tumor vessel normalization, immunostimulatory ...€¦ · checkpoint therapy (ict) offers new...

Research Article

Tumor Vessel Normalization, ImmunostimulatoryReprogramming, and Improved Survival inGlioblastoma with Combined Inhibition of PD-1,Angiopoietin-2, and VEGFMariangela Di Tacchio1,2,3, Jadranka Macas1,4, Jakob Weissenberger1,4,Kathleen Sommer1,4, Oliver B€ahr2,3,4,5, Joachim P. Steinbach2,3,4,5, Christian Senft2,3,6,Volker Seifert2,3,6, Martin Glas7,8,9, Ulrich Herrlinger10, Dietmar Krex3,11,12,Matthias Meinhardt13, Astrid Weyerbrock14, Marco Timmer15, Roland Goldbrunner15,Martina Deckert16, Andreas H. Scheel17, Reinhard B€uttner17, Oliver M. Grauer18,Jens Schittenhelm19, Ghazaleh Tabatabai3,20,21, Patrick N. Harter1,2,3,4, Stefan G€unther22,Kavi Devraj1,4, Karl H. Plate1,2,3,4, and Yvonne Reiss1,2,3,4

Abstract

Glioblastoma (GBM) is a non-T-cell–inflamed cancer char-acterized by an immunosuppressive microenvironment thatimpedes dendritic cell maturation and T-cell cytotoxicity.Proangiogenic cytokines such as VEGF and angiopoietin-2(Ang-2) have high expression in glioblastoma in a cell-specific manner and not only drive tumor angiogenesis andvascular permeability but also negatively regulate T-lymphocyteand innate immune cell responses. Consequently, the allevia-tion of immunosuppression might be a prerequisite for suc-cessful immune checkpoint therapy inGBM.Wehere combinedantiangiogenic and immune checkpoint therapy and demon-strated improved therapeutic efficacy in syngeneic, orthotopic

GBM models. We observed that blockade of VEGF, Ang-2, andprogrammed cell death protein-1 (PD-1) significantly extendedsurvival compared with vascular targeting alone. In the GBMmicroenvironment, triple therapy increased the numbers ofCTLs, which inversely correlatedwithmyeloid-derived suppres-sor cells and regulatory T cells. Transcriptome analysis of GBMmicrovessels indicated a global vascular normalization that washighest after triple therapy. Our results propose a rationale toovercome tumor immunosuppression and the current limita-tions of VEGFmonotherapy by integrating the synergistic effectsof VEGF/Ang-2 and PD-1 blockade to reinforce antitumorimmunity through a normalized vasculature.

IntroductionThe current standard therapy for GBM consisting of gross

total surgery, followed by combined radiation and chemo-

therapy with temozolomide, results in overall survival of15–25 months (1–3). The addition of antiangiogenic therapywith bevacizumab, a humanized monoclonal anti-VEGFA

1Institute of Neurology (Edinger Institute), University Hospital, Goethe Univer-sity, Frankfurt, Germany. 2German Cancer Consortium (DKTK), Partner SiteFrankfurt/Mainz, Frankfurt, Germany. 3GermanCancer Research Center (DKFZ),Heidelberg, Germany. 4Frankfurt Cancer Institute, Frankfurt, Germany. 5Senck-enberg Institute of Neurooncology, University Hospital, Goethe University,Frankfurt, Germany. 6Department of Neurosurgery, University Hospital, GoetheUniversity, Frankfurt, Germany. 7Department of Neurology, Division of ClinicalNeurooncology, University Hospital Essen, University Duisburg-Essen, Essen,Germany. 8German Cancer Consortium (DKTK), Partner Site Essen/D€usseldorf,Essen, Germany. 9DKFZ-Division Translational Neurooncology at the WestGerman Cancer Center (WTZ), University Hospital Essen, University Duis-burg-Essen, Essen, Germany. 10Department of Neurology, Division of ClinicalNeurooncology, University of Bonn Medical Centre, Bonn, Germany. 11Depart-ment of Neurosurgery, Dresden University of Technology, Dresden, Germany.12German Cancer Consortium (DKTK), Partner Site Dresden, Dresden, Germany.13Institute of Pathology, Dresden University of Technology, Dresden, Germany.14Department of Neurosurgery, Medical Center-University of Freiburg, Freiburg,Germany. 15Center for Neurosurgery, University Hospital of Cologne, Cologne,Germany. 16Institute of Neuropathology, University Hospital of Cologne,Cologne, Germany. 17Institute of Pathology, University Hospital of Cologne,Cologne, Germany. 18Department of Neurology with Institute of TranslationalNeurology, University Hospital of Muenster, Muenster, Germany. 19Departmentof Neuropathology, Institute of Pathology and Neuropathology, Eberhard-Karls

University Tuebingen, Tuebingen, Germany. 20Departments of Neurology &Neurosurgery, Interdisciplinary Division of Neuro-Oncology, Hertie Institute forClinical Brain Research, Center for CNS Tumors, Comprehensive Cancer Center,University Hospital T€ubingen, Eberhard Karls University T€ubingen, T€ubingen,Germany. 21German Cancer Consortium (DKTK), Partner Site T€ubingen,T€ubingen, Germany. 22Max Planck Institute for Heart and Lung Research,Bioinformatics and Deep Sequencing Platform, Bad Nauheim, Germany.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

K.H. Plate and Y. Reiss contributed equally to this article.

Current address for A. Weyerbrock: Department of Neurosurgery, St. GallenCantonal Hospital, St. Gallen, Switzerland.

CorrespondingAuthor:YvonneReiss, Institute ofNeurology (Edinger Institute),University Hospital, Goethe University, Heinrich-Hoffmann-Strasse 7, Frankfurt60528, Germany. Phone: 4969-6301-84155; Fax: 4969-6301-84150; E-mail:[email protected]

Cancer Immunol Res 2019;7:1910–27

doi: 10.1158/2326-6066.CIR-18-0865

�2019 American Association for Cancer Research.

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antibody, was not successful in first-line therapy despiteadvances in other cancer entities (4, 5). Hence, an urgent needexists for new treatment strategies. In this context, immunecheckpoint therapy (ICT) offers new therapeutic avenues forthe treatment of GBM (4, 6). Therapeutic targeting with immu-nomodulatory antibodies to enhance T-cell responses, such asantibodies to CTL-associated protein 4 (CTLA-4) and pro-grammed cell death protein 1 (PD-1), have demonstrated afavorable outcome in various cancer entities, with the bestefficacy in patients with advanced melanoma and lung can-cer (4, 7). Whether ICT provides a treatment option for primaryGBM is currently being investigated in clinical trials(NCT02617589, NCT02667587). In recurrent GBM, ICT hasnot met its primary endpoint compared to treatment withbevacizumab (NCT02017717; ref. 8). The unique central ner-vous system (CNS) microenvironment appears to be challeng-ing for the efficacy of ICT in GBM (5, 9). GBM is a non-T-cell–inflamed cancer whereby immunosuppressive factors such asVEGF and TGFb have been acknowledged to contribute to T-cellpaucity (4, 6, 10). Studies have demonstrated that VEGF-targeting alleviates immunosuppression and is permissive forT-lymphocyte entry (4, 11, 12) and combined VEGF/angio-poietin-2 (Ang-2) therapy efficiently targets both the vascula-ture and immune cells in GBM models (13–15). VEGF andAng-2 are known to act in concert to nourish tumor neovesselgrowth (16). Likewise, they operate as immunosuppressantsand modulate immune cell recruitment with nonoverlappingdownstream signaling pathways (9, 17). We hypothesized thatalleviating this hostile microenvironment characterized byimmunosuppression, abnormal vessel growth, hypoxia, andnecrosis might improve immunotherapy. Motivated by thesuccess of checkpoint inhibitors in melanoma, lung cancer (7),and rodent cancer models (18, 19), we analyzed tissue sectionsof patients with bevacizumab-treated GBM and identified PD-1/PD-L1 signaling as a promising target. We here identifiedantiangiogenic therapy as a treatment option to overcomeresistance to ICT in GBM. ICT led to increased survival whencombined with anti-VEGF/Ang-2 therapy by creating an immu-nostimulatory microenvironment. Bioinformatic analysis ofglioma microvessels indicated that dual antiangiogenic therapyled to a global vascular normalization that was potentiated byanti–PD-1 therapy. We provide a rational combinatorialapproach to improve the efficacy of ICT by integrating thesynergistic effects of VEGF/Ang-2 and PD-1 blockade to rein-force antitumor immunity through a normalized vasculature.

Materials and MethodsMice

Animal experiments were performed according to the princi-ples of laboratory animal care and according to German nationallaws. The study was approved by the local ethics committee(RegierungspraesidiumDarmstadt, FK/1031). In vivo experimentswere performed with 8-week-old, female C57BL/6 or B6C3F1mice that were obtained from Charles River.

Cell linesGL261 (CVCL_Y003, gift from M. Machein, Department of

Neurosurgery, Freiburg University Medical School, Freiburg,Germany), PD-L1–deficient (generated by CRISPR-Cas9, seebelow), and Tu-2449 glioma cells (CVCL_D318, generated by

J. Weissenberger; ref. 20) weremaintained inDMEM-GlutaMAX-I(Gibco) supplemented with 10% FBS (Sera Plus, Pan Biotech)and 1% penicillin and streptomycin (Sigma) at 5% CO2 at 37�C.Glioma cells were utilized one passage after thawing, authenti-cated microscopically and by their growth pattern. Cells werescreened for Mycoplasma routinely (#A3744,0020 AppliChem).

Generation of PD-L1–deficient GL261 glioblastoma cells viaCRISPR-Cas9

GL261 cells were cultivated in DMEM-GlutaMAX-I (Gibco)supplemented with 10% FBS (Sera Plus, Pan Biotech) and 1%penicillin and streptomycin (Sigma). Cells were passaged each48 hours with trypsin (Sigma) and plated at 8 � 104 cells/cm2.Cells were cultivated at subconfluence and electroporated aftercell dissociation at a cell concentration of 7 � 106 cells/mL usingBio-Rad Gene Pulser II (300 V/150 mF) with 10 mg of plasmidcoding for wild-type SpCas9-guide RNA and mCherry. Cell sort-ing was conducted with BD FACSAria III sorter (BD Biosciences)48 hours postelectroporation. mCherry-positive single cells werecultured to obtain pure clonal population. Clones #1 and #2wereamplified and isolated DNA was characterized by PCR andsequencing. PD-L1–deficient clones #1 and #2 were obtainedfollowing targetedhybridizationof sgRNA#1:GACGTCAAGCTG-CAGGACGC and sgRNA#2: GTATGGCAGCAACGTCACGA.

Immunocompetent intracranial GBM models and therapyIntracerebral implantation of GL261, PD-L1–deficient GL261,

and Tu-2449 glioblastoma cells was performed in 8-week-oldfemale C57Bl/6 or B6C3F1 (Tu-2449) mice as described previ-ously (13, 21). Mice were anesthetized by intraperitoneal injec-tion with ketamine (100 mg/kg, Pfizer) and xylazine (10 mg/kg,Bayer) and fixed in a stereotactic device (Stoelting). A total of 1�105 GL261 glioma cells in 2 mL PBS were injected in the mousestriatum using a Hamilton syringe equipped with a 30G needle.Coordinates relative to thebregmawere: 0.5mm(anterior), 2mm(mediolateral), and 3.5 mm (dorsoventral). The health status oftumor-bearing mice was checked on a daily routine. Mice weresacrificed when neurologic symptoms appeared (starting at3 weeks after intracerebral implantation). Twenty percent loss ofbody weight or prolonged weight loss accompanied with reducedwater/food uptake, or hunched back, rough coat, paresis, tremor,ataxia, and lethargia were considered as criteria for terminationaccording to the Society of Laboratory Animals (GV-Solas; http://www.gv-solas.de). Five days postimplantation, antiangiogenictherapy with AMG386 (trebananib, Ang1/Ang2 peptibody,Amgen; ref. 22) and aflibercept (zaltrap, VEGF-trap, Sanofi;ref. 23) was initiated. AMG386 and aflibercept were administeredsubcutaneously (s.c.) 2�/week at doses of 5.6 mg/kg (22) and25 mg/kg (23), respectively. Modes of action of AMG386 andaflibercept have been discussed in detail previously (13). Therapywas continued until mice became symptomatic (endpoint). Forsome experiments, animals were terminated 21 days post GL261implantation. Monotherapy with anti–PD-1 (RPM1-14, BioX-Cell, 10mg/kg) was started on day 10 by intraperitoneal injection(i.p.) 2�/week for a total of 8 injections. Untreated animalsreceived an equal amount of rat IgG (clone 2A3, BioXCell,10 mg/kg i.p.). T cells were depleted with anti-CD8 (clone 53-6.7 IgG2a; BioXCell, 10 mg/kg i.p.) on day 14 post intracranialimplantation 2�/week as an intervention therapy. Tumor-bearing brains were removed and processed for further analysesas described below.

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Lectin perfusion assayFor vascular perfusion studies, 100 mL DyLight 488-labeled

Lycopersicon Escultentum lectin (1 mg/mL, Vector Laboratories)was intravenously injected 30 minutes prior to animal sacrifice.GBM-bearing mice were then anesthetized, perfused by intracar-diac injection with 1% paraformaldehyde (PFA), and brains weresubsequently harvested and processed for cryosectioning andimmunofluorescence staining.

Immunofluorescence staining of mouse brain tumorsWhole brains were removed, embedded in Sakura Tissue-Tek

O.C.T. (Thermo Fisher Scientific), and frozen on dry ice. Ten-micron–thick cyrosections were fixed in ice-cold 95% ethanol for5 minutes and acetone at room temperature for 1 minute. Con-secutive washing was carried out in PBSA solution (150 mmol/LNaCl, 10 mmol/L Na2HPO4, 10 mmol/L KH2PO4, 1% BSA, and0.1% Triton X-100, pH 7.5). The following antibodies wereapplied for 1 hour at room temperature in antibody dilutionbuffer (0.5% BSA, 0.25% Triton X-100 in PBS, pH 7.2): rat anti-mouse CD31 (clone MEC 13.3, 1:300, BD Pharmingen), mousemonoclonal anti-desmin (clone D33, 1:300, DAKO), rat anti-mouse GLUT-1 (polyclonal, C-Terminus, 1:300, Millipore; Sup-plementary Table S1). After washing with PBSA, DyLight-labeledsecondary antibodies (Thermo Fisher Scientific; SupplementaryTable S2) were applied for 1 hour at room temperature. Slideswere postfixed in 4% PFA, counterstained with 4,6-Diamidine-2�-phenylindole dihydrochloride (DAPI; Invitrogen), and embed-ded in Aqua PolyMount (Polysciences).

Immunofluorescence staining of murine vibratome sectionsGL261-bearing mice were anesthetized and transcardially per-

fused with PBS (Gibco) for 4 minutes. Brains were dissected andpostfixed overnight in 4% paraformaldehyde. Serial 50-mm cor-onal sections were cut on a Leica Vibratome (VT 1000S). Thefollowing primary antibodies were applied: mouse monoclonalanti-desmin (1:100, clone D33, DAKO), rabbit polyclonal anti-collagen IV (1:250, Biorbyt), rabbit polyclonal anti-CD8a (1:500,Synaptic Systems), and rat monoclonal anti-CD31 (1:40, cloneSZ31, Dianova; Supplementary Table S1). Sections were incubat-ed with primary antibodies at 4�C overnight followed by incu-bation with the following secondary antibodies at room temper-ature for 2 hours: goat anti-mouse IgGAlexa FluorR 488, goat anti-rabbit IgG Alexa FluorR 568, goat anti-rabbit Alexa FluorR 488,and goat anti-rat IgG Alexa FluorR 568 (all 1:400, Thermo FisherScientific; Supplementary Table S2). Antigen retrieval was per-formed prior to the staining by heating in 10 mmol/L citric acidbuffer, pH6.0. Imageswere taken on aNikonC1 Spectral ImagingConfocal Laser Scanning Microscope System, using NIS ElementsMicroscope Imaging Software (Nikon Instruments). Video clipswere prepared using Imaris 7.6.5, Bitplane Scientific Software.

Quantification of mouse immunofluorescence stainingFive to 12 images of each complete tumor (n¼ 4–5 per group)

were taken using a Nikon C1 Spectral Imaging Confocal LaserScanning Microscope System. HALO 2.0 Image Analysis Software(Indica Labs Informed Pathology) was used for the quantifica-tion. Pericyte coveragewas defined as desminþ areanormalized to5,000mm2ofCD31þ area. Perivascular T cells were determined byspatial analysis of intratumoral CD8þ cells in relation to CD31þ

endothelial cells over a distance of 100 mm. GLUT-1 expressionwas analyzed in vessels and tumor cells [mean fluorescence

intensity (MFI) in 50 CD31þ cells and 1,000 tumor cells,respectively]. Lectin perfusion was quantified as the percentageof lectinþ area per total CD31þ area.

Hematoxylin and eosin staining of mouse tumor tissuesTwenty-one days post GL261 cell inoculation, mouse brains (n

¼ 4 each treatment group) were dissected, embedded in SakuraTissue-Tec O.C.T. Compound (Thermo Fisher Scientific), andfrozen on dry ice. Ten-micron–thick cryosections were cut, driedfor 10 minutes at 37�C, fixed in 4% PFA for 10 minutes at roomtemperature, and washed in deionized water prior to incubationin 20% Mayer hematoxylin solution for 4 minutes (Merck Che-micals). Afterwashing in running tapwater, slideswere placed in a0.25% alcoholic solution of Eosin Y for 30 seconds (Waldeck).The sections were washed in deionized water and dehydrated inincreasing ethanol solutions (2 � 70%, 2 � 96%, 2 � 100%, for10–15 seconds each). After clarification with xylene for 5minutes(VWR), samples were mounted with Eukitt mounting medium(Th. Geyer). Brightfield images were obtained using a NikonEclipse 80i microscope and NIS Elements imaging software(Nikon Instruments). Necrotic areas (percentage of completetumor) were assessed using HALO 2.0 Image Analysis Software(Indica Labs Informed Pathology; 5–12 sections/brain dependingon the tumor size).

Electron microscopy of mouse GBM samplesFor ultrastructural analysis, GL261-bearing mice were anesthe-

tized 21 days after GL261 cell inoculation and transcardiallyperfusedwith PBS for 1minute and 4%PFA/0.1mol/L cacodylatebuffer (pH 7.4) for 4minutes (n¼ 5 per treatment group). Brainswere dissected and postfixed in 4% PFA/2.5% glutaraldehyde/0.1 mol/L cacodylate buffer (pH 7.4) overnight. Small pieces oftissue containing GL261 tumors were postfixed in 1% OsO4

(Sigma-Aldrich), dehydrated in graded ethanol solutions (30%,50%, 70%, 80%, 96%, 2 � 100% for 45 minutes at roomtemperature) and propylene oxide (2 � 100% for 45 minutes atroom temperature; Sigma-Aldrich) prior to embedding in Epon(Sigma Aldrich). The polymerization was performed for 24 hoursat 60�C. Ultrathin sections were cut on a Leica Ultracut UCT(LeicaMicrosystems), contrast enhancedwith 1.5%uranyl acetatedehydrate (Serva)/0.2 mol/L sodium acetate/0.2 mol acetic acidand 3% lead citrate (Leica Microsystems) using Leica EM Stain(Leica Microsystems). Samples were analyzed using FEI TecnaiSpirit BioTWIN electron microscope at 120 kV (FEI Europe B.V.).Images were taken with an Eagle 4K CCD bottom-mount camera(FEI Europe B.V.).

Flow cytometry of mouse GL261 tumorsAfter dissection, GL261 tumors were transferred into ice cold

Hank balanced salt solution (HBSS), gently minced, and incu-bated with HBSS containing collagenase P (0.2 mg/mL, Roche),dispase II (0.8 mg/mL, Roche), DNase I (0.01 mg/mL, Sigma),and collagenase A (0.3 mg/mL, Roche) for 60 minutes at 37�C asdescribed previously (13). The digestion was stopped by addingFBS (Sera Plus, Pan Biotech) on ice and samples were spun downat 250 � g for 10 minutes at 4�C. The pellet was resuspended in25% BSA/PBS, centrifuged at 2,000 � g (30 minutes at 4�C), andthe myelin-containing supernatant was discarded. The samplewas resuspended in 1 mL HBSS, filtered through a 40-mm mesh,and washed in HBSS (centrifugation at 250� g for 10 minutes at4�C). Lysis of erythrocytes was performed for 10minutes at room

Di Tacchio et al.

Cancer Immunol Res; 7(12) December 2019 Cancer Immunology Research1912




temperature prior to staining (Red blood cell lysis buffer, Roche).Subsequently, 2 mL staining buffer (5% FBS in PBS) was addedand cells were centrifuged at 250 � g for 10 minutes at 4�C. Cellswere preincubated with rat anti-mouse FcgIII/II receptor (CD16/CD32)-blocking antibodies (�1 mg/million cells/100 mL, BDPharmingen) for 5 minutes at 4�C and stained with the fluoro-chrome-conjugated surface antibodies (0.25–1 mg, Supplemen-tary Table S3). DAPI (Invitrogen, myeloid cells) or Fixable Via-bility Dye eFluor 780 (1:1,000, eBioscience, lymphoid cells) wereused for dead cell exclusion.

For intracellular staining, cells were incubated with live/deadFixable Viability Dye eFluor 780 (1:1,000, eBioscience). Cellswerefixedwith 1mLof Fix/Perm (BDBiosciences) for 30minutes,washed twice with PermWash (BDBiosciences), and stainedwithfluorochrome-conjugated antibodies (Supplementary Table S4)for 30minutes on ice. Samples were acquired using a FACSCantoII flow cytometer (BD Biosciences) and further analyzed usingFlowJo analytic software (v. 10.0.8, FLOWJO, LLC). Backgroundfluorescence was determined by fluorescenceminus one controls.

Hypoxia of GL261 cellsSubconfluent GL261 cells were cultured in DMEM-Gluta-

MAX-I (Gibco) supplemented with 10% FBS (Sera Plus, PanBiotech) and 1% penicillin and streptomycin (Sigma) at 5%CO2 and 37�C. After 24 hours, the medium was removed andthe cells were incubated in serum-free DMEM (Gibco) adjustedto 2 mmol/L glucose (Gibco). For hypoxic exposure, cells wereplaced in sealed Gas Pak EZ pouches for anaerobic culture withindicator (Becton-Dickinson) for 48 hours. For flow cytometryanalysis, cells were detached with Accutase (Sigma), washedwith flow cytometry buffer (PBS/5% FBS), and stained with ratanti-mouse PD-L1 (clone 10F.9G2, BioLegend). Samples wereacquired using a FACSCanto II flow cytometer (BD Bios-ciences). Analysis was performed using FlowJo analytic soft-ware (v. 10.0.8, FLOWJO, LLC).

Isolation of mouse brain tumor microvesselsBrain tumors were dissected and rolled on a Whatman filter

membrane (Schleicher & Schuell) to remove meninges asdescribed previously (24, 25). In three independent experiments,GL261 tumor tissue and contralateral brain hemispheres from 3animals for each treatment group were dissected, pooled, andhomogenized in microvessel buffer (MVB; 15 mmol/L HEPES,147 mmol/L NaCl, 4 mmol/L KCl, 3 mmol/L CaCl2, 1.2 mmol/LMgCl2, 5mmol/L glucose, and 0.5%BSA, pH 7.4) using a douncehomogenizer (Wheaton, 0.025mm clearance) and centrifuged at400� g for 10minutes at 4�C. The pellet was resuspended in 25%BSA and centrifuged at 2,000 � g for 30 minutes to removemyelin. The microvessel pellet was resuspended in MVB andfiltered through a 40 mm nylon mesh (BD Biosciences) to washoff nuclei, erythrocytes, and dead cells. Microvessels trapped ontop of themesh were lysed in RLTplus buffer (Qiagen) and storedat �80�C until use. The purity of the MBMV isolation waspreviously confirmed and showed a significant enrichment ofendothelial cellmarkers (Cldn5,Cdh5) andnegligible presence ofother neurovascular unit (NVU) cell types (Aqp4, Ng2, Dcx;refs. 24, 25).

RNA sequencing of GL261 tumor microvesselsRNA was isolated from mouse brain tumor microvessels

using the mRNAeasy Micro Kit (Qiagen; GEO repository num-

ber: GSE130324). To avoid contamination by genomic DNAsamples were treated by on-column DNase digestion (DNase-Free DNase Set, Qiagen). Total RNA and library integrity wereverified with LabChip Gx Touch 24 (PerkinElmer). One-hundred nanograms of total RNA was used as input for SMAR-Ter Stranded Total RNA Sample Prep Kit - HI Mammalian(Clontech). Sequencing was performed on the NextSeq500instrument (Illumina) using v2 chemistry, resulting in averageof 25M (million) reads per library with 1 � 75 bp single-endsetup. The resulting raw reads were assessed for quality, adaptercontent, and duplication rates with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reaper version 13–100 was employed to trim reads after a quality drop below amean of Q20 (1 error in 100 base pairs) in a window of10 nucleotides (26). Only reads between 30 and 150 nucleo-tides were cleared for further analyses. Trimmed and filteredreads were aligned versus the Ensembl mouse genome versionmm10 (GRCm38) using STAR 2.4.0a with the parameter "–out-FilterMismatchNoverLmax 0.100 to increase the maximumratio of mismatches to mapped length to 10% (27). Thenumber of reads aligning to genes was counted with featureCounts 1.4.5-p1 tool from the R Subread package (Bioconduc-tor; ref. 28). Only reads mapping at least partially inside exonswere admitted and aggregated per gene. Reads overlappingmultiple genes or aligning to multiple regions were excluded.Differentially expressed genes were identified using DESeq2version 1.62 (Bioconductor; ref. 29). Only genes with a min-imum fold change of �1.5 (log2 �0.59), a maximum Benja-mini–Hochberg corrected P value of 0.05, and a minimumcombined mean of 5 reads were deemed to be significantlydifferentially expressed. The Ensemble annotation wasenriched with UniProt data (release 06.06.2014) based onEnsembl gene identifiers (Activities at the Universal ProteinResource (UniProt; www.uniprot.org). Dimension reductionanalyses (principal component analysis; PCA) were performedon DESeq2 normalized and regularized log transformed countsusing the R packages FactoMineR and factoextra. Differentiallyexpressed genes (DEGs) were submitted to gene set enrichmentanalyses with KOBAS (http://kobas.cbi.pku.edu.cn; ref. 30).The resulting bubble plot shows pathways with Benjamini–Hochberg corrected P value < 0.05 (represented by dashedline). The larger gray circles are scaled to the number of genescomprising the respective pathway, while the smaller coloredcircles represent the subset found to be DEGs.

IHC and analysis of human GBM samplesFormalin-fixed paraffin-embedded (FFPE) brain tumor tissue

specimen derived from 30 patients with GBM who receivedbevacizumab during their clinical course (see Table 1, obtainedfrom 2009 and 2015) were collected and classified according toWHO criteria by board-certified neuropathologists (P.N. Harterand K.H. Plate; ref. 2). Patients with initial brain tumor resection/biopsy and reresection/rebiopsy or autopsy after bevacizumabtreatment were included. Patients who did not receive bevacizu-mab were excluded from the study. Samples were stored at roomtemperature. Written consent was obtained from all patients. Thestudy was conducted in accordance with ethical standards such asthe Declaration of Helsinki. The study protocol was endorsed bythe local ethical committee (GS04/09 SNO-09-2018). Treatment-na€�ve and bevacizumab-treated matched-pair samples of individ-ual patients were compared in this study. Biopsies were stained





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and analyzed independently in two laboratories (Edinger Insti-tute, Frankfurt and Institute of Pathology, University of Cologne,Cologne, Germany).

Paraffin-embedded samples were cut into 3-mm sections on aLeica microtome (SM2000R, Leica Microsystems). Sections weredeparaffinized in xylene (2�10minutes, VWR)and rehydrated indecreasing concentrations of ethanol (100%, 96%, 70%2 times 5minutes each), followed by incubation in a tap water bath for 5minutes. Immunofluorescence staining with mouse anti-humanPD-L1 (clone 5H1, 1:1,000), mouse anti-human CD68 (clonePG-M1, 1:5,000), mouse anti-human CD8 (C8/144B, 1:5,000),mouse anti-human Ki67 (clone MIB-1, 1:1,000; SupplementaryTable S5) was performed using the multiplex thyramide signalamplification (TSA) system according to the manufacturer's pro-tocol (Opal 4-color IHC,NEL810001KT, PerkinElmer). The stain-ing was performed sequentially with repeating runs of antigenretrieval by heating in 0.1mol/L citric acid buffer pH6.0, (10xAR6buffer, AR6001KT, PerkinElmer) quenching of endogenous per-oxidase activity with 3% hydrogen peroxide for 30 minutes atroom temperature, incubation with blocking reagent (0.5%,FP1020, PerkinElmer) for 30 minutes at room temperature,and primary antibody incubation (for concentrations, seeSupplementary Table S5) overnight at 4�C. Species-specifichorseradish peroxidase (HRP)–conjugated secondary antibody(goat anti-mouse IgG-HRP-labeled, 10 mg/mL, NEF822001EA,PerkinElmer) was applied for 30 minutes at room temperature,followed by incubation with fluorescently labeled tyramide (1:50

in amplification diluent, NEL810001KT, PerkinElmer) for 10minutes at room temperature.

For quantitative analysis of PD-L1 expression pre- and post-bevacizumab therapy (N ¼ 30), images were obtained with aNikonEclipse 80imicroscope (Nikon Instruments, Inc.) using thesame parameter and camera settings for each image taken (10images/sample). The mean intensity of PD-L1 expression intumor cells was determined using NIS Elements MicroscopeImaging Software (Nikon Instruments, Inc.) and recorded asthree-step MFI score within 100 cells using following thresholds:< 6,5 MFI, no PD-L1 expression (�); 6,5–24,99 MFI, PD-L1expression (þ to þþþþ); � 25 MFI, high PD-L1 expression(þþþþþ).Colabeling analysis of PD-L1,CD8, andCD68expres-sion was performed on multispectral images taken at the Vectra3.0 Imaging System forQuantitative Pathology and using InFormV2.0.2 Image Analysis Software (Akoya Biosciences Inc.). Forquantitative analysis of proliferating CD8þ cells (n ¼ 10 biopsysamples),multispectral imageswere taken andunmixed using theVectra 3.0 Imaging System for Quantitative Pathology (AkoyaBiosciences Inc.), and analyzed by HALO 2.0 image analysissoftware (Indica Labs Informed Pathology).

In a second approach, GBM tissue specimens of the samepatient cohort were processed for IHC with mouse anti-humanPD-L1 clone 22C3 (Supplementary Table S5), an antibody thatwas established for PD-L1 expression scoring in non–small celllung cancer (NSCLC; ref. 31). The PD-L1 IHC 22C3 pharmDxassay (code SK006, DAKO Agilent Technologies) was employed

Table 1. Clinical data of patients with GBM treated with bevacizumab

Patient ID SexAge at tumorresection (years) MGMT status

Duration of BEVtreatment (days)

Termination of BEVtreatment prior to recurrenttumor resection (days)

Age at resectionof recurrenttumor (years)

PD-L1 expressionpre/post-BEV

1 n.a. 50 n.a. n.a. n.a. n.a. þþ/þþþþþ2 F 46 U 170 96 46 �/þ3 F 70 U 258 34 71 �/þþþþ4 M 43 U 140 31 43 �/þ5 F 62 U 210 169 63 �/�6 M 51 U 91 179 51 �/�7 M 30 U 407 204 31 �/�8 M 52 U 84 35 52 �/þ9 M 49 M 160 n.a. 49. �/�10 F 41 U n.a. n.a. n.a. �/�11 M 56 n.a. 288 45 57 þ/þ12 M 38 M n.a. n.a. n.a. �/�13 M 55 U n.a. 150 n.a. �/þ14 F 72 U 26 403 73 þ/þþþþþ15 F 63 U 280 33 64 �/þ16 M 47 U 214 133 48 �/�17 M 45 n.a. n.a. n.a. n.a. �/�18 M 49 n.a. 84 32 50 �/�19 M 41 M 217 23 42 �/�20 M 47 n.a. n.a. n.a. n.a. �/þ21 F 46 n.a. n.a. n.a. n.a. �/�22 n.a. 65 n.a. n.a. n.a. n.a. �/þþ23 F 41 n.a. n.a. n.a. n.a. �/�24 M n.a. n.a. n.a. n.a. n.a. �/�25 F 45 U 281 34 46 �/�26 M 65 n.a. 62 36 65 �/�27 F 66 M 293 0 67 �/�28 M 53 n.a. 114 27 53 �/�29 F 62 n.a. n.a. n.a. n.a. �/�30 M 51 U 90 30 51 �/�NOTE: Scoring, < 6,5 MFI, no PD-L1 expression (�); 6,5–24,99 MFI, PD-L1 expression (þ to þþþþ); � 25 MFI, high PD-L1 expression (þþþþþ); MFI, meanfluorescence intensity/100 cells.Abbreviations: BEV, bevacizumab; F, female; M, male; M, methylated; MGMT, O6-Methylguanine-DNA-Methyltransferase; n.a., not available; U, unmethylated.

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for IHC detection of PD-L1 by using the Envision FLEX visual-ization system on a DAKO Autostainer Link 48 IHC stainingplatform (DAKO Agilent Technologies) according to the manu-facturer's instruction (Dako Agilent, Code SK006, see detailedstaining procedure protocol herein). Tumor cells were consideredas PD-L1 IHC-positive if it showed linearmembranous staining ofany intensity that could be complete/circular or partial. Glioblas-toma specimens pre/post-bevacizumab tested for PD-L1 expres-sion were scored and divided into three levels based on a tumorproportion score (TPS): TPS < 1%: no PD-L1 expression; TPS 1–49%: PD-L1 expression, TPS� 50%: high PD-L1 expression (31).

Statistical analysesStatistical analyses were performed using GraphPad Prism

software (GraphPad Inc.) and JMP 11.0 software (SAS). Statisticaltests applied are indicated in the figure legends. For all nonsurvi-val statistical analyses of two experimental groups, an unpaired,two-tailed Student t test was performed. Formultiple comparisonanalyses of more than two groups, one-way ANOVA followed byTukey posttest, Wilcoxon signed-rank test, or Kruskall-Wallis testfollowed by Dunn posttest were used. Kaplan–Meier survivalcurves were analyzed using the log-rank test. P < 0.05 wereconsidered statistically significant and indicated with asterisks(�, P < 0.05; ��, P < 0.01; ��, P < 0.001; ��, P < 0.0001). Data arerepresented as mean � SEM if not otherwise indicated.

ResultsPD-L1 is a potential target for ICT in GBM

Although PD-L1 maintains immune homeostasis under nor-mal conditions, tumors often exploit the PD-L1 pathway toinhibit antitumor immune responses (7). To test whether PD-1/PD-L1 signaling is a potential therapeutic target in GBM, wedetermined PD-L1 expression in biopsies before and after bev-acizumab therapy using two different antibody approaches(Fig. 1; Table 1; Supplementary Fig. S1; SupplementaryTable S6; refs. 31, 32). In a blinded IHC study, we investigateda cohort of 30 GBMmatched-pair biopsies that were stained andanalyzed independently in two laboratories. Images of represen-tative GBM biopsies stained with anti–PD-L1 (clone 5H1; ref. 32)are displayed in Fig. 1A. Approximately 10% of resected, patientswith treatment-na€�ve GBM exhibited PD-L1 expression(Fig. 1B; Table 1). Treatment with bevacizumab resulted insignificantly increased PD-L1 expression in 36% of the patients(P < 0.001; Fig. 1B, left/bottom). Results obtained with clone22C3, an antibody that was established for PD-L1 expressionscoring in NSCLC (31), also showed increased PD-L1 expressionin post-bevacizumab specimens, althoughon a lower overall levelcompared with clone 5H1 (Supplementary Fig. S1; Supplemen-tary Table S6). Overall, anti-VEGF therapy led to a significantupregulation of PD-L1 on glioblastoma cells.

In addition to its expression on glioblastoma cells, PD-L1 wasdetected on human tumor-infiltrating macrophages (mean value¼4.33%, n¼3) andCD8þ cytotoxic T cells (mean value¼2.66%,n ¼ 3), whereby overall CD68þ macrophages numbers weresignificantly reduced and CD8þ T cells remained almostunchanged after bevacizumab therapy (medians CD68 pre/post-bevacizumab: 21.99/15.68, P < 0.0121; medians CD8 pre/post-bevacizumab: 0.24/0.26, P < 0.9003; Fig. 1B). However,CD8þ T-cell numbers pre/post-bevacizumab were heterogeneousamong individual patients, being significantly increased in 11 of

30 (P ¼ 0.0067), decreased in 10 of 30 (P ¼ 0.0059), andremaining unchanged in 9 of 30 (P ¼ 0.4436) patients(Fig. 1B). In future studies, it will be of interest to identifybiomarkers predicting CD8þ T-cell responses in bevacizumab-treated patients.

These observations were supported by data in a syngeneic GBMmousemodelwhere PD-L1wasupregulated byhypoxia anduponantiangiogenic therapy (Supplementary Fig. S2A and S2B). Thesignificant upregulation of PD-L1 post antiangiogenic therapymay identify a barrier/immune escape mechanism in the GBMmicroenvironment, arguing for PD-L1 targeted therapies in GBM.We further identified a significant contribution of tumoral PD-L1on increasing survival by employing a CRISPR/Cas9-mediatedgenome editing approach to delete PD-L1 in GL261 cells (Sup-plementary Fig. S2C). We, therefore, aimed to explore the PD-1/PD-L1 signaling pathway in experimental GBM using a therapeu-tic approach.

PD-1/PD-L1 pathway targeting in a mouse model ofglioblastoma

Although T cells are not abundant in GBM (6), we havepreviously shown that targeting VEGF leads to an improvedinfiltration of T lymphocytes, which, however, is not sufficientto elicit durable antitumor immune responses (13). Havingestablished that the PD-1/PD-L1 pathway is activated upon VEGFtargeting (Fig. 1), we aimed to test a combination of anti–PD-1and dual antiangiogenic therapy in the orthotopic, syngeneicGL261 model. GL261 cells were stereotactically implanted in thestriatum of C57BL/6 mice. Once tumors were established, anti-angiogenic therapy using aflibercept (VEGF-trap; ref. 23) andtrebananib (AMG386, an Ang-1/Ang-2 bispecific peptibody thatneutralizes both Tie2 ligands with a predominance for Ang-2 dueto a 30-fold higher binding capacity; ref. 22) was initiated 5 dayspost intracerebral implantation (see ref. 13 for more details onaflibercept and AMG386). The kinetics and treatment schedulesare displayed in Fig. 2A. Intracranial tumor experiments (Fig. 2A)were additionally performed in the syngeneic Tu-2449 gliomamodel (20, 21) with similar survival results (SupplementaryFig. S2D).

GL261-bearing mice became symptomatic 21 days post tumorcell inoculation if left untreated, and dual anti-VEGF/Ang-2treatment significantly improved survival compared withAMG386 or aflibercept monotherapies (Fig. 2B). Anti–PD-1 wasadministered 10 days after surgical implantation. Anti–PD-1monotherapy led to improved survival (median 27 days) com-paredwith controls (Fig. 2B). Combinations of either AMG386 oraflibercept with anti–PD-1 significantly improved survival, butthe therapies showed less or similar efficacy than AMG386/aflibercept combination therapy (median survival AMG386/anti–PD-1: 33.5 days; aflibercept/anti–PD-1: 46.5 days;AMG386/aflibercept: 45 days). If checkpoint blockade was com-bined with aflibercept/AMG386, survival was improved to agreater extent than with individual or dual therapies (median54 days; Fig. 2B). Flow cytometry analysis of enzymaticallydissected tumors revealed decreased numbers of live cells in theTME, which were further reflective of effective tumor targetingupon double/triple therapies (Fig. 2B). Our findings suggest thatICT in combination with dual antiangiogenic therapy offerspotential therapeutic avenues for GBM therapy.

Three weeks post tumor cell inoculation, tumor morphologydiffered in the treatment groups, evidenced by hematoxylin and






eosin (H&E) staining (Fig. 2C). Signs of necrosis were evident andsignificantly increased upon antiangiogenic treatment but werereduced upon the addition of anti–PD-1 (Fig. 2C). Comparedwith IgG control and anti–PD-1 treatment, dual antiangiogenic

and triple therapy led to vessel normalization with improvedpericyte coverage (Fig. 2D and E; Supplementary Fig. S3; Supple-mentaryMovies S1–S4), which in turnmay facilitate immune cellrecruitment and drug delivery. Tumor vessels that were

Figure 1.

Anti-VEGF (bevacizumab)–induced PD-L1 expression is atherapeutic target in human GBM.A, PD-L1, CD68, and CD8expression was determined byimmunofluorescence using theTSA amplification technique(PerkinElmer). Images ofrepresentative GBM biopsies pre-(top, scale bar, 20 mm) andpost-bevacizumab (middle andbottom, scale bars, 50 mm) aredisplayed. PD-L1 (red), CD68(white), and CD8 (green). B,Quantification of PD-L1, CD68,and CD8 expression (Wilcoxonsigned-rank test). Matched-pairanalyses of treatment-na€�ve andbevacizumab-treated human GBMspecimens (left) and thestatistical impact displayed as thedifference in PD-L1 expressionwithin this cohort (below, n ¼30). Kruskal–Wallis test followedby Dunn posttest (� , P < 0.05;�� , P < 0.01; �� , P < 0.001; ns,not significant). Bev,bevacizumab.

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normalized by aflibercept/AMG386 displayed improved perfu-sion, evidenced by intravenous injection of lectin, which wasfurther significantly increased by the addition of anti–PD-1 (tripletherapy; Fig. 2D and F). Perfusion of brain vessels from contra-lateral hemispheres was not affected by the different therapies(Supplementary Fig. S3B).Nonetheless, double and triple therapyincreased hypoxia in both endothelial cells (Fig. 2D and G) andtumors cells (triple therapy; Fig. 2D and H). Although the com-bination of angiogenesis and ICT led to a survival benefit, hypoxiamay have been caused by persistent tumor-promoting innateimmune cells (such as M2 macrophages; ref. 33). At the sametime, vessel normalization allows the recruitment of adaptiveimmune cells that may help to eradicate tumor cells (11). We,

thus, commenced detailed analyses of cells within the tumormicroenvironment (TME) by flow cytometry.

Assessing the GBM microenvironment afteranti-VEGF/Ang-2 and anti–PD-1 therapy

To understand the underlying mechanisms that led toimproved survival after anti-VEGF/Ang-2 and anti–PD-1 ther-apy, we assessed the immune cell composition in the GBMmicroenvironment. At 21 days (d) and upon the developmentof neurologic symptoms (endpoint), gliomas were dissectedand processed for flow cytometry analyses (to discriminatebetween adaptive and innate immune cells (Fig. 3). The gatingstrategy for glioma-infiltrating lymphocytes is displayed in

Figure 2.

The combination of anti-VEGF, anti–Ang-2, andanti–PD-1 therapy increases survival and leads totumor vessel normalization in GL261 glioma. A,Diagram depicting the timeline and treatmentschedule in the syngeneic, orthotopicGL261 gliomamodel. B, Kaplan–Meier survivalcurves and median survival of GL261-bearingmice after monotherapy and dual therapy withaflibercept/AMG386 and anti–PD-1 [n¼ 15 (threeindependent experiments) for IgG, anti–PD-1,aflibercept/AMG386, aflibercept/AMG386/anti–PD-1; n¼ 10 (one independent experiment) foraflibercept, AMG386, aflibercept/anti–PD-1,AMG386/anti–PD-1]. Right, flow cytometryanalysis of the cytotoxic effects of differenttherapies (IgG control, n¼ 16; anti–PD-1, n¼ 9;aflibercept/AMG386, n¼ 21; aflibercept/AMG386/anti–PD-1, n¼ 9; two–fourindependent experiments). C, RepresentativeH&E images of GL261-bearing mice 21 dayspostimplantation (n¼ 4/group) and graphdisplaying percent necrosis in the differenttreatment and control groups. D, Images andmorphologic analysis of pericyte coverage (top;desmin, red; CD31, green; DAPI nuclear staining,blue), hypoxia (middle, evidenced by GLUT-1expression; GLUT-1, green; CD31, red; DAPInuclear staining, blue) and lectin perfusion(bottom; DyLight-488 L. esculentum lectin,green; CD31, red; DAPI nuclear staining, blue) inGL261 tumors after antiangiogenic andimmunotherapy (IgG control, anti–PD-1,aflibercept/AMG386, aflibercept/AMG386/anti–PD-1) 21 days postimplantation. Single colors aredisplayed in gray scale. Scale bars, 25 mm. E–H,Analyses of pericyte coverage (desminþ), lectinperfusion, and hypoxia (GLUT-1þ) in the differenttreatment groups 21 days postimplantation (E:control, n¼ 5; anti–PD-1, n¼ 4; aflibercept/AMG386, n¼ 5; aflibercept/AMG386/anti–PD-1,n¼ 4; F: all treatments, n¼ 4; and G and H:control, n¼ 4; anti–PD-1, n¼ 4; aflibercept/AMG386, n¼ 5; aflibercept/AMG386/anti–PD-1,n¼ 4). Values are meanþ SEM (B) andmeanþSD (E–H). Statistical analyses were performedusing log-rank test (B) and one-way ANOVAmultiple comparison with Tukey posttest (B andE–H; �, P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001).






Figure 3.

Dual antiangiogenic and ICT alter the immune cell composition in the GBMmicroenvironment. A, Flow cytometry analysis showing the individual immune cellsubpopulations in GL261 tumors after employing the different therapy regimens (IgG control, anti–PD-1, aflibercept/AMG386, aflibercept/AMG386/anti–PD-1).The analysis was performed 21 days (21d) postimplantation and at the endpoint upon the development of neurologic symptoms. B, Glioma-infiltrating myeloidcells are displayed. Gating strategy was employed as described previously (13). Statistical analysis was performed using one-way ANOVAmultiple comparisonwith Tukey posttest. Values are meanþ SEM. CD45high 21d (n¼ 5/group), CD45high endpoint (control, n¼ 12; anti–PD-1, n¼ 7; aflibercept/AMG386, n¼ 10;aflibercept/AMG386/anti–PD-1, n¼ 4). CD3 21d (n¼ 5/group), CD3 endpoint (control, n¼ 14; anti–PD-1, n¼ 9; aflibercept/AMG386, n¼ 21; aflibercept/AMG386/anti–PD-1, n¼ 10). CD4 21d (n¼ 5/group), CD4 endpoint (control, n¼ 20; anti–PD-1, n¼ 9; aflibercept/AMG386, n¼ 21; aflibercept/AMG386/anti–PD-1n¼ 10). CD8 21d (n¼ 5/group), CD8 endpoint (control, n¼ 16; anti–PD-1, n¼ 9; aflibercept/AMG386, n¼ 21; aflibercept/AMG386/anti–PD-1, n¼ 10). CD19 21d(n¼ 5/group), CD19 endpoint (control, n¼ 16; anti–PD-1, n¼ 9; aflibercept/AMG386, n¼ 21; aflibercept/AMG386/anti–PD-1, n¼ 9). MDSCs 21d (n¼ 5/group),MDSC endpoint (control, n¼ 16; anti–PD-1, n¼ 8; aflibercept/AMG386, n¼ 20; aflibercept/AMG386/anti–PD-1, n¼ 9). CD206 21d (n¼ 5/group), CD206endpoint (control, n¼ 18; anti–PD-1, n¼ 8; aflibercept/AMG386, n¼ 16; aflibercept/AMG386/anti–PD-1, n¼ 11). MHCIIhigh 21d (n¼ 5/group), MHCIIhigh endpoint(control, n¼ 12; anti–PD-1, n¼ 7; aflibercept/AMG386, n¼ 15; aflibercept/AMG386/anti–PD-1, n¼ 9). MHCIIlow 21d (n¼ 5/group), MHCIIlow endpoint (control,n¼ 13; anti–PD-1, n¼ 6; aflibercept/AMG386, n¼ 15; aflibercept/AMG386/anti–PD-1, n¼ 9). CD45low 21 (n¼ 5/group), CD45low endpoint (control, n¼ 8;anti–PD-1, n¼ 4; aflibercept/AMG386, n¼ 16; aflibercept/AMG386/anti–PD-1, n¼ 9; � , P < 0.05; ��, P < 0.01; �� , P < 0.001; �� , P < 0.0001; one–fourindependent experiments).

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Supplementary Fig. S4. The number of CD45high cells signifi-cantly declined at the endpoint upon aflibercept/AMG386 andaflibercept/AMG386/anti–PD-1 treatment but not 21 dayspostsurgery. CD3þ T lymphocytes were increased in all therapygroups compared with controls (Fig. 3A; endpoint). We iden-tified an increase in CD8þ cytotoxic T cells (endpoint vs. 21d)in the anti–PD-1 and aflibercept/AMG386/anti–PD-1 therapyregimen, whereas CD4þ Th cells declined in those groups(Fig. 3A; endpoint vs. 21d). Immunosuppressive FoxP3þ cellsconstituted a major fraction of CD4þ T cells (Fig. 3A). Overall,we observed less frequent CD4/FoxP3þ regulatory T cells(Tregs) upon triple therapy (Fig. 3A; endpoint, aflibercept/AMG386/anti–PD-1 vs. IgG). Although Tregs increased signif-icantly in the control and aflibercept/AMG386 groups, this

increase was diminished by the addition of ICT, which wasalso reflected by the ratio of CD8 and FoxP3þ cells (Fig. 3A).Our flow cytometry analyses demonstrated that the addition ofanti–PD-1, single and in combination with aflibercept/AMG386, led to the infiltration of cytotoxic T cells, which mayhave glioma-eradicating capacities. This supports the survivaldata, where PD-1 targeting alone led to increased survival,which was further significantly extended when combined withanti-VEGF/Ang-2 (Fig. 2B). Our findings demonstrated that thecombination of ICT and dual antiangiogenic therapy created afavorable immune environment with increased cytotoxic T cellsthat was accompanied by a Treg decrease. Compared withcontrols at endpoint analyses, we also observed a significantincrease of B lymphocytes in the aflibercept/AMG386 and

Figure 4.

CD8 blockade reverses the survival benefit achievedby the combination of anti-VEGF/Ang-2 and anti–PD-1 immunotherapy. A, Diagram depicting the timelineand the addition of anti-CD8 treatment to the dualantiangiogenic and ICT schedule in the GL261 gliomamodel. B, Kaplan–Meier survival analysis of GL261tumor–bearing mice after different therapy regimensandmedian survival rates are displayed (IgG control,n¼ 15; anti–PD-1, n¼ 15; anti–PD-1/anti-CD8, n¼ 10;aflibercept/AMG386/anti–PD-1, n¼ 15; aflibercept/AMG386/anti–PD-1/anti-CD8, n¼ 10; two–threeindependent experiments). C, Flow cytometry plotsshowing IFNgþ, TNFaþ, PD-1þ, and Ki67þ expressionin CD8þ glioma-infiltrating T lymphocytes aftertherapy (IgG control, anti–PD-1, aflibercept/AMG386,aflibercept/AMG386/anti–PD-1). The analyses wereperformed upon the development of neurologicsymptoms (endpoint). IFNg (control, n¼ 7; anti–PD-1,n¼ 6; aflibercept/AMG386, n¼ 6; aflibercept/AMG386/anti–PD-1, n¼ 4); TNFa (control, n¼ 7;anti–PD-1, n¼ 6; aflibercept/AMG386, n¼ 6;aflibercept/AMG386/anti–PD-1; n¼ 4); PD-1 (control,n¼ 7; anti–PD-1, n¼ 6; aflibercept/AMG386, n¼ 6;aflibercept/AMG386/anti–PD-1, n¼ 4); Ki67 (control,n¼ 7; anti–PD-1, n¼ 6; aflibercept/AMG386, n¼ 6;aflibercept/AMG386/anti–PD-1, n¼ 4; oneindependent experiment). D,Morphologic andquantitative analysis of CD8þ T-lymphocyteinfiltration in GL261 glioma. Representativeimmunofluorescence images of mouse brain tumorsections 21 days postimplantation in the differenttreatment groups (CD31, green; CD8, red; DAPInuclear staining, blue). The number and distance ofCD8þ T cells to CD31þ glioma vessels in the differenttherapy regimen are displayed (n¼ 5/group). Scalebar, 20 mm. E, Representative immunofluorescenceimages of GBM biopsies [treatment-na€�ve, post-bevacizumab (Bev)] stained with anti-CD8 (green)and anti-Ki67 (red) and the quantification aredisplayed, n¼ 10. Scale bar, 10 mm. Values are meanþ SEM (C) and meanþ SD (D–E). Statistical analyseswere performed using log-rank test (B), one-wayANOVAmultiple comparison with Tukey posttest(C andD), and Student t test (E; � , P < 0.05;�� , P < 0.01; �� , P < 0.001; �� , P < 0.0001).






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aflibercept/AMG386/anti–PD-1–treated group reflective fortherapy resistance (Fig. 3A). In the context of the specializedGBM microenvironment, B lymphocytes are known to supporttumor angiogenesis and regulate macrophage phenotypes tointerfere with progression and antitumor immuneresponses (33).

We next investigated whether ICT also influenced the intratu-moral myeloid cell composition. We had previously shown thatdual antiangiogenic therapy led to tumor-associated macrophage(TAM) depletion, whereas M2-polarized macrophages remainedhigh in mouse GBM (13). TAM numbers declined after dualantiangiogenic therapy and upon the addition of anti–PD-1(21d and endpoint, compared with control; Fig. 3B). Whenanalyzing protumorigenic M2-polarized macrophages(CD45highCD11bþGr-1�F4/80þCD206þ) 21 days post tumorcell inoculation, CD206þ infiltrates were not significantly differ-ent among the treatment groups (controls vs. anti–PD1, afliber-cept/AMG386, aflibercept/AMG386/anti–PD-1; Fig. 3B). Howev-er, they significantly increased at the endpoint in all treatmentgroups, although numbers were not significantly influenced bythe anti–PD-1 therapy itself (Fig. 3B). We hypothesized thatCD206þ macrophages were potentially recruited from the circu-lation and represented reeducated TAMs as the tumor progresses.Their presence/predominance may, thus, contribute to tumorrefractoriness to therapy by the secretion of growth and immu-nosuppressive factors.M1-polarized (CD45highCD11bþGr-1�F4/80þMHCIIhigh) macrophages were present in tumors and did notfurther increase during progression (Fig. 3B). Myeloid-derivedsuppressor cell (MDSC; CD45highCD11bþGr-1þ) numbersdeclined upon anti-VEGF/Ang-2/anti–PD-1 therapy (Fig. 3B, end-point). Microglia significantly increased upon dual and tripletherapy (Fig. 3B), indicative for tumor-eradicating properties.Overall, our findings demonstrated that combined vascular andimmune checkpoint targeting led to reduced tumor-promotingMDSCs, while it did not interfere withM2-polarizedmacrophagenumbers.

Depletion of CD8þ T cells reverses the survival benefit aftertriple therapy

Having established that CD8þ cytotoxic lymphocytes wereincreased after aflibercept/AMG386/anti–PD-1 therapy, weaimed to assess whether depletion of CD8 would attenuate thesurvival benefit. GL261-bearing mice receiving anti–PD-1 andaflibercept/AMG386 treatment were additionally given anti-CD8twice per week starting from day 14 post tumor cell implantation(Fig. 4A). Using this strategy, we demonstrated that survival wasalleviated when CD8þ cells were depleted in the aflibercept/AMG386/anti–PD-1 treatment group (Fig. 4B). Median survivalwas reduced to 46 days and resembled values observed after

double therapy (Fig. 4B). This finding supports the hypothesisthat tumor-infiltrating CD8þ T cells contributed to a favorableoutcome in mouse GBM and provide a rationale for targetingadaptive immune cells in the specialized glioma TME. To revealthe mediators regulating CD8þ T-cell activity, we performed flowcytometry of tumor-infiltrating lymphocytes (TIL) and deter-mined intracellular cytokines (IFNg , TNFa) in addition to PD-1 expression, reflective for CD8þ T-cell exhaustion (Fig. 4C).Although CD8þ T-cell numbers were increased upon anti–PD-1 and aflibercept/AMG386/anti–PD-1 therapies (Fig. 3B), elevat-ed IFNg and TNFa were solely detected in double and tripletherapies but not PD-1 monotherapy, indicative of a proinflam-matory Th1 signature (Fig. 4C). Our results are in line withprevious work (18, 19) that reported increased IFNg uponanti–Ang-2 and/or anti-VEGF therapy in breast, pancreatic, andmelanoma cancer models.

Tumor-derived VEGF has previously been shown to enhancethe expression of PD-1 and other inhibitory checkpoints (34). Inline with this observation, upon anti–PD-1 treatment (i.e., whenVEGF is present in the TME), but not after aflibercept/AMG386/anti–PD-1 therapy, CD8þ TILs upregulated PD-1, indicative forexhaustion (Fig. 4C). Proliferation of cytotoxic T cells wasincreased in the aflibercept/AMG38/anti–PD-1 group (Fig. 4C),and CD8þ T cells were preferentially associated with GBM vesselsupon triple therapy (Fig. 4D). Concurrent with findings in themouse model, we observed significantly increased numbers ofproliferating CD8þ T cells in bevacizumab-treated versus treat-ment-na€�ve GBM biopsies (Fig. 4E). In conclusion, our dataestablished that increased numbers of glioma-infiltrating CD8þ

T cells are important contributors to antitumor responses in GBMby providing a proinflammatory cytokine signature particularlywhen combined with dual antiangiogenic therapy.

Triple therapy leads to global vascular normalization of GBMmicrovessels

We next analyzed the transcriptional profiles of GL261 braintumors by RNA sequencing (RNA-seq). To understand how thesynergistic effects of the combination therapies effectuated generegulation in the vascular compartment, we analyzed the vesselsof glial tumors 21 days postimplantation (Fig. 5). The tumormicrovessel isolation procedure is displayed in Fig. 5A. Purity ofthe microvessel isolation and endothelial enrichment has beendemonstrated in previous reports (24, 25). Volcano plots andheatmaps of the top 50 significant regulated genes among thetreatment groups indicated several genes thatweredysregulated inmouse GBM compared with sham-operated animals, which werealso differentially regulated upon the addition of antiangiogenictherapy or further addition of anti–PD-1 (Fig. 5B andC). PCAplotdemonstrated that the transcriptome of control tumor vessels

Figure 5.Anti–PD-1 blockade combined with antiangiogenic therapy reshapes the transcriptomes in microvessels isolated from GL261 tumors. A, Diagram of the workflowfor the isolation of brain tumor microvessels. The brain tissue was mechanically and enzymatically dissociated before removal of myelin and filtered throughcell strainers. Isolated microvessels were microscopically inspected for purity. See bottom photograph. Scale bar, 20 mm. B, Volcano plots highlighting significant(P < 0.05) genes for comparison of treatment groupswithin the dataset. The indicated conditions are (clockwise): untreated (IgG) versus sham-operated,aflibercept/AMG386-treated versus IgG, aflibercept/AMG386/anti–PD-1-treated versus IgG, and aflibercept/AMG386-treated versus aflibercept/AMG386/anti–PD-1-treated. The log2 counts were plotted versus the log2 fold change (FC). Significantly upregulated genes (log2 fold change of more than�1 andFDR < 1%) are displayed in green or red according to the groups. C, Heatmap of the top 50most regulated genes in brain microvessels from IgG versus sham,aflibercept/AMG386, aflibercept/AMG386/anti–PD-1–treated mice, and between the dual- and triple-therapy groups. The color is based on raw Z score. D, PCAof all samples within the dataset showing differences in dimension 1 (29.8%) and 2 (10.3%) for all detected genes. Each dot corresponds to the pool of three braintumors from each therapy group (of n¼ 3 independent experiments). The triple therapy is circled.






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shifted toward the profile of sham-operated animals upon doubletherapy, an effect that was more pronounced with triple therapy,suggesting a global normalization of tumor vasculature genetranscription with triple therapy (Fig. 5D; triple therapy shiftstoward sham animals, circled). Venn diagrams illustrated that inthe vasculature of untreated GL261 tumors, 7,851 genes weredifferentially regulated compared with sham controls (i.e., rep-resentative of normal brain vessels; Fig. 6A). Of these, 4,871 genesreturned to baseline after anti-VEGF/Ang2 therapy and5,331 genes after the combined treatment with anti-VEGF/Ang2and anti–PD-1 (Fig. 6A). This indicated that anti–PD-1 therapytargeted an additional set of genes (981) compared with dualantiangiogenic therapy (4,350 that were common to double andtriple therapy), and highlights the therapy benefit upon anti–PD-1 addition to vascular targeting (Fig. 6A). Molecules associatedwith a normalized vasculature or blood–brain barrier (BBB)function returned to baseline levels (Fig. 6B). For example,PDGFRB, ANGPT1, and TEK (TIE2) were downregulated inGL261 control tumors and upregulated following double andtriple therapy (Fig. 6B). A normalized vasculature with animproved pericyte coverage and perfusion was also seen viahistology (Fig. 2D; Supplementary Fig. S3A; SupplementaryMovies S1–S4) and ultrastructural analysis (SupplementaryFig. S3C). At the same time, Angpt2 (also Vegfa, and Tgfb1 tosome extent) was downregulated after double/triple therapy,respectively (Fig. 6B). Tight junctionmolecules, such as Occludin(Ocln), and other BBB-associated molecules, such as b-catenin(Ctnnb1) and GLUT-1 (Slc2a1), were upregulated upon tripletherapy (Fig. 6B). Pathway enrichment analysis using KEGGpathway database in untreated tumors (7,851 genes fromgroup 1, Fig. 6A) indicated dysregulated pathways in cancer,angiogenesis, cell adhesionmolecules, and immune cell response(Fig. 6C and D).

Corresponding analysis of genes that returned to baseline indouble and triple therapy [groups 2 (4,871) and 3(5,331); Fig. 6A] indicated that the pathways were normalizedby the respective therapy (Fig. 6C and D). To analyze the signif-icance of the genes normalized by the two therapies, common toboth or exclusive to triple therapy, we performed bioinformaticpathway analyses of these subsets comparing them to genesdysregulated in untreated tumor samples when compared withnormal shammicrovessels. We observed that cancer-related path-ways were dysregulated in untreated tumors (group 1) becausethey are above the significance threshold (Fig. 6C and D, red

bubbles above the dotted line). Several of these pathways werenormalized by the triple therapy, some also by double therapy,and a few exclusively by triple therapy, indicated by the corre-sponding bubbles closer or below the significance cutoff (dottedline). Specifically, PKG, cAMP, foxO, and insulin signaling path-ways were normalized by triple therapy and to a lesser extent bydouble therapy (purple box, Fig. 6C), whereas p53, TGFb, andHippo signaling pathways were normalized by the dual antian-giogenic therapy (green boxes, Fig. 6C). This was also the case foradherens and tight junction pathways, indicating the beneficialeffect of dual antiangiogenic therapy. Extended KEGG pathwayenrichment analysis indicated that oxytocin, MAPK, and calciumsignaling pathways were also normalized in the triple therapygroup but mediated exclusively by the addition of anti–PD-1(Fig. 6D, orange boxes). There were, however, several dysregu-lated pathways inmouseGBM that were not normalized by eithertherapy (Fig. 6D, red boxes). This group could provide candidatepathways for complete targeting in future therapies.

DiscussionImmune checkpoint blockade is currently pursued in clinical

trials inGBM, although it is anon-T-cell–inflamed cancer (10, 35).Anti–PD-1 monotherapy shows little therapeutic benefit inGBM (8). We here provided a rational combinatorial approachto improve the efficacy of immune therapy by integrating thesynergistic effects of VEGF/Ang-2 and PD-1 blockade.

Considering the failure of antiangiogenic therapy with beva-cizumab for newly diagnosed GBM and an unfavorable survivalwith standard therapy (1, 3), an urgent need exists for newtreatment options to target GBM. The therapeutic efficacy ofimmune checkpoint inhibition in other cancer modalities isappealing (7, 10) and could be an option for GBM as well.However, immune checkpoint monotherapy using PD-1 antibo-dies is not successful in recurrent GBM (36), whereas clinicalstudies in newly diagnosed glioblastoma are ongoing (ClinicalTrials.gov identifiers NCT02617589 and NCT02667587).

The aim of this study was to test in syngeneic, preclinicalGBM models whether ICT is beneficial when combined withdual antiangiogenic therapy. We demonstrated that ICT led toimproved survival following anti-VEGF/Ang-2 therapy bycreating an immunostimulatory microenvironment nourishedwith CD8þ CTLs and reduced immunosuppressive MDSCs andFoxP3þ Tregs. We provided evidence at the transcriptional level

Figure 6.Bioinformatic analysis evidenced a normalization of cancer- and barrier-related pathways in glioma vessels after anti-VEGF/Ang-2 and anti–PD-1immunotherapy. A, Venn diagrams showing the number of regulated genes between sham versus IgG (I: 7,851), sham versus double therapy (II: 3,335),and sham versus triple therapy (III: 2,915). Red, genes dysregulated in mouse GBM vessels; green, genes rescued by double therapy; purple, genesrescued by triple therapy; blue, genes rescued by both therapies (double and triple); and orange, genes rescued by triple therapy exclusively. B, Genesrelating to BBB function, such as Pdgfrb, Angpt1, Tie2, Ocln, Ctnnb1, Glut-1, and Slc2a1, that were dysregulated in tumor vessels were brought back tosham (no tumor) levels. Tumor-promoting genes (Angpt2, Vegfa, Fasl, and Tgfb1) were downregulated after double/triple therapy. Statistical analysiswas performed using DESeq2 algorithm as part of the RNA-seq analysis (P values: �� , P < 0.01; �� , P < 0.001). C and D, Bubble plot of the topcandidates according to KEGG pathway (KOBAS enrichment) analysis using genes significantly regulated in the different treatment groups [P < 0.05,absolute (log2FC) > 0.585]. Pathways significantly dysregulated in mouse GBM vessels (red bubbles) were unified with analyses from the genesnormalized in the two therapy groups, including the common and exclusive triple-therapy group. The resulting pathways were sorted by amount ofcorrelation between all contrasts (i.e., groups color coded in A). y-axis represents value of significance for each contrast, and size of bubble reflectsthe number of significant hits. Smallest size corresponds to nonsignificant test in the contrast. Red bubbles, sham versus IgG; blue bubbles, boththerapies (double and triple); green bubbles, double therapy; purple bubbles, triple therapy; and orange bubbles, triple exclusive (see also A). Specificsignificant pathways have been highlighted with the corresponding color of the therapy group (colored boxes). n.s., not significant.






that the combination of PD-1 and VEGF/Ang-2-targeting thera-pies normalized the gene expression in endothelial cells back toalmost physiologic conditions.

Numerous immunosuppressive factors such as VEGF, IL10,and TGBb promote the downregulation of the host immunesystem and lead to the exclusion of tumor-fighting immunecells (33). We and others have shown that antiangiogenic therapytargeting VEGF and Ang-2/Tie2 signaling alleviate immune sup-pression and improve survival in GBM models (13–15). How-ever, efficient targeting of tumor cells was not achieved, as theadaptive host immune system was not fully activated to promoteglioma cell eradication. We here aimed to test the hypothesis thatanti-VEGF/Ang-2 therapy was beneficial when combined withanti–PD-1 in glioma models. Basis for our hypothesis was thefinding that PD-L1 was upregulated in patients with GBM afterbevacizumab treatment. Although differential outcomes onpatient survival with regard to PD-L1 expression have beenreported (37–39), the presence of PD-L1 in GBM serves as acheckpoint and marker for therapy responses (7). PD-L1 pro-motes immunosuppression by inhibiting T-cell function (4) andmacrophage phagocytosis (40).Wehypothesized that PD-L1/PD-1 targeting could be an alternative approach to alleviate immunesuppression inGBMto turn aT-cell–poor ("cold") into T-cell–rich("hot") GBM microenvironment.

In our study, the combination of dual antiangiogenic and PD-1checkpoint therapy led to a significant increased overall survival.Compared with antiangiogenic therapy alone, the survival ofglioma-bearing mice was significantly extended upon the addi-tion of anti–PD-1. Antiangiogenic therapy with aflibercept,AMG386, and anti–PD-1 rendered a normalized vasculature withimproved pericyte coverage at the histologic and the gene expres-sion level. Disruption of vessel normalization has been reportedto reduce T-lymphocyte infiltration in transgenic mice and xeno-graft models (41). A functional vasculature is a prerequisite forleukocyte migration and, thus, provides a barrier within theTME (9, 33, 42). Proper adhesion molecules need to be presentfor effective T-cell recruitment to eradicate glioma cells (11). VEGFand Ang-2 are among the factors that create an immunosuppres-sive environment, which also affects the expression of ICAM-1and VCAM-1 (11).

In conjunction with a normalized vasculature, the increasedexpression of cell adhesionmolecules provides a basis for effectorlymphocyte recruitment.OurRNA-seq analysis showeddecreasedFasL expression with increasing therapies. FasL has previouslybeen shown to act as checkpoint at the vascular level that preventslymphocyte entry into the TME, whereby tumor-derived VEGFand IL10 cooperatively induced FasL expression in endothelialcells to promote T-effector cell death (12). The presence of FasLmight, in part, explain that CD8þ effector cells are not frequentduring early tumor progression. Indeed, CD8þ T cells increased inthe anti–PD-1 and aflibercept/AMG386/anti–PD-1–treatedgroups later during tumor progression, which demonstrated thatcheckpoint therapywas able to efficiently reinvigorate effector cellinfiltration in formerly T-cell–poor brain tumors. The survivalbenefitwas superior in the triple combination group.WhenCD8þ

T cells were depleted, the survival benefit was diminished moreeffectively in triple therapy than anti–PD-1 monotherapy, andCD8þ effector T cells were identified as crucial contributors to theextended survival upon aflibercept/AMG386/anti–PD-1 therapy,in line with findings in breast cancer models (41). CD8þ Tlymphocytes in the anti–PD-1 single treatment group, however,

showed different characteristics: they produced less IFNg , prolif-erated less, and had higher PD-1 expression, indicative of exhaus-tion, compared with triple therapy. Indeed, VEGF has beendemonstrated to contribute to T-cell exhaustion by the upregula-tion of immune checkpoints, including PD-1 (34).

Our data demonstrated a favorable outcome in the GBMmodels upon triple therapy, which is in line with literature thatsuggests an improved efficacy of antiangiogenic therapy (targetingAng-2 and/or VEGF signaling) when combined with checkpointtherapy (targeting PD-1 or PD-L1) in preclinical animal mod-els (18, 19). However, in a transgenic GBM model, the benefitwas not as efficient as in non-brain tumor models, which mayrelate to tumor-intrinsic factors of theGBMmodel applied and/orthe level of PD-L1 expression (19). In the GL261 model, which isassociated with high expression of PD-L1, the addition of anti–PD-1 led to improved survival compared with controls in GL261and even more efficiently in Tu-2449 gliomas, indicative ofdifferences in the glioma microenvironment that possibly alsoinfluence the kinetics and frequency of CD8þ TIL influx. Reportsalso indicate that PD-1 clones applied inGBMmodelsmay lead todifferent outcomes with regard to CD8þ T-cell reinvigoration,therapy, efficacy, and long-term survival (43, 44). The timing oftherapymay be crucial as suggested by a study demonstrating thatneoadjuvant administration of anti–PD-1 enhances effector cell–mediated antitumor immune responses in recurrent GBM (45).

PD-L1 is expressed on both tumor and immune cells. WhenPD-L1was eliminated by a genetic knockdown approach and PD-L1–deficient GL261 cells were implanted in the striatum ofC57BL/6 mice, we observed significantly extended survival indi-cating an essential contribution of glioma cell–derived PD-L1, inline with previous observations (46). PD-L1 was upregulatedupon aflibercept/AMG386 treatment, which leads to increasedhypoxia, a known driver of PD-L1 expression (47). PD-L1 canfurther be induced via IFNg secretion from CD8þ T cells (18, 19).In line with these observations, in the matched-pair biopsycohort, compared with treatment-na€�ve GBM, PD-L1 was signif-icantly upregulated after bevacizumab therapy. In recurrent GBM,PD-L1may provide a therapeutic target, especially in conjunctionwith vascular targeting to enhance antitumor immunity.

Because brain tumors were not eradicated completely upontriple therapy, additional components of TME are potentiallyinvolved that prevent overall tumor clearance. Indeed, althoughcells of the innate immune system were diminished with anti-angiogenic treatment, they remained high upon anti–PD-1monotherapy. The addition of anti–PD-1 had no effect onCD206þ macrophages residing in the GBM microenvironmentas immune-suppressors and effectuated fewer MDSCs andFoxP3þ Tregs, as reported previously, using the same GBMmod-els (43, 44). In patients with GBM, MDSCs play a major role inglioma-induced T-cell suppression via the PD-1/PD-L1 axis (48).Elevated hypoxia observed upon vascular targeting may be adriving force for remaining M2-polarized macrophages asreported previously (33). Thus, it might be appealing to createa less hypoxic GBM milieu to improve therapy efficacy. Simulta-neous targeting of Tie2 and Ang-2 leads to tumor vessel normal-ization, decreased hypoxia, improved drug delivery, and favor-ably alters the TME (49). Similarly, an improvement of BBBfunction in GBM may be achieved with inhibitors that activateTie2 (25, 50–52). In particular, improving endothelial cell-barrierfunctions may be useful for edema management in patients withGBM, which is a major clinical need (53).

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In an attempt to further understand the mechanisms underly-ing the survival benefit achieved by the combined targeting of thevasculature and the PD-1/PD-L1 pathway, RNA-seq analysis wasperformed on isolated tumor vessels. DEseq2-based analysiswithin the different treatment groups revealed a number ofdifferentially regulated genes, uncovering treatment-specific tran-scriptomic changes. Although barrier-associated genes weredownregulated in control tumors, genes that were associatedwithvascular stabilization [e.g., Pdgfrb, Angpt1, Tek (Tie2)] and BBBfunction were upregulated upon dual antiangiogenic or tripletherapy. Some unfavorable cytokines (such as TGFb) that weredysregulated in glioblastoma were restored close to levelsobtained in the sham control group, indicative of the efficacy ofcheckpoint therapy in the glioma model. Venn diagram analysisshowed a unique set of genes that were restored to basal levels("normalized") in the triple therapy, and were common to bothdouble and triple therapy. Bioinformatic analysis of these twosubgroups provided further insights into the molecular basis forthe increased survival in the triple therapy, and vascular normal-ization and survival benefit in the dual therapy. Several pathwayssuch as PKG, cAMP, foxO, and insulin signaling were normalizedin the triple therapy. However, oxytocin, MAPK, and calciumsignaling pathways were normalized exclusively in the tripletherapy, indicating that the effect is specific to addition ofanti–PD-1. At the same time, pathways relating to barrier functionsuch as tight- and adherens junctionpathwayswere normalized inboth therapies, including pathways relating to dysregulatedtumor cell growth, such Hippo signaling (54). However, severalpathways such as Ras, JAK/STAT, chemokine, and T-cell receptorsignaling pathways were not entirely normalized by eithertherapy.

Our analysis provides a resource for discovering therapeutictargets for potential complete eradication of tumor cells andincreased survival. Our findings support the hypothesis thatcheckpoint therapy in combination with antiangiogenic ther-apy may have an impact on the efficacy in patients with GBMby the alleviation of immunosuppression and improving can-cer immunity through a normalized vasculature that is per-missive for T-cell reinvigoration (55). Our study providesevidence that ICT efficiently improved survival in preclinicalGBM mouse models when combined with vascular-targetedtherapy. This is in line with findings in other cancer modelswhich suggest that antiangiogenic therapy targeting the angio-poietin and/or VEGF pathway elicits antitumor immunity thatis enhanced by anti–PD-1/PD-L1 therapy (18, 19). Althoughthe GBM environment facilitates immune evasion of tumorcells, for example, via TGFb, IL10, IDO1, Tregs, and MDSCs(56), the combination of immune checkpoint and vascular-targeting therapies alleviated immunosuppression in GBM.Multimodal therapies, including ICT, thus, may be the futurefor GBM disease management to invoke tumor-targetingimmune cells. Study findings imply that ICT is more effica-cious in neoadjuvant compared with adjuvant therapy inrecurrent GBM, but no data for first-line therapy are currentlyavailable (45). Clinical studies have been initiated with theaim to improve the immune–vascular crosstalk for cancerimmunotherapy and to overcome resistance that is driven bythe immunosuppressive microenvironment (35). Stratificationof patients that benefit from combination treatment couldfurther improve therapy outcomes. Studies associated PD-L1expression with the mesenchymal GBM subtype and NF1

deficiency (39). Patients with NF1 loss-of-function may thusbenefit from ICT. Hypermutation at diagnosis or at recurrencewas associated with CD8þ T-cell enrichment, indicating thatthose patients could be implemented for PD-1/PD-L1 immu-notherapy (57). Nonetheless, because PD-L1 expression inGBM is dynamic and detection is challenging, serum Ang-2might also be of value to predict response to immune check-point inhibition (58). Integrating the synergy of anti-VEGF/Ang-2 and anti–PD-1 therapy may precipitate immediate clin-ical impact. The RNA-seq datasets of isolated vessels from thecurrent work provide molecular insight into the therapeuticbenefit of triple therapy and serve as a resource for transla-tional therapies when combined with human datasets.

Disclosure of Potential Conflicts of InterestJ.P. Steinbach reports receiving a commercial research grant from Merck,

Germany, and speakers bureau honoraria from AbbVie, Boehringer, Roche,Bristol-Myers Squibb, Medac, and UCB. M. Glas is an advisory boardmember for AbbVie and Novocure and reports receiving speakers bureauhonoraria from Novocure, Novartis, Bayer, Medac, Merck, and KyowaKirin. U. Herrlinger reports receiving a commercial research grant fromRoche and speakers bureau honoraria from Medac, Bayer, Noxxon, Novar-tis, Daiichi Sankyo, Bayer, Janssen, and Bristol-Myers Squibb. R. B€uttnerreports receiving speakers bureau honoraria from MSD, Bristol-MyersSquibb, and AstraZeneca, has ownership interest (including patents) inTargos Molecular Pathology, and is a consultant/advisory board memberfor MSD, Bristol-Myers Squibb, and AstraZeneca. O.M. Grauer is a con-sultant/advisory board member for Bristol-Myers-Squibb. G. Tabatabaireports receiving commercial research grants from Roche Diagnostics,Medac, and Novocure, and speakers bureau honoraria from Medac, Novo-cure, AbbVie, and Bayer. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: K.H. Plate, Y. ReissDevelopment of methodology: M. Di Tacchio, J. Macas, J. Weissenberger,K. Devraj, K.H. Plate, Y. ReissAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Di Tacchio, J. Macas, J. Weissenberger,K. Sommer, O. B€ahr, C. Senft, U. Herrlinger, D. Krex, A. Weyerbrock,M. Timmer, R. Goldbrunner, A.H. Scheel, R. B€uttner, O.M. Grauer,G. Tabatabai, P.N. Harter, S. G€unther, K. Devraj, Y. ReissAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Di Tacchio, J. Macas, J. Weissenberger, O. B€ahr,V. Seifert, A.H. Scheel, R. B€uttner, P.N. Harter, S. G€unther, K. Devraj, K.H. Plate,Y. ReissWriting, review, and/or revision of the manuscript: M. Di Tacchio, J. Macas,J. Weissenberger, K. Sommer, O. B€ahr, J.P. Steinbach, C. Senft, M. Glas,U. Herrlinger, D. Krex, A. Weyerbrock, M. Timmer, R. Goldbrunner,O.M. Grauer, P.N. Harter, K. Devraj, K.H. Plate, Y. ReissAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Di Tacchio, K. Sommer, O. B€ahr,J.P. Steinbach, V. Seifert, M. Glas, M. Meinhardt, A. Weyerbrock, M. Deckert,J. SchittenhelmStudy supervision: K.H. Plate, Y. Reiss

AcknowledgmentsK.H. Plate and Y. Reiss thank Dr. Angela Coxon (Amgen) for providing

AMG386 (Master Agreement No. 2010537481). This work was supported bythe Clinical Translation Program "Glioma" from the Frankfurt Cancer Institute(FCI), the Collaborative Research Center "Vascular differentiation and remo-deling" (CRC/Transregio23, Project C1), the Cluster of Excellence 147 "Car-diopulmonary system" (ECCPS) from the German Research Council (DFG; toK.H. Plate and Y. Reiss), and grants from the German Cancer Consortium(DKTK, Partnersite Frankfurt/Mainz; to K.H. Plate and Y. Reiss). M. Di Tacchiowas supported by the GO-IN Goethe International Post-Doc Programme of the






Goethe University Frankfurt (PCOFUND-GA-2011-291776). The authors aregrateful for experimental support from Sonja Thom and Maryam I. Khel.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 5, 2018; revisedApril 25, 2019; acceptedOctober 1, 2019;published first October 9, 2019.

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2019;7:1910-1927. Published OnlineFirst October 9, 2019.Cancer Immunol Res Mariangela Di Tacchio, Jadranka Macas, Jakob Weissenberger, et al. PD-1, Angiopoietin-2, and VEGF

ofand Improved Survival in Glioblastoma with Combined Inhibition Tumor Vessel Normalization, Immunostimulatory Reprogramming,

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