European Journal of Cancer (2013) xxx, xxx– xxx

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Combined temozolomide and sunitinib treatment leadsto better tumour control but increased vascular resistancein O6-methylguanine methyltransferase-methylated gliomas q

M. Czabanka a, J. Bruenner a, G. Parmaksiz a, T. Broggini a, M. Topalovic a,S.H. Bayerl a, G. Auf a, I. Kremenetskaia a, M. Nieminen a, A. Jabouille b,c,S. Mueller e, U. Harms f, C. Harms e, A. Koch d, F.L. Heppner d, P. Vajkoczy a,⇑

a Department of Neurosurgery, Universitatsmedizin Charite, Berlin, Germanyb Institut National de la Sante et de la Recherche Medicale, Talence, Francec Universite de Bordeaux, Talence, Franced Department of Neuropathology, Universitatsmedizin Charite, Berlin, Germanye Center for Stroke Research, Universitatsmedizin Charite, Berlin, Germanyf Department of Neurology, Universitatsmedizin Charite, Berlin, Germany

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KEYWORDS

Antiangiogenic therapyGliomaChemotherapyNormalisation window

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l.: +49 30 450 560 002; fax:E-mail address: peter.vajko

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Abstract Introduction: Combined antiangiogenic and cytotoxic treatment represents anappealing treatment approach for malignant glioma. In this study we characterised the anti-tumoural and microvascular consequences of sunitinib (Su) and temozolomide (TMZ) therapyand verified the ideal treatment protocol, with special focus on a potential therapeutic windowfor combined scheduling.Materials and Methods: O6-Methylguanine methyltransferase (MGMT) status was analysed bypyrosequencing. Tumour growth of subcutaneous xenografts was assessed under differenttreatment protocols (TMZ, SU, SU followed by TMZ, TMZ followed by SU, combinedTMZ/SU). Intravital microscopy (dorsal skinfold chamber model) assessed microvascularconsequences. Immunohistochemistry included tumour and endothelial cell proliferation,apoptosis and vascular pericyte coverage. Real-time polymerase chain reaction (RT-PCR)analysed the expression of angiogenesis-related pathways in response to therapy.Results: Combined TMZ/SU resulted in significantly reduced tumour growth compared toeither monotreatment (TMZ: 106 ± 13mm3; SU: 114 ± 53mm3; TMZ/SU: 34 ± 7mm3) byadditional antiangiogenic effects and synergistic induction of apoptosis versus TMZ mono-treatment. Sequential treatment protocols did not show additive antitumour responses.

lsevier Ltd. All rights reserved.

19

C. Luft’ Clinical Scientist Pilot Programme funded by Volkswagen Foundation and Charite Foundation.artment of Neurosurgery, Universitatmedizin Charite, Augustenburger Platz 1, 13353 Berlin, Germany.50 560 900.ite.de (P. Vajkoczy).

M. et al., Combined temozolomide and sunitinib treatment leads to better tumour control but increased vascular

erase-methylated gliomas, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.02.019

2 M. Czabanka et al. / European Journal of Cancer xxx (2013) xxx–xxx

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resistance in O6-methylguanine methyltransf

TMZ/SU aggravated vascular resistance mechanisms characterised by significantly higherblood flow rate (TMZ: 74 ± 34 ll/s; SU: 164 ± 36 ll/s; TMZ/SU: 254 ± 95 ll/s), reduced per-meability (TMZ: 1.05 ± 0.02; SU: 0.99 ± 0.07; TMZ/SU: 0.89 ± 0.05) and recovery of peri-cyte–endothelial interactions (TMZ: 89 ± 7%; SU: 67 ± 9%, TMZ/SU:80 ± 10%) versuseither monotreatment. Vascular resistance was paralleled by an increase in Ang-1 and Tie-2

and by the downregulation of Dll4.Conclusion: Sequential application of TMZ and SU in the angiogenic window does not addantitumour efficacy to monotherapy. Simultaneous application yields beneficial tumour con-trol due to additive antiangiogenic and proapoptotic effects. Combined treatment may aggra-vate pericyte-mediated vascular resistance mechanisms by altering Ang-1–Tie-2 and Dll4/Notch pathways.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Antiangiogenic agents have shown promising resultsin clinical trials in the treatment of different metastaticcancers.1 However, clinical success for antiangiogenicagents in cancer treatment is limited by resistance mech-anisms. Clinical data demonstrate that antiangiogenictreatment exerts the best antitumour results whenapplied in combination with chemotherapy.1 Sunitinib(SU) represents a small molecular inhibitor of differenttyrosine kinases including vascular endothelial growthfactor receptor (VEGFR) and platelet-derived growthfactor receptor (PDGFR).2 Despite promising resultsin different clinical trials, SU has shown no therapeuticefficacy in glioblastoma multiforme (GBM) treatment.3

Therefore, current antiangiogenic treatment strategiesaim at combining antiangiogenic substances with che-motherapeutic agents especially in GBM treatment.4

The most effective chemotherapeutic agent in GBMtreatment is temozolomide (TMZ),5 which also exertsantiangiogenic effects in GBM.6 Combined administra-tion may overcome resistance mechanisms of GBMagainst antiangiogenesis. However, there is significantdebate about the ideal timing and sequence of combinedapplications.4 The concept of vascular normalisationpostulates a defined time window in which tumour ves-sels become normalised. Only in this limited time frameadditional administration of chemotherapy will lead tofurther therapy effects.7 Therefore the aims of our studywere (i) to investigate the antitumour effects of a combi-nation therapy consisting of TMZ and SU in a preclin-ical glioma model, (ii) to identify the ideal combinationregime to maximise antitumour efficacy and (iii) to char-acterise the effects on vascular resistance mechanisms.

2. Materials and methods

2.1. Tumour cells

SF126 and U87 human glioma cells were grown inDMEM with 4.5 g/L Glucose supplemented with 10%

a M. et al., Combined temozolomide a

erase-methylated gliomas, Eur J Cance

foetal bovine serum (PAA GmbH, Linz, Austria) at37 �C in 5% CO2 humidified incubators.

2.2. Subcutaneous xenografts

The experiments were performed in athymic nudemice (nu/nu; male, 28–32 g), bred and maintained withina specific pathogen-free environment.

For growth analysis 2 � 106 SF126 and U87 gliomacells were implanted subcutaneously in the hind flankregion (n = 6 per group). Tumour volume was assessedas reported.8

We applied TMZ (200 mg/kg body weight), SU(40 mg/kg body weight) or the combination of both sub-stances (TMZ/SU; TMZ 200 mg/kg, SU 40 mg/kg)intraperitoneally for 6 days. Treatment was startedwhen tumours reached a volume of 50 mm3. Controlanimals received placebo (NaCl 9%).

For identification of the ideal combination protocol(sequential versus continuous combination) we per-formed sequential application SU (40 mg/kg/day) andTMZ (200 mg/kg body weight) over a time period of10 days in SF126 subcutaneous xenografts. Therapywas initiated when tumours reached a size of 50 mm3.Treatment in the SU! TMZ group was started withSU and changed to TMZ after a normalisation windowof 5 days. In the TMZ! SU group, TMZ was followedby SU. In the continuous group (TMZ/SU), we appliedSU (40 mg/kg bodyweight) and low-dose TMZ (50 mg/kg body weight) constantly in combination over 10 days(Fig. 2C). Low-dose TMZ was chosen in order to restricttoxicity.

2.3. Dorsal skinfold chamber model

SF126 glioma cells were implanted in dorsal skinfoldchambers in nude mice (n = 5 per group) followed byintravital microscopy (IVM). Treatment with TMZ(200 mg/kg body weight), SU (40 mg/kg body weight)or TMZ/SU (TMZ 200 mg/kg, SU 40 mg/kg) wasstarted 5 days after tumour cell implantation. Controlanimals received placebo. Analysis was performed ondays 5, 7, 9 and 11 after tumour cell implantation.

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Fig. 1. (A and B) O6-Methylguanine methyltransferase (MGMT) promoter analysis by pyrosequencing. Whereas the tumour cells of the SF126 cellline have a distinct methylation of all five analysed CpG-Islands of the MGMT promotor, the tumour cells of the U87 have CpG-Island MGMT

promoter methylation in one of five analysed CpGs.

M. Czabanka et al. / European Journal of Cancer xxx (2013) xxx–xxx 3

2.4. Intravital microscopy

Intravital microscopy was performed according toprevious reports.8,9 Quantitative analysis included: totalintratumoural vascular density (TVD) and functionalintratumoural vascular density (FVD), defined as thelength of all newly formed red-blood cell perfusedmicrovessels. Vessel diameter (D) and microvascularpermeability (P) were analysed and measured asdescribed previously.10,11 Perfusion index (PI) was cal-culated as the ratio of FVD/TVD.11 We assessed micro-vascular red-blood cell velocity (RBCV; mm/s) andmicrovascular blood flow rate (Qv; nL/s) as describedpreviously.11 Microvascular blood flow rate (Qv) wascalculated from red-blood cell velocity and microvesseldiameter based on following formula: Qv = p� (D/2)2 -� RBCV/K, where RBCV represents red-blood cellvelocity, D the microvessel diameter and K (=1.3) theBaker Wayland factor considering the parabolic velocityprofile of blood in microvessels.12

2.5. Immunohistochemistry

Acetone-fixed cryosections (5 lm, n = 4 per group)were used for immunohistochemistry. Regions of inter-est (ROI) were defined as an area of 91 lm2 and ana-lysed in 40� magnification. Quantification wasperformed by calculating the number of positivelystained nuclei per region of interest.

We used a murine-specific anti-Ki67 antibody (Tec-3,DAKO, Hamburg, Germany) to visualise endothelialcell proliferation. An anti phosphohistone H3 antibodywas used to detect tumour cell proliferation (Upstae/Millipore, Schwalbach, Germany) at a dilution of1:500 and detection was done using the Vector EliteKit (Linaris, Wertheim, Germany) and DAB (DAKO,Hamburg, Germany) as a substrate. Tumour-associatedapoptosis was assessed by analysing caspase 3 staining.Pericyte–endothelial interactions and vessel density werevisualised using a double fluorescence immunostaining

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for CD31 and desmin and quantified as described previ-ously 13.

2.6. Methylation analysis of the O6-methylguaninemethyltransferase (MGMT) promoter

2.6.1. DNA extraction and bisulphite treatment

DNA from the glioma cell lines SF126 and U87 wasisolated from tumour tissue using the QiA-Amp DNAminikit (Qiagen, Hilden, Germany) in accordance withthe manufacturers instructions. Genomic DNA(500 ng) of the respective cell lines was treated withsodium bisulphite using the EZ DNA Methylation Goldkit (Zymo Research, Orange, CA). As internal controlgenomic DNA from white matter of non-tumourouslesions was used.

2.6.2. Pyrosequencing

The pyrosequencing methylation assay was per-formed with the PyroMarkQ24 MGMT kit (Qiagen,Hilden, Germany) on a PSQTM96 MA system (Biotage,Uppsala, Sweden). The PyroMarkQ24 MGMT kitdetects the level of methylation of five CpG sites locatedin the first exon of the MGMT gene. Cytosine not fol-lowed by guanine, and which is therefore not methyl-ated, serves as an internal control for completion ofthe bisulphite treatment.

2.7. PCR/Real-time PCR

Quantitative Real-time polymerase chain reaction(qRT-PCR) was carried out with the Applied Biosys-tems 7900H Fast Real-time PCR System. The quantifi-cation of mRNA from tumour tissue was carried outaccording to the protocol described by Nolan et al.14

mRNA was isolated as described previously.15 cDNAwas generated from mRNA according to the QuantiTectReverse Transkipion Kit instructions (Qiagen). In orderto analyse endothelial cell specific gene expression

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Fig. 2. (A) Tumour volumes of experimental SF126 tumours in different treatment groups showing a beneficial antitumour effect for thecombination therapy regime (n = 6 animals per group, *p < 0.05 versus control, §p < 0.05 versus TMZ, # = p < 0.05 versus TMZ and versus SU,One-way analysis of variance (ANOVA) followed by Bonferroni correction). (B) Tumour volumes of experimental U87 tumours in differenttreatment groups showing a beneficial antitumour effect for the combination therapy regime over TMZ monotreatment(n = 6 animals per group,*p < 0.05 versus control, #p < 0.05 versus TMZ, One-way ANOVA followed by Bonferroni correction). (C) Assessment of different applicationstrategies during the angiogenic window of SU. In SU! TMZ group animals received 40 mg/kg bodyweight SU intraperitoneally for 5 daysfollowed by TMZ 200 mg/kg bodyweight for 5 days. SU has been shown to induce vascular normalisation 5 days after treatment initiation andtherefore we expected a beneficial effect when treatment is changed to TMZ during the angiogenic window. As relative control group we startedwith TMZ (200 mg/kg bodyweight intraperitoneally for 5 days) treatment and changed to SU (40 mg/kg bodyweight intraperitoneally for 5 days).However, SU pretreatment was not superior to TMZ pretreatment. In order to analyse the effects of long term combined application of TMZ andSU we analysed the effects of SU (40 mg/kg bodyweight intraperitoneally for 10 days) in combination with low-dose TMZ (50 mg/kg bodyweightintraperitoneally for 10 days) showing a significantly reduced tumour growth to control tumours and to the SU! TMZ and TMZ! SU groups(*p < 0.05 versus control, #p < 0.05 versus SU! TMZ and versus TMZ! SU, n = 6 animal per group, One-way ANOVA followed by Bonferronicorrection). (D) Fluorescent immunohistochemistry of U87 tumours of the different treatment protocols showing apoptosis induction by Caspase 3(green) and DAPI (red) co-staining in 20-fold magnification. TMZ/SU demonstrated significantly increased apoptosis induction as compared to thecontrol or either monotreatment. (E) Quantified analysis of apoptosis induction of Caspase3-DAPI co-staining in SF 126 glioma showing additiveapoptosis induction for TMZ/SU combination treatment (n = 60 ROIs, *p < 0.05 versus control, #p < 0.05 versus TMZ and versus SU, One-wayANOVA on ranks followed by Dunn’s test). (F) Quantified analysis of apoptosis induction of Caspase3-DAPI co-staining in U87 glioma showingadditive apoptosis induction for TMZ/SU combination treatment (n = 60 ROIs, *p < 0.05 versus control, §p < 0.05 versus TMZ, #p < 0.05 versusSU, One-way ANOVA on ranks followed by Dunn’s test). (For interpretation of the references to colour in this figure legend, the reader is referredto the web version of this article.)

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murine primers were used to detect Ang-1, Tie-2, Dll4

and Gapdh control.

2.8. Statistical analysis

Quantitative data are given as mean ± SD. Meanvalues of all parameters were calculated from theaverage values in each animal. For analysis ofsubcutaneous tumour growth and for IVM analysisdifferences between groups were assessed by One-wayanalysis of variance (ANOVA) followed by Bonferronicorrection. Analysis of immunohistochemistry was

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performed using One-way ANOVA on ranks followedby Dunn’s test. Results with p < 0.05 were consideredsignificant.

3. Results

3.1. Methylation of the MGMT promotor

In both glioma cell lines the U87 as well as the SF126pyrosequencing analysis revealed a distinct methylationof at least one CpG-Island of the MGMT promoter (Fig. 1A and B).

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3.2. Combined temozolomide and sunitinib therapy

reduces tumour growth

Compared to placebo treatment SU and TMZ led toa significant reduction of tumour growth (Fig. 2A andB). There was no difference between tumours thatreceived either monotherapy. In comparison to TMZmonotherapy, combination therapy resulted in a signif-icant further reduction of tumour growth (Fig. 2A andB). Compared to SU, we observed a significant advan-tage for the TMZ/SU in SF126 s and a trend for anadditive advantage in U87 gliomas (Fig. 2A and B).

Sequential application of both substances led to a sig-nificant reduction of tumour growth compared to pla-cebo treatment ( Fig. 2C). However, a significant

Fig. 3. (A) Fluorescent immunohistochemistry of SF 126 tumours of themurine Ki-67 (green) and CD31 (red) co-staining. TMZ/SU demonstratecompared to the control or either monotreatment. (B) Quantified analysis oSF126 glioma showing inhibition of endothelial cell proliferation for TMZ/ROIs, *p < 0.05 versus control, §p < 0.05 versus TMZ, #p < 0.05 versus SU,test). (C) Quantified analysis of endothelial cell proliferation of murineendothelial cell proliferation for TMZ/SU combination treatment compa§p < 0.05 versus TMZ, #p < 0.05 versus SU, One-way ANOVA on ranks folthis figure legend, the reader is referred to the web version of this article.)

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difference between both sequential protocols could notbe observed. Only constant combined application ofboth substances led to a synergistic reduction of tumourgrowth compared to either sequential application andcompared to placebo treatment (Fig. 2C).

3.3. Combination therapy induces proapoptotic and

additive antiangiogenic effects

TMZ demonstrated a significant induction of apopto-sis in both tumour entities compared to control tumours(Figs. 2 and 3). SU resulted in a comparable induction ofapoptosis in SF126 glioma and demonstrated a superiorinduction of apoptosis in U87 glioma when compared toTMZ (Fig. 2). TMZ/SU potentiated proapoptotic effects

different treatment protocols showing endothelial cell proliferation byd significantly increased inhibition of endothelial cell proliferation asf endothelial cell proliferation of murine Ki-67 and CD31 co-staining inSU combination treatment compared to either monotreatment (n = 60One-way analysis of variance (ANOVA) on ranks followed by Dunn’sKi-67 and CD31 co-staining in U87 glioma showing inhibition of

red to either monotreatment (n = 60 ROIs, *p < 0.05 versus control,lowed by Dunn’s test). (For interpretation of the references to colour in

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Fig. 4. (A) Representative intravital microscopic images of tumour microcirculation in the different treatment groups using FITC fluorescence andgreen light epiillumination technique in 20-fold magnification. The reduced number of tumour vessels in SU treated and TMZ/SU combinationtreatment tumours is demonstrated. Resistant tumour vessels in SU and TMZ/SU treated tumours show a larger vascular diameter. Please note theextravascular fluorescence signal in the control, TMZ and SU monotreatment tumours. Extravascular fluorescence signal depicts extravasation ofFITC as a sign of increased vascular permeability. In contrast, resistant tumour vessels in TMZ/SU combination treatment tumours demonstrate asignificantly lower extravascular fluorescence signal and therefore reduced permeability compared to the remaining experimental groups. (B)Quantification of total vessel density (TVD) during IVM in SF 126 xenografts. TVD includes red-blood cell perfused and non-perfused tumourvessels and is quantified by measuring the total length of microvessels in relation to the analysed area. All treatment groups showed a significantreduction of TVD compared to control tumours. Antiangiogenic treatment with SU or with combination treatment with TMZ/SU resulted in afurther significant reduction of TVD compared to TMZ monotreatment. We did not identify a significant difference in TVD between SUmonotreatment and TMZ/SU combination treatment (*p < 0.05 versus control, #p < 0.05 versus TMZ, n = 6 animals per group, One-way analysisof variance (ANOVA) followed by Bonferroni correction). (C) Quantification of functional vessel density (FVD) during IVM. FVD includes red-blood cell perfused tumour vessels only and is quantified by measuring the total length of red-blood cell perfused microvessels in relation to theanalysed area. All treatment groups showed a significant reduction of FVD compared to control tumours. However, there was no difference inFVD between SU and TMZ treatment. Only combination treatment with TMZ/SU resulted in a further significant reduction of FVD compared toTMZ monotreatment (*p < 0.05 versus control, #p < 0.05 versus TMZ, n = 6 animals per group, One-way ANOVA followed by Bonferronicorrection). (D) Quantification of perfusion index in IVM analysis. Perfusion index is calculated as the ratio between FVD and TVD. This ratioreflects the percentage of red-blood cell perfused tumour vessels compared to all tumour vessels and therefore generates a parameter that allows theestimation of microcirculatory efficacy and functionality. In all treatment groups perfusion index significantly increased to control tumoursproviding evidence for improved microcirculatory efficacy in response to treatment. Only in the groups with advanced antiangiogenic activity (SUmonotreatment and TMZ/SU combination treatment) a further significant improvement of perfusion index was noted compared to TMZmonotreatment (*p < 0.05 versus control, #p < 0.05 versus TMZ, n = 6 animals per group, One-way ANOVA followed by Bonferroni correction).(E) Quantified results for microvascular blood flow rate in control tumour vessels and therapy resistant tumour vessels. Only advancedantiangiogenic intervention with either SU monotreatment or with TMZ/SU combination treatment resulted in a significant increase inmicrovascular blood flow rate compared to the control and compared to TMZ monotreatment (*p < 0.05 versus control, #p < 0.05 versus TMZ,n = 6 animals per group, One-way ANOVA followed by Bonferroni correction). (F) Quantified results for microvascular permeability during IVManalysis. Permeability was assessed by measuring grey values in extravascular and intravascular regions of interest. The ratio between intravasculargrey levels and extravascular grey levels was calculated as the permeability parameter. Only combination treatment induced a significant reductionof microvascular permeability compared to the control and compared to either SU or TMZ monotreatment (*p < 0.05 versus control, #p < 0.05versus TMZ and versus SU, n = 6 animals per group, One-way ANOVA followed by Bonferroni correction). (For interpretation of the referencesto colour in this figure legend, the reader is referred to the web version of this article.)

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compared to the control and compared to either mono-treatment (Figs. 2 and 3).

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resistance in O6-methylguanine methyltransferase-methylated gliomas, Eur J Cance

Even though both substances may alter tumour cellproliferation, our experiments demonstrate TMZ/SU

nd sunitinib treatment leads to better tumour control but increased vascular

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M. Czabanka et al. / European Journal of Cancer xxx (2013) xxx–xxx 7

does not show additive antiproliferative effects (data notshown).

Either monotreatment did inhibit the rate of endothe-lial cell proliferation in both tumour entities. Comparedto TMZ, TMZ/SU showed a significantly stronger inhi-bition of endothelial cell proliferation (Fig. 3). In com-parison to SU, no significant difference between TMZ/SU and SU was observed.

TVD was significantly reduced by TMZ and SU ascompared to control tumours (Fig. 4A and B). Com-pared to TMZ, we observed a significantly strongerreduction of TVD in the SU and SU/TMZ treatmentgroups ( Fig. 4B). Between SU monotreatment andTMZ/SU combination treatment no difference in TVDwas observed. Regarding FVD we observed a strongerantiangiogenic effect for the TMZ/SU. Whereas TMZand SU monotreatment did show a significant reductionof FVD compared to control tumours, only TMZ/SUdemonstrated an even further reduction of FVD (Fig. 4C). Compared to TMZ monotreatment, TMZ/SU resulted in a significantly lower FVD ( Fig. 4C).

Fig. 5. (A and B) Fluorescent immunohistochemistry of SF 126 tumours (Desmin (green) and PECAM (red) co-staining using 20-fold magnification.response to SU monotreatment and the recovery of DESMIN positive tumtreatment. The increased microvascular diameter and reduced number of tumand D) Quantification of pericyte–endothelial interactions in SF126 (C) atreatment significantly reduced the number of pericyte–endothelial inmonotreatment. However, combination treatment led to a recovery of persignificantly higher number of DESMIN positive tumour vessels (n = 60 ROway analysis of variance (ANOVA) on ranks followed by Dunn’s test). (Freader is referred to the web version of this article.)

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There were no significant differences between TMZand SU monotreatment groups.

3.4. Vascular resistance in response to combinationtreatment

TMZ/SU treatment did not result in complete eradi-cation of tumour vessels. A reduction of 70 – 80% oftumour vessels could be achieved as a maximum antian-giogenic effect. In addition, antiangiogenic treatmentwith either therapy strategy resulted in a significantincrease in perfusion index (Fig. 4D). Our study demon-strated that this effect was significantly stronger intumours that were treated with SU or with TMZ/SUas compared to TMZ monotreatment ( Fig. 4D).Improvement of perfusion index was paralleled by signif-icantly improved microvascular blood flow rate in resis-tant tumour vessels that were treated with TMZ/SU orSU monotreatment (Fig. 4E). There was no differencein microvascular blood flow rate between control tumourvessels and resistant vessels that were treated with TMZ.

A) and U87 tumours (B) showing pericyte–endothelial interactions byPlease note the reduced number of DESMIN positive tumour vessels in

our vessels in tumours that were treated with TMZ/SU combinationour vessels especially in the combination treatment group is shown. (C

nd U87 (D) tumours. SU monotreatment and TMZ/SU combinationteractions compared to control tumours and compared to TMZicyte–endothelial interactions compared to SU monotreatment with aIs, *p < 0.05 versus control and versus TMZ, #p < 0.05 versus SU, One-or interpretation of the references to colour in this figure legend, the

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TMZ/SU resistant vessels showed significantlyreduced microvascular permeability compared to eithermonotreatment or control tumours ( Fig. 4F). TMZand SU monotreatment did not induce alterations ofmicrovascular permeability. Reduction of microvascularpermeability was paralleled by reestablishment of peri-cyte coverage in tumours that received TMZ/SU ascompared to pericyte coverage in SU treated vessels (Fig. 5).

3.5. Combination treatment leads to upregulation of

Ang-1 and Tie-2

In order to identify potential molecular pathwaysinvolved in vascular resistance, we analysed angiogene-sis related gene expression using quantitative polymer-ase chain reaction (qPCR). We identified a significantupregulation of Ang-1 and a significant downregulationof Dll4 in response to TMZ/SU. The application ofTMZ induces upregulation of Ang-1 and Tie-2, whichare important mediators of pericyte-mediated survivalmechanisms. Additionally, we observed a downregula-tion of Dll4 in response to SU treatment, which was sig-nificantly more pronounced in tumours that receivedTMZ/SU (Fig. 6).

Fig. 6. Results of quantitative polymerase chain reaction (qPCR) analysis otumour entities receiving either TMZ, SU or combination (TMZ/SU) treatSU; One-way analysis of variance (ANOVA) followed by Bonferroni corr

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4. Discussion

We demonstrate that combining TMZ and SUyields enhanced antitumour effects due to synergisticapoptosis induction and additive antiangiogenic effectsif both substances are applied continuously in combi-nation. Sequential application of both substanceswithin the angiogenic window does not lead to syner-gistic antitumour responses. Surprisingly, TMZ/SUcombination therapy aggravates vascular resistancemechanisms with a reduction of vascular permeability,improvement of microvascular perfusion and reestab-lishment of pericyte–endothelial interactions potentiallyby interfering with Ang-1–Tie-2 and Dll4–Notch sig-nalling pathways.

TMZ treatment demonstrates the best antitumoureffects in tumours with the methylation of the MGMT

promoter.16 Chahal et al. showed that MGMT altersthe angiogenic profile of glioma cells emphasising thatMGMT expressing gliomas might benefit from SU ther-apy.17 Both tumour entities used in the herein presentedstudy were characterised by a methylated MGMT pro-moter. Zhou and Gallo demonstrated in preclinicalstudies, that SU increased tumour distribution ofTMZ in subcutaneous and orthotopic glioma xeno-

f murine Ang-1(A), Dll4 (B), Ang-2 (C) and Tie-2 (D) gene expression inment (n = 3 tumour per group, *p < 0.05 versus TMZ, #p < 0.05 versusection, black line indicates gene expression of control tumours).

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grafts.18 Still the ideal timing of combination treatmentremains an unsolved clinical problem. The concept ofvascular normalisation defines a short time frame, thenormalisation window, in which the addition of chemo-therapy to antiangiogenic therapy yields beneficialeffects.7 In our study, SU treatment for 5–6 days inducedvascular normalisation. When we changed the therapyto TMZ during the angiogenic window, we could notdetect a beneficial therapeutic effect. When we per-formed TMZ treatment for 6 days, which does notinduce vascular normalisation, and then changed thetreatment to SU, a comparable therapeutic effect wasobserved. Therefore, antiangiogenic pretreatment fol-lowed by a change to chemotherapy during the angio-genic window did not show beneficial antitumoureffects in our study. Only constant combined applicationshowed a synergistic antitumour effect, even though theunderlying molecular mechanism remains elusive.According to the herein presented data the antiangio-genic stimulus has to be provided constantly to maintainsynergistic antitumour effects. Improved tumour controlcan be explained by synergistic proapoptotic and antian-giogenic effects as both substances have shown proapop-totic and antiangiogenic effects as monotherapy inprevious studies.8,9,19–21 In this context combined appli-cation of TMZ/SU was paralleled by increased toxicity(weight loss) in our experiments. This may translate intoan increased risk for toxicity in patients in the case ofclinical application of this treatment strategy.

However, about 20% of tumour vessels demonstrateresistance against TMZ/SU. In resistant tumour areas,the perfusion index increased significantly. An increasein perfusion index shows a better perfusion profile withmore tumour vessels demonstrating red-blood cell per-fusion in relation to the total number of tumour vessels.In treatment resistant tumour vessels we detected highermicrovascular blood flow rates. Additionally, resistantvessels demonstrated significantly reduced permeability.These functional changes in tumour microcirculationwere paralleled by a reestablishment of pericyte–endo-thelial interactions. Tumours that were treated withSU demonstrated a significant reduction of pericyte–endothelial cell interactions. This phenomenon has beendemonstrated before and is explained by PDGFR inhi-bition.8,9 However, it remains unclear which furthermolecular interactions are responsible for the vascularphenotype that we observed as suntinib inhibits a vari-ety of angiogenic signalling pathways.2

The combination of TMZ with SU resulted in asignificant reestablishment of pericyte–endothelial inter-actions compared to SU monotreatment. Therefore,changes in microvascular blood flow and in microvascu-lar permeability could be explained by a recovery ofpericyte–endothelial interactions and by a maturationof therapy resistant tumour vessels. Contractile muralcells are known regulators of vascular blood flow and

Please cite this article in press as: Czabanka M. et al., Combined temozolomide a

resistance in O6-methylguanine methyltransferase-methylated gliomas, Eur J Cance

permeability.22 Potential pathways that guide therecruitment of mural cells to endothelial cells are theAng-1/Tie-2 and the Dll4/Notch pathway.1 In ourexperiments, endothelial cells are derived from the mur-ine host, therefore we used murine-specific primers todetect changes in Ang-1, Tie-2 and Dll4 expression.The expression of Ang-1 and Tie-2 was significantlyincreased in TMZ monotreatment and TMZ/SUcombination treatment tumours. Previous studies dem-onstrated that Ang-1 and Tie-2 mediated pericyte–endothelial interactions induce vascular survivalmechanisms.13 Potentially, the addition of TMZ to SUtreatment leads to the upregulation of Ang-1 and Tie-2

and overcomes the anti-PDGF effects of SU therapy,thereby reactivating pericyte-mediated survival mecha-nisms in tumours that are treated with the combinationregime. Supporting this hypothesis, Ang-1 and Tie-2have been shown to induce the formation of maturepericyte-covered vessels with tighter endothelial celljunctions.23 Apart from changes in the Ang-1/Tie-2pathway, we observed a significant reduction of Dll4

in tumours that received combination treatment. Therole of the Dll4/Notch signalling pathway during anti-angiogenic treatment is still unknown. Dll4/Notch sig-nalling has been shown to mediate tumour resistanceto anti-VEGF therapy in a pericyte-independent man-ner.24,25 In our study downregulation of Dll4 in responseto SU and TMZ/SU was observed. Downregulation ofDll4/Notch signalling has been shown to result in vascu-lar neoplasms resulting from uncontrolled angiogene-sis.26 Moreover, Dll4 inhibition results in extensiveangiogenesis overcoming the antiangiogenic effects ofVEGFR2 – impairment.27 Consequently, Dll4 downreg-ulation may be induced in order to maintain tumourangiogenesis in response to TMZ/SU combinationtreatment.

In conclusion the combination of TMZ and SUshows beneficial antiglioma effects in MGMT positivetumours by the synergistic induction of apoptosis andantiangiogenesis. However, long-term treatment effectsmay be hampered by aggravated vascular resistancemechanisms that are induced by a combinationtreatment.

Conflict of interest statement

None declared.

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