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Therapeutics, Targets, and Chemical Biology

Coxsackievirus B3 Is an Oncolytic Virus withImmunostimulatory Properties That Is Active against LungAdenocarcinoma

ShoheiMiyamoto1, Hiroyuki Inoue1,2, Takafumi Nakamura3, Meiko Yamada4, Chika Sakamoto1, YasuoUrata4,Toshihiko Okazaki1, Tomotoshi Marumoto1, Atsushi Takahashi1, Koichi Takayama2, Yoichi Nakanishi2,Hiroyuki Shimizu5, and Kenzaburo Tani1

AbstractAlthough oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low

tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-stepscreening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activityagainst nine human non–small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positivelycorrelated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoi-nositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK)survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injectionsof CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrationsof CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenograftson the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were welltolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundantcell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, whichare required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruitednatural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletionassays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 isa potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localizedand metastatic NSCLC. Cancer Res; 72(10); 2609–21. �2012 AACR.

IntroductionOncolytic viruses are self-replicating, tumor-selective

viruses, with an ability to directly induce cancer cell death,and have emerged as a promising treatment platform forcancer therapy (1).Over the past 2 decades, clinical trials of oncolytic virothera-

pies using a range of DNA and RNA viruses including coxsack-

ievirus A21 (CVA21), measles virus, Newcastle disease virus,and reovirus have been reported or are underway (2–5).Clinical development of these therapies has progressed to latephase trials. However, the antitumor efficacy of oncolyticviruses has yet to attain the potential anticipated in preclinicalstudies.

RNA viruses seem to be a safer modality, as most single-stranded RNA viruses replicate in the host cytosol without aDNA phase. Therefore, they lack the genotoxicity caused byintegration of the viral genome into the host DNA. Inparticular, enteroviruses, members of the Picornaviridaefamily, a diverse group of small RNA viruses have emergedas promising candidates for cancer treatment (6–9). Theiruse has several therapeutic advantages: these viruses imme-diately induce robust cytolytic changes during cell-to-cellinfection, they do not possess oncogenes that may leadto tumorigenesis, and they can be easily genetically manip-ulated by reverse genetics systems for the rescue of positive-strand RNA viruses from complementary DNA (10, 11).Furthermore, most nonpolio enteroviruses are commonand highly prevalent and are mainly associated with asymp-tomatic infection or mild disease (12). Although CVA21is reportedly a potent oncolytic enterovirus against

Authors' Affiliations: 1Division ofMolecular andClinical Genetics,MedicalInstitute of Bioregulation; 2Research Institute for Diseases of the Chest,Graduate School of Medical Sciences, Kyushu University, Fukuoka; 3CoreFacility for Therapeutic Vectors, The Institute of Medical Science, TheUniversity of Tokyo; 4OncolysBioPharma Inc.; and 5Department of VirologyII, National Institute of Infectious Diseases, Tokyo, Japan

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

S. Miyamoto and H. Inoue contributed equally to this work.

CorrespondingAuthor:KenzaburoTani, Division ofMolecular andClinicalGenetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6449; Fax: 81-92-642-6444; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3185

�2012 American Association for Cancer Research.

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miscellaneous human cancer cells (8, 9, 13), CVA21-treatedmice died of lethal myositis with paralysis (14).

Apart from direct oncolysis, another important compo-nent of the sustained therapeutic advantage of oncolyticviruses depends on their ability to trigger antitumor immuneresponses (15). Robust viral replication in tumors providesimmunologic damage-associated molecular pattern (DAMP)signals, augmenting the immunogenicity of the tumormicroenvironment (16). Previous studies showed that sev-eral oncolytic viruses could induce adaptive antitumorimmunity by tumor-specific CTL responses (17). However,little is known about how preceding innate immunity shapesthe antitumor effects and whether oncolytic viruses causeimmunogenic cancer cell death by similar mechanisms tochemotherapeutic agents, such as calreticulin (CRT) expo-sure, ATP release, or high-mobility group box 1 (HMGB1)translocation (18).

The focus of this study was to identify a novel oncolytic virusfrom screening 28 different strains of human enteroviruses,which had high tolerability when administered in mousexenograft models, and to clarify the viral cytocidal mechanismby scrutinizing the apoptotic and antitumor immunogenicpotential of the virus.

Materials and MethodsMice

Female BALB/c nude mice were purchased from CharlesRiver Japan. All animal experiments were carried out under theGuidelines for Animal Experiments of Kyushu University andLaw 105 Notification 6 of the Japanese Government.

Cell linesThe non–small cell lung cancer (NSCLC; A549, H1299, and

H460), colon cancer (Caco-2), pancreatic cancer (AsPC-1),renal cancer (A-498 and Caki-1), rhabdomyosarcoma (RD),T-cell leukemia (HuT 102), bone marrow stromal (HS-5), andnormal lung fibroblast (MRC-5) cell lines were purchased fromthe American Type Culture Collection. NSCLC cell lines (PC-9,EBC-1, and LK-2) were obtained from the Health ScienceResearch Resources Bank in Japan. Human lung squamouscell carcinoma cell lines (QG-56 and QG-95) were provided byDr Y. Ichinose (National Kyushu Cancer Center; ref. 19). Theother NSCLC cell lines (LK-87 and Sq-1) were obtained fromCell Resource Center for Biomedical Research, Tohoku Uni-versity. Human cell lines of colon cancer (DLD-1), pancreaticcancer (PANC-1), breast cancer (MCF7), cervical cancer (HeLaS3), and normal lung fibroblast (NHLF) were provided by Dr T.Fujiwara (Okayama University). No authentication besidesPCR tests that showed free from Mycoplasma contaminationwas done for all of cell lines used.

Production of enterovirusesThe 28 strains of enteroviruses tested were obtained fromH.

Shimizu (National Institute of Infectious Diseases, Japan) andwere propagated in HeLa and RD cells. The TCID50/mL onthese cell monolayers was determined as previously described(20).

Crystal violet stainingViable cells at 72-hour postinfection with enteroviruses at

appropriate multiplicity of infection (MOI) for 1 hour wereassessed by crystal violet staining as previously described (21).Cell survival rates were calculated by Multi Gauge softwareversion 3.2 (FUJIFILM).

Flow cytometry analysisCells were incubated with a monoclonal antibody against

coxsackievirus and adenovirus receptor (CAR; Upstate) fol-lowed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G, or phycoerythrin(PE)-conjugated antibody decay-accelerating factor (DAF; BDBiosciences). For analysis of surface CRT, NSCLC cells infectedwith Coxsackievirus B3 (CVB3;MOI¼ 10) was analyzed by flowcytometric analysis (22). Data were obtained with a FACS-Calibur (BD Biosciences) and analyzed by FlowJo softwareVersion 7.6 (Tree Star Inc.).

MTS assay for cell viabilityIn vitro cell viability experiments using cells infected with

CVB3 were carried out by a Cell Titer 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega) following themanufacturer's instructions.

In vitro inhibition assaysCells were pretreated with serum-free media containing the

specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002(Santa Cruz Biotechnology), the PTEN inhibitor bisperoxova-nadium (bpV, HOpic; Merck), or themitogen-activated protein(MAP)/extracellular signal-regulated kinase (ERK;MEK) inhib-itor PD0325901 (Wako) for 1 hour, and then infectedwith CVB3(MOI¼ 10) for 1 hour. For apoptosis inhibition assay, A549 cellswere pretreated with the pan-caspase inhibitor, z-VAD-fmk(100 or 200 mmol/L for 1 hour; R&D Systems), exposed to CVB3at MOI of 0.01 or 0.1 for 1 hour, and replaced in indicatedconcentrations of z-VAD-fmk for additional 48 hours.

siRNA transfection assaysiRNAs specific for human CAR and a negative control were

designed and synthesized by BONAC in Japan. Transfection ofA549 cells with siRNAs was carried out with Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol.Following 72-hour incubation after transfection, cells wereinfected with CVB3 at MOI of 0.01 for MTS assays.

Western blot analysisCell lysate samples were resolved by SDS-PAGE and immu-

noblotted (21). The primary antibodies used were monoclonalantibodies against PARP (Biovision), phospho-Akt (Ser473;BioLegend), and b-actin (Santa Cruz Biotechnology). Densi-tometry analysis was conducted with LAS3000 (FUJIFILM) andMulti Gauge software version 3.2.

Annexin V staining and cell-cycle analysisApoptotic cells after CVB3 infection were determined with

the Annexin V–PE apoptosis detection kit (BD Biosciences)according to the manufacturer's instructions. For cell-cycle

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analysis, the cells were fixed in 70% ethanol, incubated withRNase A (50 mg/mL), stained with 10 mg/mL propidium iodide(PI), and analyzed with a FACS-Calibur.

ATP release assayAfter cell death induction, secreted extracellular ATP was

measured with the luciferin-based ENLITEN ATP Assay (Pro-mega) according to the manufacturer's protocol.

Immunofluorescence microscopyCells fixed with 4% paraformaldehyde were permeabilized

with 0.3% Triton X-100 for 10 minutes, incubated with anti-HMGB1 antibody for 30 minutes followed by incubation withAlexa Fluor 488–conjugated secondary antibody (MolecularProbes), and analyzed by a fluorescence microscope BZ-9000(KEYENCE).

In vivo therapeutic studiesA549 cells (5 � 106 cells) or EBC-1 cells (3 � 106 cells) were

injected subcutaneously into the right or bilateral flanks ofnude mice. When tumors reached diameters of 0.4 to 0.6 cm,the tumors on the right flank were inoculated with CVB3 (5�106 TCID50) once on day 2 or with the same doses of CVB3 ondays 2, 4, 6, 8, and 10.The RB6-8C5 rat hybridoma cell line producing the anti-

mouse Gr-1 monoclonal antibody was provided by Dr T.Yokomizo (Kyushu University). For granulocyte depletion,nude mice implanted with 5 � 106 A549 cells were intraper-itoneally injected with 300 mg rat anti–Gr-1 antibody (99.7%elimination of circulating polymorphonuclear neutrophils).For natural killer (NK) cell depletion, nude mice were intra-peritoneally injected with rabbit polyclonal anti-asialo GM1antibody (Wako; ref. 23). The tumor volume was calculatedas length � width � width/2 and expressed as means � SEM.Animals were euthanized when the tumor diameter exceeded1.0 cm.

Tumor-infiltrating lymphocytesThe excised tumors were gently homogenized with sharp

syringes, incubated for 90 minutes in RPMI-1640 containingcollagenase (Invitrogen). For innate immunity subpopulations,the cellswere stainedwith anti-mouseDX-5-FITC, Gr-1-PE, F4/80-APC, or CD11c-APC antibodies for 30 minutes. For maturedendritic cells (DC), the cells were stained with anti-mouseCD86-FITC, CD80-PE, CCR7-PerCP, and CD11c-APC antibodyfor 30 minutes. For cytolytic effector cells, the cells werestained with anti-mouse DX-5-FITC, Gr-1-PerCP, andCD107a-PE antibody for 30 minutes (24) and analyzed by aFACS Calibur.

Statistical analysesStatistical analyses were conducted with GraphPad Prism

5.0d software package (GraphPad Software Inc.). Statisticalanalysiswas conducted using a 2-tailed unpaired Student t test,one-way ANOVA followed by Tukey multiple comparison test,or nonparametric Mann–Whitney U test. P < 0.05 was consid-ered statistically significant. Survival curves were plottedaccording to the Kaplan–Meier method (log-rank test).

ResultsSequential two-step screening identifies CVB3 as acandidate for oncolytic virus against NSCLC

To identify a safer andmore potent oncolytic enterovirus, wecarried out a large-scale screening of 28 strains of enterovirusesfor oncolytic activity. This screening was carried out in vitroagainst 12 different human cancer cell lines and the HS-5normal bone marrow stromal cell line using crystal violetstaining. Several enteroviruses displayed marked cytotoxiceffects (Fig. 1A) in a dose-dependent manner (SupplementaryFig. S1A and S1B). The Coxsackievirus B group, CVB2 (Ohio-1),CVB3 (Nancy), and CVB4 ( J.V.B.) were of particular interest,as they showed exclusive oncolytic effects on the A549 andLK-87 NSCLC cell lines (Fig. 1A). In a secondary screen, weevaluated the oncolytic efficacy of CVB2, CVB3, and CVB4against 9 additional NSCLC cell lines and 2 normal lung celllines. Unexpectedly, only CVB3 infection induced markedcytolysis in 100% (9 of 9) NSCLC cell lines of diverse histologicsubtypes, that is, adenocarcinoma (A549), squamous cell car-cinoma (EBC-1), and large cell carcinoma (H460) even at anMOI of 0.001. However, CVB3 did not induce cytolysis againstnormal lung fibroblast cell lines even at an MOI as high as 10(Fig. 1B). Accordingly, we employed CVB3 harboring potentand specific cytolytic activity as a candidate virus for oncolyticvirotherapy against NSCLC.

Correlation of CVB3-mediated cytotoxicity andexpression levels of surface receptors on NSCLC Cells

We next compared the expression level of the CVB3receptors, CAR and DAF (25) on various human NSCLC andnormal lung cell lines. NSCLC cell lines expressed moderateto high levels of CAR, whereas normal lung cell linesexpressed very low levels of CAR. The expression of DAFwas similarly high in the NSCLC and normal lung cell lines(Fig. 2A). The oncolytic activity of CVB3 significantly cor-related with a number that was equal to the percentage ofCAR-expressing cells multiplied by the percentage of DAF-expressing cells (r ¼ �0.92, P < 0.0001), whereas 2 normallung cell lines were unaffected (Fig. 2B). The results of the invitroMTS cell viability assays confirmed this correlation in atime-dependent manner (Fig. 2C). To thereby elucidate therole of CAR in the cytolytic effects of CVB3, CVB3-infectedA549 cells were analyzed in the presence of siRNA-mediatedfunctional CAR knockdown. Flow cytometric analyses con-firmed the reduction of CAR expression by 92% in A549 cellstransfected with CAR-specific siRNA (Supplementary Fig.S2). The inhibition of CAR significantly abrogated the cyto-toxicity of CVB3 (P < 0.001; Fig. 2D).

Contribution of caspase-mediated apoptosis and PI3K/Akt or MEK/ERK signaling pathways to CVB3-mediatedcytotoxicity in NSCLC cells

To determine whether CVB3 induced apoptosis in NSCLCcells treated with CVB3 (MOI¼ 0.1), we examined the cleavageof PARP by caspases, an execution marker of apoptosis.Western blot analysis revealed cleaved PARP (85 kDa) inCVB3-treated A549 and LK-2 NSCLC cells, but not in CVB3-

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treated MRC-5 normal lung cells (Fig. 3A). We analyzed exter-nalization of phosphatidylserine and DNA fragmentation, hall-marks of apoptosis. CVB3-treated A549 cells showed a greaterproportion of apoptotic (AnnexinVþ/7-AAD�) and necrotic(AnnexinVþ/7-AADþ) cells than LK-2 cells, correlating with

CAR expression (Fig. 3B). Similarly, exposure of A549 cells toCVB3 inducedmore sub-G1 cells with DNA fragmentation thanLK-2 cells, correlating with CAR expression. In contrast, nei-ther Annexin V positivity nor DNA fragmentation was inducedin CVB3-treated MRC-5 cells (Fig. 3C). To determine whether

Figure 1. Sequential 2-stepscreenings to identify a potentoncolytic virus candidate. A,various cancer cell lines and HS-5cells were infected with 28 strainsof enterovirus at an MOI of 1.0 andincubated for 1 hour. At 72 hourspostinfection, cell viability wasassessed by crystal violet staining.Black, gray, white, and hashedboxes indicate greater than 50%cytotoxicity, less than 50%cytotoxicity, no cytotoxicity, andnot analyzed, respectively.HEV-A, human enterovirus A;HEV-B, human enterovirus B;HEV-C, human enterovirus C.B, various human NSCLC andnormal lung cell lines were infectedwith CVB2, CVB3, and CVB4 for 72hours at MOIs of 0.001, 0.01, and0.1 for NSCLC cell lines and MOIsof 0.1, 1.0, and 10 for normal celllines, respectively. Cell viabilitywas assessed by crystal violetstaining. AC, adenocarcinoma;SCC, squamous cell carcinoma;LCC, large cell carcinoma.

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Figure 2. Expression profile of surface CAR and DAF on normal lung and NSCLC cells and its correlation with CVB3-mediated cytotoxicity. A, surfaceexpression of CVB3 receptors on various lung cell lines was quantified by flow cytometry. Histograms represent the measured fluorescence of cellsincubated with an isotype control antibody (unshaded) and anti-CAR or anti-DAF antibody (shaded). B, correlation between expression of CAR and DAFandcell viability at 72 hours afterCVB3 infection (r¼�0.92;P<0.0001). C, cells infectedwithCVB3at indicatedMOIswas analyzed at indicated timepoints forcell viability by MTS assay. Each value was normalized to Opti-MEM–treated cells (mock) and represents the mean � SD. D, A549 cells transfected withCAR-specific siRNA or control siRNA were infected with CVB3 (MOI ¼ 10), and assessed by MTS cell viability assay at 48 hours after CVB3 infection.Each value was normalized to mock and represents the mean � SD. ��, P < 0.001.






Figure 3. Correlation between caspase-dependent apoptosis and CVB3-mediated cytotoxicity in NSCLC cells. Lung cell lines infected with CVB3 (MOI¼ 0.1)were analyzed 24 hours later. A, each cellular lysate obtained was subjected to immunoblot analysis. Full-length PARP (116 kDa) and cleaved PARP(85 kDa) are shown. B, early apoptotic population was represented as Annexin V–PEþ/7-AAD� cells. C, each histogram PI depicts the population of cells inG1 and G2–M, and the apoptotic subdiploid peak (sub-G1). D, cells pretreated with dimethyl sulfoxide (DMSO) or z-VAD-fmk and incubated with mock orCVB3 at indicated MOIs were subjected to MTS cell viability assays. �, P < 0.05.

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CVB3-inducible apoptosis contributed to CVB3-mediatedcytotoxicity against A549 cells, we treated them with thepan-caspase inhibitor, z-VAD-fmk. CVB3-mediated cytotoxic-ity was slightly but significantly reduced when z-VAD-fmk wasadded in a dose-dependent manner (Fig. 3D).Because PI3K/Akt signaling pathways mediate CVB3 repli-

cation in human cancer cells (26), we investigated whetherCVB3 infection could activate Akt phosphorylation (p-Akt) inA549 cells. CVB3 infection induced substantial p-Akt expres-sion on the Ser-473 residue, peaking at 15 minutes postinfec-tion (Fig. 4A and B), prompting us to examine the effects ofpretreatment with LY294002, a specific PI3K inhibitor, or bpV,an inhibitor of PTEN that antagonizes PI3K activity, on p-Aktexpression levels in CVB3-treated A549 cells. LY294002

reduced but bpV enhanced the level of p-Akt induced by CVB3infection (Fig. 4C and D). In addition, LY294002 significantlydiminished but bpV significantly augmented CVB3-mediatedcytotoxicity against A549 cells at 14 hours after infection in adose-dependent manner, whereas no cytotoxicity wasobserved when either of these agents were used in the absenceof CVB3 (Fig. 4E and F). To unravel the relationship between p-Akt expression and CVB3 replication, we examined the effectsof LY294002 or bpV on release of CVB3 progeny from CVB3-treated A549 cells. The CVB3 titer was significantly reducedwhen LY294002 was added, whereas it was increased almost 2-foldwhenbpVwas added (P< 0.05; Fig. 4G), showing that PI3K/Akt pathway was involved in CVB3 replication. Furthermore,addition of a potent MEK inhibitor PD0325901, which blocks

Figure 4. Correlation between 2survival signaling pathways andCVB3-mediated cytotoxicity andviral replication in humanNSCLC celllines. A549 cells infected with CVB3(MOI ¼ 0.1) were analyzed. A and B,each cell lysate collected at indicatedtime points was subjected toWestern blotting assay for p-Akt onSer-473 detection. Resultsnormalized to mock were evaluatedby densitometric analysis usingMultiGauge. C and D, p-Akt expression inA549 cells treated with LY294002 orbpV followed by CVB3 infection wasdetermined by Western blottinganalysis. E, A549 cells were treatedwith 50 mmol/L LY294002, 1 mmol/LbpV, or DMSO for 1 hour, infectedwith CVB3 or Opti-MEM for 1 hour,incubated for 7 or 14 hours, andsubjected to MTS cell viabilityassays. �, P < 0.05. F, A549 cellstreated as above were incubated for14 hours at various concentrations ofLY294002 or bpV and subjected toMTS cell viability assays. �, P < 0.05.G, supernatants from the LY294002-or bpV-treated A549 cells after 14hours of CVB3 infection werecollected and viral titer wasdetermined. �,P < 0.05. H, A549 cellswere treated with variousconcentrations of MEK inhibitorPD0325901 or DMSO for 1 hour,followed by CVB3 infection,incubated for 14 hours, andsubjected to MTS cell viability assay.�, P < 0.05.






phosphorylation of ERK, to CVB3 infection significantlydecreased cytotoxicity against A549 cells in a dose-dependentmanner, whereas PD0325901 alone did not have any effect(Fig. 4H).

Efficacy studies in nude miceWe next evaluated in vivo oncolytic effects of CVB3 and its

tolerability in nude mice bearing subcutaneous NSCLC xeno-grafts. A single dose of intratumoral CVB3 administered intoA549 xenografts significantly suppressed tumor growth (P <0.01; Fig. 5A). In addition, the survival rate was significantlyimproved in CVB3-treated mice compared with untreatedmice (P ¼ 0.0008; Fig. 5B). To evaluate tolerability and dose-dependent oncolytic effects, 5 consecutive doses of CVB3 wereintratumorally administered into A549 xenografts. The treat-mentwithCVB3 elicited significant tumor regression in a dose-dependent manner (P < 0.05; Fig. 5C), with significantly pro-longed survival (P ¼ 0.0004). Moreover, half of the CVB3-treated mice achieved complete regression of the tumor (Fig.5D). Similarly, in the EBC-1 human squamous cell carcinomaxenograft model, intratumoral 5 administrations of CVB3revealed significant antitumor effects. All CVB3-treated miceachieved complete tumor elimination with a significantlyprolonged survival rate (Fig. 5E and F). For H1299 humanadenocarcinoma xenograft, similar potent antitumor effectswith complete tumor rejection were observed in all of CVB3-treated mice (Supplementary Fig. S3). Notably, none of themice died of side effects during these treatments.

To further investigate systemic oncolytic effects of CVB3, weused a mouse model with bilaterally preestablished subcutane-ous A549 xenografts. Five consecutive CVB3 administrationsinto the right flank tumors only significantly suppressed thegrowthofboth theCVB3-injected tumorandthedistantuntreat-ed tumorwhencomparedwithuntreatedmice (P< 0.05; Fig. 5G),anddisplayed a significantlyprolonged survival rate (P¼ 0.0021)and no lethality (Fig. 5H). To clarify the mechanism by whichCVB3 exerted antitumor effects against the contralateral tumor,we investigated whether the supernatants derived from dis-rupted untreated tumors or from CVB3-treated tumors hadoncolytic activity against A549 cells. The supernatants derivedfrom both CVB3-treated and CVB3-untreated tumors destroyedA549 cells (Fig. 5I), showing that replication-competent CVB3did exist in untreated contralateral tumors.

Immunologic assaysAs preapoptotic exposure to CRT and ATP release and

postapoptotic HMGB1 are required for immunogenic celldeath induced by chemotherapeutic agents such as anthracy-clines and oxaliplatins (18), we examined whether CVB3infection promoted similar effects on NSCLC cells. CVB3treatment induced abundant surface exposure of CRT (Fig.6A) on both A549 and H1299 cells, and active secretion ofextracellular ATP was in dose- and time-dependent manner(Fig. 6B). In addition, CVB3 infection resulted in substantialrelease of HMGB1, another determinant of immunogenicity,from the nuclei of both A549 and H1299 cells into the cytosol(Fig. 6C).

We next evaluated the effects of intratumoral CVB3 admin-istration on tumor-infiltrating immune cells in A549-bearingmice. CVB3 administration recruited significantly greateraccumulation of NK cells, granulocytes, macrophages, andDCs into the tumor bed compared with the controls (P <0.01; Fig. 7A and B). The tumor-infiltrating DCs expressedsignificantly higher levels of the costimulatory moleculesCD80, CD86, and thematurationmarker CCR7 inCVB3-treatedmice compared with untreated mice (P < 0.05; Fig. 7C). It hasbeen reported that mobilized CD107a expression, a cytolyticdegranulation marker, correlates with NK cell–mediated lyticpotential (27), and that granulocytes contribute to the antitu-mor effect of the oncolytic measles virus (28). We thereforeexplored the effects of CVB3 treatment on tumor-infiltratingCD107a-harbouring NK cells and granulocytes. A higher den-sity of NK cells as well as granulocytes mobilized CD107a totheir cell surface in CVB3-treatedmice comparedwith untreat-ed mice (Fig. 7D). We further investigated the role of endog-enous NK cells or granulocytes on the CVB3-mediated anti-tumor effect by depleting tumor-bearing mice of NK cells orgranulocytes. Depletion of either NK cells or granulocytessignificantly abrogated the therapeutic effect of CVB3 (P <0.05; Fig. 7E), illustrating their substantial contribution toCVB3-induced antitumor responses.

DiscussionLung cancer is the world's leading cause of cancer death,

resulting in more than 1 million deaths per year worldwide(29). However, standard conventional therapies have pro-duced limited cures, highlighting the need to develop noveltherapeutic modalities (30). Here, we successfully identifiedCVB3 as a useful virotherapeutic agent against NSCLC vialarge-scale screenings with 28 strains of human entero-viruses (Fig. 1).

Discriminatory mechanisms for selectively targeting cancercells are a prerequisite for therapeutic oncolytic viruses. CVB3possessed cancer-specific viral tropism, as evidenced by anextremely low infection of MOI ¼ 0.001 (Fig. 1B). In addition,CVB3-induced cytotoxicity positively correlated with theexpression levels of DAF and particularly CAR on NSCLC cells(Fig. 2). CAR is expressed on various histologic types of humanlung cancers, but not on normal alveolar epithelial cells (31).DAF counteracts the cytolytic action of the complementsystem and whose expression is upregulated on the surfaceof many tumors, seems to be a good target for oncolytic virus(32). These findings form a basis for the use of CVB3 intherapeutic applications for NSCLC patients.

Our results showed a moderate contribution of caspase-mediated apoptosis to CVB3-mediated oncolysis in humanNSCLC cells (Fig. 3D). Generally, a wide range of cancers areknown to be resistant to apoptosis following chemotherapy(33). Thus, the ability of CVB3 to induce apoptosis is anattractive property for the treatments of refractory NSCLC(34, 35). A role of PI3K/Akt signaling in accelerating CVB3replication in NSCLC cells was also shown by use of cotreat-mentwith a PTEN inhibitor andCVB3 infection (Fig. 4F andG).Overexpression of p-Akt and loss of PTEN expression inNSCLC

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patients was reported to confer poor differentiation, distantmetastasis, and poor prognosis (36). In addition, PI3K/Aktpathway–related genes are frequently activated in diversehuman cancers including NSCLC tumors (37), and cancer stem

cells after systemic chemotherapy (38, 39). These findingssuggest that CVB3 treatment may be suitable for NSCLCpatients refractory to conventional chemotherapies. In con-trast, our finding that PI3K inhibition reduced CVB3

Figure 5. In vivo oncolytic effectof intratumoral CVB3 administrationon various human NSCLCxenografts. A549 (n¼ 5 or 6; A andC)or EBC-1 (n ¼ 5; E) weresubcutaneously injected into theright flanks of nude mice. Eachmouse received either a single dose(A), or 5 doses (C and E)of CVB3 intratumorally. Tumorvolumes are expressed as means �SEM. �,P<0.05; ��,P<0.01. Kaplan–Meier survival analyses are shown forCVB3-treated mice with the singledose (B; P ¼ 0.0008) or 5 doses(D; A549, P ¼ 0.0004; F; EBC-1,P ¼ 0.0035) of the CVB3. G,administration of CVB3 to the righttumor was carried out to the nudemice with bilateral tumors (n ¼ 5).�, †, P < 0.05; ��, ††, P < 0.01. Eachsymbol represents the statisticalsignificance of right (�) and left (†)lateral tumors between untreatedand CVB3-treated mice. H, Kaplan–Meier survival analysesof the mice in G are shown(P ¼ 0.0021). I, two days after theCVB3 administration, the culturesupernatants obtained from bilateraltumors were evaluated for oncolyticactivity against cultured A549 cells.






production from NSCLC cells raises the possibility that the useof PI3K inhibitors might regulate undesired CVB3 replication.The activity of ERK, the downstream target of MEK in theMEK/ERK survival pathway, was also closely correlated withCVB3-mediated cytotoxicity. Enhanced activation of MEK/ERK has been detected in 34% of 111 primary NSCLC patientswith lower survival, but not in normal lung tissue (40, 41),providing further insight into the interplay of CVB3 replicationwith cellular components to improve antitumor efficacies.

It is noteworthy that intratumoral CVB3 administrationsshowed significant antitumor effects against A549 adenocar-cinoma xenografts resistant to radiotherapy (42) and the EGFRtyrosine kinase inhibitor, gefitinib (43), EBC-1 squamous cellcarcinoma (Fig. 5E and F), and H1299 adenocarcinoma xeno-grafts (Supplementary Fig. S3), indicating that CVB3 could be a

favorable therapeutic agent for patients with advanced NSCLCrefractory to conventional radiotherapies and/or moleculartargeted therapies.

Intratumoral CVB3 administration into one of 2 bilateralsubcutaneous xenografts elicited significant suppression ofgrowth of the distant uninjected tumors in which oncolyticCVB3 progeny could be detected. This implies that the admin-istered CVB3 replicated, and its progeny circulated via theblood or lymphatic system and infected distant tumors, whichis a beneficial for the systemic treatment of metastatic ordisseminated tumors.

For toxicity evaluation, we conducted biochemical andhistologic assays duringCVB3 treatment. CVB3 administrationinduced moderate hepatic dysfunction (SupplementaryFig. S4A), with no histologic evidence of active hepatitis

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CVB3

H1299

Figure 6. Induced immunogenicityin CVB3-infected NSCLC cells. A,CRT expression on the surface ofA549 and H1299 cells wasanalyzed at 6 hours after CVB3infection (MOI ¼ 10) by flowcytometric analysis. B, cells wereinfected with CVB3 at MOI of 1.0 or10, incubated for 6 or 9 hours, andthe concentration of extracellularATP in the culture medium wasmeasured. Results are shown asmeans � SD. �, P < 0.05;��, P < 0.01; ��, P < 0.001. C,immunofluorescence microscopyimages of HMGB1 released fromnuclei to cytosol in A549 cellsinfected with CVB3 for 7 hours.Cells were stained with anti-HMGB1 (green) antibody and DAPI(blue), and analyzed byfluorescence microscopy. Scalebars, 10 mm.

Miyamoto et al.





(Supplementary Fig. S4B). There have been no reports ofhuman hepatitis caused by CVB3 infection. Elevated creatininekinase was found in the sera of CVB3-treated mice, withhistologic evidence of mild myocarditis, but no cytopathicdamage in the lungs or kidneys (Supplementary Fig. S4B).Although human CAR mRNA is expressed in the heart (44),

CVB3 possibly causes severe myocarditis in infants only (45),suggesting its relatively mild side effects in use for adultpatients.

As oncolytic virotherapy is expected to promote inflamma-tory DAMP within the tumor, which is beneficial for effectiveantitumor immune responses, it is important to assess

Figure 7. Immunostimulatoryeffects of intratumoral CVB3administration on tumor-infiltratinglymphocytes (TIL). TILs obtained onday 2 after intratumoral CVB3administration were subjected toflow cytometry analysis. A, numberspresented in thehistogram reflect thepercentage of DX-5þ, Gr-1þ, F4/80þ,and CD11cþ cells relative to the totalcell number of TILs (top 3 rows). Thenumbers presented in 2-dimensionaldot plots reflect the percentage ofdifferent mature DC subsets relativeto the total number of DCs (bottom 2rows). B, percentages of innateimmune subpopulations of NK cells,granulocytes, macrophages, andDCs relative to the total cellcount of TILs are shown (n ¼ 5).��, P < 0.01; ��, P < 0.001. C,percentages of 3 different subtypesof mature DCs relativeto the total count of DCs are shown.Bar graphs depict the means� SEM(n ¼ 5). �, P < 0.05; ��, P < 0.001. D,percentages of granulocytes or NKcells expressing CD107a in enrichedTILs relative to total TILs are shown.�, P < 0.05. E, nude mice (n ¼ 4)bearing A549 cells were injectedintraperitoneally with anti–Gr-1antibody on days 1, 4, 7, 10, 13, 16,and 19, or anti-asialo GM1 antibodyon days 1, 7, 13, and 19 posttumorchallenge. CVB3 virus was injectedinto the right lateral tumor every otherday for a total of 5 doses (5 � 106

TCID50/dose). The tumor volumesmeasured on day 18 are shown(n ¼ 4). �, P < 0.05.






whether oncolytic viruses generate immunogenic cell death(18). CVB3 infection induced these immunogenic changes suchas CRT exposure, release of extracellular ATP, and HMGB1translocation in NSCLC cells (Fig. 6). Upon propagation ofoncolytic virus, alterations in the repertoire of immune cells intumor microenvironment can restore inherent antitumorimmunity (46), through inductions of interferons and/or cyto-kines that activate NK cells and mature DCs (47, 48). Tumor-infiltrating DCs have been shown to be impaired atmaturation(49). Media from malignant cells infected with reovirus pro-moted maturation of DCs (46). Likewise CVB3 may alter theimmunologic microenvironment by accumulating diversemature DCs into tumors (Fig. 7C), probably because thesingle-stranded RNA genome converts dysfunctional DCs intofunctional DCs after ligation of retinoic acid–inducible gene-1(RIG-1)-like receptors (50). Indeed, CVB3 intervention wasshown to shape NK cell polarization with antitumor effects,possibly through promoted recruitment of cytolytic CD107aþ

NK cells into xenografts (Fig. 7D and E). Contribution ofneutrophils to the antitumor effects may be partially due toIFN-b production following CVB3 administration, as IFN-b caninstruct neutrophils to possess an antitumor phenotype (51).These immunostimulatory properties of CVB3 on innateimmunity may subsequently prime effective generation ofadaptive immunity synergizing with direct oncolytic activities.After showing that TC-1 syngeneic mouse NSCLC cells weresusceptible to in vitro CVB3 infection (Supplementary Fig.S5A), to further evaluate the effects of T cell–mediated cellularimmunity on the oncolytic effects of CVB3, we treated immu-nocompetent mice with TC-1 tumors with a single CVB3intratumoral administration, which significantly inhibitedTC-1 tumor development in a dose-dependent manner (P <

0.05; Supplementary Fig. S5B), with significantly prolongedsurvival (Supplementary Fig. S5C). These findings indicate thatT cell–mediated antiviral immunity is not a significant barrierfor intratumoral replication of CVB3.

Notably, in all experiments, no mice manifested lethalityduring CVB3 treatments, highlighting acceptable safety char-acteristics as an oncolytic agent.

In summary, our large-scale 2-step screening identifiedCVB3 as a tumor-specific virus, which depends on apoptoticand survival signaling pathways. Furthermore, systemic andimmunostimulatory antitumor effects of CVB3 provide anencouraging avenue for future preclinical and clinical devel-opment as a promising viral agent for the treatment of NSCLCpatients.

Disclosure of Potential Conflicts of InterestK. Tani: minor stock, Oncolys BioPhama Inc. No potential conflicts were

disclosed by the other authors.

AcknowledgmentsThe authors thank Michiyo Okada, Michiko Ushijima, and Dr. Shinji Okano

(Kyushu University) for technical assistance. The authors also thank the tech-nical support from the Research Support Center, Graduate School of MedicalSciences, Kyushu University.

Grant SupportThis work was supported by grants from the Ministry of Education, Culture,

Sports, Science and Technology (17016053 and 23240133), and the Ministry ofHealth Labour and Welfare (23080101), Japan.

The costs of publication of this article were defrayed in part 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 September 23, 2011; revised February 24, 2012; accepted March 14,2012; published OnlineFirst March 29, 2012.

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2012;72:2609-2621. Published OnlineFirst March 29, 2012.Cancer Res Shohei Miyamoto, Hiroyuki Inoue, Takafumi Nakamura, et al. Properties That Is Active against Lung AdenocarcinomaCoxsackievirus B3 Is an Oncolytic Virus with Immunostimulatory

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