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cular and Cellular Pathobiology

ermal Growth Factor Receptor Expression Identifiesctionally and Molecularly Distinct Tumor-Initiating Cellsuman Glioblastoma Multiforme and Is Required

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Gliomagenesis

ia Mazzoleni1,5, Letterio S. Politi2, Mauro Pala6, Manuela Cominelli7, Alberto Franzin3,
Sergi Sergi4, Andrea Falini2, Michele De Palma4, Alessandro Bulfone6, L. Poliani7, and Rossella Galli1
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ermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognosticr of human glioblastoma multiforme (GBM). However, its functional role and biological significanceM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from theens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFRf other putative CSC markers. All these subpopulations are molecularly and functionally distinct, areigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-sing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype.ingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBMnes enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fun-tal role in gliomagenesis. These findings open up the possibility of new therapeutically relevant
damen
scenarios, as the presence of functionally heterogeneous EGFRpos and EGFRneg TIC subpopulations withinthe same tumor might affect clinical response to treatment. Cancer Res; 70(19); 7500–13. ©2010 AACR.

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he last 10 years, the identification of putative cancerells (CSC) in different types of tumors has raised manytations. CSCs represent a rare fraction of the tumorniquely responsible for tumor initiation and progres-nd, as such, are envisioned as elective targets of ther-1, 2). The CSC model of tumorigenesis has beencingly validated in many hemopoietic malignanciesd in solid tumors such as breast and colon cancers

, in other tumors, CSCs seem to make upcells within the tumor bulk and all exhibit

gene adefineand poalso bdard stheseapproHer

expresrole ipatienlines,teristilines (tiple sdistincbeingfeatur

ons: 1Neural Stem Cell Biology Unit, Division ofcine, Stem Cells and Gene Therapy, 2Neuroradiology, 3Neurosurgery Unit, and 4Angiogenesis and Tumorh Unit, Telethon Institute for Gene Therapy, Divisionedicine, Stem Cells and Gene Therapy, San Raffaele; 5Vita-Salute San Raffaele University, Milan, Italy;la, Cagliari, Italy; and 7Department of Pathology,ia-Spedali Civili of Brescia, Brescia, Italy

tary data for this article are available at Cancerhttp://cancerres.aacrjournals.org/).

uthor: Rossella Galli, Neural Stem Cell Biology Unit,rative Medicine, Stem Cells and Gene Therapy, SanInstitute, Via Olgettina 58, 20132 Milan, Italy. Phone:Fax: 39-02-2643-4621; E-mail: [email protected].

5472.CAN-10-2353

ssociation for Cancer Research.

19) October 1, 2010

Research. on April 22, 202cancerres.aacrjournals.org aded from

-initiating ability, thus being more appropriately called-initiating cells (TIC; refs. 8–11).s last model might also apply to glioblastoma multi-(GBM), as soundly shown by recent studies in culturednes (12, 13). To test whether this concept might holdspecimens from patients with primary GBM, we se-the epidermal growth factor receptor (EGFR) as a pro-ve GBM CSC marker (14, 15). EGFR expression in GBMth diagnostic and prognostic significance (16–19) andssociated with tumor progression (20). Likewise, EGFRmplification is positively correlated with molecularlyd GBM subclasses, characterized by active proliferationor prognosis (21). Valuable preclinical information haseen obtained by modulating EGFR expression in stan-erum-cultured glioma cell lines (22–25) and by usingin vitro cultures for testing different therapeuticaches (26).e, we investigated the biological significance of EGFRsion in human primary GBM by assessing its functionaln tumor cell fractions purified from several GBMt–derived specimens and by validating it in GBM CSCwhich reproduce the genotypic and phenotypic charac-cs of GBM more faithfully than standard glioma cell27, 28). By this experimental strategy, we identified mul-ubpopulations of GBM TICs, each characterized by at gene signature and tumorigenic potential, and thus
able to reproduce the diverse pathologic and functionales of GBM, such as invasive and angiogenic behavior.
1. © 2010 American Association for Cancer

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tion and processing of patients' specimensspecimens were collected from patients with histo-

iagnosis of primary GBM (WHO grade 4 glioma) in ac-nce with the protocol approved by the institutionalboard of San Raffaele Scientific Institute (01-CSC07).

r diagnosis and grading were blind-reviewed by differ-thologists at two distinct institutions. Samples that didtisfy the inclusion criteria of primary GBMs (e.g., GBMligodendroglioma features) were excluded from the. See Supplementary Experimental Procedure forsing methods.

nohistochemical stainingr-micrometer sections were cut from paraffin blocksere stained with mouse antihuman EGFR, mouseD31 (all DakoCytomation), rabbit anti-SOX2 (Abcam),anti-Bmi1, and mouse anti-CD15 (Becton Dickinson).ns were then incubated with a secondary antibodyMATE Envision Rabbit/Mouse, DakoCytomation).

ular analysisal RNA from GBM tumor specimens and from CSCas extracted using the RNeasy Micro and Mini Kitsn). cDNA was obtained by using Superscript RNaseH−

e transcriptase (Invitrogen). All cDNAs were normal-the β-actin reverse transcription-PCR (RT-PCR) product.ard RT-PCR for EGFRvIII was done as described in.

escence-activated cell sortingowing enzymatic and mechanical dissociation, cellsrimary GBM tissues and from experimental xenograftsstained with rabbit antihuman EGFR (clone EGFR.1,erotec), rat antihuman CD34/CD45/CD11b (Bectonson), antihuman AC133 (Miltenyi), mouse anti-CD15MMA, Becton Dickinson), and antihuman pan-HLAn Dickinson). Cells were sorted on a Becton Dickinsonantage SE FACSDiVa. The purity of each cell fractionbetween 90% and 99%. Fluorescence-activated cell

g (FACS) analysis of CSC lines was done as described, without enzymatic digestion.

re propagation, population analysis,loningablished GBM CSCs were cultured as previouslybed (27).

array-based gene expression profilingSupplementary Experimental Procedure for details.

the lo(Dade

acrjournals.org

Research. on April 22, 202cancerres.aacrjournals.org Downloaded from

ation of CSC tumorigenicity by orthotopicntationorigenicity was determined by injecting all the dis-

cell preparations orthotopically in nu/nu mice (27).tic resonance imaging (MRI) analysis was performedcribed in Supplementary Experimental Procedure.unofluorescence analysis was performed on cryostat

ns using mouse monoclonal antihuman EGFR (1:100;chem). Details can be found in Supplementary Data.

rn blottingates from GBM tissues and CSCs were immunoblotteding to standard protocols. The primary antibodies usedrabbit anti-EGFR, rabbit anti–phosphorylated EGFR68; Cell Signaling), rabbit anti-ErbB2 (Calbiochem) andanti-Erbb3 (Santa Cruz Biotechnology), rabbit anti–horylatedmitogen-activated protein kinase (MAPK; Celling), and rabbit anti–phosphorylated Akt (Ser473; Cell Sig-). As a loading control, a mouse anti–β-tubulin antibodysed (Sigma-Aldrich).

iral vector generation, production, andfectionan wild-type (wt) EGFR cDNA (Upstate) was cloned

he monocistronic transfer lentiviral vector pCCL.sin.PGK.GFP.WPRE11. CSCs were transduced with 1 × 107

uction units/mL of lentiviral vectors for 16 hours.tiviral vectors coding for shRNAs targeted against then EGFR were purchased from Sigma (Mission RNAi).ion of CSCs was performed according to the manufac-instructions.

nofluorescence and fluorescence in situdizationunofluorescence was performed as described in

, using mouse antihuman EGFR (Calbiochem) and rab-lyclonal anti-GFP (Molecular Probes). Fluorescencehybridization (FISH) was performed using the kit fromaccording to the manufacturer's instructions. Filamen-ctin (F-actin) was stained by TRITC-conjugated phal-(Chemicon).

acologic EGFR inhibition in CSCs478 (Calbiochem) was used at the indicated final con-tions in complete medium. DMSO was used at theconcentration as the vehicle control. Cell proliferationeasured by an MTT incorporation assay.

ion assayssion assays were performed in Matrigel-coated 8-μm-Transwell chambers (Corning Costar). EGFRpos andneg CSCs were seeded in sister cultures on the upperf the chambers in complete medium and allowed tote for 5 and 7 days, respectively, with or without8. Noninvaded cells on the upper side of the filtersor were not scraped off, and those that migrated to
wer side were fixed and stained by using DiffQuickBehring). The extent of cell migration was normalized
Cancer Res; 70(19) October 1, 2010 7501



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total number of viable cells in the correspondingped culture.

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expression identifies distinct cell subpopulationshuman primary GBM and GBM CSC lines

eral GBM surgical specimens were tested for EGFRexpression by both microarrays and semiquantitativeR and all were shown to express the transcript (Table 1).er, immunohistochemistry on corresponding paraffin-ded sections showed that, in agreement with the liter-only 50% of specimens were characterized by diffusestaining, which was particularly intense in small-sizedtumor cells (EGFRpos tumor samples; Fig. 1A, Supple-ry Fig. S1). Accordingly, the other specimens displayedible EGFR expression that was restricted mostly to-infiltrating inflammatory cells (EGFRneg tumor sam-ig. 1A). Western blotting (WB) analysis confirmedmunohistochemistry findings (Fig. 1B). Hence, EGFRn distribution identifies two main distinct subtypesan GBM (16).

FISH, a subset of EGFRpos tumor specimens showedgene amplification and expression of mutant EGFRvIIIn some patients, correlated with EGFR protein levelsC and D; Table 1). Interestingly, EGFRpos tumorsined small and thin CD31-positive vessels (Fig. 1E),as EGFRneg tumors were characterized by an activelyrating vasculature [mean vessel area (±SEM), 853.1 ±nd 2,500.2 ± 615.1 μm2 in EGFRpos and EGFRneg GBMs,tively; P > 0.05, Student's t test; n = 7; Fig. 1E]. All spec-expressed similar levels of stem cell–associated

ns, such as Sox2 and BMI1 (Fig. 1F), and of glial fibril-idic protein (GFAP; data not shown).restingly, whereas immunohistochemistry indicatedGFR was expressed in the totality of GBM cells, flowetry suggested that EGFR expression could be re-only in a smaller proportion of cells. Of note, with-EGFRpos cell fraction, a conspicuous subset of cells

led with the endothelial marker CD34 and the hema-tic markers CD45 and CD11b (Fig. 1G), indicatinghey were stromal cells. The CSC marker CD133as more expressed in proper tumor cells than inal cells in EGFRpos tumors, whereas the oppositeetected in EGFRneg tumors. On the contrary, CD15lways more expressed in the stromal cell compart-in both EGFRpos and EGFRneg tumors (refs. 31, 32;mentary Table S1).s, the frequency of EGFRpos proper tumor cells incalculated after the exclusion of stromal cells, ranged% to 35% of the total tumor cell number (Table 1). Ofsome EGFRpos proper tumor cells also labeled withand/or CD15 markers, whereas EGFRneg cells did

ig. 1F and H; Supplementary Table S2).formally show that EGFR might also play a role in they of GBM CSCs in vitro, we tested 13 GBM CSC lines
usly validated for being bona fide CSC lines (Supple-ry Fig. S2). All CSC lines expressed EGFR mRNA,
genesfractio

r Res; 70(19) October 1, 2010


as only 5 of 13 CSC lines also expressed the EGFR pro-able 2). Among the EGFRpos CSC lines, only 6% to 70%s expressed the receptor, suggesting that multiple GBMpulations could also be identified based on EGFR ex-on in CSC lines.restingly, many EGFRpos and EGFRneg CSC linesned AC133pos and CD15pos cells (Table 2; Supplemen-able S3). However, one of five EGFRpos CSC lines andf eight EGFRneg CSC lines did not contain any AC133pos

D15pos cells at all.

expression identifies GBM subpopulations with active gene signaturemolecularly analyze the above-described GBM subpo-ons, we purified them from (a) EGFRpos single patients'specimens, (b) EGFRneg specimens, and (c) EGFRpos

nes.Rpos patient samples T1224, T0131, T0222, and T0321ned (a) EGFRpos/AC133neg cells (region R1), (b) EGFRpos/pos cells (R2), and (c) EGFRneg/AC133neg (R3) cells. Twoalso contained a small proportion of EGFRpos/CD15pos

Fig. 2A and B; Supplementary Table S2). Stromal cellsxcluded from sorting.ediately after cell sorting and purity assessment,

ut any in vitro manipulation, the different GBMns were subjected to transcriptional profiling by-transcript microarrays (Fig. 2C). EGFRpos cell subpo-ons displayed a specific molecular signature clearlyuishable from that of double-negative cells (R3). Ofa subset of 163 genes was differentially expresseden EGFRpos and EGFRneg cell subpopulations, evenisolated from distinct patient specimens, suggestingGFR expression in GBM might affect common mo-r determinants (Supplementary List S1). In additionFR, the differentially expressed genes (DEG) upregu-in EGFRpos versus EGFRneg GBM fractions were relat-biological processes such as tumor invasion and

ession [e.g., periostin (POSTN), LMO3, and AQP4].rsely, genes overexpressed in EGFRneg GBM fractionsostly involved in the interaction of tumor cells with

icroenvironment [e.g., PDGFRB, Notch3, PTPRK, lumi-LUM), and endothelin receptor A (EDNRA)]. Thus,contain molecularly distinct subpopulations ofcells.

en the same freshly isolated cell fractions were cul-in vitro by the NeuroSphere Assay under limiting di-conditions (33), EGFRpos cell fractions gave rise to afrequency of secondary neurospheres than EGFRneg

Fig. 2D).l subpopulations expressing different levels of EGFRlso purified from EGFRpos CSC lines (Fig. 2E–G). Im-tely after sorting, EGFRhigh (R4), EGFRlow (R5), andneg (R6) CSC subpopulations were validated by WBH) and subjected to transcriptional profiling, which in-d that each subpopulation was molecularly distinctthe others (Fig. 2I; Supplementary List S2). Of note,
upregulated in EGFRhigh and EGFRlow versus EGFRneg
ns were also highly expressed in the most malignant

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T01 M + mT01 M + mT01 M + mT01 /F + mT03 M + mT0512 IV 75/F 1+ 0 No amplification 5–10 1+T1003 IV 65/M 1+ 0 No amplification 5–10 1+

Patient code Total EGFRpos cells (%) EGFRpos tumor cells (%) EGFRpos stromal cells (%)

EGFRpositive GBMsT0131 20.8 4.0 16.8T0222 76.8 35.2 41.6T1210 13.7 10.7 3.0T1224 78.1 20.3 57.8T0418 36.6 12.4 24.2T0625 22.4 8.9 13.5T0321 39.1 21.7 17.4

EGFRnegative GBMsT0104 4.9 0.9 4.0T0109 17.3 0.0 17.3T0125 19.0 1.6 17.4T0130 25.3 3.3 22.0T0325 0.0 0.0 0.0T0512 4.4 0.0 4.4T1003 7.5 0.0 7.5

NOTE: FISH scores: high gene amplification, >10 gene copies in >10% of the cells; low gene amplification, 6 to 10 gene copies in>10% of the cells; high polysomy, >4 gene copies in >40% of the cells; low polysomy, >4 gene copies in >10% but <40% of thecells; no amplification, 1 to 4 gene copies. Immunohistochemistry scores: 3+, very intense membrane staining; 2+, intensemembrane staining; 1+, moderate staining; 0, no staining.


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ular subclasses of primary GBM (i.e., the proliferativeesenchymal subtypes, as identified in refs. 21 and 34;ementary Fig. S3). Likewise, genes downregulated inos fractions were also downregulated in the same ma-t molecular subtypes.hown by clonogenic assays performed under limitingn conditions and by long-term population analysis,

low and EGFRneg cell fractions displayed the highestrative capacity, whereas EGFRhigh cell subpopulations

EGFRn

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acrjournals.org


d a transiently reduced growth ability that required abculturing passages to be completely restored to theting levels (Fig. 2J). All subpopulations contained thepercentage of Bmi1- and Sox2-expressing cells, whicht affected by cell differentiation (Supplementary Fig. S4).en tested immediately after sorting, EGFRhigh andow fractions contained 100% of EGFRpos cells, whereas

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31 IV 58/ 2 0 High mplification 70–90 3+ 22 IV 57 2 1 High mplification 80–100 3+ 10 IV 56/ 2 0 High mplification 80–100 3+ 24 IV 59/ 2 1 High mplification 70–90 3+ 18 IV 63/ 1 0 High olysomy 70–90 2+ M + p25 IV 74/F 1+ 0 Low amplification 70–90 2+ 21 IV 59/ 2 1 High mplification 70–90 3+ eg GBMs 04 IV 49/ 1 0 No a plification 10–20 1+ 09 IV 47/ 1 0 No a plification 10–20 1+ 25 IV 49/ 1 0 No a plification 10–20 1+ 30 IV 72 1 0 No a plification 15–25 1+ 25 IV 51/ 1 0 No a plification 0 0
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Mazzoleni et al.

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1. EGFR expression identifies distinct cell subpopulations within primary human GBM. A, EGFRpos GBM tumors show diffuse and intense reactivityR in small and round neoplastic cells (T1224). EGFRneg GBM specimens show EGFR staining restricted to tumor-infiltrating inflammatory cellsrrow; T0109). Magnification, ×400 and ×800. B, WB confirms EGFR protein levels in GBM patients' specimens. C, FISH analysis indicatesEGFR gene is amplified only in EGFRpos tumors. D, the mutant EGFRvIII can be detected in a subset of EGFRpos tumors by RT-PCR. E, EGFRpos

contain modestly proliferating vessels (arrows), whereas EGFRneg tumors show an enlarged and proliferating vasculature (arrows). H&E andtaining; magnification, ×200 and ×400. F, EGFRpos and EGFRneg tumors contain similar numbers of Sox2 and Bmi1pos cells. CD15pos proper tumorn be detected only in EGFRpos (arrow at bottom right, CD15pos monocytes within a vessel). Magnification, ×400. G, in EGFRpos tumors, manys cells are proper tumor cells, whereas in EGFRneg tumors the few EGFRpos cells are stromal/inflammatory cells. H, the EGFRpos fractions froms pos pos neg
specimens contain AC133 and CD15 cells (top middle), which are either tumor cells or stromal cells. On the contrary, EGFR fractionsht) and EGFRneg GBM tumors (bottom) contain only stromal AC133pos and CD15pos cells.
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n regenerated the same frequency of EGFRpos andeg cells found in the parental bulk population, recapit-g the original cellular heterogeneity (Fig. 2K).

expression confers increased tumorigenicity to GBM cellsassess whether EGFR expression might affect the tu-enic ability of GBM cells, R1, R2, and R3 fractions,d from EGFRpos GBM specimens, were transplantede brain of nu/nu mice, without any prior in vitro ma-tion (Fig. 3A; Supplementary Fig. S1). MRI-basedetric analysis showed that EGFRpos cells were charac-by enhanced tumorigenic capacity (Fig. 3A). How-GFRneg/AC133neg cells were also capable of initiatings, although these developed significantly more slowlyhose generated by EGFRpos GBM cells (Kaplan-Meieris; Fig. 3B).ably, EGFR expression was retrieved not only in xeno-derived from EGFRpos cells but also in those derivedEGFRneg fractions (Fig. 3C–E). Importantly, the latterrafts never contained either AC133pos cells (Fig. 3E)15pos cells (Fig. 3F, Supplementary Fig. S1). Thus, as re-shown for other stem cell markers in melanoma (35),expression in GBM TICs is dynamically regulated,that EGFRneg cells can become EGFRpos on transplan-
. Most importantly, EGFR expression positively corre-ith gliomagenesis.
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acrjournals.org


ough R1-, R2-, and R3-derived xenografts showed theexpression of SOX2 and Bmi1 (Fig. 3G and data not), they were molecularly different (Fig. 3H). By micro-based analysis, proinvasive genes were overexpressedFRpos cell–derived xenografts, whereas genes involvediogenesis were upregulated in EGFRneg cell–derivedrafts (Supplementary Fig. S5).greement with these latter findings and with molec-ata obtained on EGFRpos and EGFRneg subpopulationsC), xenografts from EGFRpos cells were shown to beinvasive, with cells spreading preferentially along theatter while avoiding white matter fiber tracts (Fig. 3C; Supplementary Figs. S1 and S6). On the contrary,s formed by EGFRneg cells showed well-demarcatedaries, with cells migrating preferentially along whiter tracts (Fig. 3C and G; Supplementary Figs. S1 andccordingly, EGFRpos cell–induced xenografts wereterized by thin, regularly shaped blood vessels andproliferative index, whereas tumors from EGFRneg

contained enlarged and proliferating vessels and afrequency of mitotic cells (Supplementary Figs. S1

6).s, EGFR expression confers different invasive, angio-and tumorigenic abilities to GBM cells in vivo.ably, the same functional differences observed between

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27 2 High mplification 2+ 25.8 3.5 42.9 15.2 22.3 2.9 04 2 High olysomy 0 7.7 1.2 5.9 0.9 2.7 1.0 06 2 Low mplification 3+ 21.1 3.0 0 28.1 2.9 + 0 a ± 0. ±01 2+ 0 High polysomy 1+ 79.1 ± 3.9 3.1 ± 1.5 13.7 ± 0.9 05 2 Low olysomy 0 8.5 2.8 0 0 egative CSC lines 04 1 No a plification 0 2.0 0.8 30.1 13.8 50.8 3.2 23 1 No a plification 3+ 0 0 10.7 1.3 25 1 No a plification 1+ 0 0 0 10 1 No a plification 0 2.4 1.2 0 0 14 1 No a plification 3+ 0 0 5.9 1.7 25 1 No a plification 0 0 0 0 03 + 0 m .0 0. .0
0512 1+ 0 No amplification 3+ 1.7 ± 0.6 2.1 ± 1.4 11.7 ± 2.90418 1+ 0 No amplification 0 0.0 18.2 ± 4.5 0.0

E: EGFR wt and EGFRvIII mRNA levels were measured by semiquantitative RT-PCR. EGFR, AC133, and CD15 expression wassured by flow cytometry. High gene amplification, >10 gene copies in >10% of the cells; low gene amplification, 6 to 10 geneies in >10% of the cells; high polysomy, >4 gene copies in >40% of the cells; low polysomy, >4 gene copies in >10% but <40%he cells; no amplification, 1 to 4 gene copies. The frequency of EGFRpos cells within EGFRpos CSC lines remained stableughout extensive subculturing. Likewise, EGFRneg CSC lines were never shown to comprise EGFRpos cells even at very high

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2. EGFR expression identifies GBMsubpopulationswith distinctive gene signatures and different growth characteristics in vitro. A andB, EGFRpos/AC133neg

FRpos/AC133pos (R2), and EGFRneg/AC133neg (R3) cells were isolated from EGFRpos tumor specimens by FACS (IgGs, isotype controls). C, global geneiondata in R1, R2, and R3 fractions purified from patients' specimens (left). The expression of a selection of coregulated genes is highlighted in the heatmaps, limiting dilution clonogenic assay of R1, R2, and R3 fractions immediately after sorting. E, different CSC lines comprise variable amounts of EGFRpos

agnification, ×200 (insets, ×600). F, some EGFRpos CSCs display EGFR gene amplification. G, different CSC subpopulations could be isolated by FACSFRpos CSC lines based on their EGFR expression (region R4, EGFRhigh CSCs; R5, EGFRlow CSCs; R6, EGFRneg CSCs; L0627). H, WB analysis shows thatactivated in EGFRhigh and EGFRlow CSCs but is absent in EGFRneg CSCs. I, global gene expression data of R4, R5, and R6 GBM fractions purified from

s
CSC lines.SemiquantitativeRT-PCRvalidationofDEGs. J, clonogenic assayand long-termgrowthcurvesofR4,R5, andR6CSCs immediately after sortingr in vitro culture. K, EGFR expression in CSC fractions immediately after sorting (top) and on in vitro culture (bottom; EGFR, green; magnification, ×200).



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3. EGFR expression identifies TICs with different tumorigenic capacities. A, EGFRpos cell–derived tumors, independently of the coexpression ofwere significantly larger than tumors induced from EGFRnegAC133neg cells. P < 0.05, one-way ANOVA with Bonferroni test (n = 5, six independentents). B, Kaplan-Meier survival curves (P < 0.01). C, EGFR expression detected by an antihuman EGFR is retrieved in all xenografts, includinggAC133neg cell–derived tumors (EGFR, green). EGFRpos cell–derived tumors are characterized by an infiltrative pattern of growth. Tumor cellse within the gray matter, avoiding the corpus callosum (white arrow). EGFRnegAC133neg cells give rise to expanding tumors, mostly confined withinction site, which migrate and invade along white matter tracts (white arrowhead). D, WB confirms EGFR protein expression in all xenografts.ame distinct EGFRpos and EGFRneg subpopulations as detected in the parental tumor were retrieved in the corresponding xenografts by flowtry. CD15pos cells (F) were not retrieved in any xenografts by immunohistochemistry. G, a similar frequency of Sox2 and Bmi1 immunoreactive cellse retrieved in R1- and R3-derived xenografts. H, global gene expression data of R1- and R3-derived xenografts. I to J, EGFR expressionenhanced tumorigenic potential to EGFRpos fractions from EGFRpos CSC lines (one-way ANOVA with Bonferroni test, n = 5, three independent
ents). P < 0.01, Kaplan-Meier survival curves. K, EGFR expression was retrieved in all xenografts, whereas ErbB2 was highly expressedEGFRhigh CSC–derived tumors (WB).
Cancer Res; 70(19) October 1, 2010acrjournals.org 7507



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ted by in vitro expanded EGFRpos and EGFRneg CSCSupplementary Fig. S2; ref. 36).ause EGFR expression in EGFRneg cell–derived xeno-might be due to contaminating EGFRpos tumor cells,rified EGFRneg tumor cells from EGFRneg specimensere totally devoid of EGFRpos, AC133pos, and CD15pos

r tumor cells (Fig. 1A, G, and H; SupplementaryS1). FACS-purified EGFRneg/AC133neg cells generatedrafts that expressed EGFR but were negative forand CD15, thus confirming that EGFR might indeedprominent role in glioma formation (Supplementary). Notably, both in vivo limiting dilution analysis andtransplantation proved that all GBM subpopulationsied based on EGFR expression could be consideredfide TICs (Supplementary Fig. S8).tumorigenic potential of EGFRhigh, EGFRlow, and

eg cells purified from different EGFRpos CSC lines2G and 3I and J and Supplementary Fig. S9 forand Supplementary Fig. S9 for L0605) was also. As observed for xenografts obtained from GBMen–derived subpopulations (Fig. 3A–E), tumors de-from EGFRhigh and EGFRlow CSCs were larger thangenerated from EGFRneg cell fractions, which, again,yed tumor-initiating ability (Fig. 3I and J). Again,neg CSC fraction–derived tumors expressed EGFR,ming that EGFR upregulation occurred during ex-ental tumorigenesis (Fig. 3K). ErbB2 expressionetected only in EGFRhigh cell–derived tumors, sug-g that the xenografts were molecularly heterogeneousK).understand the relationship between the expression ofand that of the CSC markers CD15 and AC133 inof tumor-initiating potential, EGFRposCD15neg (R4),

posCD15pos (R5), EGFRnegCD15pos (R6), and EGFRneg

eg (R7) as well as EGFRposCD15negAC133neg (R1) andegCD15negAC133neg (R3) fractions were purified fromand L0605 (Supplementary Fig. S9). Interestingly,os subpopulations, independent of the coexpression133 and/or CD15, generated tumors that developedearlier and grew larger than those generated byeg fractions. Consistent with previous findings, CSCubpopulations negative for EGFR, AC133, and CD15ated xenografts that did not express either of thearkers in vivo but did express the EGFR (Supplemen-igs. S2 and S9).s, all of our GBM CSC lines, taken as a whole or frac-ed into subpopulations and independent of their basalexpression, gave rise to xenografts that upregulatedGFR significantly (Fig. 3K; Supplementary Figs. S29). To understand the mechanism underlying EGFRsion in EGFRneg cell–derived xenografts, we hypothe-that because EGFRneg GBM cells retain EGFR tran-expression (Tables 1 and 2; Fig. 2C and I), in vivoexpression might be regulated posttranscriptionally., when we simulated in vitro the mitogen-reducedbrain microenvironment by starvation, EGFRneg CSCs
ntly reexpressed the EGFR protein (Supplementary). Accordingly, when the same CSCs were reexposed
increamice

r Res; 70(19) October 1, 2010


ndard mitogen concentrations, they downregulatedexpression, as may occur in vivo during tumor pro-n when tumor-infiltrating stromal cells secrete mito-n a paracrine fashion (37).

ced EGFR expression by gene transfer increasesalignant behavior of CSCs in vitro and in vivotiviral vector–mediated EGFR expression in threet EGFRneg CSC lines allowed the expression of a ful-ctional receptor (Fig. 4A). Of note, the majority oftransduced cells acquired a fibroblastoid shape andped stress fibers and actin-driven lamellipodia, allmarkers of epithelial-mesenchymal transition and

ced malignancy (Fig. 4B; Supplementary Table S4).tently, the average cell size was increased in mosttransduced cells (Fig. 4C; Supplementary Table S4).e expression profiling of naïve, mock, and EGFR-pressing CSC lines identified a common subset ofthat was altered in both L0104 and L0125 CSC lines,sting that enforced EGFR expression affected themolecular determinants in different patient-derivedines (Fig. 4D and E). In addition to EGFR, LIF, and, genes previously shown to be also highly expressedCS-purified EGFRpos CSC fractions (Fig. 2I; Supple-ry List S2), proinvasive genes such as CXCL10 and(38, 39) were upregulated in EGFR-transduced CSCsD–E) and also overexpressed in the proliferative andchymal subtypes of human GBM (ref. 2I; Supplemen-ig. S3). Accordingly, tumor suppressors such asL1 (Fig. 2I), ERBB4, ASCL1, and EDNRB, which wereegulated in EGFR-transduced CSCs, were also down-ted in GBM malignant subtypes.ably, all EGFR-transduced CSC lines formed experi-l tumors that were larger than those induced by theponding mock lines (Fig. 4F).

ic loss of function of EGFR reduces thenancy of CSCs in vitro and in vivosilence EGFR expression stably, we exploited lineand line L0627, which express high levels of EGFRand 30% of the cells, respectively (Table 2). Theclones 204 and 206 induced EGFR protein knock-with an efficiency of 60% and 70% in lines L0201A) and L0627 (data not shown), respectively. WB anal-nfirmed the flow cytometry results and indicated antion of downstream signaling pathways on EGFRing (Fig. 5B).R-knockdown CSCs displayed morphologic changeso, with neurospheres appearing as well-differentiatedFig. 5C). Consistently, the frequency of differentiatedetected in shRNA-transduced CSCs increased underroliferative (Fig. 5D) and differentiative culture condi-(data not shown).note, tumors generated from EGFR-knockdown CSCsped more slowly and were significantly smaller thangenerated by the CTRL subclone, thus resulting in the
sed survival of EGFR-knockdown CSC–transplanted(Fig. 5E).
Cancer Research



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4. Overexpression of EGFR enhances the tumorigenic potential of CSCs. A, lentiviral-mediated EGFR gene transfer resulted in the expression ofional EGFR, as indicated by phosphorylation at residue Tyr1068 and by the hyperactivation of downstream pathways. B, enforced EGFR expressionneg CSCs (EGFR, red) correlates with changes in cell morphology, which are absent in GFP-transduced CSCs (GFP, green). F-actin staining

din, red). Magnifications: top, ×400; insets, ×1,000; bottom, ×1,000. C, EGFR overexpression in EGFRneg CSCs induces a significant increase in theirC-A and SSC-A. D, global gene expression data in naïve, mock-transduced, and EGFR-transduced CSC lines identify a subset of DEGs thategulated in different patient-derived CSC lines and modulated by enforced EGFR expression (EGFR gene signature). E, semiquantitative RT-PCR

neg
on of DEGs. F, ectopic expression of EGFR in EGFR CSC lines increases their tumorigenic potential and reduces mouse survival (Student's= 5, three independent experiments).
Cancer Res; 70(19) October 1, 2010acrjournals.org 7509



FigureshRNAC and DE, EGFP < 0.0responinhibitioPTEN e

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5. Inhibition of EGFR expression and activity negatively affects the malignant behavior of CSCs. A and B, flow cytometry and WB of L0201204 and shRNA 206 CSC subclones indicate efficient knockdown of EGFR protein and downregulation of MAPK and Akt downstream pathways., EGFR knockdown induces CSC differentiation. Tuj1, red; GFAP, green. Magnifications: top, ×200; bottom, ×400. *, P > 0.05, Student's t test.

R silencing in CSCs significantly decreases their tumorigenicity (one-way ANOVA with Bonferroni test, n = 4, two independent experiments).1, Kaplan-Meier survival curves. F, AG1478 treatment inhibits the phosphorylation status of EGFR and Akt in CSCs (WB). G, EGFRpos CSC linesd to treatment by decreasing their proliferation/survival, whereas EGFRneg CSC do not. *, P > 0.05; **, P > 0.01, Student's t test. H and I, EGFR
n by AG1478 negatively affects the invasive ability of CSCs. **, P > 0.01, Student's t test. J, CSC responsiveness to TKI is independent ofxpression (WB).



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pos and EGFRneg CSC lines differentially respondarmacologic inhibition of EGFRpharmacologically inhibit EGFR activity, we exploitedFR tyrosine kinase inhibitor (TKI) AG1478. WB analy-two different EGFRpos CSCs that were exposed to8 for 2, 5, and 16 hours showed effective inhibitionFR autophosphorylation and a significant decrease inhosphorylation, a hallmark of drug sensitivity (Fig. 5F).rmacologic treatment of EGFRpos CSC lines with8 for up to 72 hours in vitro resulted in a significantse in CSC survival/proliferation (Fig. 5G). Relevantly,rvival and proliferation of EGFRneg CSC lines werefected by AG1478, even when the same lines wereuced with EGFR-coding vectors before treatment, sug-g that EGFR-overexpressing EGFRneg CSC lines did note addicted to EGFR (data not shown). Likewise, expo-f EGFRpos CSCs to AG1478 strongly reduced their inva-apacity as compared with controls (Fig. 5H and I).the invasive ability of EGFRneg CSC lines was not af-by AG1478 exposure.restingly, all the EGFRpos CSC lines responded to phar-ogic treatment independently of the expression ofa putative molecular determinant of TKI responsive-

n patients with GBM (Fig. 5J; ref. 40).
s, EGFRpos CSCs are responsive to pharmacologic inhi- In
the EmightEGFRcessivwe simtion. IGBMadditileculaTICs.sine kfractiosynergperimtion hthat dthe laPDGFEGF

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ssion

expression contributes to GBM heterogeneityis characterized by a high degree of heterogeneity,

is efficaciously reflected in the definition “multiforme.”er, the intrinsic heterogeneity of GBM might refer notthe well-known molecular diversity of GBM subtypes

so to the existence of genetically and functionally dis-ohorts of TICs within the same tumor.ine with recent reports that provide new interpreta-of the process of tumorigenesis in mouse hemato-c malignancies (8), mouse colon cancer (41), humanoma (10, 11), and brain tumors (42), our findings indi-at the initiation and progression of GBM may not de-n the presence of a single and rare fraction of CSCs, assed by the hierarchical model of tumor developmentn the contrary, our data propose a different biologicalt, in which all GBM cells can be considered TICs withnt degrees of stemness as proven by their different tu-enic ability and by their distinct phenotypic and molec-eatures (44). In fact, all of our GBM-derived TICpulations satisfy the requirements for bona fide CSCs:elf-renew in vitro and in vivo and give rise to experi-l tumors that recapitulate the phenotypic traits ofmor of origin under limiting dilution conditions andial transplantation.ed on these observations, GBM tissue heterogeneitybe explained by the presence of a hierarchy of distinct
as recently suggested for in vitro generated GBM CSC13). These distinct TICs distribute along a gradient of
GBMson rec

acrjournals.org


igenic potential that is strictly associated with EGFRsion.s, our study provides conclusive evidence that the ma-of GBM cells have tumorigenic capacity and that thechical model of tumorigenesis might not fully applyM.

anisms underlying EGFR-dependentheterogeneityor cells might modify their phenotype during tumorssion, with multiple cohorts of TICs being generatedn this study, we have shown that EGFR expressionbutes to GBM cell diversity by generating differentopulations. As to the mechanisms involved in theation of EGFR-dependent phenotypic heterogeneityM, the contribution of tumor microenvironmentto be taken into consideration. Paracrine EGFR li-, such as epidermal growth factor, are released bystromal cells that, in this way, contribute to the ac-n of the EGFR signaling in both proper tumor cellsndothelial cells (37). When stroma-produced epider-rowth factor is secreted, EGFR protein downregula-ccurs in target cells by receptor internalization,ed by lysosomal degradation.line with this view, it is tempting to speculate thatGFRneg cell component detected in EGFRpos GBMbe generated from GBM cells that were originally

pos through receptor downregulation induced by ex-e ligand stimulation during tumor progression, asulated in vitro by modulation of mitogen concentra-n addition, termination of EGFR signaling in EGFRneg

cells might lead to the acquisition of compensatoryonal genetic events, which might account for the mo-r differences detected between EGFRpos and EGFRneg

For instance, the expression of other receptor tyro-inases, such as PDGFRs, observed in EGFRneg TICns might contribute to their tumorigenic ability byizing with the EGFR re-upregulation required for ex-ental gliomagenesis. Indeed, PDGFR pathway activa-as been recently shown to identify a GBM subtypeoes not express the EGFR (46), thus suggesting thattter GBM subtype might be sustained by EGFRneg

Rpos TICs.Rpos and EGFRneg TICs can both give rise to experi-l tumors that phenocopy the tumor of origin, andore, they both can be considered bona fide TICs.ver, because EGFRpos TICs are characterized by en-d tumorigenic potential and highly invasive behavior,might be envisioned as the “actual” GBM CSCs.rsely, EGFRneg TICs, which form tumors with lowncy and need to re-upregulate the EGFR to be tumor-, might be better defined as “potential” GBM CSCs,might be kept in a dormancy-like state by EGFRegulation, being reactivated on exposure to appropri-imuli when exposed to the in vivo microenvironment.e with this notion, it has been recently shown that
, which were EGFRneg in origin, expressed the EGFRurrence (47).



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peutic implications of EGFR-dependentheterogeneityrole of EGFR in the malignant progression of GBMs effect on patient survival have been highly debated.te the controversial prognostic significance of EGFRsion in GBM, pharmacologic targeting of EGFR byof TKIs has been proposed as a possible therapeuticy (40).date, single-agent EGFR inhibition in GBM did note complete therapeutic efficacy. This failure has beened primarily to the maintenance of a high level of Akt-dent signaling in PTEN-mutated tumors (48) and tovation in the same tumor cell of multiple receptor ty-kinases, which might compensate for the effective in-n of EGFR-dependent pathways (49).complementing these explanations, our findings sug-at the simultaneous presence of distinct TICs withinme tumor might also influence the outcome of EGFR-ed therapies in GBM. Indeed, whereas effective inhi-of EGFR in our EGFRpos CSC lines can be achievedendently of PTEN expression, EGFRneg CSCs do notd to treatment. By translating these results into a clin-rspective, in spite of the inhibition of the EGFRpos cell
nent in EGFRpos GBM, the nonresponsiveness of the
en R, Nishimura MC, Bumbaca SM, et al. A hierarchy of self-ewing tumor-initiating cell types in glioblastoma. Cancer Cell06;17:362–75.

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o survive treatment and to support tumor relapse.fore, an effective therapeutic regimen should take intoeration the role of residual, nonresponder TICs. Tod, the development of a combination therapy targetedly against the most aggressive “actual” TIC popula-ut also against the less malignant “potential” TICs willessary to improve the management of GBM.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Alessio Palini and Chiara Villa for technical assistance with FACS.lla Iadanza for technical assistance with MRI, and Carol Stayton forof the manuscript.

Support

pagnia S. Paolo grants CSP-2002.0735 and CSP-2005.0153, the Oncologyh Programme-Istituto Superiore di Sanità, and the Fondazione Cariploi).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement in

eg TIC fraction to EGFR in
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2010;70:7500-7513. Published OnlineFirst September 21, 2010.Cancer Res Stefania Mazzoleni, Letterio S. Politi, Mauro Pala, et al. GliomagenesisHuman Glioblastoma Multiforme and Is Required forFunctionally and Molecularly Distinct Tumor-Initiating Cells in Epidermal Growth Factor Receptor Expression Identifies

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