interferon-binduceslossofspherogenicityandovercomes...

Cancer Biology and Signal Transduction

Interferon-b Induces Loss of Spherogenicity and OvercomesTherapy Resistance of Glioblastoma Stem Cells

Caroline Happold1, Patrick Roth1, Manuela Silginer1, Ana-Maria Florea4, Katrin Lamszus3, Karl Frei2,Rene Deenen4, Guido Reifenberger4,5, and Michael Weller1

AbstractGlioblastoma is the most common malignant brain tumor in adults and characterized by a poor prognosis.

Glioma cells expressing O6-methylguanine DNA methyltransferase (MGMT) exhibit a higher level of resis-

tance toward alkylating agents, including the standard of care chemotherapeutic agent temozolomide. Here,

we demonstrate that long-term glioma cell lines (LTL) as well as glioma-initiating cell lines (GIC) express

receptors for the immune modulatory cytokine IFN-b and respond to IFN-b with induction of STAT-3

phosphorylation. Exposure to IFN-b induces a minor loss of viability, but strongly interferes with sphere

formation in GIC cultures. Furthermore, IFN-b sensitizes LTL andGIC to temozolomide and irradiation. RNA

interference confirmed that both IFN-b receptors, R1 and R2, are required for IFN-b–mediated sensitization,

but that sensitization is independent of MGMT or TP53. Most GIC lines are highly temozolomide-resistant,

mediated byMGMT expression, but nevertheless susceptible to IFN-b sensitization. Gene expression profiling

following IFN-b treatment revealed strong upregulation of IFN-b–associated genes, including a proapoptotic

gene cluster, but did not alter stemness-associated expression signatures. Caspase activity and inhibition

studies revealed the proapoptotic genes to mediate glioma cell sensitization to exogenous death ligands by

IFN-b, but not to temozolomide or irradiation, indicating distinct pathways of death sensitizationmediated by

IFN-b. Thus, IFN-b is a potential adjunct to glioblastoma treatment that may target the GIC population. IFN-boperates independently of MGMT-mediated resistance, classical apoptosis-regulatory networks, and stem-

ness-associated gene clusters. Mol Cancer Ther; 13(4); 948–61. �2014 AACR.

IntroductionGlioblastomas are characterized by infiltrative growth

and resistance to cell death induction.Despitemultimodaltherapy, tumor progression occurs inevitably and surviv-al remains in the range of months (1–3). Early therapyfailure is associatedwith the expression of O6-methylgua-nine-DNA-methyltransferase (MGMT), a DNA repairprotein that accounts for glioma cell resistance by counter-acting the effects of alkylating chemotherapy (4). MGMThas become an important molecular marker and is nowimplemented in clinical diagnostics as predictive bio-marker for benefit from alkylating chemotherapy and

clinical outcome (5–7). Patients with a nonmethylatedMGMT promoter are prone to therapy failure with stan-dard alkylating chemotherapy, and to date, effectiveapproaches for this large group of patients, comprisingmore than half of all the patients with glioblastoma, arestill lacking, including dose intensification of temozolo-mide (8).

Recently, a subfraction of glioma cells exhibiting stemcell–like properties (stem-like glioma cells), referred to asglioma-initiating cells (GIC), has been identified (9, 10).GICs are thought to have the ability of self-renewal, tumorinitiation, and pluripotency and have been proposed toaccount for the ultimately lethal nature of glioblastoma.They may contribute to therapy resistance in vivo andtherefore promote tumor progression. The value ofGIC cultures and their MGMT status as a model to studyresistance to temozolomide in glioblastoma has remainedcontroversial. Among a panel of 20 GIC lines, somewere sensitive to temozolomide, which was associatedwith lowMGMT protein levels, but notMGMT promotermethylation, and the MGMT promoter status was thusnot strongly predictive of response to temozolomide (11).High temozolomide sensitivity of GIC cultures lackingMGMTexpressionhasbeendescribed in vitro (12). Finally,the frequency of MGMT promoter-methylated allelesin glioblastomas may range from 10% to 90%, but

Authors' Affiliations: 1Laboratory of Molecular Neuro-Oncology, Depart-ment of Neurology, and 2Department of Neurosurgery, University HospitalZurich and Neuroscience Center Zurich, Zurich, Switzerland; 3Laboratoryfor Brain Tumor Biology, Department of Neurosurgery, University HospitalHamburg-Eppendorf, Hamburg; 4Department of Neuropathology andCenter for Biological and Medical Research, Heinrich Heine University,D€usseldorf; and 5German Cancer Consortium (DKTK), German CancerResearch Center (DKFZ), Heidelberg, Germany

Corresponding Author: Michael Weller, Department of Neurology,University Hospital Zurich, Frauenklinikstrasse 26, 8091 Zurich, Switzer-land. Phone: 410-44255-5500; Fax: 410-44255-4507; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0772

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(4) April 2014948

on December 31, 2019. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

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methylated alleles were enriched in GIC cultures (13).Considering the limited activity of current standards ofcare, new approaches should take into account this noveltarget cell population.IFN-b, a member of the IFN class I family, exerts num-

erous functions for cellular differentiation, cell growth,and immune responses. IFN-b signaling is mediatedthrough binding to a type II cytokine receptor, involvingheterodimerization of 2 IFN-a/b receptor subunits,IFNAR-1 and IFNAR-2. Downstream signaling path-ways involve the Janus kinase/signal Transducers andActivators of Transcription (JAK/STAT) pathway (14)and lead to accumulation of myxovirus resistance 1(MxA) protein, a GTPase interfering with the cytoskel-etal structure (15, 16). MxA induction is an establishedmarker for responsiveness to IFN-a/b (17, 18).IFN-b has gained great clinical impact in the treatment

of multiple sclerosis. Its safety profile in patients withbrain disease has therefore been firmly established.More-over, IFN-a/b were the first agents to show a significantsurvival benefit in randomized trials in malignant mela-noma (19). The mechanisms mediating antitumor effectsof IFN may involve direct cytostatic, antiangiogenic, orimmunomodulatory activities. IFN-b has been reportedto sensitize human glioma cell lines to temozolomide ina TP53- and MGMT-dependent manner (20, 21). Thesereports, however, were based on findings in T98G andU251MG, both being TP53-mutant cell lines (22, 23).Moreover, IFN-b has been attributed anti–stem cell prop-erties in glioma models (24). Finally, clinical trials explor-ing the addition of IFN-b to radiotherapy plus temozolo-mide have been initiated and interpreted as promising(25, 26). These data prompted us to evaluate the potentialrole of IFN-b as a component of future, innovative treat-ment approaches for glioblastoma inmore detail. In brief,we demonstrate profound anti-GIC activity of IFN-b aswell as IFN-b–mediated sensitization to temozolomideand irradiation, but exclude MGMT and TP53 as primarymediators of these effects. IFN-b exposure induces char-acteristic IFN-b–associated gene clusters, but does notalter the stemness signature of GIC. A proapoptotic genesignature induced by IFN-b is functionally relevant, butmediates neither anti-GIC properties nor sensitivity totemozolomide or radiotherapy.

Materials and MethodsMaterials and cell linesThe human U87MG and T98G glioma cell lines, K562

leukemia cells, and MCF-7 breast adenocarcinoma cellswere purchased from the American Type Culture Collec-tion. The other long-term cell lines (LTL) were kindlyprovided byN. de Tribolet (Department ofNeurosurgery,University Hospital, Lausanne, Switzerland). LNT-229cells cultured in our laboratory express TP53 wild-typetranscriptional activity (23). The generation of LNT-229cells depleted of TP53 or overexpressingMGMT has beendescribed (23, 27).MGMT-silenced LN-18 cells were gen-

erated by transfection with pSUPER puro encodingMGMT short hairpin (sh) RNA using Metafectene Protransfection reagent (Biontex LaboratoriesGmbH; ref. 28).The GIC cell lines GS-2, GS-5, GS-7, GS-8, and GS-9 havebeen characterized elsewhere (29). LTL were cultured inDulbecco’s modified Eagle medium containing 10% fetalcalf serum (FCS), 2mmol/L glutamine and penicillin (100IU/mL)/streptomycin (100 mg/mL). GS sphere cultureswere maintained in neurobasal medium supplementedwith 2%B-27 supplement, 1%GlutaMAX, 20 ng/mLEGF,20ng/mLfibroblast growth factor, and32 IE/mLheparin.K-562 cells were maintained in RPMI-1640 supplementedwith 1 mmol/L sodium pyruvate (Gibco Life Technolo-gies) and 10% FCS. Temozolomide was provided bySchering-Plough (Kenilworth). A stock solution of temo-zolomide at 200 mmol/L was prepared in dimethyl sulf-oxide. Recombinant IFN-b1b was purchased from AbDSerotec, reconstituted to a concentration of 1,000,000 IU/mL with distilled water. The following antibodies wereused: STAT-3 and phospho-STAT-3, caspase-3, andLC3A/B fromCell Signaling Technology; glyceraldehyde3-phosphate dehydrogenase (GAPDH) from Everest Bio-tech; TNF-related apoptosis inducing ligand (TRAIL)and XIAP-associated factor 1 (XAF-1) from Santa CruzBiotechnology; caspase-8 from Enzo Life Science; Glialfibrillary acidic protein (GFAP) from Dako; Nestin fromZytomed Systems GmbH; b-III tubulin from Abcam.Mega-Fas-ligand (MFL)was provided by Topotarget. TheMxA antibody was kindly provided by O. Haller andG.Kochs (Department ofVirology,University of Freiburg,Germany). All other reagents, including anti–20, 30-cyclicnucleotide 30-phosphodiesterase (CNPase) antibody,O6-benzylguanine (O6-BG), and salinomycin, were pur-chased from Sigma. For irradiation experiments, cellswere irradiated in a Co-radiation source (Gebr€uderSulzer, Thermische Energiesysteme, 60-Co) at 1, 3, and5 Gy at 24 hours after IFN-b exposure.

Polymerase chain reactionTotal RNA was prepared using the NucleoSpin System

(Macherey-Nagel AG) and transcribed using random pri-mers (Bioconcept/NEB, Bioconcept) and Superscript IIreverse transcriptase (Invitrogen). For reverse transcrip-tase PCR, the conditionswere: 35 cycles, 94�C/45 seconds,specific annealing temperature (54�C for IFNAR-1, 52�Cfor IFNAR-2, 51�C for GAPDH)/45 seconds, 72�C/1 min-ute. For real-time (quantitative) qPCR, cDNA amplifica-tion was monitored using SYBRGreen chemistry onthe 7300 Real time PCR System (Applied Biosystems).Theconditionswereas follows: 40cycles, 95�C/15seconds,60�C/1 minute. Data analysis was done using the DDCt

method for relative quantification. The following specificprimers were used: GAPDH forward: 50-CTCTCTGCT-CCTCCTGTTCGAC-30, GAPDH reverse: 50-TGAGC-GATGTGGCTCGGCT-30; IFNAR-1 forward: 50-TAT GCTGCGAAAGTC TTC TTGAG-30; IFNAR-1 reverse: 50-TCTTGG CTA GTT TGG GAA CTG TA-30; IFNAR-2 forward:50-TCT TGA GGC AAG GTC TCG CTA-30; IFNAR-2

Antiglioma Activity of Interferon-b

www.aacrjournals.org Mol Cancer Ther; 13(4) April 2014 949




reverse: 50-CAG GGA TGC ACG CTT GTA ATC-30; XAF1forward: 50-AGC AGG TTG GGT GTA CGA TG-30; XAF1reverse: 50-TGA GCT GCA TGT CCA GTT TG-30; TRAILforward: 50-TGCGTGCTGATCGTGATCTTC-30; TRAILreverse: 50-GCT CGT TGG TAA AGT ACA CGT A-30.

Flow cytometry, cell cycle, and viability assaysDifferentiated glioma cells and sphere cultures were

detached respectively dissociated using Accutase (PAALaboratories) and blocked with 2% FCS in PBS. The cellswere incubated for 30 minutes on ice using the followingPE-labeled antibodies: anti–IFNAR-1 or anti–IFNAR-2antibodies (PBL interferon source) for IFNAR experi-ments and anti-CD133/2-PE (Miltenyi Biotec) for stemcell marker experiments. Flow cytometry was performedwith a CyAn flow cytometer (Beckman Coulter). Signalintensity was calculated as the ratio of the mean fluores-cence of the specific antibody and the isotype controlantibody (specific fluorescence index). Dead cells weregated out. For some analyses, cells were permeabilized byFix/Perm Buffer Set (Lucerna Chem AG, Biolegend). Foranalysis of cell death, cells were grown in 6-well plates,incubated with IFN-b at 150 IU/mL for 24 hours,washed in PBS, and allowed to grow for 48 hours.Annexin (Anx) V-fluorescein isothiocyanate (1:100) andpropidium iodide (PI; 50 mg/ml) were added, andfluorescence in a total of 10,000 events (cells) per con-dition was recorded in a CyAn flow cytometer. AnnexinV- or PI-positive cells were counted as dead cells, theremaining cells were designated the surviving cell frac-tion. In some experiments, loss of viability was alsoconfirmed by Trypan blue dye exclusion. Autophagiccell death was assessed by immunoblot and immunos-taining. In brief, for the latter, the cells were exposed toIFN-b as indicated and Cytospin samples were pre-pared, fixed in 4% paraformaldehyde (PFA), blockedin TBS containing 0.2% Triton X-100, 5% goat serum and5% horse serum, and exposed to the primary LC3A/Bantibody (Cell Signaling Technology) overnight at 4�Cand 1 hour at room temperature for secondaryantibodies.

Clonogenicity assaysFor LTL, clonogenicity assays were performed by seed-

ing 100 cells (LNT-229, LN-18) per well in 96-well plates,allowed to adhere overnight, and exposed to IFN-b at 150IU/mL for 24 hours in fresh medium. After removal ofIFN-b, the cells were exposed to temozolomide at theindicated concentrations for 24 hours in serum-free medi-um, followed by an agent-free observation period for 7 to14 days in serum-containing medium. Cell density wasassessed by crystal violet staining. For sphere cultures, thecells were seeded at 500 cells per well in neurobasalmedium and treated consecutively as indicated above inneurobasal medium. Lower cell numbers resulted in inef-ficient sphere formation. Cell growth was assessed byMTT assay. Initially, we confirmed that crystal violetassay and MTT assay were good surrogate markers of

the number of colonies respectively spheres in theseassays, but easier to standardize for large-scale concen-tration response analyses.

Immunoblot analysisThe cells were treated as indicated and lysed in lysis

buffer containing 50 mmol/L Tris-HCl, 120 mmol/LNaCl, 5 mmol/L EDTA and 0.5% NP-40. Protein (20mg/lane) was separated on 10% acrylamide gels. Aftertransfer to nitrocellulose (Bio-Rad), the blotswere blockedin Tris-buffered saline containing 5%skimmilk and 0.05%Tween 20 and incubated overnight at 4�C with primaryantibodies and 1 hour at room temperature for secondaryantibodies. Visualization of protein bands was accom-plished using horseradish peroxidase-coupled secondaryantibodies (Santa Cruz Healthcare) and enhanced chemi-luminescence (Pierce/Thermo Fisher).

Immunocytochemistry and immunofluorescencemicroscopy

For stemness experiments, the cells were exposed toIFN-b (150 IU/mL) for 48 hours and cytospin sampleswere prepared, fixed in 4% PFA, and blocked in eitherblocking solution (Candor Bioscience GmbH) for GFAP,b-III tubulin or CNPase, or PBS containing 10% swineserum and 0.3% Triton X for nestin, and exposed to theprimary antibody overnight at 4�C and 30 minutes atroom temperature for secondary antibodies. Detectionwas performed with DAB detection systems (Dako) for5 minutes at room temperature, and slides were mountedin Eukitt (Sigma-Aldrich). Counterstaining was per-formed with hemalum. For IFNAR receptor staining,cytospin samples of LTL or GIC were prepared asdescribed above. Staining was performed as suggestedby the manufacturer, using IFNAR-1 antibody from anti-bodies-online.com and IFNAR-2 antibody from Abcam.

RNA interference-mediated gene silencingFor transient transfections, 2.5 � 105 glioma cells were

seeded in a 6-well plate and transfected with 100 nmol/Lof specific or scrambled control small interfering (si) RNA,using Metafectene Transfection reagent (Biontex). siRNAwas purchased from Dharmacon/Thermo Fisher usingsiGENOME SMARTpool targeting human IFNAR-1 andhuman IFNAR-2. Samples were collected 48 hours aftertransfection and processed for further treatment.

Reporter assayDual luciferase/Renilla assays were carried out with

cotransfection of 150 ng of the specific reporter constructand 20 ng of the Renilla reniformis-CMV (pRL-CMV)control plasmid (Promega). Luciferase activity was nor-malized to constitutive Renilla activity. The pGL2-LucMGMT construct (30) was a kind gift from Dr. S. Mitra(Sealy Center for Molecular Science and Department ofHuman Biological Chemistry and Genetics, University ofTexas Medical Branch, Galveston, TX). The TP53-Lucconstruct has been described elsewhere (31).

Happold et al.

Mol Cancer Ther; 13(4) April 2014 Molecular Cancer Therapeutics950




Caspase activity assayCells were seeded at a density of 1,000 cells per well in a

6-well plate and treated as described above with eitherIFN-b and temozolomide alone or in combination with orwithout ZVAD-fmk, with either MFL (Mega-FAS-ligand)or staurosporine (STS) as positive control. Cells wereincubated in lysis buffer (25 mmol/L Tris/HCL, 60mmol/L NaCl, 2.5 mmol/L EDTA, 0.25% NP40) for 10minutes, and the substrate Ac-DEVD-amc was added at aconcentration of 20 mmol/L. Fluorescence was assessed at360 nm wavelength every 15 minutes until extinction offluorescence (32).

Microarray-based gene expression profilingTechnical details are provided in the Supplementary

Methods. Array records are deposited under GEO acces-sion number GSE53213.

Statistical analysisData are representative of experiments performed three

times with similar results. Where indicated, analysis ofsignificancewas performed using the two-tailed Student ttest. Synergy of irradiation or temozolomide and IFN-bwas assessed by the fractional productmethod (33) whereindicated and differences of 10% of observed versus pre-dicted (additive) effect were considered synergistic. Forthe analysis of functional gene interactions, the SearchTool for the Retrieval of Interacting Genes/Proteins(STRING) Version 9.05 at http://string-db.org (34) wasused. Highest confidence settings were applied, integrat-ing combined scores higher than 0.900. Cluster analysiswas performed by application of the Markov Clusteralgorithm (MCL). Disconnected nodes were hidden fromthe image.

ResultsIFNAR are expressed in human glioma cellsIn total, 12 LTL and 5 GIC were assessed for mRNA

expression of IFNAR-1 and -2 by RT-PCR. All linesexpressed transcripts for both receptor subunits(Fig. 1A). IFNAR-1, in contrast to IFNAR-2, was notdetected at the surface on any glioma cell line by flowcytometry (Fig. 1B, left), but was detected by immunos-taining (Fig. 1B, right). Permeabilization of the cells orscraping of cells instead of using accutase did not producea specific signal for IFNAR-1 on flow cytometry either.Specific fluorescence intensity (SFI) values for IFNAR-2above 3 were never seen in GIC, but in all LTL (data notshown).

IFN-b signaling in glioma cells involves MxA andSTAT-3To ensure that IFN-b induces classical signaling path-

ways in glioma cells, we investigated whether STAT-3,the proposed signal transducer in response to IFN-b ingliomas, and the IFN-a/b–regulated protein MxA, a clas-sical marker for cellular responsiveness to IFN-b, were

induced. Immunoblot confirmed that STAT-3 phosphor-ylation levels increased shortly after exposure to IFN-b,with a peak at 30–60 minutes, with no relevant concen-tration dependency between 50 and 500 IU/mL, in LTLandGIC (Fig. 1C). Likewise, the exposure to IFN-b led to aconcentration-dependent increase of MxA protein inLNT-229 (Fig. 1D, top row) or LN-18 cells (data notshown), in a time-dependent manner (Fig. 1D, bottomrow), with first marked elevation of protein levels at aconcentration of 150 IU/mL at 24 hours.

IFN-b induces cell-cycle arrest in glioma cells andcauses sphere disruption in GIC

Within the concentration and time ranges of our experi-ments, IFN-b induced a reduction of G1 cells in all celllines, associated with increases in S cells or G2–M cells orboth. Moreover, there was an increase in sub-G1 cells,indicating minor cell death induction (Fig. 2A). A mod-erate induction of necrotic rather than apoptotic cell deathwas confirmed by Annexin V/PI flow cytometry, definedby an increase of the PI-positive fraction (Fig. 2B). Cellularstaining and immunoblot for autophagy were negative inresponse to IFN-b (Fig. 2C and D).

We also assessed a possiblemodulation of spherogeni-city of GIC by IFN-b.When exposed to 150 IU/mL IFN-b,singularized GIC cultures showed a reduced sphereformation at 1,000–10,000 cells per well; lower cell num-bers did not result in efficient sphere formation, inde-pendent of IFN-b exposure (Fig. 2E). Conversely, expo-sure of fully formed spheres to 150 IU/mL IFN-b led tosphere disaggregation, with lesser and significantlysmaller spheres remaining after 2 weeks of culture, asassessed by sphere count and absorbance (Fig. 2F). Thesingle cells detaching from the spherewere viable duringthe first days after sphere disaggregation, but underwentcell death after a week, as assessed by Trypan blue assay(data not shown).

To assess the possible impact of IFN-b on stem celldifferentiation, we analyzed GS-2, 5, 7, and 9 cells forchanges in the expression of different stem cell markers.GS-2 and GS-9 cells expressed CD133, but expressionremained unchanged after exposure to 150 IU/mL IFN-b for 48 hours. GS-5 and GS-7 did not express CD133(Fig. 2G). Moreover, IFN-b–treated GS-2, GS-5, and GS-9cells showed no changes of GFAP, nestin, b-III-Tubulin,or CNPase staining (shown for GS-2; Fig. 2H).

Requirement for IFNAR-1 and IFNAR-2 for IFN-b–mediated sensitization to irradiation andtemozolomide

To verify the role of IFNAR-1 and IFNAR-2 for IFN-b–mediated signal transduction and sensitization totemozolomide and irradiation, we silenced the expres-sion of both receptors by siRNA. Efficacy of gene silenc-ing was assessed by qPCR for IFNAR-1, which could notbe assessed by flow cytometry (Fig. 1), and by qPCR andflow cytometry for IFNAR-2 (Fig. 3A). In the settingof temozolomide exposure (Fig. 3B) or irradiation






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Figure 1. IFNAR-1 and -2 expression and responsiveness to IFN-b in human LTL and GIC. A and B, the cells were assessed for expression of IFNAR-1and IFNAR-2 mRNA by PCR with GAPDH as control (A) and of protein by cell surface flow cytometry (B, left; representative profiles for K562as a positive control and LNT-229, LN-18, LN-308, GS-2, and GS-9 GIC cells; black curve, isotype control; gray curve, IFNAR antibody), orimmunofluorescence staining (B, right panel; MCF-7 and K562 were used as control). C, phosphorylation levels of STAT-3 in a concentration- (left) andtime-dependent (right) manner, shown representatively for LNT-229 cells (top row), and for GS-7 and GS-9 GIC in a time-dependent manner(bottom row). D, responsiveness to IFN-b was assessed through detection of MxA protein in a concentration- (top row) and time-dependent (bottomrow) manner, shown representatively for LNT-229 cells.

Happold et al.





(Fig. 3C), silencing of IFNAR-1 or -2 or both attenuatedthe sensitizing effect of IFN-b in LNT-229 cells and LN-18 cells, confirming a role for IFNAR-mediated signaltransduction pathways in the IFN-b–induced sensitiza-tion process. IFNAR-2 gene silencing alone did notabrogate the effect of IFN-b on temozolomide activityto the same extent as IFNAR-1 gene silencing alone.

Response to temozolomide, irradiation, and IFN inGIC linesSince GIC are proposed to be the major source of tumor

relapse and resistance, we next focused on these models.All investigated GIC, except GS-9, expressed MGMT andwere highly resistant to temozolomide (SupplementaryNote S1 and Supplementary Fig. S1). IFN-b had no majoreffect on cell density of LTL cells, but a significant concen-tration-dependent impact of IFN-b alone was observed inGIC cells (Fig. 4A), as expected from the data shown in Fig.2F. To assess whether IFN-b also partially overcomestemozolomide resistance of GIC, we pre-exposed the cellsto increasing concentrations of IFN-b (Fig. 4B, left), or to150 IU/mL IFN-b for increasing periods of time (Fig. 4B,right), before treatment with temozolomide at EC50 con-centrations. All investigated GIC lines showed sensitiza-tion to temozolomide after pre-exposure to IFN-b. Sensi-tization was significant in all cell lines when treated at aconcentration of 150 IU/mLand for an exposure time of 24hours. Exposure of GS-2 cells to IFN-b for 24 hours beforeadministration of temozolomide induced a strong growth-inhibiting effect on the sphere cultures (Fig. 4C). IFN-b–treated cells showed a decrease in clonogenic survivalexceeding 50%comparedwith temozolomide only-treatedcells, and exhibited a progressive loss of the sphere struc-ture (see also Fig. 2D). Similar effects, albeit with lessimpressive disaggregation, were observed for GS-5, GS-7, and GS-9 spheres, where cooperative effects were mostprominent when IFN-b was given with temozolomide atapproximately EC50 concentrations.In the setting of irradiation experiments, GIC cells were

not more resistant than LTL, with GS-2 displaying thestrongest radioresistance of the panel, andGS-5 theweak-est, possibly because of the TP53 wild-type status of thiscell line (ref. 29; Fig. 4D). Pre-exposure to increasingconcentrations of IFN-b followed by low dose irradiationat 1Gy resulted at least in additive inhibitionof clonogenicsurvival. Again, in the rather radioresistant GS-2, a sig-nificant impact of IFN-b alonewas observed (Fig. 4E, left);preexposure to IFN-b before irradiation led to a reductionof cell density fulfilling criteria of synergy, with an addi-tional reduction of cell density of almost 50% whenexposed to 150 IU/mL of IFN-b. GS-5 and GS-7 displayedsynergistic effects at 150 IU/mL IFN-b; no synergy wasdetermined in GS-9 (Fig. 4E).

Gene expression analysesMicroarray-based gene expression profiling demon-

strated IFN-b–dependent significant differential regula-tion of 509 genes after 6 hours of IFN-b treatment in all

three glioma cells lines investigated. Gene expressionprofiling after 24 hours revealed significant differentialregulation of 522 genes. The combined analysis, compar-ing the 6- and 24-hour data, resulted in an overlap of 132differentially expressed genes upon exposure to IFN-b inall three cell lines. In addition to the statistical analysis,where IFN-b treatment effects were studied globallyacross all three cell lines, the impact of IFN-bwas assessedindividually for every single cell line. Single analysisprobe lists of all three cell lines were compared at 6 and24 hours, respectively (Supplementary Fig. S2), definingoverlapping lists of regulated genes as well as lists ofgenes exclusively regulated in each individual cell line.Multiple transcripts known to be regulated by IFN-bwerefound, including STAT1, interferon regulatory factors(IRF), and Mx1 (MxA; Supplementary Table S1). More-over, we noted that a number of genes predicted topromote apoptotic signaling were induced by IFN-b,(Supplementary Table S1), with an overlap of 6 genesannotated to both gene groups. Submission of the differ-entially expressed IFN-regulated genes to the Search Toolfor the Retrieval of Interacting Genes/Proteins (STRING)identified a single main cluster of genes with stronginteraction profile (Supplementary Fig. S3A). Submissionof the differentially expressed apoptosis-related genesrevealed 3 main clusters, one cluster involving tumornecrosis factor-related apoptosis-inducing ligand(TRAIL/TNFSF10)-interacting genes (yellow), oneinvolving XIAP-associated factor 1 (XAF1)-interactinggenes (blue), both connected via a third cluster centeredaround STAT1 (green; Supplementary Fig. S3B). Induc-tion of TRAIL and XAF1 was confirmed by qPCR andimmunoblot analysis (Fig. 5). As we had observed differ-ential reaction of the LTL cell line LNT-229 and the GICcell lines GS-2 and GS-9 in clonogenic growth assays afterexposure to IFN-b alone, we performed a gene ontology(GO) analysis of thegenes exclusively regulated (FC> 2) inLNT-229, or theGIC lines (Supplementary Fig. S2, periph-eral regions), and identified several downregulated genesinvolved in proliferation and cell growth as well as upre-gulated genes involved in negative regulation of prolif-eration or cell-cycle arrest (Supplementary Table S2).Notably, more genes were regulated in the GIC lines,with 2main clusters of negative regulators of proliferationinteracting mainly through JUN (blue cluster) and IL-8,IL-6, and JAK2 (red cluster; Supplementary Fig. S3C).

Altered expression of apoptosis-regulating genesdoes not mediate enhanced clonogenic cell deathinduced by temozolomide or irradiation

To define a role for altered expression of apoptoticmachinery genes in the biologic effects of IFN-b, weexamined whether IFN-b alone or when combined withdifferent death stimuli promoted caspase activationunder the conditions that induced synergistic inhibitionof clonogenic survival (Fig. 4). As a positive control, weused MFL since we previously observed IFN-a–inducedsensitization toCD95Lalthough themechanismremained






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Figure 2. Assessment of IFN-b effects on GIC cells. A and B, LTL or GIC cells were exposed to IFN-b at 150 IU/mL for 24 hours, cultured for an additional48 hours in serum-enriched medium, and flow cytometric cell-cycle analysis (A) or Annexin (AnxV)/PI flow cytometry (B) was performed. Celldistributions are shown as bar graphs (striped, sub-G1; black, G1; white, S; gray, G2–M). Cell lines in B match those shown in A. C. LNT-229 or GS-2 cellswere exposed to IFN-b (150 IU/mL), ddH2O aqua (negative control) or salinomycin (SAL; 4 mmol/L; positive control) for 24 hours. D, LNT-229 andGS-2 cells were treated as in C for protein lysates and assessed for LC3 A/B cleavage. GAPDH was used as control. E, the effect of IFN-b (150 IU/mL,24 hours) on sphere formation over a range of 5–10,000 single cells was assessed by sphere count and is shown for GS-2 (black square,IFN-b; white diamond, vehicle). Data, mean � SEM (n ¼ 6; ��, P < 0.001). (Continued on the following page.)

Happold et al.





unclear then (35). MFL, but neither IFN-b nor temozolo-mide, induced the cleavage of caspases 8 or 3 or DEVD-amc-cleaving caspase activity (Fig. 6A–C). Similar resultswere obtained for irradiation assays (data not shown).Preexposure to IFN-b did not result in caspase processingin cells exposed to temozolomide either. Cell death in theinvestigated settings was confirmed by Trypan blue dye

exclusion (Fig. 6D). Finally, we performed clonogenic celldeath assays similar to those in Fig. 4 in the absence orpresence of the broad-spectrum caspase inhibitor, zVAD-fmk, to explore whether caspase inhibition attenuated orabrogated the effects in clonogenic cell death assays ofIFN-b or temozolomide alone or in combination (Fig. 6E).These assays confirmed the absence of a role for caspases,

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Figure 3. Abrogation of the sensitizing effect of IFN-b by IFNAR gene silencing. A, gene silencing was performed by siRNA and confirmed by qPCR forIFNAR-1 and IFNAR-2 (left), and by flow cytometry for IFNAR-2 (right). B, control-transfected LNT-229 or LN-18 cells, siIFNAR-1-, siIFNAR-2-, ordouble-knockdown cells were exposed to IFN-b at 150 IU/mL and to temozolomide in the respective EC50 concentration range for 24 hours each, at 48 hoursafter transfection. Cell density was assessed by crystal violet staining after 2 weeks. Sensitizing effect of IFN-b is represented as delta of IFN-b–exposedversus vehicle-exposed cells. C, control-transfected, siIFNAR-1-, siIFNAR-2-, or double-knockdown LNT-229 (left) or LN-18 (right) cells were exposedto IFN-b at 150 IU/mL at 48 hours after transfection, irradiated with 5 Gy and allowed to grow for 2 weeks after irradiation. The sensitizing effect of IFN-b isrepresented as delta of IFN-b–exposed versus vehicle-exposed cells. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

(Continued.) Photomicrographs corresponding to 1,000, 5,000, and 10,000 cells at 150 IU/mL IFN-b or control are included (original magnification �40).F, the impact of IFN-b (150 IU/mL, 24 hours exposure) on established sphere cultures was assessed after a period of 10 days (top diagram).Composite quantification of the sphere number and size after exposure to IFN-b 150 IU/mL for 24 hours was performed by MTT absorbance measurementas a surrogate marker (bottom diagram). Data, mean � SEM (n ¼ 3; ��, P < 0.001). GS-2 sphere cells were visualized by bright field microscopywith (right) or without (left) IFN-b exposure for 24 hours (original magnification �40). G and H, GS cells were exposed to ddH2O or IFN-b at 150 IU/mL for48 hours and assessed for the expression of stem cell markers by flow cytometry (black curve, isotype control; gray curve, specific antibody; SFI valuesdisplayed in the diagram) or immunostaining (left, ddH20 control; right, IFN-b exposure; insert, isotype control; Scale bars, 100 mm).






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Happold et al.





either upon single agent exposure or combination. Thebiologic activity of zVAD-fmkunder these conditionswasconfirmed by the inhibition of death receptor-mediatedapoptosis as previously reported (36).

DiscussionEven after the implementation of temozolomide as

the first active chemotherapeutic agent when combinedwith standard radiotherapy, glioblastomas remain amajor challenge in the field of neuro-oncology as theyoften relapse early and follow an invariably fatal clini-cal course. The latter is also true for the subgroup ofpatients with MGMT promoter methylation who derivemost benefit from temozolomide (4, 6). Large effortshave been made to improve prognosis by investigatingnew agents, either at recurrence, but recently more oftenearly in development already in the paradigm of con-

comitant and adjuvant temozolomide plus radiotherapy(3, 37, 38).

Here we asked whether the immune modulatory cyto-kine, IFN-b, acts on glioma LTL and GIC lines, andsensitizes for the anticlonogenic effects of temozolomideor irradiation.Wedetected the expression of IFNAR-1 and-2 mRNA in all glioma cells, making them a potentialtarget for IFN-b (Fig. 1A). Although detection of IFNAR-1protein turned out to be challenging and was possible byimmunofluorescence microscopy only, but not by flowcytometry, the biologic effects of IFNAR-1 gene silencingwere prominent: siRNA-mediated knockdown of the sin-gle IFNAR-1 chain inhibited IFN-b–mediated sensitiza-tion (Fig. 3), thereby confirming its biologic function.Silencing of the IFNAR-2 chain alone inhibited IFN-bsignaling only to a moderate level whereas silencing ofIFNAR-1 was additionally required to abort the intracel-lular signal cascade enough to lead to a highly significant

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Figure 5. Gene clusters induced by IFN-b. The proapoptotic proteins XAF-1 and TRAIL were assessed by qPCR for RNA and by immunoblot for protein at6 and 24 hours after exposure to IFN-b in increasing doses (time points as assessed by microarray profiling), in LNT-229 (left) and GS-2 (right).

Figure 4. GIC sensitization to temozolomide and irradiation by IFN-b. A, LTL or GIC cells were exposed to IFN-b in a concentration-dependent manner andclonogenic survival was assessed by crystal violet (LTL) or MTT (GIC) assay. B, LTL or GIC cells were exposed to IFN-b in a concentration-dependent (left) ortime-dependent (right) manner before a 24-hour pulse of temozolomide in an EC50 concentration range. Clonogenic survival was assessed by crystalviolet or MTT assay. C, GS-2, GS-5, GS-7, or GS-9 cells pre-exposed to IFN-b (150 IU/mL, 24 hours) were treated with temozolomide as shown in Fig. 3A andallowed to grow in neurobasal medium for 2 weeks before the assessment of clonogenicity byMTT assay (control: white bar; IFN-b: black bar). Data, mean�SEM (�, P < 0.05; ��, P < 0.01; ��, P < 0.001, for the effect of IFN-b þ temozolomide versus temozolomide alone). D, GS-2, GS-5, GS-7, or GS-9 cellswere irradiated at 1, 3, or 5 Gy and monitored for clonogenic survival. Cell density was assessed by MTT assay after 2 weeks. E, GS-2, GS-5, GS-7,or GS-9 cells were pre-exposed to increasing concentrations of IFN-b for 24 hours and subjected to irradiation with 1 Gy. Cell density was assessed after 2weeks of clonogenic growth in neurobasal medium by MTT assay. Data, mean � SEM normalized to untreated cells (�, P < 0.05; ��,P < 0.01;��, P < 0.001, relative to nonirradiated cells;þ, synergistic values, defined as >10%difference between observed and predicted effect). Data (for A, B and D),mean � SEM (n ¼ 3; �, P < 0.05; ��, P < 0.01; ��, P < 0.001) relative to untreated cells.






Figure 6. Role of apoptosis-regulatory genes in IFN-b–induced sensitization. A and B, immunoblots were performed after exposure of LNT-229 (left) or GS-2(right) to IFN-b for 6 hours (top row) or 24 hours (bottom row) and the levels of caspases 8 and 3were assessed either for IFN-b exposure alone, temozolomidealone, or in combination as described. MFL and STS were used as a positive control (MFL: 1 mg/mL; STS: 1mmol/L). C, DEVD-amc cleaving assays wereperformed after 6 hours of exposure (shown for 2-hour fluorescence measurement). D, cell death was assessed by Trypan blue exclusion after 6 hours ofexposure to IFN-b at 150 or 300 IU/mL, IFN-b þ temozolomide (10 mmol/L for LNT-229, 500 mmol/L for GS-2), or MFL and STS as positive controls, asdescribed in C; ddH2Owas used as negative control (white bars, alive cells; black bars, dead cells; normalized to 100%cells). E, clonogenic cell death assayswere performed for LNT-229 andGS-2 and analyzed by crystal violet staining (for LNT-229) orMTT assay (for GS-2) at day 10 after exposure to either IFN-b ortemozolomide alone, or combinations at indicated concentrations, with or without ZVAD-fmk (15 mmol/L).

Happold et al.





reduction in inhibition of clonogenic survival after IFN-bexposure (Fig. 3B and C). This may be due to the fact thatwhile IFNAR-2 holds only ligand-binding capacities,IFNAR-1, mainly inducing the intracellular signaling cas-cades, has also been described to have weak ligand bind-ing activity, too, and may interact with other proteins inthe absence of IFNAR-2 (39, 40). Alternatively, althoughboth siRNA pools reduced receptor mRNA expression toa similar extend (Fig. 3A), differential protein stabilitymay account for different biologic efficiency of genesilencing in these experiments. Responsiveness of the cellswas assessed by monitoring the expression of targetproteins of the IFN-b signaling pathways (Fig. 1D).STAT-3 has been shown to be involved in antiprolifera-

tive functions when induced by IFN-a/b in human Daudicells (41) and, in glioma cells,may act as an IFN-b–inducedtumor inhibitor through negative modulation of miR-21(42). Conversely, there is also accumulating evidencethat STAT-3 is a driver of the malignant phenotype ofglioblastoma (43). In our study, STAT-3 was uniformlyphosphorylated in both LTL and GIC cell lines shortlyafter exposure to IFN-b (Fig. 1C). Thus, the biologic con-sequences of STAT-3 phosphorylation may be context-dependent and need to be interpreted in the natural courseor the therapeutic setting in which they are observed.Since GIC cells cultured under sphere conditions are

considered to be a more resistant subpopulation ofglioma cells (44–46), we characterized cell-cycle pro-gression, cell death induction, and sphere formationcapacity of GIC in the presence of IFN-b. Similar toLTL, we observed a cell-cycle arrest with a reduced G1

phase and an increase of the sub-G1 population, reflect-ing a moderate increase in the necrotic fraction onAnnexin V/PI flow cytometry (Fig. 2A and B). Exposureof singularized cells isolated from sphere cultures toIFN-b led to reduced sphere formation; moreover, fullyformed spheres exposed to IFN-b for 24 hours disag-gregated in the first days (Fig. 2E and F), and the cellsunderwent delayed cell death after more than a week asassessed by Trypan blue dye exclusion (data notshown). This sphere disruption effect with delayed celldeath induction appears to be a major effect of IFN-b onGIC survival and to play a more important role thanearly classical apoptotic pathways that were not shownto be activated in the first 48 hours after exposure inAnnexin V/PI flow cytometry. Accordingly, Affymetrixarray data demonstrated upregulation of several apo-ptosis-related genes (Supplementary Table S1; Supple-mentary Fig. S3B), but ZVAD-fmk-controlled clonogeni-city assays or DEVD-amc–cleaving assays did again notconfirm an induction of classical caspase-determinedapoptotic pathways (Fig. 6C and E).We demonstrated that IFN-b facilitated the loss of

clonogenicity induced by the standard chemotherapeu-tic agent for glioblastoma, temozolomide. Since a sen-sitizing mechanism mediated through MGMT and TP53had been described (20), we assessed IFN-b effects on apanel of cells with a heterogeneous pattern of MGMT

and TP53 status (Supplementary note S2 and Supple-mentary Fig. S4A). In our experimental settings, neitherTP53 nor the MGMT status was important for thesensitizing effects of IFN-b. Specifically, we did notobserve an induction of TP53 expression in cells withTP53 wild-type transcriptional activity (LNT-229) or adownregulation of MGMT expression in MGMT-expressing cells (LN-18) (Supplementary note S2 andSupplementary Fig. S4C and S4D), nor did we observeMGMT- or TP53-associated changes in clonogenic sur-vival assays, where sensitization by IFN-bwas achievedindependent of MGMT or TP53 status (Supplementarynote S2, Supplementary Fig. S4A and S4B). Finally,Affymetrix chip analyses did neither detect inductionof TP53 nor downregulation of MGMT in the cell lineexpressing the respective gene products (TP53: LNT-229, GS-2; MGMT: GS-2; data not shown). The sensiti-zation in the presence of MGMT is particularly attrac-tive in view of the urgent clinical need for novel strat-egies for the majority of glioblastoma patients withtumors without MGMT promoter methylation. More-over, we confirmed that preexposure to IFN-b sensi-tized different LTL to the anticlonogenic effects of singledoses of irradiation, too (Supplementary Fig. S4B), andthat these effects were mediated by the known receptorsof IFN-b, IFNAR-1, and IFNAR-2 (Fig. 3).

GIC lines, which were confirmed here to be highlyresistant to temozolomide due to MGMT expression,appeared to be a special target to IFN-b which impairedsphere formation when administered alone, in theabsence of major cell death induction (Fig. 2).

Accordingly, we observed a growth-inhibiting effect ofIFN-b in GIC cultures evenwhen applied as a single agent(Fig. 4A), even in MGMT-expressing and highly temozo-lomide-resistant models (Fig. 4B–E). These chemosensi-tizing effects of IFN-b were concentration- and time-dependent, with significant results assessed at a concen-tration of 150 IU/mL, and a 24-hour application (Fig. 4BandE).Of note, themolecular pattern inGIC cellswas alsodiverse with regard to TP53 status (29), confirming thatTP53 is not a major mediator of IFN-b–induced sensiti-zation in glioma cells.

All gliomamodels investigated herewere sensitized atconcentrations of IFN-b reached in the serum in clinicalsettings following intravenous application, whereranges of more than 1,000 IU/mLwere reported at dosesof 18� 106 IE i.v. in healthy volunteers (47–49). IFN-b hasbeen applied intravenously before in several clinicalstudies and was shown to be well tolerated withoutsignificant side effects (50), making it an interestingcandidate for adjuvant glioblastoma therapy, close topractice. Thus, the present laboratory evidence, togetherwith the wealth of data on the safety and tolerability ofIFN-b in patients withmultiple sclerosis, justifies furtherclinical trials of IFN-b in combination with radiotherapyand temozolomide in patients with newly diagnosedglioblastoma, specifically those patients suffering fromMGMT-unmethylated tumors.






Disclosure of Potential Conflicts of InterestC. Happold is a consultant/advisory board member for MSD. P. Roth

is a consultant/advisory board member for MSD, Roche, and MolecularPartners. G. Reifenberger has received honoraria from the speakers’bureau of Merck Serono. M. Weller has commercial research grants fromBayer, Antisense Pharma, Merck Serono, and Roche; has received hono-raria from the speakers’ bureaus ofMSD, Roche, andMerck Serono; and isa consultant/advisory board member for Magforce. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: C. Happold, P. Roth, M. WellerDevelopment of methodology: C. Happold, P. Roth, M. WellerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Happold, M. Silginer, A.-M. Florea, K. Lams-zus, R. Deenen, G. Reifenberger, M. WellerAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): C. Happold, P. Roth, M. Silginer,A.-M. Florea, R. Deenen, G. Reifenberger, M. WellerWriting, review, and/or revision of the manuscript: C. Happold, P. Roth,A.-M. Florea, K. Frei, G. Reifenberger, M. Weller

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Lamszus, K. Frei, M. WellerStudy supervision: M. Weller

AcknowledgmentsThe authors thank Dr. Sankar Mitra and Dr. Kishor Bhakat for provid-

ing the MGMT reporter plasmid, as well as Jasmin Buchs, Nadine Lauin-ger, and Silvia Dolski for excellent technical assistance.

Grant SupportThis work was supported by a grant from the Swiss National Science

Foundation (31003A_130122) to M. Weller.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 13, 2013; revised February 3, 2014; acceptedFebruary 8, 2014; published OnlineFirst February 13, 2014.

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2014;13:948-961. Published OnlineFirst February 13, 2014.Mol Cancer Ther Caroline Happold, Patrick Roth, Manuela Silginer, et al. Therapy Resistance of Glioblastoma Stem Cells

Induces Loss of Spherogenicity and OvercomesβInterferon-

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