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Microenvironment and Immunology

RAGE Expression in Tumor-Associated MacrophagesPromotes Angiogenesis in Glioma

Xuebo Chen1, Leying Zhang2, Ian Y. Zhang2, Junling Liang3, Huaqing Wang4, Mao Ouyang5,Shihua Wu3, Anna Carolina Carvalho da Fonseca6, Lihong Weng7, Yasuhiko Yamamoto8,Hiroshi Yamamoto8, Rama Natarajan9, and Behnam Badie2,10

AbstractInteraction of RAGE (the receptor for advanced glycation endproducts) with its ligands can promote tumor

progression, invasion, and angiogenesis. Although blocking RAGE signaling has been proposed as a potentialanticancer strategy, functional contributions of RAGEexpression in the tumormicroenvironment (TME) havenotbeen investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth ofgliomas. In both invasive and noninvasive glioma models, animal survival was prolonged in RAGE knockout(Ager�/�) mice. However, the improvement in survival in Ager�/�mice was not due to changes in tumor growthrate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TMEabrogated angiogenesis by downregulating the expression of proangiogenic factors, which prevented normalvessel formation, thereby generating a leaky vasculature. These alterations were most prominent in noninvasivegliomas, in which the expression of VEGF and proinflammatory cytokines were also lower in tumor-associatedmacrophages (TAM) in Ager�/� mice. Interestingly, reconstitution of Ager�/� TAM with wild-type microglia ormacrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associatedmicroglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis.Cancer Res; 74(24); 7285–97. �2014 AACR.

IntroductionWith the development of targeted therapies against gliomas,

the contribution of tumor microenvironment (TME) to treat-ment response is becoming clearer. Glioma-associated stromalcells like astrocytes, endothelial cells, mesenchymal cells andinfiltrating inflammatory cells play an important role in tumor-

igenesis, angiogenesis, invasion, and immune evasion. Amongthese cells, infiltrating microglia andmacrophages (referred toas tumor-associated macrophages or TAMs) have receivedrecent attention due to their involvement in glioma escapefrom antiangiogenic agents (1). As components of the innateimmune system, TAMs are a heterogeneous cell populationthat are derived from resident brain microglia and myeloid-derivedmonocytes and express a variety of pattern recognitionreceptors that constantly monitor changes in TME. One suchreceptor is the receptor for advanced glycation endproducts(RAGE) that was initially discovered as a membrane proteinthat binds glycosylated macromolecules.

RAGE has been implicated in a variety of human diseaseprocesses (2, 3). Under normal physiologic conditions, RAGE isexpressed at high levels in the lungs, and at lower levels in avariety of other cell types, including immune cells, neurons,activated endothelial, and vascular smooth muscle cells. How-ever, in pathophysiologic settings such as diabetes, chronicinflammation, or neurodegenerative disorders, RAGE expres-sion is increased drastically in other tissues such as vasculature,hematopoietic cells, and the central nervous system (CNS; ref. 4).

RAGE binds to a variety of ligands, including advancedglycation endproducts (AGE) and S100 proteins. Furthermore,as a pattern recognition receptor, RAGE binds to proteins suchas high-mobility group box 1 (HMGB1) that originate fromdamaged cells and activate the immune system (3). Engage-ment of RAGEby its ligands results in the activation ofmultipledownstream signaling pathways, which eventually lead to theactivation of NF-kB, ERK1/2, p38, and STAT3 and consequent

1Department of General Surgery, China Japan Union Hospital of JilinUniversity, Changchun, Jilin Province, P.R. China. 2Division of Neurosurgery,City of Hope Beckman Research Institute, Duarte, California. 3ResearchCenter of Siyuan Natural Pharmacy and Biotoxicology, College of LifeSciences, Zhejiang University, Hangzhou, P.R. China. 4Department ofEmergency Surgery, Qilu Hospital of Shandong University, Jinan, ShandongProvince, P.R. China. 5Department of Cardiology, Third Xiangya Hospital,Central South University, Changsha Hunan, P.R. China. 6Laborat�orio deMorfogenese Celular, Instituto de Ciencias Biom�edicas, Universidade Fed-eral doRiodeJaneiro (BolsistadoCNPq), RiodeJaneiro,Brazil. 7Departmentof Hematology and Hematopoietic Cell Transplantation, City of Hope Beck-man Research Institute, Duarte, California. 8Department of Biochemistry andMolecular Vascular Biology, Kanazawa University, Kanazawa, Japan. 9Divi-sion of Molecular Diabetes Research, City of Hope Beckman ResearchInstitute, Duarte, California. 10Department of Cancer Immunotherapeuticsand Tumor Immunology, City of Hope Beckman Research Institute, Duarte,California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

X. Chen and L. Zhang contributed equally to this article.

Corresponding Author: Behnam Badie, Division of Neurosurgery, City ofHope Medical Center, 1500 East Duarte Road, Duarte, CA 91010. Phone:626-471-7100; Fax: 626-471-7344; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-1240

�2014 American Association for Cancer Research.

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regulation of cytokines, chemokines, adhesion molecules, andpathways that regulate cell proliferation, survival, differentia-tion, migration, phagocytosis, and authophagy (4). Thus, it isnot surprising that RAGE signaling has also been implicated intumorigenesis.

By facilitating the maintenance of a chronic inflammatorystate, upregulation of RAGE and its ligands has been linked tothe development and progression of several neoplasms, includ-ing gastric cancer (5), colon cancer (6), pancreatic cancer (7),and prostate cancer (8). Furthermore, blockade of RAGEsignaling has been used to inhibit tumor growth, invasion,and angiogenesis in a variety of cancers (9–12). One of theearliest reports to demonstrate the therapeutic efficacy of thisapproach was in gliomas in which blockade of RAGE interac-tion with HMGB1 was shown to inhibit tumor growth andinvasion (13). Most studies that have evaluated RAGE signalingin tumors, however, have focused on the RAGE signaling inneoplastic cells, and the contribution of RAGE expression inthe TME to tumorigenesis has not been investigated in detail.

The goal of this study was to evaluate the effect of geneticdepletion of RAGE in TME on the growth of gliomas. Becausegliomas are heterogeneous tumors with both invasive andnoninvasive "bulky" phenotypes, two different syngeneic glio-ma models were used for these experiments. In both gliomamodels, animal survival was prolonged in Ager�/� mice. Thisimprovement in animal survival, however, was not due tochanges in tumor growth, but to a reduction in tumor-asso-ciated inflammation in Ager�/� mice. Furthermore, RAGEablation in TME abrogated angiogenesis by downregulatingthe expression of proangiogenic factors that prevented normalvessel formation. These vascular alterations, however, weremost prominent in noninvasive gliomas in which the expres-sion of VEGF and proinflammatory cytokines was lower inAger�/� TAMs. Interestingly, reconstitution of either RAGEnull microglia or macrophages with corresponding WT cellsnormalized tumor angiogenesis. These findings support therole of TAM RAGE signaling in glioma angiogenesis.

Materials and MethodsReagents and cell lines

Murine GL261 glioma cells were obtained from the labora-tory of Dr. Karen Aboody (City of Hope Beckman ResearchInstitute, Duarte, CA) in 2006 and stably transfected withfirefly luciferase expression vector as described previously(14, 15). Positive clones (GL261-luc) were selected using zeocin(1 mg/mL) and G418. Luciferase-expressing KR158B cells (orK-Luc), an invasive glioma cell line that was derived fromspontaneous gliomas in Trp53/Nf1 double-mutant mice inthe laboratory of Dr. Tyler Jacks (Massachusetts Instituteof Technology, Cambridge, MA), were a generous gift fromDr. John Sampson (Duke University, Durham, NC) in 2011 (16).Both GL261 and K-Luc cells were cultured in DMEM mediumsupplemented with 10% FBS (BioWhittaker), 100 U/mL pen-icillin-G, 100mg/mL streptomycin, and 0.01mol/LHepes buffer(Life Technologies) in a humidified 5% CO2 atmosphere, andtheir tumorigenicity was authenticated by histologic charac-terization of intracranial gliomas in mice.

Tumor implantation and imagingMice were housed and handled in accordance with the

guidelines and approval of City of Hope Institutional AnimalCare and Use Committee under pathogen-free conditions.All mice were on C57BL/6J background. CX3CR1

GFP knock-inmice that express EGFP under control of the endogenousCx3cr1 locus were purchased from The Jackson Laboratory.Ager�/� mice, a generous gift from Dr. Yasuhiko Yamamoto(Kanazawa University, Kanazawa, Japan), were bred at ourinstitution and PCR genotyped using tail DNA (17). Intra-cranial tumor implantation was performed stereotacticallyas described previously (18). Briefly, GL261 or K-Luc gliomacells were harvested by trypsinization, counted, and resus-pended in culture medium. Female C57BL/6 mice (TheJackson Laboratory) weighing 15 to 25 g were anesthetizedby i.p. administration of ketamine (132 mg/kg) and xylazine(8.8 mg/kg), and implanted with 105 tumor cells using astereotactic head frame at a depth of 3 mm through a burhole placed 2-mm lateral and 0.5-mm anterior to the breg-ma. Tumor growth was assessed by the Xenogen IVIS In VivoImaging System (Xenogen) as previously described (15).

Real time RT-PCR and Western blot analysisReal-time quantitative PCR (qPCR) was performed with

corresponding primers (Supplementary Table S1) in a TaqMan5700 Sequence Detection System (Applied Biosystems) asdescribed previously (18).

Western blots were performed as described previously (18)using primary antibodies specific for S100B (Abcam), full-length RAGE (FL-RAGE; Abcam), b-actin (Santa Cruz Biotech-nology), S100A9 (R&D Systems), HMGB1 and GAPDH (CellSignaling Technology).

Cathepsin S assayPrimary bone marrow–derived monocytes (BMM) were

harvested from WT and Ager�/� mice as described previously(19) and cultured in DMEM supplemented with 1% FBS, 100U/mL penicillin-G, 100 mg/mL streptomycin, and 0.01 mol/LHepes. Cathepsin S assay was performed according to themanufacturer's instructions (AnaSpec Inc.; catalog# 72099).Briefly, 106 BMM from WT and Ager�/� mice were plated in12-well plated for 24 hours. Cells were then treated with IFNg(1 mg/mL), or conditioned medium (CM) from GL261or K-Luccells. Cell lysates were collected at different time points andtested for Cathepsin S activity.

RAGE promoter activity assayA fragment contains the 50-flanking region (�2000 bp) of the

mouse Ager (RAGE gene) was generated from mouse genomicDNA by PCR using the following primers: forward, 50-attgc-tagcgggaggtcagatatacagtc-30 and reverse, 50-attaagcttccatctcc-catctcgttc-30. This product was cloned into the NheI andHindIII sites of the pGL3-basic vector (Promega), and thegenerated plasmid was designated pRAGE-Luc.

Primary BMM from WT and Ager�/� mice were culturedin 6-well plates as described above. Cells were then trans-fected with pRAGE-Luc by lipofectamine Reagent (Invitro-gen) for 36 hours and then incubated with CM from GL261

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or K-Luc cells for 12 hours. Cells were harvested by manualscraping in lysis buffer (Promega) to yield lysates that werethen assayed for luciferase activity using the LuciferaseAssay System (Promega). The luciferase activity was mea-sured in a luminometer.

Vascular permeabilityMice bearing 2-week-old intracranial GL261 or K-Luc

tumors were injected with TRITC-dextran 150 (Life Technol-ogy) solution (100 mg/kg i.v.). Brains were harvested after2 hours, and fixed in paraformaldehyde for 4 hours beforestorage in 30% sucrose solution. Brains were then embedded inO.C.T. (optimal cutting temperature compound; Tissue-Tek),sectioned (10 mm), and baked at 37�C . Images were capturedwith anAX-70fluorescentmicroscopy (LeicaMicrosystems Inc.)and analyzed by Zeiss LSM Image Browser software.For Evans blue permeability assay, tumor-bearingmicewere

anesthetized and injected with Evans blue dye (100 mL of a 1%solution in 0.9% NaCl; Sigma-Aldrich) into the retro-orbitalplexus. Thirty minutes later, mice were sacrificed and perfusedwith PBS. Brains were removed, imaged, and dried in 60�Covernight. The Evans blue dye was then extracted using 1 mLformamide at 55�C for 16 hours and quantified with a spec-trophotometer at 630 nm (20).

Vascular densityVascular density was calculated bymeasuring average vessel

diameter over four �10 fields obtained by AX-70 fluorescentmicroscopy (Leica Microsystems Inc.) and prepared by ZeissLSM Image Browser software.

Flow-cytometry analysisFor in vivo staining, tumor tissue was minced and digested

with trypsin for 20 minutes at 37�C. Tissue homogenate wasthenfiltered through a 40-mmfilter andprepped usingfixation/permeabilization solution according to the manufacturer'sinstructions (BD Pharmingen). Multiple-color FACS analyseswere performed at the City of Hope FACS facility using a 3-laserCyAn immunocytometry system (Dako Cytomation), and datawere analyzed using FlowJo software (TreeStar) as describedpreviously (19). PerCP-conjugated antibodies to mouse CD45(cat. no. 557235) were purchased from BD Pharmingen, allo-phycocyanin-conjugated anti-mouse CD11b (cat. no. 17-0112-82) and the primary rabbit-anti-mouse RAGE (cat. no. ab3611-100) were purchased fromAbcam. Secondary goat anti–rabbit-FITC (cat. no. sc-2012) and secondary goat anti–mouse-FITC(cat. no. sc-2010) were purchased from Santa Cruz Biotech-nology. TAMs (CD11bhigh CD45high) and microglia (CD11bhigh

CD45low) were distinguished on the basis of previouslydescribed FACS analysis (21).

Bone marrow transplantationLethally irradiated recipient mice (two doses of 550 cGy

every 4 hours) were injected via a tail vein with 5 � 106 bonemarrow (BM) cells freshly collected from donor mice. Thedonor mice were euthanized and BM cells were asepticallyharvested by flushing femurs with Dulbecco's PBS (DPBS)containing 2% FBS. The samples are combined, filtered

through a 40-mmnylonmesh, centrifuged, and passed througha 25 gauge needle. Recovered cells are resuspended in DPBS ata concentration of 5 � 106 viable nucleated cells per 200 mL.All animals were given autoclaved water with sulfatrim from2 days before 2 weeks after transplantation. Six to 8 weeksafter transplantation, mice were implanted intracranially withGL261 gliomas.

Immunofluorescence stainingFrozen brain sections were prepared from na€�ve and tumor-

bearing mice. Immediately after harvest, brains were fixed inparaformaldehyde for 4 hours before storage in 30% sucrosesolution. Brains were embedded in O.C.T. (Tissue-Tek) and10-mm sections were cut using cryostat (Leica MicrosystemInc.). Before immunofluorescence staining, slides were bakedat 37�C and permeabilized in methanol for 15 minutes. After a1-hour block, slides were incubated with Hypoxyprobe-F6(1:200, Hypoxyprobe), RAGE (1:200; rabbit anti-mouse RAGE;Abcam), CD11b (1:20; rat anti-mouse; Abcam), or CD31 (1:20;rat anti-mouse; Abcam) primary antibodies for 1 hour. Slideswere washedwith PBS three times for 5minutes and incubatedwith secondary antibody (Goat anti-rabbit Alexa Fluor 555orGoat anti-mouse Alexa Fluor 555, 1:200 dilution Life Techno-logies) for another hour. Tissue sections were mounted inVectashield mounting medium containing 40,60-diamidino-2-phenylindole (DAPI; Vector), imaged with AX-70 fluorescentmicroscopy (Leica Microsystems Inc.), and prepared by ZeissLSM Image Browser software.

For human studies, tumor samples from 7 patients withglioblastoma and peritumoral whitematter tissue from1patientwere collected under an Institutional Review Board (IRB)-approved protocol (IRB 07074), flash-frozen, sectioned (8-mmthick), blocked with BSA (0.5% for 1 hour) before overnightincubation with primary RAGE (1:300; cat. no. 3611; Abcam),CD34 (1:100; M7165; Dako), or CD163 (1:100; VP-C374; Vector)antibodies. After washing, slides were incubated with secondaryantibodies (donkey anti-mouse—A488 and anti-rabbit—A555)for 1 hour and counterstained with DAPI for 5 minutes.

Hypoxia stainingWT or Ager�/� mice bearing 2-week-old intracranial GL261

or K-Luc tumors were injected once with 100 mg/kg pimoni-dazole HCl (100 mg/kg in PBS, i.v.) 2 hours before tissueanalysis. Tissue sections were incubated with Hypoxyprobebefore imaging.

Statistical analysisStatistical comparison in all different experimental condi-

tions was performed with the GraphPad Prism software usingtwo-way ANOVA or the Student t test. Survival was plottedusing a Kaplan–Meier survival curve and statistical signifi-cance was determined by the log-rank (Mantel–Cox) test. A Pvalue of less than 0.05 was considered significant.

ResultsRAGE ablation prolongs survival

To evaluate the role of RAGE depletion in TME on gliomagrowth, two different syngeneic orthotopic glioma models

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were studied. GL261 gliomas grow as "bulky" tumors with onlymicroscopic invasion, whereas the K-Luc model has a moreinvasive phenotype. In both models, animal survival was

modestly, but significantly prolonged in Ager�/� mice whencompared with WT controls (Fig. 1A, bottom). This improvedsurvival correlated with slower tumor growth in the GL261, but

Figure 1. Impact ofRAGEablation in TMEongliomagrowth. A, Kaplan–Meier plots(top) and tumor luciferase activity(bottom) in wild-type (WT) and RAGEknockout (Ager�/�) mice implanted witheither intracranial GL261 or K-Lucgliomas (n ¼ 7–8 mice/group; MS,median survival). B, despiteimprovement in survival, tumor size (2weeks after implantation) was similar inWT and Ager�/� mice. Cross sectionsthrough the largest tumor area wereused for size calculations (n ¼ 4 mice/group � SD). C, except for increasedcentral necrosis in the GL261 tumorsin Ager�/� mice, tumor histology andinvasion pattern were similar in bothstrains. Experimental results arerepresentative of three separateexperiments.

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not the K-Luc model (Fig. 1A, bottom). Interestingly, even withimprovement in animal survival, tumor sizes were similar(Fig. 1B), and except for increased necrosis in the Ager�/�

GL261 gliomas, tumor histology and invasion pattern wereunchanged in the Ager�/� mice (Fig. 1C). These findingssuggested that tumor response to RAGE ablation in TME wasdifferent in each glioma model, and other factors besidestumor growth were responsible for improved survival oftumor-bearingAger�/�mice. BecauseRAGE signaling has beenimplicated in tumorigenesis, we next evaluated the expressionof RAGE and its ligands in each model.

Expression of RAGE and its ligands in gliomasRAGE engagement by its ligands can lead to induction of

NF-kB and activation of inflammatory pathways that furtherupregulate RAGE expression and its ligands in a feed-forwardmechanism (3). As a result, ablation of RAGE in TME mayimpair RAGE/ligand interactions in tumor leukocytes andabrogate tumor inflammation. Alternatively, RAGE ablationinTMEmay also decrease the release of soluble RAGE (sRAGE).

sRAGE serves as a decoy receptor and by binding to ligands inthe extracellular space antagonizes the activation of RAGE(22). Thus, by reducing sRAGE in TME, more ligands will beavailable to activate tumor RAGE in an autocrine fashion. Tostudy this complex interaction in vivo, we first confirmed theexpression of RAGE and its ligands in each glioma model. Asexpected, RAGE expression was markedly lower in the lungsand brains of Ager�/� mice (Fig. 2A). Very low levels of RAGEstaining in the Ager�/� brains (neurons) and lungs most likelyrepresented expression of other RAGE isoforms that were notdepleted in the Ager�/� model or may have been due tononspecific binding of the primary RAGE antibody (Fig. 2A,bottom). Although each cell line expressed similar levels of FL-RAGE in vitro (Fig. 2B), RAGE expression in intracranial tumorswas different in eachmouse strain. In GL261 tumors, FL-RAGElevels were similar in WT and Ager�/� mice (Fig. 2C, left), butin the K-Luc tumors, they were slightly higher in Ager�/� mice(Fig. 2C, right). Considering that nuclear RAGE levels weresimilar in WT and Ager�/� tumors (Fig. 2D), higher FL-RAGEprotein in Ager�/� K-Luc tumors (Fig. 2C) most likely

Figure 2. Expression of RAGE and its ligands in gliomas. A, PCR (top) and immunostaining (bottom) confirming lower RAGE expression in the lungs andbrains of nontumor bearing Ager�/� mice. B, Western blot analysis demonstrating in vitro expression of full-length (FL)-RAGE by each cell line beforeimplantation. C, FL-RAGE Western blot analysis. D, immunostaining of intracranial GL261 and K-Luc gliomas 2 weeks after implantation into either WTor Ager�/� mice (n ¼ 3 mice/group � SD). E, Western blot analysis comparing the expression of RAGE ligands by each tumor type 2 weeks afterintracranial implantation. Experimental results are representative of two separate experiments.






represented higher membrane-bound RAGE or sRAGE levelsthat were not detectable by immunohistochemistry. BecauseRAGE signaling can directly modulate the expression of itsligands, we next measured the levels of known glioma RAGEligands in these models.

To assess RAGE ligands, intracranial tumors were sepa-rated from surrounding brain tissue and analyzed by West-ern blot analysis. In the normal brains, levels of all theligands were slightly lower in Ager�/� mice, confirming theabrogation of the RAGE feed-forward regulation of its ligandproduction (Fig. 2E). Interestingly, the expression profile ofRAGE ligands was different between the two glioma models;although K-Luc tumors expressed lower S100B levels thanGL261 gliomas, these tumors had higher levels of proin-flammatory ligands such as HMGB1 and S100A9. High levelsof HMGB1 and proinflammatory S100 proteins have beenimplicated in tumor invasion (23), possibly accounting forthe phenotypic differences between these two models. Fur-thermore, the expression of common RAGE ligands was

similar in WT and Ager�/� mice in both tumor models.Thus, the impact of RAGE ablation on animal survival wasmost likely due to modulation of TME and not to alterationsof RAGE signaling in tumor cells. Because inflammation hasbeen shown to promote tumor progression in a variety ofcancers (24), we next studied leukocyte trafficking in eachglioma model.

Impact of RAGE ablation on tumor inflammatoryresponse

To evaluate the role of RAGE in leukocyte trafficking intotumors, Ager�/� CX3CR1

GFP mice were generated andimplanted with each glioma model. Surprisingly, RAGEablation in TME did not alter leukocyte trafficking ordistribution in either GL261 or K-Luc tumors (Fig. 3A), andthe proportion of inflammatory cells (i.e., lymphocytes,microglia, and macrophages) in each tumor type was similarin the WT and Ager�/� mice (Fig. 3B). This suggested thatRAGE did not play an important role in leukocyte

Figure 3. Impact of RAGE ablation on tumor inflammatory response. A, infiltration of CD11bþ cells (microglia and macrophages) into GL261 and K-Lucgliomas 2 weeks after intracranial implantation into WT or Ager�/� CX3CR1

GFP mice. B, representative flow-cytometry contour plots (top) andquantification (bottom) of tumor-associated leukocytes in 2-week-old intracranial GL261 and K-Luc tumors in WT or Ager�/� mice. (lymphocytes,CD45þ, CD11b�; microglia, CD45intermediate, CD11bhigh; macrophages, CD45þ, CD11bhigh; n ¼ 3 mice/group � SD). C, qPCR of proinflammatorycytokines in normal brain (NB) and intracranial GL261 and K-Luc tumors 2 weeks after implantation (n ¼ 4 mice/group � SD); �, P < 0.05; experimentalresults are representative of three separate experiments.

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chemoattraction in these glioma models. However, whentumor cytokines were examined, both tumor models hadlower expression of proinflammatory cytokines (IL6, IL1b,and TNFa) in Ager�/� mice (Fig. 3C). These findings areconsistent with the proinflammatory functions of RAGE andindicate that suppression of inflammation may have abro-gated tumor growth and accounted for the improved sur-vival of glioma-bearing Ager�/� mice. Because RAGE signal-ing has also been implicated in tumor angiogenesis, we nextevaluated the expression of angiogenic factors in eachmodel.

Impact of RAGE on angiogenesisBesides proinflammatory cytokines, tumors in Ager�/�

mice also expressed lower levels of proangiogenic factors(Fig. 4A). Although MMP2, a metalloproteinase that isinvolved in glioma invasion and angiogenesis (25), was also

expressed at lower levels in Ager�/� mice, tumor-invasivephenotypes were similar in both mouse strains (Fig. 1C).Because histologic analysis of tumors had demonstratedRAGE expression in tumor vessels (Supplementary Fig.S1), we next evaluated the vascular density in each tumortype. Although proanigogenic factors appeared to be sup-pressed in both glioma models in Ager�/� mice, only theGL261 tumors had an obvious vascular phenotypic changewith large dilated vessels (Fig. 4B and Supplementary Fig.S2). Furthermore, GL261 tumors that typically grow as a"bulky" mass appeared to be more hypoxic in Ager�/� micethan in WT animals (Fig. 4C). The more invasive K-Lucgliomas, on the other hand, had no signs of hypoxia andexhibited normal vascular characteristics. This finding wasconsistent with the histologic data demonstrating centralnecrosis in large GL261 gliomas in Ager�/� mice (Fig. 1C).Furthermore, poor perfusion of the GL261 gliomas in

Figure 4. RAGE ablation abrogates tumor angiogenesis. A, qPCR of normal brain (NB) and intracranial GL261 and K-Luc gliomas demonstrating lowerexpression of tumor proangiogenic factors in Ager�/� mice (n ¼ 3 mice/group � SD). B, morphology (top) and quantification (bottom) of GL261 and K-Luc blood vessels in WT and Ager�/� mice (n ¼ 4 mice/group � SD). C, tumor hypoxia (green staining) was measured by injecting glioma-bearing micewith pimonidazole 2 hours before tissue examination. Intracranial tumors were harvested 2 weeks after implantation. D, effect of RAGE ablation onvascular permeability. Mice bearing 2-week-old intracranial GL261 or K-Luc were injected with TRITC-dextran 150 2 hours before image analysis.Vascular leakage is evident as dextran extravasation into perivascular space (arrows). E, tumor vascular permeability was also assessed with Evans blueassay. Tumor-bearing mice were injected with Evans blue dye 30 minutes before sample collection. The dye was extracted and quantified with aspectrophotometer; (n ¼ 4 mice/group � SD); �, P < 0.05; ��, P < 0.001. Experimental results are representative of two separate experiments.






Ager�/� mice may have prevented the uptake of luciferin bytumors, thus accounting for the discrepancy between thetumor luciferase activity (Fig. 1A) and tumor size (Fig. 1B).

In addition to changes in vascular morphology, GL261vessels also were more permeable in Ager�/� mice (Fig. 4Dand E). K-Luc vessels also appeared to be leakier in Ager�/�

mice, but this higher permeability did not translate into astatistically significant increase in overall tumor permeability(Fig. 4D and E).

In summary, the impact of RAGE ablation in TME on thegrowth of gliomas appeared to be due to a decrease in tumorinflammation and impairment of angiogenesis. The latterphenomenon, however, was more apparent in the "bulky"GL261 glioma model that relies on angiogenesis and not themore invasive K-Luc model. To assess the significance of thesefindings to human tumors, we then evaluated RAGEexpressionin freshly harvested malignant glioma tumor samples.

RAGE expression in human malignant gliomasRAGE expression was evaluated in seven histologically

confirmed glioblastoma samples (grade 4 astrocytomas) andwhite matter tissue harvested from "tumor edge." All gliomas,but not cells in the "tumor edge," expressed high levels of RAGE(Fig. 5A and B). RAGE expression was also detected in bothtumor vessels (CD34þ) and TAMs (CD163þ) in every glioma

sample (Fig. 5C). Nearly half of TAMs expressed RAGE in eachsample. Because TAMs are a prominent component of TMEand play an important role in tumor angiogenesis (26),we evaluated their angiogenic properties following RAGEablation.

Role of TAM RAGE expression on angiogenesisAs we reported previously, RAGE was expressed by both

CNS microglia and myeloid-derived macrophages in GL261gliomas (Fig. 6A; ref. 18). Furthermore, the proportion ofRAGEþ microglia (50%–60%) and macrophages (�20%) inthe GL261 model was very similar to the average proportionof RAGEþ TAMs in the human glioblastoma samples (50%–60%). To evaluate the impact of RAGE ablation in these cells,TAMs from intracranial GL261 gliomas were isolated andanalyzed by qPCR. As expected, TAMs that originated fromAger�/� mice had lower expression of IL6 and VEGFa(Fig. 6B). Also, as compared with WT cells, BMM isolatedfrom Ager�/� mice expressed lower levels of MMP9 (Fig. 6C)and Cathepsin S (Fig. 6D) when they were exposed to GL261CM but not K-Luc CM. Finally, RAGE signaling was signif-icantly attenuated in Ager�/� macrophages in response toCM from either GL261 or K-Luc cells (Fig. 6C), confirmingactivation of RAGE by glioma-derived factors in both mod-els. However, in comparison with Ager�/� BMM that were

Figure 5. RAGE expression in human glioblastomas. A, representative immunohistochemistry of a glioblastoma tumor sample (left) and tumor edge(right) from the same patient demonstrating RAGE expression in tumor and not peritumoral white matter. B, RAGE expression in another glioblastomatissue sample demonstrating nuclear (1), membranous (2), and cytoplasmic (3) RAGE expression in tumor cells (T). C, expression of RAGE in tumor-associated vessels (CD34þ, arrows) and macrophages (CD163þ, arrows) in a representative glioblastoma sample. Nearly half of the TAMs expressedRAGE in every tumor.

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exposed to GL261 factors, Ager promoter activity was notcompletely abolished in the cells that were incubated withK-Luc CM, most likely due to activation of other RAGE-independent pathways by proinflammatory RAGE ligands(like HMGB1). Overall, these findings suggest that TAMRAGE activation was more important in angiogenesis inGL261 tumor, but not in the more invasive K-Luc gliomas.TAMs in gliomas are derived from CNS microglia and

circulating myeloid-derived cells such as monocytes. Toevaluate the angiogenic function of RAGE in each cell type,chimeric mice were generated before tumor implantation.The feasibility of this technique was first assessed bycross transplanting BM from CX3CR1

GFP and WT mice. Afterrecovery, mice were implanted with GL261 tumors andanalyzed by histochemistry and flow cytometry. Gliomamacrophages were identified as CD45high CD11bhigh cellsas we reported previously (21), and appeared to infiltrateinto the tumors (Fig. 7A, left). In the reverse transplantation

experiments, in which recipient mice were CX3CR1GFP, tumor

microglia were identified as CD45intermediate CD11bhigh

cells and mostly remained within the margin of the tumor(Fig. 7A, right).

To evaluate the impact of macrophage RAGE expressionon tumor angiogenesis, Ager�/� mice were then used asdonor BM. Total tumor VEGFa expression was significantlylower in these mice (Fig. 7B), but this VEGF decline was notas profound as when tumors were propagated in Ager�/�

mice (Fig. 4A), suggesting that other myeloid-derived cells(like microglia) from WT recipient mice may have alsocontributed to tumor VEGFa production. To confirm this,tumor vascular density was compared in cross transplantexperiments (Fig. 7C). Interestingly, only mice that lackedRAGE expression in both microglia and macrophagesdemonstrated large dilated vessels, confirming that RAGEexpression by either tumor microglia or macrophages wassufficient to normalize angiogenesis in GL261 tumors.

Figure 6. Effect of RAGE ablation in TAMs. A, representative immunohistochemistry (left) and flow cytometry (right) of intracranial GL261 tumor in WTmice confirming RAGE expression in TAMs (left, CD11bþ, arrows; right, red events). Nearly 20% of tumor macrophages (CD45high CD11bhigh) and 50%of tumor microglia (CD45intermediate CD11bhigh) expressed RAGE. B, TAM expression of IL6 and VEGFa was suppressed in Ager�/� GL261gliomas. TAMs were isolated by Percoll gradient from intracranial GL261 gliomas 2 weeks after implantation (n ¼ 4 mice/group � SD); �, P < 0.05;��, P < 0.01. C, expression of MMP9 was lower in Ager�/� primary BMM after incubation with CM from GL261 cells; �, P < 0.05; ��, P < 0.001.D, cathepsin S expression was modestly lower when Ager�/� BMM was incubated with IFNg and GL261 CM, but not K-Luc CM; �, P < 0.05.E, RAGE promoter activity was measured in WT and Ager�/� BMM after incubation with CM from GL261 and K-Luc cells. Although Ager promoteractivity was lower in Ager�/� BMM under both conditions, its activity was not completely abolished when cells were exposed to K-Luc CM(n ¼ 4 þ SD); ��, P < 0.01; ��, P < 0.001; ns, not significant as compared with cells transfected with control vector. Representative data from twoseparate experiments are shown.






Discussion

Interaction of RAGE with its ligands through both autocrineand paracrine mechanisms can enhance tumor progression,invasion, and angiogenesis, and blockage of this interactionhas been proposed as a potential anticancer therapy (27). Inthis study, we demonstrate that genetic ablation of RAGE inTME also prolonged survival of glioma-bearing mice by reduc-ing tumor-associated inflammation and angiogenesis. Thesurvival benefit of RAGE ablation in TME, however, wasmodest and its function in angiogenesis was dependent ontumor phenotype. In less invasive GL261 gliomas that grow as"bulky" tumors, RAGE ablation in TAMs reduced vascularremodeling, leading to dilated vessels, tumor hypoxia, andnecrosis. In the more invasive K-Lucmodel, on the other hand,RAGE blockade did not have a significant impact on tumorangiogenesis. To our knowledge, this is the first report todemonstrate the proangiogenic function of RAGE signaling intumormacrophages. Our findings also emphasize the complexrole of RAGE and its ligands in gliomagenesis.

We expected RAGE ablation in TME to inhibit leukocytetrafficking into gliomas because RAGE may function as an

adhesion molecule (28) and aid leukocyte recruitment bybinding to CD11b (29, 30). Furthermore, RAGE has been shownto be important in S100B-mediated chemoattraction of micro-glia (31). In this study, however, RAGE expression did notinhibit TAM infiltration in gliomas. This finding is consistentwith our recent report demonstrating that S100B-mediatedpromotion of leukocyte trafficking into gliomaswas not depen-dent on RAGE expression in TME, but was mediated throughupregulation of CCL2 (32). Similarly, Heijmans and colleagues(33) reported that lack of RAGE in colon polyps did notinfluencemacrophage trafficking but instead caused amarkedincrease in infiltrating mast cells. These results support thepresence of multiple overlapping pathways that promote TAMchemoattraction into tumors.

Although RAGE did not affect macrophage trafficking, itsblockade abrogated tumor inflammation and improved sur-vival of two phenotypically different glioma models. As amultiligand receptor, activation of RAGE can upregulate theexpression of proinflammatory cytokines (4). Consistent withthese reports, we noted a decrease in inflammatory cytokinesin both invasive and noninvasive gliomas in Ager�/� mice.Suppression of tumor inflammation was most likely due to

Figure 7. RAGE ablation in TAMs abrogates angiogenesis in GL261 gliomas. A, to demonstrate the feasibility of generating chimeric mice, BMcross transplant experiments between wild-type (WT) and CX3CR1

GFP mice were performed. After recovery, mice were implanted with GL261 tumorsand analyzed by histochemistry and flow cytometry. Glioma macrophages were identified as CD45high CD11bhigh cells (green cells and green events indot plot) and appeared to infiltrate throughout the tumors (left). In the reverse transplantation experiments (right) in which recipient mice wereCX3CR1

GFP, tumor microglia were identified as CD45intermediate CD11bhigh cells (green events and cells) that mostly remained within the margin of thetumor. B, qPCR demonstrating suppression of VEGFa expression in GL261 tumors that were implanted into WT mice transplanted with Ager�/�

BM. C, tumor vessel characteristics in GL261 gliomas implanted into chimeric mice demonstrating normalization of dilated vessels in mice witheither WT microglia or WT macrophages. Representative data from two separate experiments are shown; (n ¼ 3 � SD); �, P < 0.05.

Chen et al.





inactivation of RAGE on TAMs and not tomodulation of RAGEin tumor cells because levels of RAGE ligands did not signif-icantly change in Ager�/� mice. Nevertheless, reduction oftumor inflammation was sufficient to modestly improve sur-vival of glioma-bearing Ager�/� mice.RAGE ablation in TME also affected angiogenesis in both

glioma models. Tumors in Ager�/� mice had more perme-able blood vessels and lower expression of proangiogenicfactors. Circulating AGEs can contribute to vascular remo-deling processes associated with inflammation and cancer(23, 34). Furthermore, RAGE is expressed by vascular smoothmuscle and endothelial cells in which its activation by RAGEligands results in their proliferation, migration, and tubeformation (35–37). Also, Spiekerkoetter and colleagues (38)reported that S100A4 (a RAGE ligand) activates the MAPKpathway via RAGE, leading to the expression of MMP2,which is essential for vascular smooth muscle cell migration.Thus, in both glioma models, development of permeablevessels may have been due to direct RAGE ablation invascular smooth muscle and endothelial cells. However,although tumor vessels were more permeable in Ager�/�

mice, angiogenesis was more perturbed in the GL261 modelin which vascular remodeling was diminished, resulting inpoor tumor perfusion and central necrosis.In contrast with K-Luc tumors, GL261 gliomas in Ager�/�

mice were more hypoxic and necrotic due to their "bulky"phenotype and dependency on angiogenesis for growth. Inthese tumors, RAGE expression was necessary for vascularnormalization and remodeling that was essential to tumorperfusion. The exact mechanism by which RAGE signaling inGL261 TAMs regulated angiogenesis remains unclear, butour data suggest that expression of proteinases may play arole in this process. BMM from Ager�/� mice expressedlower levels of MMP9 and Cathepsin S when incubated withGL261 CM. These proteases are secreted by microglia andmacrophages (26, 39, 40), are important in CNS tissueremodeling, and have been shown to play a role in gliomaangiogenesis and invasion (25, 41, 42). Interestingly, inGL261 gliomas, RAGE expression by both resident microgliaand infiltrating monocytes was important for angiogenesisin these tumors, suggesting that therapeutic strategies thatonly target infiltrating monocytes (and not microglia) maynot be adequate in preventing angiogenesis in these tumors.In addition to RAGE activation in TAMs, variation in the

expression of RAGE ligands may have differently affectedtumor angiogenesis in response to RAGE ablation in eachmodel. The K-Luc model expressed high levels of HMGB1 andS100A9, which enhance tumor invasion, migration, and angio-

genesis (43). High levels of HMGB1 expression by K-Lucgliomas may have overcome the dependency on RAGE signal-ing for angiogenesis in this model. Besides RAGE, HMGB1 hasbeen shown to bind other receptors such as TLR4 and TLR2(44, 45). Activation of these receptors by HMGB1 that wassecreted by K-Luc cells may have been responsible for stim-ulation of the RAGE pathway through a RAGE-independentmechanism. Perhaps inhibition of HMGB1 production in thismodel may be a more potent antiangiogenic strategy thaninhibiting RAGE signaling.

In summary, this study demonstrates the role of TAMRAGE expression in glioma angiogenesis and highlights thediversity of RAGE signaling in these heterogeneous tumors.Although RAGE ablation in TME by itself may not be a viabletreatment for malignant gliomas, targeting the interaction ofRAGE ligands with their receptors may have therapeuticpotential.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Zhang, R. Natarajan, B. BadieDevelopment of methodology: L. Zhang, I.Y. Zhang, H. Wang, M. Ouyang,S. Wu, B. BadieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Chen, L. Zhang, J. Liang, H. Wang, M. Ouyang,S. Wu, A.C.C. da Fonseca, L. Weng, Y. Yamamoto, H. Yamamoto, B. BadieAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Chen, L. Zhang, I.Y. Zhang, H. Wang, M. Ouyang,B. BadieWriting, review, and/or revision of the manuscript: I.Y. Zhang, S. Wu,R. Natarajan, B. BadieAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): X. Chen, J. Liang, H. Wang, M. Ouyang,B. BadieStudy supervision: B. Badie

AcknowledgmentsThe authors thank Brian Armstrong for assisting with fluorescent

microscopy.

Grant SupportThis work was supported by R01 CA155769, R21 NS081594, and R21 CA189223

(B. Badie), and R01DK065073 (R. Natarajan). The City of Hope Flow CytometryCore was equipped in part through funding provided by ONR N00014-02-1 0958,DOD 1435-04-03GT-73134, and NSF DBI-9970143.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received April 22, 2014; revised August 22, 2014; accepted October 1, 2014;published OnlineFirst October 17, 2014.

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2014;74:7285-7297. Published OnlineFirst October 17, 2014.Cancer Res Xuebo Chen, Leying Zhang, Ian Y. Zhang, et al. Angiogenesis in GliomaRAGE Expression in Tumor-Associated Macrophages Promotes

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