The Impact of Macrophage- and Microglia- Secreted TNFa on ......(January 2015), LN229 (July 2013), GB30 (January 2015), and U251 (January 2015) cells were authenticated by the University

Cancer Therapy: Preclinical

The Impact of Macrophage- and Microglia-Secreted TNFa on Oncolytic HSV-1 Therapyin the Glioblastoma Tumor MicroenvironmentW. Hans Meisen1, Eric S.Wohleb2, Alena Cristina Jaime-Ramirez1, Chelsea Bolyard1,Ji Young Yoo1, Luke Russell1, Jayson Hardcastle3, Samuel Dubin1, Kamaldeen Muili1,Jianhua Yu4, Michael Caligiuri4, Jonathan Godbout5, and Balveen Kaur1

Abstract

Purpose: Oncolytic herpes simplex viruses (oHSV) repre-sent a promising therapy for glioblastoma (GBM), but theirclinical success has been limited. Early innate immuneresponses to viral infection reduce oHSV replication, tumordestruction, and efficacy. Here, we characterized the antiviraleffects of macrophages and microglia on viral therapy forGBM.

Experimental Design:Quantitative flow cytometry of micewith intracranial gliomas (�oHSV) was used to examinemacrophage/microglia infiltration and activation. In vitrococulture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSVreplication. Macrophages from TNFa-knockout mice andblocking antibodies were used to evaluate the biologiceffects of TNFa on virus replication. TNFa blocking anti-bodies were used to evaluate the impact of TNFa on oHSVtherapy in vivo.

Results: Flow-cytometry analysis revealed a 7.9-fold increasein macrophage infiltration after virus treatment. Tumor-infil-trating macrophages/microglia were polarized toward a M1,proinflammatory phenotype, and they expressed high levels ofCD86, MHCII, and Ly6C. Macrophages/microglia producedsignificant amounts of TNFa in response to infected gliomacells in vitro and in vivo. Using TNFa-blocking antibodies andmacrophages derived from TNFa-knockout mice, we discov-ered TNFa-induced apoptosis in infected tumor cells andinhibited virus replication. Finally, we demonstrated the tran-sient blockade of TNFa from the tumor microenvironmentwith TNFa-blocking antibodies significantly enhanced virusreplication and survival in GBM intracranial tumors.

Conclusions: The results of these studies suggest that FDAapproved TNFa inhibitors may significantly improve the effi-cacy of oncolytic virus therapy. Clin Cancer Res; 21(14); 3274–85.�2015 AACR.

IntroductionGlioblastoma (GBM) is one of the most common and deadly

types of primary brain tumors. These tumors are characterized bywidespread invasion, extensive angiogenesis, and resistance to celldeath (1). These features along with a restrictive blood–brainbarrier severely limit treatment options and result in a medianpatient survival of 15 months (2).

Oncolytic herpes simplex viruses (oHSV) are viruses genet-ically modified to specifically infect, replicate in, and target

cancer cells for destruction. oHSVs represent a promising treat-ment modality for patients with GBM, and in clinical trialsthese viruses are safe and well tolerated (3). Early-phase clinicaltrials have produced promising results, and there is currently aphase III clinical trial for patients with advanced melanoma(NCT00769704; refs. 4–6).

The success of oHSV-derived therapeutics is thought todepend on the oncolytic destruction of tumor cells and theactivation of antitumor immune responses that can potentiallylead to long-term cancer remission. However, the proinflam-matory immune responses generated by viral infection can alsoantagonize oHSV replication and spread. Innate immuneresponses destroy replicating virus and reduce tumor cell kill-ing, and several studies have demonstrated the negative effectsof innate immune responses to oHSV treatment (7–9).

Microglia and infiltrating macrophages are thought to besignificant mediators of the innate immune response to viralinfection in the CNS (10–14). Depletion of these cells withclodronate liposomes or cyclophosphamide (CPA) reducesantiviral responses and improves oHSV efficacy (15–20). Asa result of these preclinical studies, the combination of onco-lytic measles virus with CPA is currently being evaluated ina phase I clinical trial for multiple myeloma (ClinicalTrials.govIdentifier: NCT00450814). Although these studies highlightthe importance of modulating early immune responses tooHSV infection, the depletion of all phagocytic cells with

1Department of Neurological Surgery, James Comprehensive CancerCenter, The Ohio State University Medical Center, Columbus, Ohio.2Department of Psychiatry, Yale University School of Medicine, NewHaven, Connecticut. 3Department of Oncology, Mayo Clinic, Roche-ster, Minnesota. 4Division of Hematology, James ComprehensiveCan-cer Center,TheOhio State University Medical Center, Columbus,Ohio.5Department of Neuroscience, James Comprehensive Cancer Center,The Ohio State University Medical Center, Columbus, Ohio.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:Balveen Kaur, Department of Neurological Surgery, TheOhio State University, 385-D OSUCCC, 410 West 12th Avenue, Columbus, OH43210. Phone: 614-292-3984; Fax: 614-688-4882; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-3118

�2015 American Association for Cancer Research.

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clodronate liposomes or total immune suppression with highdoses of CPA does not specifically address the mechanism bywhich macrophages and microglia limit oHSV replication,spread, and efficacy.

In this study, we investigated the impact of microglia andmacrophages in oHSV therapy for GBM. Quantitative flow-cytometry analysis of mice with intracranial gliomas treatedwith oHSV revealed significant changes in the activation andinfiltration of macrophages and microglia in oHSV-treatedanimals relative to untreated mice. To evaluate the impact ofthese cells on oHSV propagation, we developed an in vitrococulture system with infected glioma cells and microglia/macrophages. In these studies, macrophages and microgliasignificantly reduced virus replication. Furthermore, we identi-fied microglia/macrophage–secreted TNFa as a major factor thatreduces viral replication through the induction of apoptosisin infected cells. In coculture assays, we were able to rescuechanges in virus replication with TNFa-knockout macrophagesor TNFa function–blocking antibodies. Finally, we demonstrat-ed that the specific inhibition of TNFa produced by the tumormicroenvironment could significantly enhance virus replicationand efficacy in vivo. The results of these studies suggest thatFDA approved TNFa inhibitors may significantly enhancepatient responses in oHSV clinical trials.

Materials and MethodsCell lines

Vero, LN229, U87DEGFR, U251-T2, and U251-T3-mCherrycells were maintained in DMEM supplemented with 10% FBS.U251-T2 and U251-T3-mCherry cells were created in our labo-ratory (May 2009) as tumorigenic clones of U251 cells by seriallypassaging these cells two and three times in mice, respectively.Monkey kidney epithelial–derived Vero cells andU87DEGFR cellswere obtained in April 2005 from Dr. E. Antonio Chiocca (OhioState University, Columbus, OH). LN229 cells were obtained inJanuary 2005 from Erwin Van Meir (Emory University, Atlanta,Georgia). GB30 neurospheres were originally received in 2012from Dr. EA Chiocca (Ohio State University, Columbus, OH).

GB30 neurospheres were maintained as tumor spheres in Neu-robasal Medium supplemented with 2% B27, human EGF(50 ng/mL), and basic fibroblast growth factor (50 ng/mL) inlow-attachment cell culture flasks as previously described (21).Vero cells have not been authenticated since receipt. U87DEGFR(January 2015), LN229 (July 2013), GB30 (January 2015), andU251 (January 2015) cells were authenticated by the Universityof Arizona Genetics Core via STR profiling. Murine BV2 micro-glia were maintained in DMEM supplemented with 2% FBS. BV2cells were obtained in January 2009 from J. Godbout (Ohio StateUniversity, Columbus, OH). Murine RAW264.7 macrophageswere obtained in RPMI supplemented with 10% FBS. RAW264.7macrophages were received in June 2010 from S. Tridandapani(Ohio State University, Columbus, OH). Murine BV2 andRAW264.7 cells have not been authenticated since receipt. Allcells were incubated at 37�C in an atmosphere with 5% carbondioxide and maintained with 100 U of penicillin/mL, and0.1 mg of streptomycin/mL. All cells are routinely monitoredfor changes in morphology and growth rate. All cells are negativefor Mycoplasma.

Viruses and virus replication assayrHSVQ1, rHSVQ1-Luciferase, 1716, hrR3, and rQNestin34.5

were prepared and titered on Vero cells via a standard plaqueforming unit assay as previously described (22).

Coculture assaysGlioma cells (550,000) were plated in 6-well Falcon

tissue culture plates and infected with virus at a multiplicityof infection (MOI) of 1 or 2 in DMEM supplemented with0.05% FBS. Cells were washed three times over the course ofan hour to remove unbound virus. Infected cells were thenoverlaid with 1,000,000 microglia or macrophages (2:1 ratioof macrophages/microglia to glioma cells) for 12 hours (pre-virus burst). For TNFa-blocking antibody assays, 1,800 ng/mLof mouse TNFa-neutralizing antibody (D2H4; Cell SignalingTechnology) or an isotype control was used. Concentrations ofantibody were determined experimentally based on the man-ufacturer's specifications.

Western blotsCells were cultured with virus, TNFa, and/or microglia/macro-

phages as described above. BCA analysis (Pierce Biotechnology)was used to determine protein concentration. Equal amounts ofprotein were separated on a 4% to 20% Tris-HCL gel and trans-ferred to a polyvinylidene difluoride membrane. Caspase-8,cleaved caspase-3, cleaved PARP, and GAPDH (Cell signalingTechnology) were used at 1:1,000 except GAPDH (1:5,000),which was used as a loading control.

Microglia and macrophage antibody stainingStaining of surface antigens were performed as previously

described (23, 24). Briefly, Fc receptors were blocked with anti-CD16/CD32 antibody (eBioscience). Cells were then incubatedwith the appropriate antibodies: CD45, CD11b, MHCII, CD86,LY6C, LY6G, and CD160 (eBioscience) for 45minutes. Cells wereresuspended in FACS buffer (2% FBS in HBSS with 1 mg/mLsodium azide) for analysis. Nonspecific binding was assessedvia isotype-matched antibodies. Antigen expression was deter-mined using a Becton-Dickinson FACS Caliber four color cyto-meter. Ten thousand events were recorded for each sample and

Translational Relevance

Glioblastoma is one of the most common and deadly typesof primary brain tumors, and patients diagnosed with thesetumors have a median survival of only 15 months. Oncolyticherpes simplex viruses (oHSV) represent a promising therapyfor glioblastoma, and these viruses are currently being testedin patients for safety and efficacy. Innate immune responsesto viral infection are thought to reduce oHSV replication,tumor destruction, and efficacy. In this study, we investigatedthe antiviral functions of microglia and macrophages inoHSV therapy for glioblastoma. We identified microglia/macrophage–secreted TNFa as a major factor that reducesviral replication through the induction of apoptosis in infectedcells. We demonstrated that the inhibition of TNFa couldsignificantly enhance virus replication and efficacy in vivo. Theresults of these studies suggest that FDA approved TNFainhibitors may significantly enhance patient outcomes inoHSV clinical trials.

Macrophage- and Microglia-Secreted TNFa Inhibits oHSV Efficacy

www.aacrjournals.org Clin Cancer Res; 21(14) July 15, 2015 3275




isotype-matched conjugate. Data were analyzed using FlowJosoftware (Tree Star).

Image acquisitionFluorescent and bright field images were acquired using

an Olympus IX81 epi-fluorescence microscope equippedwith a QImaging Retiga 2000R FAST camera and Olympusobjectives. Image-Pro software (Version 6.2) was used forimage acquisition. For luciferase imaging, mice received ani.p. injection of luciferin (Caliper Life Sciences) and theluciferase signal was visualized/quantified using an IVISLumina II imaging system.

Animal surgeryAll animal experiments were performed in accordance with

the Subcommittee on Research Animal Care of The Ohio StateUniversity guidelines and were approved by the InstitutionalReview Board. Six- to 8-week-old female athymic nude mice(NCI) were used for in vivo tumor studies. Intracranial surgerieswere performed as previously described with stereotacticimplantation of 100,000 U87DEGFR (22). Tumors were treatedwith HBSS, rQNestin34.5, or rHSVQ1-Luciferase virus at thelocation of tumor implantation. For antibody studies, micewere treated via i.p. injection at the days indicated with 400 mgof anti-murine TNFa antibody (XT3.11) or isotype controlantibody (BE0094; BE0088) from BioXCell. Animals wereeuthanized when they showed signs of morbidity.

Statistical analysisThe Student t test or one-way ANOVA with Bonferroni mul-

tiple comparision post hoc tests were used to analyze changes incell killing, viral plaque forming assays, luciferase imagingexperiments, and flow-cytometry assays. In survival assays,Kaplan–Meier curves were plotted and the log-rank test wasused to determine statistical significance. All statistical analyseswere performed with the use of GraphPad Prism software(version 5.01). A P value of <0.05 was considered statisti-cally significant. Derived P values are identified as �, P < 0.05;��, P < 0.01; ��, P < 0.001.

See Supplementary Materials and Methods.

ResultsoHSV Therapy activates microglia in vivo

In GBM animal models, microglia comprise 13% to 34% ofall viable cells in the tumor (25). Similar ranges are seen inhuman tumors, and these observations underscore the impor-tance of this cell type in the context of oHSV therapy (26, 27).The ability of microglia in the tumor microenvironment toswitch from a glioma-supportive role to an antiviral statefollowing oHSV treatment has not been well studied. Toexamine changes in microglia activation following oHSV infec-tion in vivo, we treated mice with established U87DEGFRintracranial tumors with oHSV or PBS (injection control).These mice were euthanized 3 days following treatment, andwe analyzed the tumor- and nontumor-bearing hemispheresfor microglia (CD11bþCD45lo) MHCII expression (Fig. 1A).We observed an 8.75-fold increase in microglia MHCII expres-sion in oHSV-treated mice compared with PBS-treated animals(P < 0.001; Fig. 1B). Interestingly, oHSV therapy upregulatedmean microglia MHCII expression in both the tumor- and

nontumor-bearing hemispheres of the brain, but this increasewas higher in the tumor bearing hemisphere (16.33%MHCIIþ)compared with the nontumor-bearing hemisphere (8.23%MHCIIþ; Fig. 1C).

oHSV therapy increases macrophage infiltration into the braintumor microenvironment

Microglia activation induces the expression of various cyto-kines and chemokines that can stimulate the migration ofimmune cells into the CNS (14). Macrophages are importantmediators of this innate immune response to viral infection,but the extent of macrophage infiltration into the CNS follow-ing oHSV therapy is unknown. To quantify the impact ofoHSV-induced macrophage migration, we treated mice withestablished intracranial U87DEGFR tumors with oHSV or PBSas described earlier. We observed a 7.96- and 5.70-fold increasein macrophage (CD11bþCD45hi) infiltration into the tumor-and nontumor-bearing hemispheres following oHSV infection,respectively (n ¼ 5/group; P < 0.001 and P < 0.05; Fig. 1D andE). Although oHSV therapy strongly induced macrophageinfiltration into both hemispheres, this increase was signifi-cantly higher in the tumor-bearing hemisphere (P < 0.001;Fig. 1D and E). Although macrophages comprised the bulkof the innate immune cell infiltrate, other innate immunecells populations are known are known to migrate into theCNS following viral infection (7, 28). We examined the percollisolated cell populations for Ly6Gþ neutrophils and CD160þ

natural killer (NK) cells, and we found few NK cells or neu-trophils in the CD11bþCD45þ populations at this time point(Supplementary Fig. S1).

oHSV therapy increases macrophage activation in the braintumor microenvironment

oHSV therapy induced significant macrophage infiltrationinto the brain tumor microenvironment, but the phenotypeand activation of these cells remained unknown. Dependingon their polarization, macrophages can promote an immuno-suppressive or proinflammatory tumor microenvironment.The activation status of these infiltrating cells is crucial tounderstanding how these cells contribute to oHSV therapyfor GBM. To determine the polarization status of infiltratingmacrophages, we evaluated the expression of the classic acti-vation markers CD86, Ly6C, and MHCII. We observed signi-ficant increases in the percentages and cell numbers ofmacrophages (CD11bþCD45hi) positive for CD86þ andLY6Cþ following oHSV treatment compared with PBS treat-ment (P < 0.001; P < 0.001, respectively; Fig. 2A and B).Although the percentages of MHCII-positive macrophages(CD11bþCD45hi) in the tumor environment between oHSVand control treatments did not change, we observed a 9-foldincrease in the total numbers of MHCII-positive macrophagesinfiltrating the tumor-bearing hemisphere following oHSVtherapy compared with control-treated mice (P < 0.001;Fig. 2C). The surface expression of these three activationmarkers increased on macrophages(CD11bþCD45hi) in boththe treated and untreated hemispheres, but the treated hemi-spheres contained higher percentages of macrophages thatexpressed CD86 and LY6C (Fig. 2A–C). Together, these datasuggested that the infiltrating macrophages were polarizedtoward a proinflammatory state.

Meisen et al.

Clin Cancer Res; 21(14) July 15, 2015 Clinical Cancer Research3276




Coculture of oHSV-infected tumor cells with microglia ormacrophages reduces viral replication in vitro

oHSV treatment significantly activated microglia/macrophagesin vivo, but the effects of these polarized immune cells on virusreplication remained unknown. To determine the functional con-sequences of this microglia/macrophage activation, we developedan in vitro coculture system.Humanglioma cellswere infectedwithoHSV at anMOI of 2, washed to remove unbound virus, and thenoverlaid with murine macrophages (RAW264.7) or microglia(BV2; Fig. 3A). Figure 3A is representative of co-cultures done withmurine macrophages (RAW264.7) or microglia (BV2). To specif-ically examine the microglia/macrophage response towardinfected cells and not toward free virus, the infected cells werecultured for less than 12 hours to prevent the lytic burst of tumorcells and the infection of microglia/macrophages. Compared withinfected glioma cells alone, culturing infected cells with microgliaor macrophages reduced viral titers by 37.28% and 69.99%,respectively (P < 0.01; P < 0.001; Fig. 3B and C). This decreasein virus replication was also accompanied by significant pheno-typic changes12hoursafter infection.Uninfectedgliomacellswereadherent with extensive filopodia. Following infection, these cells

became rounded, but remained adherent (Fig. 3D). Similarly,uninfected glioma cultured with microglia/macrophages revealedno significant changes inmorphology. Interestingly, when infectedglioma cells were cultured with macrophages or microglia, themicroglia/macrophages surrounded the infected tumor cells andformed tight rosette-like clusters that became nonadherent (Fig.3D; Supplementary Fig. S2A and S2B).

Macrophage and microglia secreted TNFa inhibits virusreplication

Culturing infected glioma cells with microglia or macro-phages significantly decreased virus replication, but how thesecells reduced virus propagation remained to be elucidated.TNFa is a pleiotropic cytokine whose expression is signi-ficantly upregulated in response to viral CNS infections(14, 29). To test whether TNFa produced by activated macro-phages/microglia could limit viral replication in glioma cells,we determined the levels of TNFa secreted by microglia andmacrophages in our coculture system using a species-specificELISA (Supplementary Fig. S3). Using a murine-specific TNFaELISA, we observed that microglia and macrophages produced

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Figure 1.oHSV treatment increases microglia activation and induces macrophage infiltration into the tumor microenvironment. A, diagram of mice with intracranialU87DEGFR tumors (black dot) treated with 1 � 105 pfu of oHSV (rQNestin34.5) or PBS 7 days after tumor cell implantation. Three days after oHSVtreatment, the mice were euthanized and the tumor and nontumor-bearing hemispheres were separated via gross dissection (midline drawn betweentwo hemispheres). B, quantification of MHCII expression of the tumor-bearing hemispheres of PBS- and oHSV-treated mice. Data shown are meanpercentage of positive � SD (n ¼ 5/group). C, the representative scatter plot of MHCII expression on microglia (CD11bþCD45lo) in tumor and nontumor-bearing mice treated with PBS or oHSV. D, quantification of macrophage infiltration into the tumor-bearing hemisphere following oHSV therapy orPBS injection. Data shown are mean percentage of positive � SD (n ¼ 5/group; P < 0.001). E, representative scatter plot of macrophage (CD11bþCD45hi)infiltration following oHSV therapy. �� , P < 0.001.






significant amounts of TNFa in response to infected tumorcells. Compared with uninfected cocultures, oHSV infectionincreased macrophage- and microglia-secreted TNFa by 35.42-and 9.00-fold, respectively (P < 0.001; P < 0.001; Fig. 4A and B).Interestingly, we observed a 57.11% and 33.66% decrease inmacrophage- and microglia-secreted TNFa when these cellswere cultured with uninfected tumor cells compared with beingcultured alone, respectively (Fig. 4A and B). In support of thesein vitro data, we also observed a significant increase in murine-secreted TNFa in the brain and serum following oHSV treat-ment in intracranial xenografts (P < 0.001; P < 0.01, respec-tively; Supplementary Fig. S4A and S4B). This result is concor-dant with previously published work demonstrating thatmacrophages and microglia produce large amounts of TNFain response to HSV infection in the CNS (29). Next, wedetermined whether the levels of TNFa produced in thesecocultures were sufficient to reduce virus replication in gliomacells. Treatment of infected cells with 1,000 or 2,000 pg/mL ofrecombinant human TNFa resulted in 34.29% and 40.73%reductions in virus replication, respectively (P < 0.05; P < 0.01;Fig. 4C). Similar results were obtained when infected gliomacells were treated with recombinant murine TNFa (Supple-mentary Fig. S5A). Visual inspection of infected cells treated

with soluble TNFa also revealed surprising morphologicchanges. oHSV-infected glioma cells treated with TNFa becamerounded and nonadherent. The cells resembled infected cul-tures with microglia/macrophages (Fig. 4D, Fig. 3D and Sup-plementary Fig. S5B). We did not observe any morphologicchanges or reductions in cell viability in multiple uninfectedglioma cell lines treated with TNFa (Supplementary Fig. S6Aand S6B). Additional experiments with uninfected glioma cellstreated with varying doses of TNFa for 60 hours also did notreduce cell proliferation (P < 0.001 for all doses; Supplemen-tary Fig. S6C).

Secreted TNFa induces apoptosis in oHSV-infected cellsMacrophage- and microglia-secreted TNFa significantly

reduced virus replication in oHSV-infected cells, but the mecha-nism of TNFa-directed virus inhibition remained to be deter-mined. High-magnification images of oHSV-infected cells treatedwith TNFa revealed significant changes in cell morphology.Unlike oHSV-infected cells alone, the addition of TNFa resultedin significant membrane blebbing (white arrows), cell shrinkage,and a loss of adherence, all features characteristic of cells under-going apoptosis (Fig. 4D). On the basis of these observations, wehypothesized TNFa-induced apoptosis in infected cells resulting

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Figure 2.oHSV treatment increases macrophage (CD11bþCD45hi) activation. A to C, left, representative scatter plots of CD86þ (A), LY6Cþ (B), and MHCIIþ (C)macrophages in the tumor and nontumor-bearing hemispheres following oHSV (rQNestin34.5) or PBS treatment (n ¼ 5/group). Right, quantificationof the percentage and cell numbers of macrophages (CD11bþCD45hi) expressing the indicated marker following oHSV or PBS treatment in the tumor- andnontumor-bearing hemisphere (n ¼ 5/group). Quantified data are mean values � SD. �� , P < 0.001.

Meisen et al.





in reduced virus titers. To investigate whether TNFawas inducingapoptosis in infected cells, we conducted immunoblot assays forcaspase-8, cleaved caspase-3, and cleaved PARP. We observedsignificant caspase-8, caspase-3, and PARP activation in cellstreated with oHSV and TNFa. We did not observe significantactivation of these proteins in glioma cells treated with TNFa oroHSV alone (Fig. 4E; Supplementary Fig. S7).

Inhibition of macrophage- or microglia-secreted TNFaincreases oHSV replication in vitro

Because macrophage- and microglia-secreted TNFa reducedvirus replication by inducing apoptosis in infected cells, wehypothesized that the inhibition of macrophage/microglia–pro-duced TNFa would significantly improve virus replication. Todetermine whether inhibiting macrophage TNFa was sufficient torescue virus replication, we conducted coculture assays with fresh-ly isolated wild-type or TNFa-knockout (TNFa�/�) bone mar-row–derived macrophages (BMDM). Phase-contrast microscopy

of these cultures revealed significant morphologic differencesbetween the two groups. Consistent with our previous results, amajority of the infected glioma cells cultured with wild-typeBMDMs formed nonadherent clusters and exhibited significantmembrane blebbing indicative of apoptosis. In contrast with theseobservations, infected glioma cells cultured with TNFa�/�

BMDMs were adherent and showed substantially less membraneblebbing (Fig. 5A). These observations correlated with changesin virus titers; culturing infected glioma cells with TNFa�/�

BMDMs significantly rescued virus replication compared wild-type BMDMs (P < 0.05; Fig. 5B). In a similar experiment, wefound the addition of murine-specific TNFa-blocking antibodiesrescued the reduction in virus replication in infected glioma cellswhen cultured with BV2 microglia (P < 0.05; Fig. 5C and D).

Inhibition of TNFa increases virus replication in vivoBlockade or knockout of macrophage/microglia–secreted

TNFa significantly enhanced oHSV replication in vitro. To assess

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Figure 3.Microglia and macrophages reduce virus replication in tumor cells in vitro. A, schematic of microglia/macrophage and tumor cell cocultures. Tumor cells (oval-shaped cells) were infected with oHSV (rHSVQ1; black dots) at an MOI of 2. Unbound virus was washed away and microglia or macrophages(branched cells) were overlaid on the infected glioma cells. The cells were cultured for 12 hours and the viral titers were determined by a standardplaque formation assay. B and C, viral titers of glioma cells infected alone or cultured with BV2 microglia (n ¼ 4/group; B) or RAW264.7 macrophages(n ¼ 4/group; C). Data shown are mean virus titer � SD. D, representative images of glioma cells cultured with microglia and macrophages with andwithout oHSV infection 12 hours after infection (n ¼ 4/group). ��, P < 0.01; �� , P < 0.001.






the translational relevance of these results for oHSV therapy,we tested whether TNFa blockade could enhance virus repli-cation in vivo. In these experiments, athymic nude mice wereimplanted s.c. with U87DEGFR human GBM tumors. Whenthe tumors reached an average volume of 143 mm3 the micewere treated with a single dose of oncolytic virus. Mice werealso administered a murine-specific TNFa-blocking antibody ora control antibody. Mice were given antibody 1 day before virusinjection, the day of virus administration, and on days 1, 3, and5 after virus treatment. In these studies, a luciferase-expressingoHSV was used to visualize virus replication. We observed asignificant enhancement in virus propagation in vivo (as mea-sured by luciferase encoded by virus) in mice treated with aTNFa-blocking antibody as compared with a control IgG anti-body on days 1, 2, and 3 following oHSV administration

(n ¼ 5/group; P < 0.02; P < 0.01; P < 0.02, respectively;Fig. 6A and B). These results suggested that the inhibition ofTNFa produced by macrophages and the tumor microenviron-ment was sufficient to increase virus replication in vivo.

Finally, we conducted intracranial GBM studies to determinewhether TNFa blockade could enhance the survival of micetreated with oHSV. In these studies, mice were implanted intra-cranially with U87DEGFR human GBM cells and treated withoHSV 8 days later (2 � 105 pfu rHSVQ1-luc). The antibodydosing regimen from the subcutaneous tumor experiments wasused in this study. Mice treated with oHSV and a murine-specificTNFa-blocking antibody lived significantly than those treatedwith oHSV and an isotype control antibody (P ¼ 0.026),TNFa-blocking antibody alone (P ¼ 0.0003), or with an isotypecontrol antibody alone (P ¼ 0.0003; Fig. 6C). These results

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Figure 4.Microglia- and macrophage-secreted TNFa inhibits virus (rHSVQ1) replication in vitro. A and B, quantification of TNFa secreted by BV2 microglia (A)or RAW264.7 macrophages (B) alone, and when cultured with uninfected or infected U251-T2 glioma cells for 12 hours. Data shown are meanconcentration TNFa � SD. C, viral titers of glioma cells infected at an MOI of 2 alone, with recombinant human TNFa (1,000 or 2,000 pg/mL), or withBV2 microglia for 12 hours. Data shown are mean virus titer � SD. D, representative images of U251-T2 glioma cells infected with oHSV (rHSVQ1) atan MOI of 2 with vehicle (left) or with TNFa (2,000 pg/mL; right) for 12 hours. White arrows indicated membrane blebbing in infected cells treatedwith TNFa. E, Western blot analysis of U251-T2 glioma cells alone, treated with TNFa (5,000 pg/mL), infected with oHSV at an MOI of 2, or treated withTNFa and oHSV for 12 hours. Infected U251-T2 glioma cells cultured with BV2 microglia is also shown. Caspase-8, cleaved caspase-3, cleaved PARP, andGAPDH are shown. Caspase-8 blot shows full-length protein (1), cleaved intermediate protein (2), and active protein (3). �, P < 0.05; �� , P < 0.01; �� , P < 0.001.

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suggested that the combination of TNFa-blocking antibodiesmay enhance oHSV therapeutic efficacy for GBM.

DiscussionoHSV therapy is a promising treatment modality for GBM. The

success of oHSV-derived therapeutics depends on both the onco-lytic destruction of tumor cells and the activation of long-term,antitumor immune responses. Although the innate immuneresponse is important for activating adaptive responses, the innateresponses to oHSV therapy can also inhibit virus replication andoncolytic tumor cell killing. Depletion of macrophages andmicroglia with clodronate liposomes and CPA has previouslybeen shown to reduce antiviral responses and improve oncolyticvirus efficacy for GBM (9, 15–20, 30–32). The combination ofoncolytic measles virus with CPA is currently being evaluated in aphase I clinical trial for multiple myeloma (ClinicalTrials.govIdentifier: NCT00450814). Recently, NK cells were shown to helpcoordinate the innate immune response to oHSV therapy, and the

depletion of these cells was found to enhance oncolytic virus(OV) efficacy for GBM (7). Neutrophils have also been shown tolimit OV dissemination in part through the release of neutrophilextracellular traps (33). Collectively, these studies suggest mod-ulating early innate immune responses to achieve the optimalbalance between viral replication and inflammation is critical tothe clinical success of oHSV therapies.

Although microglia and infiltrating macrophages are thoughtto be the primary mediators of the innate immune response tooHSV infection for GBM, the mechanism by which these cellslimit virus replication and therapeutic efficacy has not been wellstudied (16). Here, we quantified the extent of microglia/macro-phage activation and infiltration following oHSV treatment.Although microglia are the resident immune cells of the CNS, inthis study, we observed infiltrating macrophages outnumberedmicroglia more than 2:1 in the tumor microenvironment follow-ing oHSV infection. These results suggested that monocyte-derivedmacrophages may be the dominant cell type that controlsoHSV infection.

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Figure 5.Inhibition of microglia/macrophage–secreted TNFa increases virus (rHSVQ1) replication in vitro. A, representative images of U251-T2 glioma cells infected atan MOI of 2 cultured with bone marrow–derived macrophages derived from wild-type or TNFa knockout mice for 12 hours. Large representativeimages are taken at a �4 magnification with the insets taken at a �20 magnification (white arrows indicate blebbing). B, 12 hour viral titers of culturesdescribed in A. Data shown are mean virus titer � SD. C, schematic of experimental setup using murine specific TNFa antibodies to block microglia(branched cells) secreted TNFa in cocultures with infected glioma cells (oval-shaped cells). D, quantification of virus titer obtained from infected gliomacells cultured with BV2 microglia with IgG or anti-murine TNFa–blocking antibody (1,800 ng/mL). Data shown are mean virus titer � SD. � , P < 0.05;�� , P < 0.01.






Although infiltrating macrophages primarily increased in thetumor-bearing hemisphere, there was also significant activationand infiltration of immune cells in the contralateral hemi-sphere. These results suggested that oHSV infection induceda global inflammatory response in the CNS rather than alocalized immune response confined to the tumor. Activationsignals such as TNFa are propagated throughout the CNS inresponse to inflammatory stimuli. Although this study focuseson the antiviral effects of TNFa, in response to virus infectionmany signals such as IL1b, IL6, IFNs, and nitric oxide arereleased to control oHSV infection (11). These proinflamma-tory mediators signal in an autocrine and paracrine mannerto activate immune cells such as macrophages and enhancetheir ability to respond to viral infection. oHSV-associatedinflammation in the nontumor-bearing hemisphere and sur-rounding healthy brain parenchyma has not been well studied.These observations may have implications in the treatment ofbrain tumor patients with oHSVs where unchecked inflamma-tion can be detrimental.

In these studies, we observed a significant inflammatoryresponse to viral infection until at least 3 days after treatment.In vivo flow-cytometry experiments indicated that microglia andinfiltrating macrophages were polarized toward an M1, proin-flammatory state. We demonstrated the antiviral consequence of

microglia and macrophage activation in coculture studies andfound both macrophages andmicroglia reduced virus replicationin glioma cells. Together these results confirmed the antiviralcapabilities of these cell types in modulating oHSV replicationin vivo. These data also support previous studies that identifythe antiviral activity of macrophages and microglia againstwild-type herpes simplex virus 1 (HSV-1) infections (34, 35).

In this study, we identified TNFa as a major macrophage/microglia–secreted factor that reduces oHSV replication. TNFais a pleotropic cytokine important for the recruitment and acti-vation of immune cells. Macrophages and microglia are alsomajor producers of TNFa. TNFa is known to limit wild-type HSVreplication in the CNS, and it has previously been shown tomediate antiviral effects in studies with wild-type vesicular sto-matitis virus, adenovirus-2, encephalomyocarditis virus, HSV-1,HSV-2, respiratory syncytial virus, and influenza through a varietyof mechanisms (29, 36–43).

Although TNFa is detrimental to virus replication, TNFa sig-naling in cancer cells can result in increased tumor cell growth,angiogenesis, invasion, and progression (44–46). Higher levels ofthe antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 as well asdecreased levels of apoptotic proteins such as BAX are commonlyobserved in recurrent GBM and demonstrate the ability of thesetumors to resist caspase-mediated cell death (47). Consistent with

IgG Anti-mTNFa

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Figure 6.TNFa inhibition increases virusreplication and efficacy in vivo. Nudemice with U87DEGFR subcutaneoustumors were treated with 1 � 106 pfuof an oHSV expressing luciferase(rHSVQ1-luc). Murine-specific TNFaor isotype control antibodies wereadministered on days�1, 0, 1, 3, and 5after oHSV therapy. A, data shown arequantification of virally expressedluciferase gene activity in U87DEGFRsubcutaneous tumors treated withcontrol or TNFa-blocking antibodieson the days indicated after rHSVQ1-luciferase virus treatment. Datashown are total flux in each mouse(n ¼ 5/group). B, representativeluciferase images of oHSV-treatedmice with TNFa-blocking or isotypecontrol antibodies at the daysindicated (n ¼ 5/group). C, Kaplan–Meier survival curve of mice bearingU87DEGFR intracranial tumorstreated with PBS or 2 � 105 pfurHSVQ1 with IgG or TNFa-blockingantibody (IgG þ saline n ¼ 10; anti-TNFa þ saline n ¼ 11; IgG þ rHSVQ1n ¼ 14; anti-TNFa þ rHSVQ1 n ¼ 15).

Meisen et al.





these published studies, we observed that TNFa was not toxic touninfectedGBMcells in vitro. In infected glioma cells, however, weobserved that TNFa activated the extrinsic apoptotic pathwayresulting in premature cell death, reduced virus replication, anddecreased antitumor efficacy. The precise mechanism of how thecombination TNFa with oHSV induces apoptosis is unclear.Although HSV-1 has been shown to inhibit apoptosis, previouswork has demonstrated the inability of HSV-1 to prevent apo-ptosis in infected cells exposed to environmental stimuli such asTNFa (48). TNFa-induced cell death was found to be cell-typedependent, and in the case of glioma, this processmay depend onthe expression of pro- and antiapoptotic proteins within the cells.

Oncolytic HSVs expressing TNFa have previously been testedfor their ability to enhance oHSV antitumor efficacy (49). Inthese studies, TNFa-expressing viruses did not enhance antitu-mor efficacy in an immune-competent lymphoma model com-pared with a control oHSV that did not express TNFa. Inaddition, in human squamous carcinoma xenografts, the anti-tumor efficacy of an oHSV-expressing high levels of TNFa wassignificantly less than an oHSV-expressing low levels of TNFa.In support of our findings, these results suggest that TNFaelicits strong antiviral responses that may be detrimental tooncolytic HSV therapy.

Although TNFa blockade lead to increased virus propaga-tion, its effect on toxicity in the context of HSV-1 infections isnot clear. Both virus-mediated and immune-mediated mechan-isms contribute toward the pathology of HSV-1 infections. Instudies with mice infected with wild-type HSV-1, TNFa-knock-out mice had higher virus titers and were more susceptible tofatal HSV encephalitis than wild-type mice. These results high-light the protective, antiviral functions of TNFa (29, 36).Although TNFa is important for controlling virus replication,high levels of TNFa have also been shown to induce blood–brain barrier disruption leading to increased inflammation(50). Interestingly, HSV-1–infected mice treated with TNFa-blocking antibody showed reduced signs of viral encephalitisand lived longer than those treated with virus alone (51). Thus,a transient blockade of TNFa during virotherapy could increasevirus replication and reduce neurotoxicity due to acute inflam-mation while still allowing for an immune response to even-tually clear the infection. Importantly, in our studies, weobserved no toxicity associated with TNFa antibody adminis-tration in combination with our attenuated, oncolytic virus.

Radio- and chemotherapy also induce the production of cyto-kines such as TNFa (52–54). Oncolytic virotherapy for GBM isoften administered following tumor resection and concurrentlywith radio- and chemotherapy. As a result, patients may benefitfrom the transient use of TNFa inhibitors before oHSV admin-istration to enhance oncolytic tumor cell killing and reduce CNSinflammation. The TNFa inhibitors etanercept, adalimumab,certolizumab, and golimumab are currently FDA approved fora variety of diseases and could be readily used in oHSV clinicaltrials. These inhibitors may be more effective than generalimmune suppressants, such as high-dose myeloablative CPA,which can have significant toxicities in patients. The combination

of oHSV with TNFa inhibitors could enhance virus replication,reduce TNFa-driven tumor proliferation, angiogenesis, and inva-sion, as well counter the negative effects of chemotherapy/radio-therapy–induced inflammation. This transient inhibition ofTNFa could then be removed to allow for the activation oflong-term, antitumor immune responses thatmaybemore potentdue to increased virus-mediated cell killing and antigen release. Insubcutaneous and intracranial tumor studies, we found that theinhibition of TNFa secreted by the tumor microenvironmentsignificantly enhanced virus replication and therapeutic efficacy.In these experiments, we used a TNFa-blocking antibody becausethe current FDA approved TNFa inhibitors are antibody based.Although the integrity of the blood–brain barrier is disrupted inglioblastoma, the ability of therapeutic antibodies to cross theblood–tumor barrier (BTB) is thought to be limited (55, 56).Although we observed up to 9-fold increases in viral luciferaseexpression in subcutaneous tumors, the therapeutic effect in theintracranial tumor studies was more modest. In addition toantibody penetration into the brain tumor microenvironmentfollowing oHSV therapy, we hypothesize that the increase inanimal survival may have also been through the ability of theantibody to bind TNFa in the serum following oHSV therapy.The future development of specific, soluble TNFa inhibitorsthat better penetrate the BTB may further increase the antitumorefficacy we observed. These experiments support the future useof TNFa inhibitors in combination with oHSV for GBM.

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

Authors' ContributionsConception and design: W.H. Meisen, A.C. Jaime-Ramirez, J. Yu, J. Godbout,B. KaurDevelopment ofmethodology:W.H.Meisen, E.S.Wohleb, A.C. Jaime-Ramirez,J.Y. Yoo, L. Russell, J. Hardcastle, K. Muili, J. Godbout, B. KaurAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W.H. Meisen, E.S. Wohleb, A.C. Jaime-Ramirez,C. Bolyard, L. Russell, S. Dubin, K. MuiliAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W.H. Meisen, E.S. Wohleb, A.C. Jaime-Ramirez,S. Dubin, K. Muili, J. Godbout, B. KaurWriting, review, and/or revision of themanuscript:W.H.Meisen, E.S.Wohleb,A.C. Jaime-Ramirez, J.Y. Yoo, S. Dubin, K. Muili, J. Yu, J. Godbout, B. KaurAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W.H. Meisen, J.Y. YooStudy supervision: W.H. Meisen, J. Yu, M. Caligiuri, B. Kaur

Grant SupportThis work was supported in part by: NIH grants R01NS064607,

R01CA150153, P30NS045758, and P01CA163205 (to B. Kaur) and PelotoniaFellowships (to A.C. Jaime-Ramirez and S. Dubin).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 11, 2014; revised February 26, 2015; accepted March 24,2015; published OnlineFirst March 31, 2015.

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2015;21:3274-3285. Published OnlineFirst March 31, 2015.Clin Cancer Res W. Hans Meisen, Eric S. Wohleb, Alena Cristina Jaime-Ramirez, et al. MicroenvironmentOncolytic HSV-1 Therapy in the Glioblastoma Tumor

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