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UTMJ • Volume 87, Number 3, May 2010156

generate secondary spheres.20-22 The presence of NSCs in theneurospheres is confirmed by their simultaneous expression of neural (Figure 2, B and C) and stem-cell specific markers by immunohistochemistry.20-22 The multipotency of NSCs can alsobe tested in culture; when plated on adhesive substrates without

the mitogens EGF and bFGF, NSCs differentiate into neurons,astrocytes and oligodendrocytes.2,20-22

Figure 2. Neural stem cells (NSC) and their characterization in culture

systems. NSCs undergo highly regulated, self-renewing divisions to

regenerate themselves (Panel A). They are multipotent and generate line-

age-committed progenitor cells that can then differentiate into the three

main neural cell types: neurons, astrocytes and oligodendrocytes. NSCs

derived from healthy adult brains can be grown in culture to give rise to

floating aggregates or neurospheres (inset). NSCs in the spheres can bedouble immunolabeled with antibodies against astrocyte markers (glial fib-

rillary acidic protein (GFAP), blue, Panel B) and neuronal markers (b-III

tubulin, green, Panel C). Adapted from: Singh SK, Clarke ID, Hide T, Dirks

PB. Cancer stem cells in nervous system tumors. Oncogene 2004;23:7267-

727; and Ignatova TN, Kukekov VG, Laywell ED et al. Human cortical glial

tumors contain neural stem-like cells expressing astroglial and neuronal

markers in vitro. Glia 2002;39:193-206, with permission.

Stem-like Cells can be Isolated from GBM Tumours

To give credibility to the NSC→CSC theory, it was important to show that GBM tumour specimens contained populations

with stem-cell like properties. To this end, several groups showedthat a small fraction of cells from dissociated GBM tumourscould proliferate in suspension as neurospheres that were mor-

phologically indistinguishable from normal NSC derived neu-rospheres.22-28 These cell aggregates could be dissociated repeat-edly at the level of a single cell to give rise to new neurospheres(subspheres),22,23,25-28 establishing their self-renewal ability.Unlike normal NSCs, tumour-derived cells were alive longer,23

with enhanced abilities to give rise to new subspheres.25 Further, when differentiated cells were switched to proliferating condi-tions, only GBM derived cells gave rise to new neurospheres,demonstrating their efficiency in maintaining the CSC pool.25

Immunohistochemistry was used to compare molecular markersexpressed by clonal neurospheres grown under proliferation anddifferentiation conditions. Undifferentiated tumour derived

neurospheres could be stained for the NSC markers Nestin andMusashi-2 (Figure 3, A and B).14,22-28 Salmaggi et al. used ELISAs toshow that GBM neurospheres secreted vascular endothelial growthfactor (VEGF),28 which is implicated in endothelial cell migrationand may be important for tumour angiogenesis.2,29 Like NSCs,

clonally derived GBM cells underwent differentiation on mitogenremoval.22-28 The majority of clones from each tumour gave rise tocells resembling both neurons (expressing b-III-tubulin) and glia(expressing glial fibrillary acidic protein, or GFAP) (Figure 3C).22-

28 The NSC, neural, and astrocytic markers could also be seen inthe original tumour tissue (Figure 3, D and E).22-28 Interestingly,only tumour derived cells gave rise to neurospheres with abnor-mal, dual phenotype cells expressing b-III-tubulin and GFAP(Figure 3C).22-25 The percentage of differentiated cell types gener-ated varied considerably from one tumour to another,15,23 whichmay be related to the heterogeneity of GBM tumours seen in vivo .

Figure 3. Cultured tumour-derived neurospheres are multipotent, andretain immuno-histochemical properties of original tumour samples.Adapted from: Hemmati HD, Nakano I, Lazareff JA et al. Cancerous stemcells can arise from pediatric brain tumors. PNAS 2003;100:15178-15183,

with permission.

Review

Does Glioblastoma arise from Oncogenic Transformation of Neural Stem Cells into Cancer Stem Cells?

A B

CTumour Derived Neurospheres:

Undifferentiated neurospheres from a representative tumour could be

immunostained for expression of the NSC markers Nestin (green, Panel A)

and Musashi (green, Panel B). Under differentiating culture conditions

(Panel C), neural and glial cells could be detected in the neurospheres, by

staining with anti b-III tubulin (Tuj1, red) and anti-GFAP (green) antibodies.

Some cells (indicated by arrows) simultaneously expressed both neural

and glial markers.

Original Tumours:

Paraffin-embedded sections of original tumours could also be labelled with

antibodies to Nestin (green, panel D), Tuj1 (red, Panel E) and GFAP (green,

Panel E). Counterstaining for nuclei with DAPI (blue, Panels B, D, and E) is

indicated.

Semi-quantitative RT-PCR was used to analyze expression of stem cell

markers in a representative tumour and tumour derived neurospheres

(NS). Like control (normal brain) tissue, GBM tumours expressed the stem

cell marker CD133. However, tumour NS over expressed genes involved in

NSC proliferation, i.e. BMI-1 and SOX2 (-, no/minor signal; +, weak signal;

++, moderate signal; +++, strong signal).

A B C

D E C

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Figure 4. Tumour forming ability of intracranially injected neurosphere

and non-neurosphere forming cells. Adapted from: Yuan X, Curtin J,

Xiong Y et al. Isolation of cancer stem cells from adult glioblastoma mul-

tiforme. Oncogene 2004;23:9392-9400, with permission.

CD133+ CSCs May Comprise the Tumour-Initiating Cells

As the above assays represent retrospective analyses of CSCphenotype and function, a few groups also prospectively isolatedCSCs to confirm their presence in tumour specimens.

Dissociated brain tumour cells were fractionated into CD133+and CD133- populations by fluorescence activated cell sorting(FACS); in culture, only the CD133+ cells proliferated togenerate self-renewing neurospheres that, when differentiated,recapitulated the phenotype of the parental tumour.27,30-32

CD133, a cell surface protein that was originally described as ahaematopoietic stem cell marker,9,14,17 may also be useful to iden-tify brain CSCs, as these studies have shown. The numbers of CD133+ cells derived from different brain cancer samplesshowed correlation with the aggressiveness of the originaltumour.17,31,32 For example, neurospheres derived from low-grade gliomas contained ≤5% CD133+ cells, whereas up to 30%of the cells in GBM derived spheres expressed the marker.32

When affinity purified with an antibody against CD133, as few as

100 CD133+ cells from the tumour neurospheres initiatedserially transplantable tumours in NOD/SCID mice (Figure 5A),

whereas up to 100,000 injected CD133- cells failed to inducetumour formation.30-32 Further, transplantations of constant amounts of tumour cells with increasing percentages of CD133+cells increased the growth and vascularization of resultant tumours (Figure 5B).31

UTMJ • Volume 87, Number 3, May 2010 157

GBM Neurosphere Derived Cells Aberrantly Express

NSC-specific Genes

RT-PCR based genetic profiling studies showed that GBMtumour specimens had normal expression of the stem cellmarker CD133 , but overexpressed genes required for NSC pro-

liferation, including SOX2 (Figure 3F).16,25,26 Interestingly, someCD133 expression was detected in undifferentiated tumour-derived, but not control neurospheres. This suggests that theculture conditions may have selected for and allowed expansionof stem-like cells in the tumour samples. Tumour-derivedneurospheres also overexpressed the NSC genes BMI-1 and SOX2 .Overexpression of BMI-1 was previously shown to modulate theSonic Hedgehog growth signalling pathway, by transcriptionalrepression of the tumour suppressors INK4A and ARF.2,6,14

Accordingly, deletions of the INK4A and ARF loci induced GBMin the NSC containing regions of murine brains.8 The functionof the transcription factor SOX2 is not clear, although it isbelieved to be crucial to NSC maintenance, based on impairedneurogenesis and neurological defects in SOX2 knockout mice.6

The molecular signatures of undifferentiated GBM-derived cellsmirrored those of the parental tumours (Figure 3F), and wereunique from patient to patient,26 which suggests that geneexpression profiles specific to a given tumour could help developindividualized therapies for GBM patients.

GBM Neurosphere Derived Cells Can Initiate Tumours

in Immunocompromised Mice

To validate the hypothesis that CSCs were indeed isolatedfrom the GBM specimens, several groups demonstrated that these cells had tumourigenic potential. Neurospheres derivedfrom pediatric GBM tumours were shown to be able to engraft,migrate, proliferate, and differentiate in neonatal rat brains.11

This finding was expanded in non-obese, diabetic, severe com-bined immunodeficient (NOD/SCID) mice; transplanted GBM

neurospheres reproducibly gave rise to heterogeneous, GBM-like tumours,25,26,28 whereas normal human NSCs,28 commercial-ly available GBM cell lines,26 and non-neurosphere formingcells25 did not (Figure 4, A-I). The success of engraftment wasgreater when neurosphere-derived cells were injected ortho-topically (as opposed to subcutaneously),26 which emphasizesthe importance of the right microenvironment to support tumour growth. Two groups also demonstrated the tumouri-genic potential of the GBM-neuropsheres in serial transplanta-tion assays.25,26 Cells isolated from tumours in primary recipients

were re-cultured to give rise to new neurospheres that retainedmultipotentiality, could be identified by human specificmarkers, and resembled their parental line in gene expressionprofile.25,26 When transplanted into secondary recipients, these

cells could recapitulate tumour formation.25,26

Review


Cells isolated from GBM neurospheres were able to form GBM-like tumours

(Panels A, B), but non-sphere forming cells did not (H&E stained in Panel C),

even when implanted at 50-fold excess of the GBM sphere cells.

Tumours developed in the mice brains could be stained by human-

specific antibodies against NSC markers Nestin (green, Panels D and E),

and CD133 (red, Panel G). Panel I is a merged version of Panels G and H.

Counterstaining for nuclei with DAPI (blue, Panels F, H) is also indicated.

A B C

D E F

G H I

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Review

Figure 5. GBM derived CD133+ cells form tumours on intracranial trans-

plantation into NOD/SCID mice. Adapted from: Bao S, Wu Q, McLendon

RE et al. Glioma stem cells promote radioresistance by preferential acti-

vation of the DNA damage response. Nature 2006;444:756-760; and

Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour-

initiating cells. Nature 2004;432:396-401, with permission.

CD133+ CSCs May Comprise the Treatment Resistant

Cell Population

It is tempting to propose that acquired mutations in any tumour cell that confer treatment resistance may lead to selectiveclonal expansion of that cell. However, the alternate hypothesisthat CD133+ CSCs may comprise the tumour-initiating, treatment-resistant subpopulation of cells in GBM has been supported by recent studies. Liu et al. exposed CD133+ and CD133- CSCs (FACSsorted from GBM cultures) to conventional chemotherapeuticagents, i.e., temozolomide, and showed that cell proliferation of the CD133+ cells was less sensitive to the drugs.27 The CD133+pool also overexpressed stem cell genes known to enhance drug-

resistance, i.e., the multi-drug resistance (MDR) gene BCRP-1,DNA-mismatch repair genes including MGMT, and apoptosis-inhibiting genes including BCL-2.27 These findings were corrobo-rated by FACS analyses showing significantly higher expression of MDR proteins by GBM tumour neurosphere cells compared tothose derived from control NSC neurospheres.28

The importance of characterizing the CD133+ CSC to developGBM treatment strategies was highlighted by a study that identified surface receptors for bone morphogenic proteins(BMPs), soluble factors that induce NSC→astrocyte differentia-tion, on CD133+ human GBM cells.33 In culture, the addition of exogenous BMP reduced the proliferation of GBM-derived

CD133+ cells, and led to reduced tumour size/malignancy whentreated cells were grafted into immunodeficient mice.33 In thisstudy, the small size of the secondary tumours precluded isola-tion of enough CD133+ cells for further characterization or seri-al transplantation studies.33 Nonetheless, the data implied that

GBM tumours contain hierarchically organized, stem-cell-basedpopulations.

The mechanism of radiation resistance of the CD133+ cells was recently evaluated by Bao et al., who isolated tumour cellsfrom human GBM specimens and murine xenograft tumours.31

On exposure to high dose radiation ex vivo , the percentage of CD133+ in cultured neurospheres increased. When serially transplanted, the irradiated CD133+ cells yielded more aggres-sive tumours.31 CD133+ cells from original and transplantedtumours showed enhanced activation of checkpoint proteins inresponse to radiation-induced DNA damage, repaired theirdamaged DNA damage more efficiently than CD133- cells, andshowed resistance to apoptosis.31 Further, their radiationresistance could be pharmacologically inhibited by blocking the

checkpoint kinases Chk1 and Chk2.

31

These studies may haveidentified potentially targetable vulnerabilities in CSCs. Forfuture studies, it should be interesting to evaluate whether thesupportive stroma of NSC niches contributes to the radiationresistance of CD133+ CSCs.

NSCs as Candidates for Oncogenic Mutations in GBM

Indirect evidence implicating NSCs, even before their disco- very, as the cells of origin in GBM came from animal studies where the exposure to oncogenic viruses or administration of carcinogens resulted in greater numbers of tumour formation inthe SVZ2,8,14 – a NSC niche. Further indirect support is providedby the long lifespan and self-renewal abilities of NSCs,6,8,20 whichincrease the possibility for these cells to acquire mutations.

Although astrocyte and oligodendrocyte generations have simi-

larly long lifespans, they are also quiescent in vivo ,20 makingthem unlikely targets for transforming mutations. Genetic pro-filing studies have shown that oncogenic mutations in GBM aremost frequent in genes that encode regulatory proteins involvedin growth factor signaling cascades (i.e., Sonic Hedgehog path-

way) or cell cycle control (i.e., Chk1 and Chk2) known to mod-ulate NSC proliferation.2,6,16,25,26 Further support for NSCs asGBM cells of origin was seen in a transgenic mouse model,

where genes encoding activated forms of Ras and Akt were trans-ferred to astrocytes and neural progenitors in a tissue-specificmanner.34 The combination of Ras and Akt induced GBM only after transducing neural progenitor cells, but not astrocytes.34

Although the disruption of regulatory mechanisms may indeed be involved NSC→CSC pathway, these correlational

findings may merely represent mechanistic similarities betweenNSC and CSC renewal. Several murine studies also suggest that,given the right combination of genetic aberrations that interrupt critical signalling pathways, mature neural cells can also causeGBM. Bachoo et al. retrovirally transfected a constitutively activeform of epidermal growth factor (EGF) receptor in either NSCsor astrocytes from Ink4a/Arf-/- mice.4,8,35 Both the mutated NSCsand astrocytes were capable of inducing high-grade gliomas

when they were transplanted in the brains of NOD/SCID mice.35

In another study, a human astrocyte cell line was retrovirally transfected with human telomerase reverse transcriptase, humanpapillomavirus 16 E6/E7 (to inactivate tumour suppressors


Patient Xenograft

M A P 2

G F A P

M I B - 1

N e s t i n

H & E

Panel A: CD133+ cells

from a GBM patient

transplanted in a NOD/SCID

recipient resembled the

original patient tumour.

H&E stained sections of thexenograft showed

histological features of GBM.

Immunohistochemically,

both the xenograft and the

patient’s original tumour

showed expression of

Nestin, had high proliferative

indices (MIB-1/Ki67

staining), and expression of

both astrocytic (GFAP) and

neuronal (MAP2) markers.

Panel B: CD133+ expressing

cells from a human GBM

specimen were sorted by

FACS and mixed withCD133- cells in different

ratios, for injection into

mouse brains (100,000 total

cells per mouse). Gross

morphology of mouse

brains bearing

secondary tumours showed

correlation between the

percentage of CD133+ cells

injected, with tumour size

and vascularity.

A

B

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UTMJ • Volume 87, Number 3, May 2010 159

p53 and Rb), and activated Ras/Akt.36 When intracranially trans-planted into immuno-compromised rats, the transformed astro-cytes were able to initiate GBM-like tumours.36

Study Limitations

A number of studies have shown that a stem-cell like popula-tion can be isolated from GBM tumour specimens, and that these cells can initiate GBM when transplanted into immuno-compromised mice. However, a number of questions still needto be addressed. It is unclear whether the tumour heterogeneity in GBM is caused by differentiation of CSCs or merely repre-sents the unstable gene expression pattern inherent in cancercells.12 This complexity is compounded by the fact that,although most studies showed cancer-initiation by CSC enrichedcell populations, CD133+ CSCs were not purified to homogene-ity from GBM samples, based partially on the lack of availablemarkers to identify these cells.27,30-32 Additionally, these cells havenot been shown to be conclusively of neural origin,27,30-32 likewise

with the relative lack of characterization of the newly identified

NSCs.It is difficult to accept findings from studies based largely oncell culture systems as a layer of cells in a culture dish is vastly dif-ferent than the dynamic environment of the human brain andmay lack key players involved in disease pathogenesis in vivo .The current studies extensively used neurosphere cultures toassess CSC presence in GBM tumour samples. Cell proliferationin this system is dependent on their responsiveness to EGF andbFGF,37 which may not represent the niche where GBM tumoursare growing in the brain. Depending on plating density, neu-rosphere formation was previously shown to result from sponta-neous cell aggregation of mature cells.37 The multipotency of neurosphere-forming cells was assessed by immunohistochemi-cal staining, without functionally confirming the identity of thedifferentiated cells.37 The formation of self-renewing neu-

rospheres that differentiated into multiple cell types thus repre-sent supportive, but not conclusive data on CSC isolation fromGBM tumours.

The use of immuno-compromised or transgenic mice tomodel human GBM adds some difficulty to data interpretation.This is due, firstly, to the well known differences between miceand humans, allowing limited extrapolation between species.Secondly, the tumourigenic potential of CSCs was shown by xenotransplantation in mice lacking an immune system, whosebrain microenvironments may differ significantly from thosesupporting CSC generation or tumour growth in healthy sub-

jects. Lastly, in studies that generated transgenic mice, often-times multiple proteins were mutated to manifest the full spec-trum of clinical abnormalities. It is currently unclear how close-

ly these genetic backgrounds resemble those actually found inhuman GBM.5 The use of non-human primates or other largeanimals for future studies may be helpful for characterizing theinvolvement of CSCs in GBM.

There is some evidence to suggest that NSCs may comprisethe CSC population in GBM, although conflicting data showedthat differentiated cells can also give rise to cancer, given theright combination of mutations.4,8,35,37 These findings suggest that, during tumour progression, CSCs could spontaneously arise at any time from a cell acquiring de-differentiating muta-tions. Further questions regarding NSCs as the CSC source inGBM are raised by the possibility that CSCs from other tissues

can migrate to and initiate cancer in the brain.38 For example,adult haematopoietic stem cells injected into the brain wereshown to home towards distant brain tumour sites,4,20 throughinteractions between CXCR4 on their surface and thechemokine SDF-1 (stromal cell derived factor 1).20 Interestingly,

circulating hematopoietic stem cells have been shown to fuse with various target cells, including cardiac myocytes and oligo-dendrocytes, producing hybrid cells with abnormal gene expres-sion patterns.4 In GBM, fusion between migratory hematopoiet-ic cells with normal neural cells could endow resultant hybrids

with CSC properties,4 including enhanced proliferation, infiltra-tion, and radiation/chemotherapy resistance.

Conclusion

A CSC population appears to play an important role in seed-ing and maintaining tumours in GBM. This was shown by theirprospective isolation from tumour specimens, demonstration of their self-renewing and multipotent properties, and recapitula-tion of heterogeneous tumours when CSCs were serially trans-

planted into NOD/SCID mice. The theory that GBM tumoursdepend on a small population of CSCs for their long-termgrowth and propagation provides an explanation for why con-

ventional therapies fail. Retrospective characterization of CSCsidentifed possible mechanisms for their treatment resistance,and identified potentially targetable genetic abnormalities inthese cells. However, given the available data, it is not clear

whether CSCs arise from NSCs, or mature neural cells that de-differentiated to unmask latent stem cell properties.

Alternatively, a migratory stem cell from another organ may act as or fuse with a normal brain cell to become a CSC. For clinicaltranslation, further characterization of CSCs is needed to iden-tify ways in which they differ from normal stem cells. Insight intothe molecular characteristics of CSCs, including the identifica-tion of cell-specific markers, analysis of their gene expression

profile, identification of the signalling pathways and microenvi-ronment required to support their growth and maintenance,could potentially result in effective drugs for this currently incur-able malignancy.

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