safe and effective treatment of experimental neuroblastoma and glioblastoma … · toma multiforme...

Cancer Therapy: Preclinical

Safe and Effective Treatment of ExperimentalNeuroblastoma and Glioblastoma UsingSystemically Delivered Triple MicroRNA-Detargeted Oncolytic Semliki Forest VirusMohanraj Ramachandran1, Di Yu1, Matheus Dyczynski1, Sathishkumar Baskaran1,Lei Zhang1, Aleksei Lulla2, Valeria Lulla2, Sirle Saul2, Sven Nelander1, Anna Dimberg1,Andres Merits2, Justyna Leja-Jarblad1, and Magnus Essand1

Abstract

Background: Glioblastoma multiforme and high-risk neuro-blastoma are cancers with poor outcome. Immunotherapy in theform of neurotropic oncolytic viruses is a promising therapeuticapproach for these malignancies. Here we evaluate the oncolyticcapacity of the neurovirulent and partly IFNb-resistant SemlikiForest virus (SFV)-4 in glioblastoma multiformes and neuroblas-tomas. To reduce neurovirulence we constructed SFV4miRT,which is attenuated in normal central nervous system (CNS) cellsthrough insertion of microRNA target sequences for miR124,miR125, miR134.

Methods: Oncolytic activity of SFV4miRT was examined inmouse neuroblastoma and glioblastoma multiforme cell linesand inpatient-derivedhumanglioblastoma cell cultures (HGCC).In vivo neurovirulence and therapeutic efficacy was evaluated intwo syngeneic orthotopic glioma models (CT-2A, GL261) and asyngeneic subcutaneous neuroblastoma model (NXS2). The roleof IFNb in inhibiting therapeutic efficacy was investigated.

Results: The introduction of miRNA target sequencesreduced neurovirulence of SFV4 in terms of attenuated rep-lication in mouse CNS cells and ability to cause encephalitiswhen administered intravenously. A single intravenous injec-tion of SFV4miRT prolonged survival and cured four of eightmice (50%) with NXS2 and three of 11 mice (27%) with CT-2A, but not for GL261 tumor-bearing mice. In vivo therapeuticefficacy in different tumor models inversely correlated tosecretion of IFNb by respective cells upon SFV4 infectionin vitro. Similarly, killing efficacy of HGCC lines inverselycorrelated to IFNb response and interferon-a/b receptor-1expression.

Conclusions: SFV4miRT has reduced neurovirulence, whileretaining its oncolytic capacity. SFV4miRT is an excellentcandidate for treatment of glioblastoma multiforme and neu-roblastoma with low IFN-b secretion. Clin Cancer Res; 1–12.�2016 AACR.

IntroductionMalignant tumors arising from the central nervous system

(CNS) are among the most feared types of cancer. Glioblas-toma multiforme is the most common, malignant form ofprimary brain tumor with poor prognosis and 1-year survivalrate of only 35.7% (1). Surgical resection combined withradiotherapy and adjuvant temozolomide is the current stan-dard of care therapy, which extends survival but is not

curative (2). Glioblastoma multiforme is more common inadults but can also arise in children. Neuroblastoma is themost common extracranial solid cancer in children arisingfrom neural crest cells. Low-risk neuroblastomas are associ-ated with good outcome but children with high-risk neuro-blastoma often relapse even after multimodal therapy (3).Conditionally replicating oncolytic viruses are an attractiveoption for treatment of cancers, as viruses have a naturalability to replicate inside dividing tumor cells and kill themand simultaneously induce an immune response against thetumor. Herpes simplex virus, adenovirus, and Newcastledisease virus have been evaluated in phases I and II clinicaltrials for treatment of glioblastoma multiforme. They showexcellent safety data but have so far not resulted in successfultumor shrinkage and prolonged survival (4). Adenovirus hasbeen used to treat children with neuroblastoma with encour-aging results (5, 6).

Semliki Forest virus (SFV) is an Alphavirus belonging to theTogaviridae family (7). It is an enveloped, positive strand RNAvirus, having high replication efficacy and ability to kill a varietyof tumor cells (7–10). It has a natural neurotropism (11),making it an attractive candidate for use as an oncolyticimmunotherapy agent to treat neuroblastoma and glioblasto-ma multiforme. Several strains of SFV have been described,

1Department of Immunology, Genetics and Pathology, Science for Life Labora-tory, Uppsala University, Uppsala, Sweden. 2Institute of Technology, Universityof Tartu, Tartu, Estonia.

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

M. Ramachandran, D. Yu, J. Leja-Jarblad, andM. Essand have contributed equallyto this article.

Current affiliation for M. Dyczynski: Department of Oncology and Pathology,CCK, Karolinska Institute, Stockholm, Sweden.

Corresponding Author:Magnus Essand, Uppsala University, SE-75185 Uppsala,Sweden. Phone: 46 18 471 4535; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0925

�2016 American Association for Cancer Research.

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including the neurovirulent strain SFV4 and the avirulent strainSFV-A(7)74 (referred to as A7/74 from now; ref. 7). Theneurovirulence of SFV4 is mainly due to initial replication inneurons and oligodendrocytes, and subsequent spread to therest of the brain resulting in encephalitis (12, 13). A7/74 isusually preferred for use as an oncolytic agent due to its naturalavirulence in adult rodents. However, type-I IFN a/b (IFNa/b)responses elicited by the host in response to virus infectionimpede A7/74 efficacy (14, 15). The nonstructural proteinsnsP3-nsP4 of SFV4 are involved in reducing STAT1 Tyr701phosphorylation (P-STAT1) upon stimulation of virus infectedcells with exogenous IFNb, and it confers its ability to partlyresist type-I antiviral defense (16). Thus, SFV4 can be a prom-ising alternative to treat IFNa/b responsive tumors if its neu-rovirulence can be attenuated.

miRNA are small noncoding RNAs involved in posttranscrip-tional regulation of gene expression. miRNA expression is cell-type restricted, which has been taken advantage of whendesigning oncolytic virus to prevent their replication in healthytissues (17–20). This is achieved by introduction of miRNAtarget sequences (miRT) in the viral genome, leading to thatviral messenger RNA (mRNA; in the case of positive stand RNAviruses it is also genomic RNA) degradation in cells where thematching miRNA are expressed. Regarding miRNAs in the CNS,miR124 and miR125 are highly expressed in mouse brain (21),whereas miR134 is expressed in the dendrites present in thehippocampal neurons (22). Their expression in glioblastomamultiforme and neuroblastoma are partly unknown and inves-tigated herein.

In this article we evaluate the efficacy of SFV immunother-apy with respect to type-I IFN response in patient-derivedhuman glioblastoma cell cultures (HGCC) along with preclin-ical in vivo glioma and neuroblastoma models. We improvedthe safety of SFV4 immunotherapy by insertion of targetsequences for miR124, miR125, and miR134 in its genome(SFV4miRT) to reduce neurovirulence in mice but retain rep-lication potential and oncolytic capacity in glioma and neu-roblastoma cells.

Materials and MethodsQuantitative real-time PCR

Total RNA from cell lines was isolated for miRNA detectionusing miRNeasy Mini Kit (Qiagen). To isolate RNA from unin-fected mouse tissues: brain, spinal cord, and dorsal root ganglion(DRG) were excised, dissected, immersed in RNAlater (Ambion,Thermo Fisher Scientific) and homogenized before total RNAwasprepared by TRizol extraction (Invitrogen). cDNA was synthe-sized using QuantiMir RT Kit (System Biosciences). miRNAswere quantified from cDNA by SYBR green based real-time PCRusing the forward primers miR124.F: 50-TAAGGCACGCGGTGA-ATGCC-30, miR125.F: 50-TCCCTGAGACCCTTTAACCTGTGA-30,miR134.F: 50-TGTGACTGGTTGACCAGAGGGG-30, and the uni-versal reverse primer provided in the QuantiMir RT Kit. Human(hU6.F: 50-CGCAAGGATGACACGCAATTC-30) and mouse(mU6.F: 50-TGGCCCCTGCGCAAGGATG-30) U6 snRNA wasused to normalize expression levels of miRNAs of interest.

Total RNA from15mgmouse brain tissue infectedwith SFV4orSFV4miRT was extracted using RNeasy-Mini Kit (Qiagen). RNAsamples (1 mL) at the concentration of 100 ng/mL were useddirectly to determine viral genome copies, IFNa and IFNbmRNAby real-time PCR analysis using iScript One-Step RT-PCR Kit(BioRad). Viral genomes were determined by using primersannealing to nsP1 encoding region: nsP1.F 50-CGCCAAAA-GATTTTGTTCCA-30; nsP1.R, 50-CCATCGTGGGTGGTTAATCT-30. Primers used for murine IFNa detection were mIFNa.F: 50-AGGACAGGAAGGATTTTGGA-30, mIFNa.R: 50-GCTGCTGATG-GAGGTCATT-30, for murine IFNb detection were mIFNb.F: 50-CACAGCCCTCTCCATCAACT-30, mIFN-b.R: 50-GCATCTTCTCC-GTCATCTCC-30, and for house-keeping murine hypoxanthine-guanine phosphoribosyltransferase (HPRT1) detection wereHPRT.F: 50-CATAACCTGGTTCATCATCGC-30, HPRT.R: 50- GG-AGCGGTAGCACCTCCT-30. All primers were from Sigma-Aldrich. Relative expression of murine IFNa and murine IFNbwas calculated relative to HPRT1 expression levels. Data wereevaluated using the 2�DDCT method (23).

In vitro cell killing assay using GL261, CT-2A, NXS2, andHGCCcells

Mouse glioma cell lines GL261, CT-2A, and mouse neuroblas-toma cell line NXS2were seeded at densities 20,000 cells/well (96well plate) and infected with SFV4, SFV4miRT, A7/74, or A7/74miRT at multiplicity of infection (MOIs) 0.001 to 10 plaqueforming units (PFU)/cell. Cells infected with SFV4miRT werepretreated with murine IFNb (mIFNb; R&D Systems) at concen-trations 1 ng/mL, 0.01 ng/mL, or only medium. Cell viability wasanalyzed at 3 days post virus infectionusingMTSaqueous cell titerreagent (Promega).

Human glioblastoma HGCC cell lines were seeded at density5,000 cells/well (96-well plate) and infected with SFV4miRT atMOIs 0.01 to 1 PFU/cell 24 hours after seeding. Cells werepretreated with human IFNb (hIFNb; Peprotech) at concentra-tions 10, 1, 0.01 ng/mL, or only medium before virus infection.Cell viability was analyzed at 2 and 3 days postinfection usingAlamarBlue Cell viability reagent (Thermo Fisher Scientific).

IFNb ELISA and flow cytometryMurine GL261, CT-2A, NXS2, and human HGCC cells were

plated at density 50,000 cells/well (24-well plate) in 250 mLmedium, infected with SFV4 at MOI-0.01 or 1 PFU/cell; control

Translational Relevance

With Talimogene laherparepvec, the first oncolytic virusapproved for treatment of cancer, oncolytic virus immuno-therapy has a very potential future. Glioblastoma multiformeis a rapidly growing neoplasm that is very difficult to cure withthe current standard therapies. Oncolytic viruses have beenpreviously tested in the clinic, but without much therapeuticbenefit. We have engineered a safe oncolytic Semliki Forestvirus (SFV) detargeted using three central nervous system(CNS)-related microRNAs (SFV4miRT) that has potential tokill malignant gliomas and neuroblastomas in preclinicalmurine models. Because SFV4 is partly type-I IFN insensitive,the SFV4miRT virus could kill also tumors cells withmoderatesecretion of type-I IFNs. By using the newly established humanglioblastoma cell culture (HGCC) resource (www.hgcc.se), wedemonstrate that SFV4miRT could kill human glioblastomacell cultures of all molecular subtypes (proneural, classical,mesenchymal, neural), which indicates the potential for itsclinical translational.

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cells were uninfected. Supernatants were collected after 24 hours.mIFNbwasquantifiedusing LEGENDMAXMouse IFNbELISAKit(BioLegend). IFNb secretion by HGCC cells was quantified usingVeriKine Human Interferon Beta ELISA Kit (PBL Assay Science).Murine cells were stained at 24 hours postinfection using biotin-labeled anti-mouse IFNa/b receptor (IFNAR)-1 antibody (BioLe-gend) and streptavidin-conjugated Alexa Fluor 488 (ThermoFisher Scientific), whereas HGCC cells were stained with phyco-erythrin (PE)-conjugated human IFNAR1 antibody (R&D Sys-tems). Stained cells were analyzed in BD FACS Canto II (BDBiosciences).

Animal experimentsFour to five weeks old outbred NMRI female mice (Janvier

Labs) were injected intravenously or intraperitoneally with SFV4-nLuc or SFV4-nLucmiRT (1 � 107 PFU in 100 mL PBS/mouse).Mice were sedated using isoflurane (Isofluran Baxter, BaxterMedical AB); sites of virus replication were revealed bymeasuringnano-luciferase (nLuc) activity using Nano-Glo Luciferase Assaysubstrate (100 ml i.v., diluted 1:40 in 1� PBS; Promega) andNightOWL in vivo imaging system (Berthold Technologies). Somemice were sacrificed 3, 4, or 5 days post virus injection and brainsamples were either snap frozen or fixed with cold 4% parafor-maldehyde overnight. Fixed tissues were stored in 70% ethanoluntil paraffin embedment.

Four to five weeks old female, A/J mice (Taconic) wereimplanted with NXS2 cells (2 � 106 cells in 100 mL PBS) subcu-taneously in the hind flank and were treated intratumorally (5 �107 PFU in 50 mL) or intravenous (1 � 107 PFU in 100 mL)injections of either SVF4miRT or A7/74miRT on day 7 posttumorinoculation. Tumor growth was monitored by caliper measure-ments and tumor size was calculated using the ellipsoid volumeformula (length � width � depth � p/6).

Four to five weeks old female C57BL/6NRj mice (Janvier Labs)were used for the orthotopic gliomamodels. GL261-Fluc (2� 104

cells in 2 mL DPBS) or CT-2A-Fluc (5 � 104 cells in 2 mL DPBS)were injected intracranially 1mm anterior and 1.5mm right frombregma at 2.7 mm depth using a Hamilton syringe and stereo-tactic equipment (Agnthos AB). Tumor growth was measured asbioluminescence signal using D-luciferin (150 mg/kg i.p.; Perki-nElmer) and NightOwl imaging. All mice that had stable lucif-erase signal 7 or 8 days after tumor inoculation received a singledose of 4 � 107 PFU or 1 � 108 PFU of SFV4miRT virus or PBSintravenously. Mice were sacrificed upon appearance of symp-toms like paralysis, hunchback, loss of more than 10% of bodyweight, or notable distress.

ImmunohistochemistryParaffin-embedded brain tissues were sliced into 6 mm sections

and deparafinized. Antigen revival was done by heating the slidesfor 20 min at 121�C in antigen revival solution (Vector Labora-tories). Sections were blocked with goat serum (Vector Labora-tories) and stained with rabbit antibody (diluted 1:3,000 in PBS)against SFV structural proteins (kind gift formDr. Ari Hinkkanen,University of Eastern Finland, Finland). Mouse brain cell typeswere detected usingmouse anti-MAP2 (Sigma),mouse anti-GFAP(Sigma), and mouse anti-CNPase (Sigma) antibodies. IFNb wasdetected using rabbit anti-IFNb polyclonal antibody (ThermoFisher Scientific). Primary antibody staining was detected byprobing with goat-anti-rabbit-HRP or goat-anti-rabbit-AF647 ordonkey-anti-mouse-AF555 secondary antibodies (Thermo Fisher

Scientific). The sections were imaged in Zeiss AxioImager micro-scope (Zeiss).

Statistical analysisOne-wayANAOVA post hoc Tukey test formultiple comparisons

was used for statistical comparison of means between more thantwo experimental groups in an experiment. Statistical comparisonof Kaplan–Meier survival curves of mice treated with differentviruses was performed by Log-rank (Mantel–Cox) test. Associa-tion of cell viability with IFNb secretion and IFNAR1 expressionwas evaluated by fitting a linear regression model by backwardselection. Associations with P-value <0.05 was considered asstatistically significant. Results were analyzed using GraphPad-Prism 6 (Graph-Pad Software) and R statistical programmingsoftware.

Biosafety level and ethics declarationSwedish Work Environment Authority has approved the work

with genetic modification of SFV [ID number 202100-2932v66a14 (laboratory) and v67a10 (mice)]. All experiments regard-ing modified SFV were conducted under Biosafety level 2. Thelocal animal ethics committee in Stockholm (N164/15, N170/13) approved the animal studies. All human glioblastoma multi-forme samples were collected in accordance with protocolsapproved by the regional ethical review board (2007/353) andafter obtaining written consent by all of the patients.

Other related material and methods can be found in thesupplementary document.

ResultsmiRNAsmiR124,miR125, andmiR134 are expressed inhealthyCNS cells but not in glioblastoma multiforme andneuroblastoma cells

Expression ofmiR124,miR125, andmiR134were examined ina variety of cells and tissues. All healthy murine tissues from theCNS (brain, spine, and DRG) expressed miR124 and miR125whereas miR134 was expressed only in healthy mouse brain. Invitro cultured murine neural stem cells (NSC) had low expressionof all three miRNAs, differentiated neurons expressed miR124,oligodendrocytes expressedmiR134, and astrocytes had relativelylow expression of all three miRNAs (Fig. 1A, miR expressionvalues normalized to U6 snRNA; Supplementary Table S1). Mosthuman andmouse neuroblastoma and glioblastomamultiformecells had lower expression of all three miRNAs in comparison tohealthy murine neural tissues. Exceptions were the U3034HGCCcells, which expressed moderate levels of all three miRNAs, andU3024 with expression of miR134 (Fig. 1A). Non-CNS-relatedtumor cell lines HeLa (cervical cancer) and mel526 (melanoma)had low expression of all three miRNAs as expected (Fig. 1A).

miRT-detargeted SFV retains replication ability in tumor cellsbut is attenuated in in vitro differentiated murine CNS cells

To prevent neurovirulence of SFV, we inserted two copies ofsequences complementary to each of miR124, miR125, andmiR134 (hereafter referred to as target sequences, SFV4miRT orA7/74miRT) in the beginning of 30UTR of the SFV genome thatwas modified to contain duplicated subgenomic (SG) promoter(Fig. 1B). The oncolytic capacity of SFV4miRTwas compared to itswild-type counterpart (SFV4) in vitro. Neuroblastoma and gliomacells were infected with SFV4miRT or SFV4, using amount of viruscorresponding to MOIs from 0.001 to 10 PFU/cell (titrated on

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BHK-21 cells). No difference was observed between viruses withandwithoutmiRTs to kill murine neuroblastomaNXS2 (Fig. 1C),murine glioma GL261 (Fig. 1D), or CT-2A cells (Fig. 1E). CT-2Acells were more susceptible to viral oncolysis, which is probablydue to more susceptibility to virus infection whereas GL261 weremore resistant to virus killing, whichmay reflect that these cells arepoorly infected by SFV viruses. The same trendwas observedwhenA7/74 and A7/74miRT strains were tested (Supplementary Fig.S3A–S3C). Also, all four viruses efficiently killed human neuro-blastoma cell lines in a dose dependent manner (SupplementaryFig. S2A–S2D).

In vitro cultured NSC, differentiated neurons, astrocytes, andoligodendrocytes were infected with SFV4-GFP or SFV4-GFPmiRT to assess toxicity in healthy CNS cells. Virus replica-tion (GFP expression as an indirect measure of viral mRNAtranslation) for the SFV4-GFPmiRT virus was largely attenuatedin neurons, astrocytes, and oligodendrocytes but not in NSC(Fig. 1F and G). There was also a significant reduction in thepercentage of relative GFP positive BHK-21 cells for the SFV4-GFPmiRT and A7/74-GFPmiRT infected cells when transfectedexogenous with miR124, miR125, or miR134, whereas SFV4-

GFP and A7/74-GFP were not affected by the addition ofmiRNAs (Supplementary Fig. S1).

SFV4miRT has reduced replication in adult mousebrain, neurovirulence, and type-I IFN responsecompared to SFV4

Infection and replication characteristics of SFV4miRT wereassessed in vivo by infecting intravenously with 1 � 107 PFU ofSFV4 and SFV4miRT. Mice infected with SFV4 start developingvirus-related symptoms like mild paralysis at day 3 postinfectionand had to be sacrificed due to severe neurological symptoms atday 5 (Fig. 2A and C). Mice infected with SFV4miRT did notdevelop any visible virus-related symptoms (Fig. 2A and D).Infected mice were sacrificed on day 3 or 5 postinfection andvirus replication in mouse brain was quantified. SFV4-infectedmice had very high viral RNA copy numbers at day 3 (>106 viralgenome copies/g) and that increased even further by day 5 (>107

viral genome copies/g; Fig. 2B). The average viral genome copynumber in the brain for SFV4miRT-infectedmice was in the rangeof 103 viral genome copies per gram on day 3 and 104 viralgenome copies on day 5 (Fig. 2B), which is around 1,000 times

Figure 1.

miRNA screening and oncolytic potential ofmiRT-tagged SFV inmurine tumor cell lines.A,Heatmap representing expression pattern ofmiR124, miR125, andmiR134in normal mouse CNS tissues and in several murine and human neuroblastoma and GBM cell lines. Expression levels of miRNA were normalized to controlmurine or human U6 snRNA, respectively. The heat map was plotted by taking Z-scores for each miRNA across all the samples to equalize the scale. B, Schematicrepresentation of SFV vector construction. Two copies of sequences complementary tomiR124, miR125, andmiR134were inserted at the 30 UTR of themodified SFVgenome to construct miRT-tagged viruses. Reporter transgenes, when included, were inserted after a duplicated copy of the subgenomic (SG) promoter. C–E,In vitro killing ability of SFV4 and SFV4miRT onmurine neuroblastoma cells NXS2 (C), murine glioma cells GL261 (D), andmurine glioma cells CT-2A (E) at MOIs 0.001to 10. Cell viability was measured using MTS at 72 hours postinfection and values represent viability normalized to that of un-infected cells. Data are presented asmean � SD (n ¼ 2, with three internal replicates). F, In vitro cultured and differentiated primary murine CNS cells were infected with SFV4-GFP or SFV4-GFPmiRT(2000PFU). GFP-positive cellswerequantified 16hours postinfection usingflowcytometry. Data are presented as%mean�SDGFPþ cells of total (n¼ 3). Statisticalcomparison of means was assessed using two-way ANOVA, with Sidak posttest for correcting multiple comparisons (�� , P < 0.0001; �� , P < 0.001; �� , P < 0.01; n.s,P > 0.05). G, Representative images of murine neural stem cells (Nestin), neurons (MAP2), astrocytes (GFAP), and oligodendrocytes (CNPase) infectedwith either SFV4-GFP or SFV4-GFPmiRT (63� magnification, scale bar ¼ 20 mm). NSC, murine neural stem cells.

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lower compared to the neurovirulent SFV4. Mice infected withnanoluciferase-encoding virus SFV4-nLuc either intraperitoneally(Fig. 2E) or intravenously (Fig. 2F) displayed wide distribution ofvirus infection at day-1 postinfection, which remained on day-4.SFV4-nLucmiRT infectedmice had very low luminescence signalday-1 postinfection and luminescence signal was completelylost by day-4 except in the tail region of the mice, who wereintravenously-injected in the tail vein (Fig. 2E and F).SFV4miRT induced significantly lower IFNa and IFNb mRNAlevels in the brain as compared to the wild type SFV4 coun-terpart both at 3 and 5 days postinjection (Fig. 2G and H). IFN-b protein levels were also verified by IHC in SFV4 andSFV4miRT-infected mouse brain at day 5 (Fig. 2I and J). IHCanalysis also confirmed the observation made by qRT-PCRanalysis of the IFNb mRNA levels in the brain.

Brain sections fromSFV4-infected, nontumor-bearingmicehadpositive staining for SFV proteins (Fig. 3B). Mice infected withSFV4miRT did not have any strong staining patterns for viralproteins above the background staining observed in PBS-treatedmice (Fig. 2A and C). SFV-infected mouse brains were alsocostained for neuron-specific (MAP2), oligodendrocyte-specific(CNPase), and astrocyte-specific (GFAP) markers. SFV4 primarilyinfected neurons (Fig. 3E), seen as colocalized SFV (red) andMAP2 (green) staining, also infected oligodendrocytes to someextent (Fig. 3H) but clearly did not infect astrocytes (Fig. 3K). TheSFV4miRT replication was attenuated in all the three cell types;neurons (Fig. 3F), oligodendrocytes (Fig. 3I), and astrocytes (Fig.3L), and the staining pattern resembled that of brain from PBS-treated mice (Fig. 3D, G, and J). These data confirm that miRT-tagged virus replication was strongly attenuated in mouse brain.

Figure 2.

SFV4miRT has reduced neurovirulence, does not cause virus-associated neurological symptoms to adult mice, and elicits lower host IFNb response compared toSFV4.A, Survival curves of mice infected intravenously with SFV4-nLuc or SFV4-nLucmiRT at dose 1� 107 PFU (n¼ 8).B,Virus genome copy numbersmeasured byqRT-PCR in brain tissues collected from groups of mice infected intravenously with SFV4 or SFV4miRT (n¼ 4) at day 3 and 5 postinfection. C and D, Evaluation ofvirus-associated symptoms in mice infected intravenously with 1 � 107 PFU/mice SFV4 (C) or SFV4miRT (D). Score for the symptoms are as follows: 0, nosymptom; 0.1, mild limping or minor in coordination; 0.2, paralysis and/or marked in coordination; �0.3, severe paralysis or death of the mouse. E and F,Biodistribution of virus in adult mice injected with SFV4-nLuc or SFV4-nLucmiRT at dose 1 � 108 PFU either i.p. (E) or i.v. (F) at day 1 and 4 postinfection. nLucexpression was monitored following intravenous injection (100 mL) of Nano-Glo Luciferase Assay substrate and imaging mice using NightOWL in vivo imagingsystem. G and H, Relative mRNA expression of IFNa (G) and IFNb (H) compared to the HPRT1 housekeeping gene in brain tissues collected from groups of miceinfected intravenouslywith SFV4 or SFV4miRT (n¼4) at day 3 and 5 postinfection. Baseline (BSL)means relativemRNA expression of IFNa and IFNb in brain tissuescollected fromun-infectedmice. I and J, IHC analysis for IFNbprotein expression inbrain tissueofmice injected intravenouslywith SFV4 (I) or SFV4miRT (J). Sampleswere collected 5 days postinfection and stained with antimouse IFNbmonoclonal antibody (Tile scan images: 10�magnification, scale bar¼ 2mm; zoomed images:20�magnification, scale bar¼ 100 mm). Red arrows indicate positive staining for IFNb. Statistical comparison of means was assessed using one-way ANOVA, withTukey posttest for correcting multiple comparisons (�� , P < 0.0001; �� , P < 0.001; �� , P < 0. 01; n.s, P > 0.05).






SFV4miRT prolonged survival of mice bearing subcutaneousneuroblastoma tumors

Therapeutic efficacy of SFV4-miRT was evaluated in A/J miceimplanted s.c. with syngeneic murine neuroblastoma NXS2.Tumors grew vigorously in the PBS-treated group and all micewere sacrificed before day 20. Tumor growth for individualmice (Fig. 4A and C) was significantly delayed and a significantsurvival benefit (Fig. 4B and D; 50% remained tumor free) was

observed in mice treated with SFV4miRT irrespective of route ofvirus administration. None of SFV4-miRT treated animalsdeveloped encephalitis and the mice were sacrificed due totumor growth (volume > 1,000 mm3). All mice that remainedtumor free after day 60 were re-challenged with NXS2 cellsand none of them developed tumors (data not shown), indi-cating that immunological memory was formed. The miRNA-detargeted IFN-sensitive SFV strain A7/74-miRT also showed

Figure 3.

SFV4miRT has reduced replication in mouse brain. A–C, IHC analysis for SFV proteins in mouse brain tissue in mice injected intravenously with PBS (A), SFV4 (B), orSFV4miRT (C). Sampleswere collected 5 days after injection and stainedwith a polyclonal anti-SFV antibody (Tile scan images: 10�magnification, scale bar¼ 2mm;zoomed images: 20� magnification, scale bar ¼ 100 mm). D–L, Immunofluorescent analysis for SFV proteins (in red) and brain cell types (in green),neurons (D–F), oligodendrocytes (G–I), and astrocytes (J–L) in mice injected intravenously with PBS (D, G, J), SFV4 (E, H, K), or SFV4miRT (F, I, L). Sampleswere collected 5 days after injection and stained with a polyclonal anti-SFV and anti-MAP2 (neuron), anti-CNPase (oligodendrocyte), or anti-GFAP (astrocyte)antibodies (tile scan images: 20� magnification, scale bar ¼ 200 mm; zoomed images: 40� magnification, scale bar ¼ 20 mm).

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similar therapeutic efficacy on curing mice bearing NXS2tumors, regardless of administration route (SupplementaryFig. S3D–S3G).

SFV4miRT prolongs survival of mice bearing orthotopic CT-2Aglioma but not GL261 glioma

Therapeutic efficacy of SFV4miRT was evaluated in two ortho-topic murine gliomas, GL261 and CT-2A, in syngeneic C57BL/6mice. All mice inoculated with GL261 had tumor growth at day 8,the time of treatment. GL261 tumor-bearing mice treated withSFV4miRT did not have statistically significant survival benefitcompared to PBS treated group.Only onemouse in the SFV4miRTtreatment group survived (Fig. 5A) and one mouse had tumorregression but later relapsed (Fig. 5C). Around 85% of miceinoculated with CT-2A cells developed tumors at day 8 aftertumor implantation, and only those with tumors were includedand treated. CT-2A tumor-bearing mice treated with SFV4miRThad a significant survival benefit and delayed tumor growth (3/11mice survived, 27.3% survival) compared to the PBS-treatedgroup (Fig. 5B). Representative pictures of mice with lumines-cence signal and immunohistochemistry from GL261 and CT-2Atumor-bearing mice treated with SFV4miRT are shown (Fig. 5C–H). CT-2A tumor-bearingmice treated with SFV4miRT had strongstaining for SFV proteins (Fig. 5H) in the tumor, whereas theGL261 tumor bearing mice did not have any staining for SFVproteins (Fig. 5G).

Therapeutic efficacy of SFV4miRT treatment negativelycorrelates with IFNb secretion by tumor cells upon virusinfection

To analyze mechanisms leading to difference in susceptibil-ity to SFV-killing in vitro and correspondingly very differenttherapeutic efficiencies in the three in vivo models, the abilities

of NXS2, GL261, and CT-2A cell lines to mount type-I IFNresponse upon SFV4miRT infection was measured. Cells wereinfected with SFV4miRT at MOI-0.01 or 1 PFU/cell and 24hours later their ability to secrete IFNb and regulate IFNAR1expression was analyzed. Uninfected cells and cells infected atMOI-0.01 secreted undetectable amounts of IFNb. Upon infec-tion at MOI-1 GL261 cells secreted high amounts of IFNbwhereas CT-2A cells secreted moderate amounts of IFNb andNXS2 cells secreted undetectable amounts of IFNb (Fig. 6A).There was no significant change in the surface expression ofIFNAR1 upon infection of cells with SFV4miRT for both MOIsevaluated (Fig. 6B). The reduced expression of IFNAR1observed on CT-2A cells at MOI-1 is due to cell death causedby virus infection (Fig. 6B).

Previous studies have revealed that SFV4 is partly resistant toantiviral effects of type-I IFN (16). Therefore, cell-killing assayswere performed on NXS2, GL261, and CT-2A cells pretreatedwith high amounts of exogenous recombinant murine IFNb.Lower concentration of IFNb (0.01 ng/mL) was sufficient toreduce death of CT-2A cells at MOI-0.01 and 0.001 but hadvirtually no effect on survival of NXS2 cells (Fig. 6C). At a highvirus dose (MOI-10), no protective effect of exogenous IFNbwas observed for NXS2 and CT-2A (Fig. 6C). GL261 cell linewas more resistant to virus killing compared to NXS2 and CT-2A even in the absence of exogenous IFNb. However, in thepresence of exogenous IFNb (0.01 and 1 ng/mL), GL261 cellswere protected from SFV4miRT killing even at the highest virusdose (MOI-10; Fig. 6C).

SFV4miRT efficiently kills patient-derived primaryHGCC in vitro

Oncolytic effect of SFV4miRT in 11 HGCC lines either inthe presence or absence of exogenous human IFNb was also

Figure 4.

NXS2 tumor growth and survival ofmice after a single treatment withSFV4miRT. NXS2 neuroblastoma cellswere injected subcutaneously in theright hind flank of femaleA/Jmice. Micewere treated when they had palpabletumors 7 days after tumor inoculation.A, Tumor size of individual mice and B,Kaplan–Meier survival curves for micetreated intratumorally either with 50 mLPBS (n ¼ 10) or SFV4miRT (n ¼ 7, 5 �107 PFU). C, Tumor size of individualmice and D, Kaplan–Meier survivalcurves for mice treated intravenoulsywith either with 100 mL PBS (n ¼ 10) orSFV4miRT (n ¼ 8, 1 � 107 PFU). Thesurvival curves were compared withPBS-treated mice by performinga log-rank (Mantel–Cox) test(�� , P < 0.0001).






Figure 5.

Orthotopic glioma tumor growth and survival of miceafter a single intravenous treatment with SFV4miRT.GL261-Fluc or CT-2A-Fluc murine glioma cells wereinjected intracranially in femaleC57BL/6NRjmice.Micethat developed tumors (as detected by luminescence)were treated intravenously with SFV4miRT (1 � 108

PFU/in 100 mL) or PBS 7 days after tumor inoculation.Tumor growth was monitored by measuring luciferaseexpression using NightOWL imaging system.A, Kaplan–Meier survival curves for GL261 tumor-bearing mice treated with either SFV4miRT (n¼ 15) orPBS (n¼ 13).B,Kaplan–Meier survival curves for CT-2Atumor-bearing mice treated with either SFV4miRT(n ¼ 11) or PBS (n ¼ 7). The survival curves werecompared by performing a log-rank (Mantel–Cox) test(�� , P < 0.001; n.s, P > 0.05). C and D, Representativein vivo luminescence images ofmice-bearingGL261 (C)and CT-2A (D) tumors treated with SFV4miRT havingcomplete response (CR), relapse, or no response. E–H,IHC analysis for SFV proteins in tumor tissue, GL261(E, G) or CT-2A (F, H) injected intravenously with PBS(E, F), or SFV4miRT (G, H). Samples were collected3 or 4 days after injection and stained with a polyclonalanti-SFV antibody (tile scan images: 20�magnification, scale bar ¼ 200 mm; zoomed images:63� magnification, scale bar ¼10 mm).

Ramachandran et al.





evaluated. The cohort of cell lines used represented all foursubtypes of glioblastoma multiforme and detailed informationis mentioned in Supplementary Table S2. After 72 hours,most HGCC cell lines were efficiently killed in a dose-depen-dent manner by SFV4miRT (Supplementary Fig. S4). The effi-cacy of virus-mediated tumor cell killing decreased in thepresence of exogenous hIFNb in a concentration-dependentmanner (Supplementary Fig. S4). Figure 6D represents mean-relative viability across different viral doses (MOI-0.01 to 1PFU/cell) in the absence or presence of 1 ng/mL exogenoushIFNb. The presence of IFNb inhibited cell killing, withU3024 being an exception having unchanged cell viabilitywhen hIFNb was added. We believe that this is attributed tothe high miR134 expression of U3024, leading to virus genomedegradation (Fig. 1A).

IFNb secretion from the HGCC cell lines was measured beforeandafter SFV4miRT infection.Noneof theuninfectedHGCC linessecreted IFNb (Fig. 6E). Only U3024 and U3213 secreted detect-able levels of IFNb when infected by SFV4miRT at low virus dose(MOI-0.01). However, at high virus dose (MOI-1), a majority ofthe HGCC lines (U3013, U3021, U3024, U3028, U3034, U3053,U3054, and U3213) secreted varied amounts of IFNb (Fig. 6F).Most of the HGCC lines that secreted IFNb were less effectivelykilled by SFV4miRT in the absence of exogenous hIFNb (blacksquares above dotted line; Fig. 6D). Furthermore, the meanrelative viability of cells after SFV4miRT infection correlated withthe ability of cells to secrete IFNb (P ¼ 0.01164; Fig. 6G). TheHGCC line U3034 secreted very low IFNb upon virus infection,but had high cell viability (Fig. 6G, grey dot), which may bebecause U3034 had expression of all three miRNAs (Fig. 1A).Change in IFNAR1 expression was measured 24 hours afterSFV4miRT infection by flow cytometry. All HGCC cells expressedbasal levels of IFNAR1 (Fig. 6F, white bars) and had only feeblechange in receptor expression after virus infection. Mean relativecell viability after SFV4miRT infection correlated to IFNAR1expression (MFI values) after SFV4miRT infection (P ¼0.00205; Fig. 6H). Taken together, efficacy of SFV4miRT to killHGCC lines was negatively associated with the amount of IFNbsecreted by cells upon virus infection and levels of IFNAR1expression.

DiscussionMany oncolytic viruses have been tested and proven to be safe

in the clinic for glioblastoma multiforme treatment (4, 24).Despite encouraging safety results there are many factors likevirus delivery, anti-virus immune response, intratumoral spreadof infection that still need to be addressed to significantly improveefficacy, and make oncolytic virus therapies for glioblastomamultiforme a reality (25, 26). An important factor impedingdevelopment of successful therapies for glioblastomamultiformeis lack of models closely representing the characteristics of tumorin patients. We utilized the HGCC biobank to test efficacy ofoncolytic SFV therapy. HGCC lines are cultured in serum-freemedium and retain the characteristics of their original tumors ascompared to the traditionally used human glioblastoma multi-forme cell models (27, 28). They also express NESTIN and SOX2,markers that are usually associated with glioblastomamultiformestem cells. It is commonly believed that glioblastomamultiformestem cells are not efficiently killed by conventional therapies (29,30). Hence, efficacy of SFV and other potential therapies to kill

HGCC lines may aid in better evaluating its clinical translationalability.

The SFV A7/74 strain has been successfully tested safe for use asan oncolytic agent in several preclinical models for glioblastomamultiforme and other cancer types (8, 31, 32). However, type-IIFN responsive gliomas are resistant to A7/74 virotherapy inmurine models (14). The neurovirulent strain SFV4 is interestingbecause it has the ability to partly resist type-I antiviral defense(33) and this phenotype is important for treatment of IFNa/bresponsive tumors (16). To attenuate neurovirulence of the lethalSFV4 strain, we inserted target sequences complementary tomiR124, miR125, and miR134 into the SFV genome (Fig. 1B).The initial choice of these miRTs was based on their expression inCNS reported in the literature. miR124 is highly expressed in allbrain regions, predominantly in the neurons except for thepituitary gland (21, 34, 35), miR125 is expressed in mid-hind-brain boundary and neurons in mouse (21, 36) and miR134 is abrain-specific miRNA, localized in dendrites and synaptic sites(22). We also confirmed that these miRNAs are highly expressedin normal mouse CNS cells, including in vitro-differentiatedneurons, and are downregulated in several murine gliomas andneuroblastomas (Fig. 1A). Most HGCC lines also had low expres-sion of thesemiRNAs, whichmakes thesemiRNAs suitable targetsto be used to attenuate virus replication in normal CNS cellshowever, expression profiles in healthy human brain cells need tobe confirmed before clinical translation. Previously, miRT124-tagged SFV4 was shown to possess significantly reduced neuro-virulence (16, 20). But, a few mice still developed severe neuro-logical symptoms, with virus replication detected in the oligo-dendrocytes of corpus callosum and spine (16, 20). The triplemiRT de-targeted SFV4 virus (SFV4miRT) presented herein hadattenuated replication in mouse brain cells specifically neurons,and oligodendrocytes (Fig. 3). SFV4 did not infect astrocytes (Fig.3J–L), which is in accordance with previous reports (37). Inimmune competent mice SFV4 is also known not to infect anyother organ except CNS (37), therefore we believe that SFV4miRThas an improved safety profile.

Insertion of miRTs did not inhibit virus replication in tumorcells lines that did not express any of the miRNAs (Fig. 1C–Eand Supplementary Figs. S2 and S3). But in cell lines where oneor more of these miRNAs were expressed (U3024 and U3034),virus replication was attenuated when administered at lowMOIs (Supplementary Fig. S4). Nontumor-bearing brain sam-ples from SFV4miRT-infected mice had no or very little IFN-a/bsecretion compared to samples from mice infected with SFV4(Fig. 2G–J). The levels of IFNa/b secretion also correlated withvirus load in the brain, that is, more IFNa/b on day 5 postin-fection, where the titers of the SFV4 virus in the brain were alsohigher (Fig. 2B, G–J). This finding is in accordance with pre-vious reports, where miRT124 attenuated SFV4 was used (20)and confirms that IFNb secretion depends on the efficiency ofvirus replication in the brain (38).

Successful biological outcomes of type-I IFN response dependson expression of all components in the IFN signaling pathway likeIFNa/b itself, IFN receptor (IFNAR), signaling molecules JAK1,TYK2, STAT1, STAT2, and Interferon Stimulated Genes (ISG;ref. 39). In three murine tumors models tested, the neuroblasto-ma cell line NXS2 did not secrete IFNb upon virus infection, butthe two glioma cell lines CT-2A and GL261 secreted IFNb uponvirus infection, with GL261 secreting the highest amount of IFNb(Fig. 6A). In the murine tumor models, IFNb secretion levels






Figure 6.

The tumor cell killing ability of SFV4miRT negativelycorrelates to IFNb secretion by tumor cells. NXS2, GL261,and CT-2A cells were left uninfected or infected withSFV4miRT at MOIs 0.01 and 1. A, IFNb secretion wasmeasured 24 hours postinfection in cell culturesupernatant by ELISA. Data are presented as mean � SD(three internal replicates, supernatants from twoindividual experiments were pooled). B, IFNAR1expression was measured 24 hours postinfection by flowcytometry through staining of cells with a biotinylatedantimouse IFNAR1 antibody followed by a streptavidin-conjugated Alexa Fluor 488 secondary antibody. Cellsfrom two individual experiments were pooled andanalyzed by flow cytometry. C, In vitro killing ability ofSFV4miRT on NXS2, GL261, and CT-2A cells pretreatedwithmurine IFNb (1 ng/mL, 0.01 ng/mLor no IFNb) atMOIs0.01 to 10. Cell viability was measured using MTS at 72hours postinfection and values represent viabilitynormalized to un-infected cells. Data are presented asmean�SD (n¼ 2,with three internal replicates).D, Invitrokilling ability of SFV4miRT on HGCC pretreated withhuman IFNb (1 ng/mL or no IFNb). Cell viability wasmeasured using Alamar Blue reagent at 72 hourspostinfection and values represent mean cell viabilityacross MOIs 0.01 to 1 of SFV4miRT. Data are presented asmean � SD (n ¼ 2, with three internal replicates). E, IFNbsecretion was measured in cell culture supernatant byELISA 24hours post infection. Data are presented asmean� SD (two internal replicates, supernatants from twoindividual experiments were pooled). F, IFNAR1expression was measured 24 hours postinfection in a flowcytometer by staining cells with PE-conjugated antibodyagainst human IFNa/bR1. Cells from two individualexperiments were pooled and analyzed in the flowcytometer. G, Scatter plot representing mean viability ofcells and IFNb secretion after SFV4miRT infection (MOI-10) for the respective HGCC lines. H, Scatter plotrepresenting mean viability of cells at MOI-1 infection andIFNAR1 expression for the respective HGCC lines.

Ramachandran et al.





(autocrine effect) negatively correlatedwith therapeutic efficacy invivo (Figs. 4–6). NXS2-bearing mice benefitted the most (50%survival) from treatment with SFV4miRT (Fig. 4) followed by CT-2A tumor-bearing mice, then GL261 tumor-bearing mice (Fig. 5Aand B). It should be noted that NXS2 model used was subcuta-neous, whereas orthotopic brainmodels were used forGL261 andCT-2A, and therapeutic efficiencies can differ because of thelocation of the tumor. GL261-bearing mice treated with theIFNb-sensitive strain A7/74miRTdid not have any survival benefit(data not shown). This is in accordancewith previously publisheddata showing that even low amount of IFNb is enough to inhibitreplication of A7/74 strain in both GL261 and CT-2Amodels (14,16). SFV4miRT efficiently killed most of the HGCC lines in vitroand efficacy depended on the amount of IFNb secreted by the cellsupon virus infection (autocrine effect; Fig. 6G), which is accor-dance with the murine cell line data.

In the presence of exogenous IFNb, SFV4miRT killed NXS2and CT-2A cells in a dose-dependent manner in vitro (Fig. 6C).However, GL261 were resistant to SFV4miRT oncolysis in thepresence of exogenous IFNb, even at the highest virus dosetested (Fig. 6C). As previously observed, in clones of mousecolon carcinoma, CT26WT and CT26LacZ differential expres-sion of ISGs can contribute to dramatic difference in the efficacyof SFV therapy (40). ISG expression in CT-2A and GL261 isunknown, which may be a reason for the observed difference inoncolytic response. In HGCC lines, addition of exogenous IFNbdecreased SFV4miRT oncolysis, indicating that all HGCC linesare responsive to IFNb (Fig. 6D). However, at high SFV4miRTdose, IFNb did not inhibit viral oncolysis, confirming theability of SFV4 to partly resist type-I antiviral defense (Supple-mentary Fig. S3), as previously reported (33).

The ability of tumors to initiate antiviral defense is an impor-tant factor that determines the therapeutic efficacy of SFV4miRT.This emphasizes the need to test oncolytic potential of SFV inimmune competent models as compared to xenografts whereexcellent efficacy is observed (41). It may still be possible to usethe avirulent, type-I IFN-sensitive A7/74 strain for treatment ofnon-IFNb responsive tumors like NXS2 but our data stresses theneed for an IFN-insensitive SFV strain for successful treatment ofIFN-responsive tumors. Recent studies suggest that viral-vaccinescombined with checkpoint inhibition (aPD-1 or aCTLA4 anti-bodies) is beneficial for treatment of aggressive tumors likeGL261(42). Oncolytic viruses are naturally immunogenic; combinationof SFV4miRT with checkpoint inhibition for treatment of glio-

blastoma multiforme would be worth to investigate. In conclu-sion, our results show that, insertion of multiple miRTs reducesSFV4 neurovirulence and yields an improved safety profile ofSFV4 as an antitumor agent.

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

Authors' ContributionsConception and design: M. Ramachandran, D. Yu, M. Dyczynski, A. Lulla,J. Leja-Jarblad, M. EssandDevelopment of methodology: M. Ramachandran, D. Yu, M. Dyczynski,L. Zhang, A. Lulla, V. Lulla, S. Saul, A. MeritsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M. Ramachandran, D. Yu, M. Dyczynski, S. Baskaran,V. Lulla, S. Saul, A. Dimberg, J. Leja-JarbladAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M. Ramachandran, D. Yu,M. Dyczynski, S. Baskaran,S. Nelander, A. Merits, J. Leja-Jarblad, M. EssandWriting, review, and/or revision of the manuscript: M. Ramachandran,D. Yu, M. Dyczynski, S. Baskaran, S. Saul, S. Nelander, A. Dimberg, A. Merits,J. Leja-Jarblad, M. EssandAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Ramachandran, D. Yu, M. DyczynskiStudy supervision: D. Yu, S. Nelander, J. Leja-Jarblad, M. Essand

AcknowledgmentsThe authors wish to thank Patrik Johansson (UppsalaUniversity) for tips and

guidancewith statistical analysis, BerithNilsson, Grammatiki Fotaki, Chuan Jin,and Tina Sar�en (Uppsala University) for assistance in the lab, Dr. MiikaMartikainen for fruitful discussions and reviewing the manuscript. We alsothank Prof. Ari Hinkkanen (University of Eastern Finland) for providing usa-SFV antibodies and Dr. Markus V€ah€a-Koskela (Ottawa Hospital ResearchInstitute) for providing the CT-2A cell lines.

Grant SupportThe Swedish Children Cancer Foundation (PROJ12/082), the Swedish

Cancer Society (CAN2013/373), and the Swedish Research Council (K2013-22191-01-3) supported this work (to M. Essand). Estonian Research Council(Eesti Teadusagentuur) and grant number 20-27 (to A. Merit). The funders hadno role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 11, 2016; revised August 10, 2016; accepted August 31, 2016;published OnlineFirst September 16, 2016.

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Ramachandran et al.




Published OnlineFirst September 16, 2016.Clin Cancer Res Mohanraj Ramachandran, Di Yu, Matheus Dyczynski, et al. MicroRNA-Detargeted Oncolytic Semliki Forest Virusand Glioblastoma Using Systemically Delivered Triple Safe and Effective Treatment of Experimental Neuroblastoma

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