majority of neoplastic cellswere positive for NG2. This begs the question of therole of NG2 in paediatric brain tumours. A further studyon the precise nature of the ECM in these tumours ascompared with their adult counterpart intrinsic braintumours is planned. A second interesting finding alsoemerged where supratentorial PNETs were invariablyNG2 positive while in infratentorial medulloblastomasstaining appeared to be confined to the vascular com-ponent. This is, perhaps, suggestive of a different pro-genitor cell origin between the histologically similarsupratentorial PNETs and medulloblastomas.

Precursor cells in tumour targeted therapy

The cornerstone of conventional treatment of malignantprimary brain tumours has been a combination of sur-gical debulking, radiation therapy and chemotherapy.Due to the limited successes of these therapeutic strate-gies, alternative approaches such as immunotherapyand anti-angiogenic therapy are currently under eval-uation in pre-clinical and clinical trials.

MONOCLONAL ANTIBODY DIRECTED THERAPY

The anti-tumour effects of monoclonal antibodies havebeen achieved by (1) blocking tumour-dependentgrowth factor activation, (2) by enhancing immune-mediated cytotoxicity and (3) by specifically delivering

Table 2. Pre-clinical and clinical immuno-therapeuticapproaches targeting NG2 epitope.

Antibody Class Conjugate Species

9.2.27 γ 2A 125I, Diptheria Toxin, HumanDoxorubicin

F(ab′ )2 Mel-14 IgG2A 125I; 131I; 123I; Murine211Astatine

Phage decapeptides – – Synthetic

toxins and radioisotopes to tumour cells (Ashley et al.,1997). The therapeutic efficacy of antibody directed im-munotherapy has been limited by several shortcomings(Epenetos et al., 1986), including (1) the loss or down-regulation of antigenic epitopes on tumour cells, (2)lack of tumour specific antigens, (3) antibody toxicity,(4) failure of antibodies to cross the blood brain bar-rier (Neuwelt et al., 1987) (5) high interstitial pressureleading to poor diffusion of antibodies into all parts ofthe tumour (Jain & Baxter, 1988); (6) dehalogenation orremoval of radioactive label from mAb, (7) enzymaticremoval of mAb from tumours, and (8) lack of mAbhumanisation. Nonetheless, clinical trials and severalpre-clinical experimental studies targeting the tumourand vessel associated NG2 have been developed, (seeTable 2).

The 9.2.27 mAb belongs to the γ 2a immunoglob-ulin subclass and recognises the NG2 proteoglycan(Morgan et al., 1981; Bumol & Reisfeld, 1982), alsoknown as the human melanoma proteoglycan (HMP),because the majority of melanomas extensively expressit. The antibody recognises the human NG2 proteogly-can, whose amino acid sequence is 84% homologousto the rat NG2. The NG2/HMP epitope is highly ex-pressed in vivo, where it has been detected on >90% ofmelanoma cells (Harper & Reisfeld, 1983; Bumol et al.,1983; Burg et al., 1998). The 9.2.27 mAb has been cho-sen as a drug-targeting device because it effectively tar-gets and has a high affinity for melanoma cells. Theantibody does not cause antigen modulation in vivoand expression of the NG2/HMP antigen it recognizesis not cell cycle dependent (Lindmo et al., 1984). Theresults of phase 1 clinical trials indicated that whenadministered intravenously in excess of 250 mg tomelanoma patients, it effectively covered all skin le-sions. The first experiments that explored the use of the9.2.27 mAb to treat human melanomas radio-labelledit with 125I and assessed the radio-localisation to thetumour compared to normal tissues in mice (Hwanget al., 1985). The 125I-9.2.27 localised preferentially inhigh antigen-expressing melanomas but not in low anti-gen expressing tumours. In addition, the uptake of theradioactive antibody into the tumour was dependenton the size of the tumour, where larger tumours re-tained a greater amount of the antibody compared to

516 CHEKENYA and PILKINGTON

the small tumours. Conversely, the specific radioactiv-ity (cpm/mg of tumour) was inversely proportionalto tumour size, probably because either (1) a smalltumour would be exposed to more mAb or, (2) thefocal necroses in large tumours may decrease the to-tal antigen/cell and thus the specific radioactivity or,(3) the larger and more necrotic tumours may have ahigher proteolytic activity leading to degradation ofantigen, resulting in loss of the bound mAb. Althoughthe 125I-9.2.27 mAb was capable of selective localisationin vivo, the proportion of the total injected dose that ac-cumulated in the tumour mass was quite small andit was converted into a non-immunologically reactivemonomeric form. The conclusion was that, not only washigh antigen density required but also, a radioisotopewith high activity emissions was needed. Indeed, with125I-9.2.27 conjugates, tumour sizes below 6-8mm couldnot be imaged but when the 9.2.27 mAb was conju-gated to 111diethyleletetraaminepentaaceticacid, barelypalpable tumours could be imaged by scintigraphy(Epenetos et al., 1984).

Parallel studies to the above radio-immuno conju-gates, investigated the possibility that a diphtheriatoxin A chain (DTA) and mAb 9.2.27 conjugate couldbe used in immunotherapy of NG2/HMP expressingmelanomas in athymic nude mice (Bumol et al., 1983).Specifically, they compared the serological reactivity,toxic activity, and tumour-growth inhibitory proper-ties of the MAb and its DTA conjugate. They foundthat although both 9.2.27 IgG and 9.2.27-DTA conju-gates had high serological reactivity and suppressed tu-mour growth in vivo, the latter only exhibited a 2–3 daymaximum in vitro toxicity toward the melanoma targetcells. The experimental immunotherapy only partiallysuppressed the growth of established tumours in vivo,due to difficulties in optimising suitable delivery routesand the maintenance of stable 9.2.27-DTA conjugatesin vivo.

Meanwhile, Yang and co-workers conjugated thechemotherapeutic drug, doxorubicin (DXR) to the9.2.27 mAb via an acid-sensitive linker, cis-acotinic an-hydride (Yang & Reisfeld, 1988a, b), which preservesboth antibody and DXR activities (Diener et al., 1986;Shen & Ryser, 1981). The 9.2.27-DXR conjugates specif-ically suppressed the growth of established humanmelanoma xenografts in athymic mice while markedlyincreasing the life-span of the animals. 30 days aftertumour cell innoculation, four nude mice in the exper-imental group treated with mAb 9.2.27-DXR remainedtumour free while the remaining eight animals devel-oped small lesions at a much slower rate than controlanimals. These animals also showed an 81% increasein life-span compared to 27% with DXR treatmentalone.

Colapinto and co-workers utilised the F(ab′)2 frag-ment of the mAb Mel-14 (Colapinto et al., 1988).Me1-14 is a murine IgG2a monoclonal antibody that

recognizes the NG2/HMP proteoglycan and it reactswith most melanoma, glioma, and medulloblastomacell lines (Carrel et al., 1980). The Mel-14 mAb bindsto tumour and vascular cells in biopsy samples of hu-man glioblastoma and to most primary and metastaticmelanomas (Behnke et al., 1988). It does not react withnormal human brain (Behnke et al., 1988). The F(ab′)2fragment of Me1-14 localizes specifically in paired-labelstudies to human melanoma and glioma xenografts inathymic mice (Buchegger et al., 1986) and has been ad-ministered and shown to localise specifically and simi-larly in human gliomas (Zalutsky et al., 1990; Bucheggeret al., 1986; Behnke et al., 1988). Doses of up to 20 mgof the Me1-14 F(ab′)2 fragment, trace labeled with 131I,125I, or 123I, have been administered intravenously orin the carotid artery to glioma patients with no toxic-ity. Therapeutic efficacy of systemically administered131I-labeled Me1-14 F(ab′)2 has been shown by survivalprolongation in mice bearing intracerebral human D54MG human glioblastoma xenografts, following admin-istration of up to 2 mCi per animal of radiolabeledMe1-14 F(ab′)2 (Colapinto et al., 1990). Estimated ra-diation dosimetry revealed that the 131I-labeled Me1-14F(ab′)2 delivered substantially higher radiation dosesto the tumour than to the liver, spleen and blood(Colapinto et al., 1988). Unfortunately, the small sizesof the experimental animals precluded the collection ofbone marrow for Fc receptor binding analyses.

Therapeutic efficacy of 131I-conjugated Mel-14 mAbhas been evaluated in Phase I clinical trials for thetreatment of residual disease after surgical resection inglioma patients (Bigner et al., 1995). Long term survivalhas recently been reported in a Phase I trial of a patientwith neoplastic meningitis secondary to melanoma,treated with 131I-labeled Me1-14 F(ab′)2 (Cokgor et al.,2001). Currently, the patient remains neurologicallynormal except for a mild bilateral hearing loss morethan 4 years after treatment and has no radiographicevidence of neoplastic meningitis.

Although encouraging results were obtained in somepatients using 131I-radiolabeled Me1-14 F(ab′)2, treat-ment with Astatine-211 radionuclide (211At) labelledMe1-14 F(ab′)2 mAb was expected to be more effica-cious due to the short range and high linear energytransfer (LET) of the α particles emitted by 211At. Thecytotoxicity and microdosimetry of 211At labelled Me1-14 F(ab′)2 was investigated for glioma and melanomacells in vitro (Larsen et al., 1998). Further studiesare required before the utility of this radioisotope isascertained.

PHAGE DISPLAY DECAPEPTIDE DIRECTED THERAPY

In order to circumvent the problems associated withantibody directed immunotherapeutic strategies, analternative approach can involve using small pep-tides that are capable of targeting both the tumour

NG2 expression in malignant brain tumours 517

cell and their neovasculature. Phage display of ran-dom peptide libraries has enabled isolation of peptidescapable of binding to integrin receptors (Murayamaet al., 1996; Koivunen et al., 1995, 1999a, b), growth fac-tors (Yanofsky et al., 1996) and other tumour associatedproteins (Goodson et al., 1994; Pennington et al., 1996).NG2 binding decapeptides that home directly to theangiogenic neovasculature of melanoma xenografts inmice have been developed (Burg et al., 1999). The lo-calisation and accessibility of NG2 on pericytes sug-gested a potential use of NG2 homing sequences for tar-geted delivery of therapeutic agents to tumours. Severalreports have suggested that pericytes play an impor-tant role in controlling endothelial cell proliferationand vessel stabilisation during angiogenesis (Lindahl &Betsholtz, 1998; Lindahl et al., 1997; Sims, 1986; Hirschi& D’Amore, 1997). Thus, anticancer strategies target-ing pericytes in the angiogenic vasculature may com-plement approaches targeting endothelial cells. SinceNG2 is also expressed on tumour cells (Behm et al., 1996;Leger et al., 1994; Schrappe et al., 1991), the peptidescould deliver therapeutic agents both to the tumourcells and their vasculature. This offers several advan-tages over therapies that are strictly tumour-directed.Probes that target tumour cells, are limited by both theheterogeneous expression of the antigens within the tu-mour, as well as by the high rate of tumour cell mutation(Burrows & Thorpe, 1993; Folkman, 1995). In contrastcells that comprise tumour vasculature are relativelyhomogenous and lack the problems associated withdrug resistance (Kerbel, 1991, 1997; Boehm et al., 1997).The small peptides may prove superior to antibodies interms of penetration into tumours.

Other methods for targeted knock-down of NG2in brain tumours and other pathological conditionsthat over express NG2 could be attempted by an-tisense technologies. Pharmacological protocols thatlimit the macrophage or OPC response to injury wouldbe contra-indicated in brain tumour patients as sup-pression of the immune system is a serious side-effectof most treatment regimens.

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