Enhancement of the Proapoptotic Properties of Newcastle ... › content › molcanther › 14 › 5 › 1247.full.pdfpase 8 or InSolution Caspase 9 (EMD Millipore; Ref 218840 and 218841,

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Enhancement of the Proapoptotic Properties ofNewcastle Disease Virus Promotes TumorRemission in Syngeneic Murine Cancer ModelsSara Cuadrado-Castano1,2, Juan Ayllon2,3, Mena Mansour4, Janis de la Iglesia-Vicente4,Stefan Jordan5, Shashank Tripathi2, Adolfo García-Sastre2,3,6, and Enrique Villar1

Abstract

Newcastle disease virus (NDV) is considered a promisingagent for cancer therapy due to its oncolytic properties. Theseinclude preferential replication in transformed cells, inductionof innate and adaptive immune responses within tumors, andcytopathic effects in infected tumor cells due to the activationof apoptosis. To enhance the latter and thus possibly enhancethe overall oncolytic activity of NDV, we generated a recom-binant NDV encoding the human TNF receptor Fas (rNDV-B1/Fas). rNDV-B1/Fas replicates to similar titers as its wild-type(rNDV-B1) counterpart; however, overexpression of Fas ininfected cells leads to higher levels of cytotoxicity correlated

with faster and increased apoptosis responses, in whichboth the intrinsic and extrinsic pathways are activated earlier.Furthermore, in vivo studies in syngeneic murine melanomamodels show an enhancement of the oncolytic properties ofrNDV-B1/Fas, with major improvements in survival and tumorremission. Altogether, our data suggest that upregulation ofthe proapoptotic function of NDV is a viable approach toenhance its antitumor properties and adds to the currentlyknown, rationally based strategies to design optimized thera-peutic viral vectors for the treatment of cancer. Mol Cancer Ther;14(5); 1247–58. �2015 AACR.

IntroductionNewcastle disease virus (NDV) is a negative sense single-

stranded RNA virus classified as an avian paramyxovirus in theAvulavirus genus of the Family Paramyxoviridae (1). In theabsence of vaccination, NDV outbreaks in poultry can causedevastating economic losses. However, NDV infections inhumans are infrequent, and only cause mild conjunctivitis.The antitumor potential of NDV was first described during the1960s (2). Since then, its natural oncolytic capabilities havebeen demonstrated in different mammalian cancer cell lines,animal tumor models, and clinical trials (3–5). NDV selectivelyreplicates in cancer cells inducing cell death and stimulatinginnate and adaptive immune responses against tumor cells.Together with the lack of preexisting immunity in the general

population, NDV is a suitable candidate to be used as anoncolytic therapeutic agent (6). As with many oncolytic viruses,the establishment of reverse genetics systems for NDV facili-tated the development of new genetically modified recombi-nant NDV viruses with improved antitumor properties (7, 8).The principal strategy followed by most research groupsincluding ours has been focused on the enhancement of NDVimmunostimulatory properties through the generation ofrecombinant NDV viruses that express cytokines (IL2, IFNg ,TNFa), tumor-associated antigens, or tumor-specific anti-bodies (9–13). In our quest to design an optimized therapeuticNDV vector, here we focused our efforts on enhancing thecancer killing potential of NDV by increasing its potential forapoptosis activation.

Apoptosis is a highly regulated form of death that cells undergoafter activation by different, but specific, external or internalstimulation (14). There are two main apoptosis pathways: theextrinsic or death receptor pathway and the intrinsic or mito-chondrial pathway (15). Each pathway is executed in a caspase-dependent manner, and both pathways communicate by crosssignaling in such a way that molecules from one pathway caninfluence the other. Apoptosis resistance is a major hallmark ofmost, if not all, types of cancers due to defects in the apoptoticpathways (16, 17).

Indeed apoptosis resistance has recently been identified as amarker for resistance to chemotherapy and poor prognosis incancer patients (18, 19). Nevertheless, the oncolytic activity ofNDV has been shown to correlate with activation of the intrinsicor mitochondrial apoptosis pathway even in cancer cells resistantto apoptosis induction (20–23).

In this study, we propose a novel approach to enhance theoncolytic properties of NDV. By generating a recombinant NDV

1Department of Biochemistry and Molecular Biology, University ofSalamanca, Salamanca, Spain. 2Department of Microbiology, IcahnSchool ofMedicine atMountSinai,NewYork,NewYork. 3GlobalHealthand Emerging Pathogens Institute, Icahn School of Medicine at MountSinai, NewYork, NewYork. 4Department of Pathology, IcahnSchool ofMedicine at Mount Sinai, New York, New York. 5Department of Onco-logical Sciences, Immunology Institute and the Tisch Cancer Institute,Icahn School of Medicine at Mount Sinai, New York, New York. 6Divi-sion of Infectious Disease, Department of Medicine, Icahn School ofMedicine at Mount Sinai, New York, New York.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Adolfo García-Sastre, Icahn School of Medicine atMount Sinai, 1468 Madison Avenue, New York, NY 10029. Phone: 212-241-7318; Fax: 212-534-1684; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0913

�2015 American Association for Cancer Research.

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that encodes the human tumor necrosis factor receptor Fas, wehypothesized that we would enhance the apoptotic and antitu-mor properties of NDV (24). Fas is one of themost important andbetter characterized death receptors due to its role in homeostasis,elimination of pathogen-infected cells, and activation of theimmune response (25–29). The transduction of the Fas-depen-dent death signal initiates from binding of its ligand FasL thatresults in receptor-mediated apoptosis signaling complex forma-tion and caspase 8 activation (30–33). Fas-mediated cytotoxicityis not only restricted to the activation of the extrinsic pathway, butis also required for CTL-mediated perforin–granzyme cytotoxicity(34). Defects in the Fas-FasL system have been documented inmany tumor types as a major feature of malignant progression,tumor immune evasion, and resistance to cytostatic drug treat-ment (35, 36).

In our current study, we evaluated the antitumor potential of anewly generated rNDV-B1/Fas virus. We postulated that by over-expressing Fas in NDV-infected cells we will introduce a strongextrinsic proapoptotic stimulus that will synergize for apoptosisinductionwith the intrinsic pathways activated byNDV infection,translating into an enhancement of its antitumor phenotypein vivo.

Materials and MethodsCell lines, antibodies, and other reagents

Vero (African greenmonkey kidney epithelial cells; ATCC Cat#CCL-81, 2014), B16-F10 (mouse skinmelanoma cells; ATCCCat#CRL-6475, 2013), HeLa cells (human cervical adenocarcinomaepithelial cells; ATCC Cat# CCL-2, 2012), NIH/3T3 (murineembryonic fibroblast; ATCC Cat# CRL-1658, 2014), HuH-7(human hepatocarcinoma; genteelly provided by Dr. MatthewEvans research group (Department ofMicrobiology, Icahn Schoolof Medicine at Mount Sinai, New York, NY, 2014), and A549 cells(human lung carcinoma; ATCC Cat# CCL-185, 2014) were main-tained in DMEM medium supplemented with 10% FBS, L-Glu-tamine (1% Glutamax-100X; Invitrogen), penicillin and strepto-mycin (DMEM10%FBSP/S). CT26 cells (ATCCCat#2638, 2013)were maintained in RPMI-NaHCO3 5% medium supplementedwith 10% FBS, penicillin and streptomycin. C-33A cells (humancervical carcinoma epithelial cells; ATCC Cat# HTB-31, 2012)weremaintained in EMEMmedium supplemented with 10% FBSand 5% of penicillin and streptomycin (Gibco by Life Technol-ogies; Thermo Scientific). A master cell-bank was created for eachcell line after purchase, and early-passage cells were thawed inevery experimental step. Once in culture, cells were maintainednot longer than 2 months to guarantee genotypic stability andweremonitored bymicroscopy. As a specific feature of the HuH-7cells, ability to support HCV replication was demonstrated byMatthew Evans group (37).

Cell lysates for SDS-Page were obtained using the ProteoJETMammalian Cell Lysis reagent purchased from Fermentas(Thermo Scientific). ECL Western blotting system and horse-radish peroxidase (HRP)–conjugated secondary antibodieswere purchased from GE Healthcare. ECL Western BlottingSubstrate (Thermo Scientific) was used for detection of HRPin immunoblots.

Polyclonal antibodies to human/Fas (C18C12), anti-caspase 3,anti-caspase 9, and anti-PARP, as well as monoclonal antibody tocaspase 8, were purchased from Cell Signaling. Monoclonal anti–b-actin (AC-15) was purchased from Abcam. Rabbit polyclonal

serum to NDV was previously described (38). Monoclonalantibody to human APO-1/Fas was for Bender MedSystems.Monoclonal mouse anti-IgG Alexa Fluor 568 and polyclonalrabbit anti-IgG Alexa Fluor 488 were purchased from Invitrogen(Molecular Probes). Hoechst 33258 nuclear staining reagent waspurchased from Invitrogen (Molecular Probes). MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagentwas purchased from Sigma.

Generation of recombinant NDVPlasmid pNDV-B1, encoding the full-length antigenomic

cDNA of Hitchner B1 lentogenic strain of NDV, has been previ-ously described (8). The open reading frame (ORF) of human Fasreceptor was amplified by PCR using specific primers includingthe required regulatory signals for its functional integration intotheNDV genome and then cloned into the XbaI site between the Pand M genes of pNDV-B1. Recombinant rNDV-B1/Fas was res-cued using previously described techniques (8). Insert fidelity wasevaluated by amplification of the Fas sequence by reverse tran-scription PCR and virus genomic RNA followed by sequencing.Viral stocks of rNDV-B1/Fas and rNDV-B1 were propagated in 9-day embryonated chicken eggs and purified from the allantoicfluid in a potassium tartrate gradient.

Fluorescence microscopyFor indirect immunofluorescence staining, cells seeded in 24-

well standard plates or glass-bottomed 12-well plates wereinfected for 1 hour at an multiplicity of infection (MOI) of 1PFU/cell, after which the inoculum was removed and replacedwith 1mLofDMEMwith10%FBSP/S. At 20hours after infection,cells were fixed with 2.5% paraformaldehyde for 15 minutes andblocked in PBS with 1% BSA for 1 hour. Primary antibodies wereincubated with the samples for 1 hour at room temperature.

Secondary antibodies (goat anti-mouse Alexa Fluor 568 or 633or goat anti-rabbit Alexa Fluor 488; purchased from Invitrogen)were used at a 1:1,000 dilution for 45 minutes before imagingusing an Olympus IX41 microscope or a Zeiss LSM 510 Metaconfocal microscope.

Growth curves and titersHeLa, Vero, A549,HuH-7,C-33A, andB16-F10 cellmonolayers

in 6-well plates were infected with the virus suspension at anMOIof 0.01 PFU/cell in OPTIMEM-I. After 1 hour, the infectionmediawere removed and the cells were incubated with 3 mL of DMEMwith 0.3% BSA and 1 mg of TPCK-treated trypsin/mL to allowthe production of fusion-competent viruses. Supernatants werecollected at 24, 36, 48, and 64 hours (72 hours in A549, HuH-7,C-33A, and B16-F10) after infection and titrated by immunoflu-orescence assay on Vero cells seeded on 96-well plates by using apolyclonal anti-NDV serum.

MTT cytotoxicity assayHeLa, A549, HuH-7, C-33A, and B16-F10 cells were cultured at

a confluence of 50% in 24-well dishes and infected at anMOI of 1PFU/cell. Infection media were removed 1 hour after infection,and cells were incubated 24, 36, and 48 hours in 1 mL ofsupplemented DMEM (or EMEM for C-33A). At each time point,themedia were aspirated and cells were subsequently incubated 1hour 15minutes with 300 mL of 2.5mg/mLMTT solution at 37�Cand under restricted light conditions. Subsequently, the MTTsolution was aspirated, and cells were incubated with 500 mL of

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Isopropanol for 10 minutes in a shaker. The absorbance of eachsample was recorded at 570 nm using a BioTek plate reader.

Annexin-V/propidium iodide flow cytometric analysisCells were plated in 35-mm dishes and infected an MOI of 1

PFU/cell. Infection media were removed 1 hour after infectionand cells were incubated 24 and 48 hours in supplementedDMEM. Apoptosis induction was determined using the Immuno-step DY-634 Annexin-V Apoptosis Detection Kit (Immunostep),according to themanufacturer's instructions. Cell acquisition wasmade on a FACSCalibur flow cytometer (Becton Dickinson).

Data analysis was performed using CellQuest, from BD Bios-ciences, and FlowJo software (Tree Star).

Caspase activity assayHeLa cells were plated into 96-well plates and infected at an

MOI of 1 PFU/cell. One hour after infection, the media werereplaced by conventional DMEM or DMEM supplemented withcaspase-specific inhibitor at different final concentrations. Forspecific caspase activity inhibition, the reagents InSolution Cas-pase 8 or InSolution Caspase 9 (EMDMillipore; Ref 218840 and218841, respectively) were used. At 24 hours after infection,caspase 8, 9, and 3 activity was quantified by Caspase-Glo 8, 9,or 3/7 assay systems (Promega; G8210, G8211, and G8090,respectively) following the manufacturer's instructions.

Interferon response assayB16-F10 and NIH/373 cell monolayers in 6-well plates were

infected with the virus suspension at an MOI of 1 PFU/cell inOPTIMEM-I. After 1 hour, the infection media were removed andthe cells were incubated with 3 mL of DMEM. Total RNAs fromcultured cells were isolated 8 hours after infection with a QiagenRNeasyMini kit (Qiagen). Mean n-fold expression levels of cDNAfrom three individual biologic samples, each measured in tripli-cate, were normalized to 18S rRNA levels and calibrated to mock-treated samples according to the 2�DDCT method (39).

The primer sequences were as follows: for the murine geneIFNb, the forward primer was 50CAGCTCCAAGAAAGGACGAAC-30 and the reverse primer was 50GGCAGTGTAACTCTTCTG CAT-30. For murine IFIT1, the forward primer was 50CTGAGAT-GTCACTTCACATGGAA-30 and the reverse was 50GTGCATC-CCCAATGGGTTCT-30. For murine gene OAS1, the forward prim-er was 50ATGGAGCACGGACTCAGGA-30 and the reverse was50TCACACACGACATTGACGGC-30. For murine IRF7 gene, theforward primer was 50GAGACTGGCTATTGGGGGAG-30 and thereverse primer was 50GACCGAAATGCTTCCAGGG-30.

The 18S forward primer was 50-GTAACCCGTTGAACCCCATT-30, and the 18S reverse primer was 50-CCATCCAATCGGTAG-TAGCG-30.

Syngeneic melanoma tumor modelC57/BL6J female mice 4 to 6 weeks of age used in all our in vivo

studies were purchased from The Jackson Laboratory.A B16-F10 cell suspension (5� 105 cells in 100 mL of PBS) was

intradermally inoculated into theflank of the right posterior leg ofeach C57/BL6J mouse. After 10 days, the mice were treated byintratumoral injection of 50 mL of 5 � 106 PFU of the indicatedrecombinant NDV viruses or PBS. The intratumoral injectionswere administered every 24 hours for a total of three treatmentdoses. Tumor volume was monitored every 48 hours or every 24hours when the last volume estimation was approaching the

experimental endpoint of 1,000 mm3. Mice were humanelyeuthanized the day in which the volume exceeded the predefinedendpoint. Tumor measurement was determined using a digitalcaliper, and total volumewas calculatedusing the formula: Tumorvolume (V) ¼ L � W2, where L, or tumor length, is the largerdiameter, and W, or tumor width, is the smallest diameter.

Immunohistochemistry and immunofluorescence staining oftumor samples

A suspension of B16-F10 cells (5 � 105 cells in 100 mL ofPBS) was intradermally inoculated into the flank of the rightposterior leg of C57/BL6J mice. After 10 days, one intratumoralinjection of PBS or recombinant NDV virus suspension (5 �106 PFU in 50 mL of PBS) was administrated. At 24 hours afterinoculation, the tumors were removed and preserved by for-malin fixation and paraffin embedding for immunohistochem-istry (IHC) analysis.

IHC staining for active caspase 3 was performed on 5-mm-thicktumor sections. The slides were incubated in H2O2 solution for15minutes, and antigen retrieval was performed by steamheatingin 10 mmol/L citrate buffer (pH 6.0) for 45 minutes.

After epitope recovery, the slides were then treated with 10% ofnormal goat serum for 60 minutes, followed by incubation withcaspase 3 antibody (1:500 dilution; Cell Signaling; ref. 9664)incubation overnight at 4�C. The slides were washed and incu-bated with secondary biotinylated anti-Rabbit IgG antibody(HþL; Vector Laboratories, Inc.) at 1:500 dilution for 1 hourfollowed by incubation with avidin–biotin conjugate (1:25 dilu-tion; ABC complex; Vector Laboratories, Inc.) incubation for 30minutes. The samples were treated with the chromogen diami-nobenzidine for antigen detection, and the final counterstainingwas performed with hematoxylin.

Analysis ofmyeloid cell populations present in infected tumorsFor the characterization of the immune cells within the tumor

in response to the virus treatment, B16-F10 melanoma syngeneicmodel was carried out in C57/BL6J female mice as describedbefore. The animals received a total of three intratumoral injec-tions of PBS or recombinant NDV virus suspension (5� 106 PFUin 50mL of PBS), one every 24 hours, and the tumorswere isolated24 hours after the last injection.

For the immune cell isolation, the tumors were minced,digested with collagenase IV (Roche) for 1 hour at 37�C, andpassed through 70-mm cell strainers to obtain single-cell suspen-sions. Cells were layered in a 40% and 90% Percoll gradient (GEHealthcare) and centrifuged at 1,260 � g for 40 minutes withoutbrake. The interphase was collected and analyzed by flowcytometry.

Flow cytometry analysis: fluorochrome-conjugated antibodiesagainst CD44 (clone IM7) were purchased from BD Pharmingen,against Ly-6C (AL-21) from BD Biosciences, against CD4 (RM4-5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418), CD25(PC61.5), CD62L (MEL-14), and Foxp3 (FJK-16s) fromeBioscience, against CD3 (17A2), CD45 (30-F11), CD64 (X54-5/7.1), CD103 (2E7), I-A/I-E (M5/114.15.2), and Ly-6G (1A8)from BioLegend, against CCR2 (475301) from R&D Systems.Cells were incubated with specific antibodies in DPBS containing0.5%BSAand2mmol/L EDTA for 20minutes at 4�C. Intracellularstaining for Foxp3 was performed using the transcriptionfactor fixation/permeabilization concentrate and diluent fromeBioscience. Samples were acquired on a BD LSRFortessa (Becton

Oncolytic Activity of rNDV-B1/Fas

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Dickinson) using the FACSDiva Software and analyzed withFlowJo software (Tree Star).

Statistical analysisStatistical significance between results from triplicate samples

was determined by the two-tailed Student t test. The results areexpressed as mean values � SDs. The comparison of survivalcurves for the data obtained in the B16-F10 melanoma syngeneicmodel was performed using the long-rank (Mantel–Cox) test. Theanalysis of the myeloid cell populations presented within thetreated tumors was performed using a one-way ANOVA (DunnMultiple comparison test).

Ethics statementAll animal procedures performed in this study are in accordance

with Institutional Animal Care and Use Committee (IACUC)guidelines, and have been approved by the IACUC of IcahnSchool of Medicine at Mount Sinai.

ResultsDetection of human fas receptor expression in rNDV-B1/Fas–infected cells

In our study, a new recombinant NDV virus, rNDV-B1/Fas, wasgenerated and assessed for improved oncolytic potential. For thispurpose, the ORF of the human Fas receptor was inserted into thebackbone of the lentogenic NDV-B1 virus genome between the Pand M genes (Fig. 1A) to ensure high level of protein expressionduring virus replication (1). Insert stability within the viralgenome was assessed after six viral growth passages in eggs, andthefidelity of its sequencewas confirmedbyRT-PCR. Comparisonof growth properties of rNDV-B1/Fas with those of the parentalrNDV-B1 in the human cancer cell lines HeLa, A549, HuH-7, andC-33A, as well as in Vero cells showed no differences (Fig. 1B),with both viruses replicating to similar titers and kinetics. Specificexpression of Fas receptor after infectionwith the newly generatedviruswas detected by immunofluorescence inHeLa cells (Fig. 1C),as well as in other human (A549, HBL, C-33A, Hep2, HuH, PC3)cancer–derived cell lines, and in the African Green monkey cellline Vero (data not shown). As expected, the Fas protein wasbroadly distributedon the cell surface,where areas of high-densityreceptor accumulation could be clearly distinguished (Fig. 1D,top).

The intracellular localization of the receptor during rNDV-B1/Fas infection was determined by confocal microscopy fol-lowing immunofluorescent labeling (Fig. 1D, bottom). Insidethe cell, Fas could be found colocalizing with the early endo-some marker Rab5 in the cytoplasmic endosomal compart-ment, one of the hallmarks of Fas activation. These localizationpatterns suggest that overexpression of Fas receptor by therecombinant virus results in its activation in the absence of itsligand.

Enhanced cytopathic effect in rNDV-B1/Fas–infected cellscorrelates with an early activation of the apoptosis response

To determine whether overexpression of Fas receptor duringrNDV-B1/Fas virus infection could enhance the inherent proa-poptotic activity of NDV, we studied specific morphologic andbiochemical features of this type of cell death. First, apoptosis-specificmorphologicmodifications in rNDV-B1or rNDV-B1/Fas–infected cells were monitored by microscopy. At 24 hours after

infection, the number of adherent cells was dramaticallyreduced during rNDV-B1/Fas virus infection as compared withthose infected with rNDV-B1. The remaining attached cellspresented typical apoptosis morphologic landmarks like cyto-plasmic membrane blebbing and DNA fragmentation asobserved by immunostaining (Fig. 2A). Cells that undergoapoptosis gradually lose metabolic functions leading to celldeath. To quantify rNDV-B1/Fas and rNDV-B1 replication-associated cytotoxicity, we performed an MTT viability assaythat measures the activity of mitochondrial reductase enzymeswhich are only active in living cells. rNDV-B1/Fas virus infec-tion resulted in more than 50% reduction in HeLa cells viabilityat 24 hours after infection (Fig. 2B, right). Even at the latesttime point of our study, 48 hours after infection, rNDV-B1 virusdid not promote more than 50% reduction in cell viability.Both cytomorphologic and viability studies suggest that cellsinfected with rNDV-B1/Fas undergo an earlier and more potentapoptosis response. This enhanced cytotoxicity was also vali-dated in other human (A549, HuH-7, C-33A) and murine(CT26) cancer-derived cell lines (Fig. 2B, left).

To further characterize the apoptosis response induced byrNDV-B1/Fas infection, we performed Annexin-V/propidiumiodide (PI) stains in HeLa-infected cells. Since Annexin-V stainingprecedes PI staining during apoptosis, late stages of apoptosis arecharacterized by double Annexin-V/PI stain (Fig. 2C). As early as24 hours after infection, rNDV-B1/Fas–infected cells showedhigher number of cells progressing into the final stages of lateapoptosis than cells infected with rNDV-B1.

Overexpression of Fas leads to activation of both intrinsic andextrinsic pathways in rNDV-B1/Fas–infected cells

We next studied the caspase activation pattern during infectionwith either rNDV-B1/Fas or rNDV-B1 (Fig. 3A). Protein extractscollected at different time points after infection were tested todetect active forms of the principal caspases involved in bothextrinsic and intrinsic pathways. As compared with rNDV-B1–infected cells extracts, rNDV-B1/Fas–infected cells showed acti-vation of caspases 8, 9, and 3 at 24 hours, which is in contrast withtheir late (48 hours) activation in rNDV-B1–infected cells. Thiswas also accompanied by the presence of cleaved Poly (ADPribose) polymerase PARP that is a marker for the final stages ofprogrammed cell death.

We next wanted to investigate the contribution of the extrinsic(caspase 8–dependent) pathway into the newmodified apoptoticresponse due to Fas overexpression. Adding specific caspaseinhibitors to the postinfection media, we could observe that after24 hours the inhibition of caspase 8 had the strongest effectrestricting both caspase 9 and 3 activities in rNDV-B1/Fas–infected cells (Fig. 3B). However, caspase 9 inhibition led to aslight inhibition of caspase 8 and 3 activities. No effects were seenin rNDV-B1–infected cells, since at 24 hours after infection, thereis still no detectable caspase 3, 8, and 9 activation. The apoptosisactivation pattern seen in rNDV-B1/Fas–infected cells indicatesthat the upregulation of Fas receptor results in coactivation andcooperation in cell death of both the extrinsic (caspase 8–depen-dent) and intrinsic (caspase 9–dependent) pathways with animportant and earlier contribution of the Fas-mediated caspase8–dependent extrinsic pathway. This cooperative effect is likelyresponsible for the increase in the general apoptosis responseobserved during rNDV-B1/Fas infection as compared with rNDV-B1 infection.






rNDV-B1/Fas virus infection leads to interferon responseactivation and enhances apoptosis induction in murine B16-F10 melanoma cells

Previously to the evaluation of the potential therapeutic effectof the rNDV-B1/Fas virus in vivo, we wanted to know if theoncolytic properties displayed by the rNDV-B1/Fas virus inhuman cancer cell lines in vitro would be preserved also in cancercells of murine origin.

Both, rNDV-B1 and rNDV-B1/Fas viruses were able to replicateat similar levels in B16-F10 showing similar titers and kinetics(Fig. 4B). By confocal microscopy, we could observe that thehuman Fas receptor was highly expressed and broadly distributedon the cell surface as well as in the cytoplasmic endosomecompartment of the murine cell line (Fig. 4A), same way as we

described before for the human cancer cell line HeLa (Fig. 1C andD). rNDV-B1/Fas virus exerted higher and earlier cytotoxicity inthemurine cancer cell line (Fig. 4C), and this cytotoxicity was alsocorrelated with an earlier activation of the apoptosis response,with the presence of active forms of caspase 3 detected 24 hoursafter infection (Fig. 4D).

Because the NDV therapeutic effect in vivo involves an activeinterferon response induction upon virus infection (5), wewanted to evaluate the potential stimulation of the rNDV-B1/Fasvirus in the murine B16-F10 melanoma cells. To assess thisquestion, B16-F10 cells with rNDV-B1 or rNDV-B1/Fas virusesat anMOI of 1 and total RNAwere isolated 8 hours after infection.Immortalized NIH/3T3 murine fibroblasts were infected in sim-ilar conditions to be used as a positive control. The levels of INFb

Figure 1.Generation and characterization of recombinant rNDV-B1/Fas. A, schematic representation of the genomes of rNDV-B1 and rNDV-B1/Fas, showing theposition between the P/V andMgenes inwhich the newORFwas inserted. B,multicycle growth curves of rNDV-B1 and rNDV-B1/Fas. HeLa, A549, HuH-7, C-33A, andVero cell monolayers were infected at an MOI of 0.01 PFU/cell. At different time points after infection, viral titers in the supernatant were determined. Datapoints show mean values from three replicates with error bars representing SDs. C, immunofluorescence detection of the expression of human Fas receptorin rNDV-B1/Fas–infected cells. HeLa cells were infected at an MOI of 1 PFU/cell, fixed 24 hours after infection, and stainedwith amonoclonal antibody against humanFas (red), polyclonal serum against NDV (green), and Hoechst for nuclear contrast (blue). Scale bar, 50 mm. D, cellular distribution of recombinant Fas receptorupon rNDV-B1/Fas infection. Cells were infected at an MOI of 1 PFU/cell, fixed 20 hours after infection, and stained with monoclonal anti-human Fas antibody (red),polyclonal serum anti NDV (green, top), or antibody against the early endosome marker rab5 (green, bottom) and Hoechst for nuclear contrast. Top,cytoplasmicmembrane location of Fas receptor. Main top, white arrows pointing at higher density receptor areas at themembrane surface. Scale bar, 25mm.Bottom,Fas receptor endosomal compartment location. Main bottom, colocalization of human Fas receptor and Rab5 into endosomal compartment. Scale bar, 50 mm.






mRNA as well as those for the interferon stimulated genes IFT1,IRF7, and OAS1 were evaluated by qPCR (Fig. 4E). No differencewas found between the viruses, and the B16-F10 cells showedsimilar levels of interferon induction as the immortalized fibro-blasts, 8 hours upon infection.

Intratumoral treatment with rNDV-B1/Fas virus enhancessurvival, promotes complete tumor remission and protectionagainst rechallenge in melanoma syngeneic murine model

To assess whether the improvement of the proapoptoticactivity of rNDV-B1/Fas would enhance the inherent oncolyticproperties of NDV in vivo, we tested its antitumor capacity in

melanoma syngeneic tumor models. Murine melanoma B16-F10 cells were subcutaneously implanted in the leg flanks ofC57BL/6 mice. Tumors were allowed to develop until palpable,and a total of three intratumoral injections of each virus or PBSwere administrated, as described in Materials and Methods.Treatment with rNDV-B1/Fas virus restricted tumor progression(Fig. 4F), leading to a significant improvement in survivalcompared with mice treated with rNDV-B1 or PBS. rNDV-B1treatment delayed tumor growth, but only resulted in completetumor remission in 12% of treated animals. However, completetumor remission was observed in 83% of mice treated withrNDV-B1/Fas (Fig. 4F). These remaining tumor-free animals

Figure 2.rNDV-B1/Fas infection induces higher cytotoxicity and an earlier apoptotic response in cancer cells. A, cytopathic effect due to rNDV-B1/Fas infection. Confocalmicroscopy images of HeLa cells infected with rNDV-B1/Fas. Cells were infected at an MOI of 1 PFU/cell, fixed 20 hours after infection, and stained withmonoclonal anti-human Fas antibody (red), polyclonal anti-NDV serum (green), and Hoechst for nuclear contrast. Left, composite Z-stack of six optical slicesshowingmembrane and intracellular distribution of Fas receptor. Left plots show different late apoptotic features, likemembrane blebbing and DNA fragmentation,observed among the infected population. Scale bar, 50 mm. B, cytotoxicity. HeLa, A549, HUH-7, C-33A, and Ct26 cells were infected at an MOI of 1 PFU/cell,and their viability was determined byMTT viability assay at different time points (24, 36, and 48 hours after infection; n¼ 3; � , P <0.05; �� , P <0.005; �� , P <0.0005;�� , P < 0.0001. C, apoptosis induction during rNDV-B1 and rNDV-B1/Fas infection. HeLa cells were infected at an MOI of 1 PFU/cell, collected at different timesafter infection, and double stained with Annexin-V/PI. Early (Annexin-V–positive) and late (Annexin-V/PI–double positive) apoptotic populations wereassessed by flow cytometric analysis. Internal axes were defined using Annexin-V/PI data from mock-uninfected HeLa cells. Density plots from one of threeindependent experiments are shown. The percentage distribution of the different apoptotic stages is also shown (n ¼ 3; � , P < 0.05; ��, P < 0.0005).






did not show tumor recurrence, loss of weight, or any other signof sickness.

We also wanted to know if the enhancement of the apoptoticresponse could also influence the emergence of a long-timeprotection against cancer relapse. To assess this question, a newset of animals were used to induce a syngeneic melanomamodel,andonce theypresented tumors, the animalswere subjected to thesame treatment conditions described before. The rNDV-B1/Fasvirus treatment induced an overall enhancement of survival of43%, and complete tumor revision was also reported within theexperimental group (Fig. 4G). However, in this study, none of theanimals treated with the wild-type virus underwent tumor remis-sion. The survivors treated by the rNDV-B1/Fas virus that dem-onstrated complete recovery for up to 30 days were then rechal-lenged against melanoma by subcutaneous reinjection of B16-F10 melanoma cells in the flank of the opposite leg. Thoseanimals, as a part of a long-term study, were under periodicalobservation, and no sign of tumor reversion or any other sign ofsickness was reported for up to 6 months.

Early and enhanced apoptosis response during rNDV-B1/Fasvirotherapy could be a key point for a more specific immuneresponse against the tumor

We wanted to know if the earlier proapoptotic activity exertedby rNDV-B1/Fas virus in vitro could be also a main feature of therNDV-B1/Fas–treated tumors in vivo. For that propose, murinemelanoma B16-F10 cells were intradermally implanted on theflank of the leg in C57BL/6 mice. After 10 days, the generatedtumors were intratumorally treated with a single dose of PBS orvirus suspension. At 24 hours after inoculation, the mice wereculled and the tumors were removed and processed for generalhistopathology analysis, virus immunodetection, and apoptosismarkers determination (Fig 5A, B, C and Supplementary Fig. S1Aand S1B). At this time point (24 hours after treatment), tumor

histopathology analysis did not show any relevant differencesin markers of advanced melanoma progression, such as highvascularization and necrosis, between treatment groups (PBS,rNDV-B1, or rNDV-B1/Fas–treated tumors), as monitored afterhematoxylin and eosin staining (Fig. 5A). Only rNDV-B1– orrNDV-B1/Fas–infected tumors were positive for anti-NDV Fprotein detection (Fig. 5B), but there were no significant differ-ences related to infection distribution or virus spread betweenthese two groups. However, we could detect a notable presenceof active caspase 3–positive cells in rNDV-B1/Fas–treated sam-ples compared with both PBS and wild-type virus treatments(Fig. 5C). This indicates that the earlier and improved apoptosisresponse previously described in vitro for rNDV-B1/Fas (Figs.2, 3, and 4) also occurs after intratumoral inoculation in vivo(Fig. 4A and B).

Last, we wanted to refine the characterization of the therapeuticeffect exerted by the recombinant rNDV-B1/Fas in the early phaseof the treatment by analyzing the innate immune cells popula-tions resident within the tumors upon virus treatment. For thatpropose, we carried out a new syngeneic melanoma assay inC57BL/6 mice, following the same proceedings previouslydescribed. In this particular experiment, the animals received thecomplete treatment (3 doses of PBS or recombinant virus injectedintratumorally) andwere culled 24 hours after the administrationof the last dose.

The tumors were removed and specifically processed for theisolation and analysis by Flow cytometry of the different myeloidcells lineages, as is mentioned in Materials and Methods. Asexpected, the response to both viruses demonstrated to be moreimmunogenic compared with the PBS treatment, with a decreasein dendritic cells and an increase in proinflammatory macro-phages and neutrophils resident at that moment in the infectedtumors (Fig. 5D). Further investigations would be needed toevaluate the contribution of the innate immune response in our

Figure 3.rNDV-B1/Fas–infected cells display a modified NDV-induced apoptotic response. A, time course of caspase activation during rNDV-B1/Fas and rNDV-B1 virusesinfection and Western blot. HeLa cells were infected with rNDV-B1/Fas and rNDV-B1 at an MOI of 1 PFU/cell, and lysates were obtained at different timepoints after infection. Differential caspase activation pattern was assessed by Western blot using specific anti-caspase 8, 3, 9, and PARP antibodies. Fas receptorexpression was detected using an anti-human Fas monoclonal antibody. Viral replication (NP levels) was detected using an anti-NDV polyclonal serum. B, extrinsicand intrinsic pathways interdependence. HeLa cells were infected with rNDV-B1/Fas and rNDV-B1 at an MOI of 1 PFU/cell, and different amount of specificcaspase 8 (Z-IETD-FMK) or caspase 9 (Z-LEHD-FMK) inhibitors were added to the postinfection media. After 24 hours postinfection, caspase 8, caspase 9, andcaspase 3 activities were evaluated using a luciferase-based caspase activity reporter assay (n ¼ 3; � , P < 0.05).






Figure 4.rNDV-B1/Fas virus exerts higher oncolytic capacity and increases survival in a syngeneic melanoma murine model. A, immunofluorescence detection of human Fasreceptor in rNDV-B1/Fas-B16-F10–infected cells. Confocal microscopy image of murine melanoma B16-F10 cells infected with rNDV-B1/Fas. Cells were infectedat an MOI of 1 PFU/cell, fixed 20 hours after infection, and stained with monoclonal anti-human Fas antibody (red), polyclonal anti-NDV serum (green), andHoechst for nuclear contrast. Scale bar, 50 mm.White arrow, endosome compartment localization of the recombinant human Fas receptor. B, multicycle growth curves.B16-F10 monolayers were infected at an MOI of 0.01 PFU/cell. At different time points after infection, viral titers in the supernatant were determined. Data pointsshow mean values from three replicates with error bars representing SDs. C, cytotoxicity. B16-F10 cells were infected at an MOI of 1 PFU/cell, and their viability wasdetermined by MTT viability assay at different time points (24, 36, and 48 hours after infection; n ¼ 3, �� , P < 0.0005). D, time course of caspases activationand Western blot. B16-F10 cells were infected with rNDV-B1/Fas and rNDV-B1 at an MOI of 1 PFU/cell, and lysates were obtained at different time points afterinfection. Apoptosis activation was assessed by Western blot using a specific anti-caspase 3 antibody. Human Fas receptor expression was detected using ananti-human Fas monoclonal antibody. Viral replication (NP levels) was detected using an anti-NDV polyclonal serum. (Continued on the following page.)






model. An improved and more specific innate immune responsedue a better immunological cell death of the tumor cells would bethemajor determinant in the long-termprotectionobserved in therNDV-B1/Fas–treated mice.

DiscussionDuring the last decade, new studies exploring NDV antitumor

characteristics have emerged, and a new generation of recombi-nant NDVs has been engineered attempting to enhance its naturaloncolytic capacity (4). In our current study, to design an improvedtherapeutic agent, we attempted to increase cell death induced inNDV-infected tumors. To do so,we generated a recombinantNDVencoding the human tumor necrosis factor receptor Fas(TNFRSF6, CD95). Neither Fas nor its ligand FasL had come outin the list of host elements involved in the NDV-stimulatedapoptosis response. Our rationale in designing such vector is thatoverexpression of Fas receptor in infected cells would lead to theactivation of the extrinsic apoptosis pathway which would bothcomplement and increase the proapoptotic capacity of NDVotherwise mainly mediated by the activation of the intrinsicpathway (30–32). This may enhance the antitumor activity ofNDV in several ways, first enhancing tumor cell death duringdirect infection, second, increasing immune-mediated cell deathof infected cells, and third, promoting the release of proinflam-matory mediators that promote immune activation of antitumorresponses.

At this moment, we do not know which of these factors orcombination of factors is the main driver in the increased ther-apeutic effect of rNDV-B1/Fas.

Our in vitro studieswith rNDV-B1/Fas demonstrated high levelsof the recombinant human Fas receptor expression in differenthuman- and mouse-infected cancer cell lines. Fas receptorexpressed by NDV exhibited widespread distribution throughoutthe cell membrane with areas of receptor aggregates, consistentwith Fas receptor activation by overexpression. The recruitmentand stabilization of preassembled receptors in signaling proteinoligomerization structures (SPOTS) in the cytoplasmic mem-brane are necessary to initiate signal transduction. This is phys-iologicallymediated through Fas/FasL interaction (40). However,during rNDV-B1/Fas infection, the overexpressed receptor wasable to form these proapoptotic SPOTS in the cytoplasmic mem-brane in absence of FasL stimulus. A similar phenomenon waspreviously described in other approaches in which the mainaim was to increase or stabilize the receptor at the membranesurface (41–45). Also, we could detect the presence of Fasreceptor in the cytoplasmic endosomal compartment, previ-ously described to be a secondary and necessary location for theamplification of caspase-mediated signaling (46). These resultssupport that in rNDV-B1/Fas–infected cells, the required envi-

ronment and cellular compartmentalization necessary for Fasreceptor activity has been achieved by overexpression in aligand-independent manner.

Furthermore, the rNDV-B1/Fas–infected cells display a differ-ent caspase activation profile than the one in rNDV-B1–infectedcells. According to previous studies, wild-type NDV-infected cellsundergo apoptotic cell death at a late step during virus replicationthrough the activationof both the intrinsic and extrinsic apoptosispathways (20–23).We confirmed this by assessing the presence ofcaspase 9, caspase 8, and caspase 3 48 hours after infection inrNDV-B1–infected cells. In contrast, after rNDV-B1/Fas infection,we detected the presence of the active forms of the extrinsicpathway initiator caspase 8 and the executor caspase 3 earlier onduring infection. To our knowledge, no other tested strain ofNDVhas shown a similar apoptosis induction profile, with only onereport of a velogenic strain inducing an early activation of theextrinsic pathway (47), which preceded the standard intrinsicactivation andwas apparently independent of it. Interestingly andin blatant contrast, during rNDV-B1/Fas infection, the presence ofactive forms of the intrinsic pathway initiator caspase 9 wasdetected at the same time as caspase 8 and caspase 3, whichsuggest the possibility of an early coactivation of both pathways.The interdependence between pathways was assessed by inhibi-tion of the activity of the principal initiators caspase 8 and caspase9. We observed that the downregulation of caspase 8 activitystrongly repressed not only the execution phase caspase 3–depen-dent but also caspase 9 activity, which suggests that in rNDV-B1/Fas–infected cells, the strongest proapoptotic stimuli come fromthe extrinsic signaling pathway. Taking under consideration thepreviously known elements involved in NDV-mediated apopto-sis, we propose that overexpression of Fas receptor during rNDV-B1/Fas infection acts as an early apoptotic trigger that promotesupregulation of the extrinsic pathway and increased levels of celldeath.

In our in vivo studies, we tested the oncolytic activity ofrNDV-B1/Fas and rNDV-B1 against B16 melanoma, one of themore aggressive syngeneic murine tumor models. rNDV-B1/Fasvirotherapy demonstrated an extraordinary efficacy, not onlyimproving survival time but also inducing complete tumorremission and long-term protection against tumor relapse. Asingle dose of the virus suspension was enough to unleash astrong apoptosis response in tumor cells perceptible as early as24 hours after virus administration. Supported by our in vitroobservations, the in vivo results strongly suggest that an earlyand enhanced apoptosis response might play a key role in therNDV-B1/Fas successful virotherapy. This enrichment in apo-ptotic cell death within the tumor in early stages during thetreatment and the consequent modification of the tumormicroenvironment could be boosting a better and more specificinnate immune response against the tumor and therefore

(Continued.) E, interferon response induction in infected cells. Monolayers of B16-F10 and NIH/373 cells were infected with the virus suspension at an MOI of 1PFU/cell. Total RNAs from cultured cells were isolated 8 hours after infection. Mean n-Log10 fold expression levels of cDNA from three individualbiologic samples, each measured in triplicate, were normalized to 18S rRNA levels and calibrated to mock-treated samples. mRNA expression levels for INFb,IFT1, IFR7, and OAS1 were evaluated in both cell lines (ns > 0.05). F, oncolytic capacity of rNDV-B1/Fas and rNDV-B1 viruses in syngeneic murinemelanoma tumor model. Tumor growth curves and long-term survival report. B16-F10 cells were implanted in the flank of the posterior right leg ofC57BL/6 mice. Starting on day 10 after tumor cell line injection, the animals were intratumoral treated every other day with a total of three doses of 5 � 106

PFU of rNDV-B1/Fas, rNDV-B1, or PBS for control mice. Tumor volume was monitored every 48 hours or every 24 hours when approaching theexperimental end point of 1,000 mm3, after which mice were euthanized (�� , P < 0.0001). G, long-term survival and protection against melanomarechallenge. Syngeneic melanoma tumors and treatment were performed as described in F. After 30 days of absence of tumor, B16-F10 cells werereimplanted in the flank of the opposite leg. The new development or relapse of tumors was reviewed periodically up to 6 months (�� , P < 0.05).






provide the perfect scenario to the emergence of a long-termadaptive immune response.

An improvement in survival and complete tumor remissionwas also achieved by rNDV-B1/Fas treatment of colon carcinomasyngeneicmurine tumormodel (data not shown).Melanoma andcolon carcinoma are some of the human cancers with the worstprognosis due to their metastatic capacity and resistance to

chemotherapeutic drugs in which defects on the Fas/FasL systemare known to be responsible for the tumor progression(35, 48, 49). The list of malignant tumors in which Fas/FasLdeficits have been implicated also includes pancreatic, thyroidand lung carcinomas, breast and ovarian cancer, and bloodcancers, among others (35, 50). Our results obtained in themurine model open the possibility of a new approach for the

Figure 5.rNDV-B1/Fas intratumoral treatment leads to earlier and enhanced apoptotic response and differential immune infiltration in vivo. A, histopathology. Hematoxylinand eosin staining of 50-mm-thick sections from rNDV-B1 or rNDV-B1/Fas–treated tumors. White squares note magnified areas corresponding with 1 and2 lower panels. Black scale bar, 200 mm. White scale bar, 100 mm. B, virus immunodetection. Microscopy images of tumor-treated samples showing virus infection24 hours after intratumoral administration. Sections (5-mm-thick) from rNDV-B1– or rNDV-B1/Fas–treated tumors were stained with monoclonal anti-NDVF protein (red) and Dapi (blue) for nuclear contrast. White square notes magnified area. White scale bar, 100 mm. C, apoptosis detection in fixed tumor samples.Active caspase 3 immunodetection in tumor-treated samples 24 hours after injection. Sections (5-mm-thick) from PBS, rNDV-B1, or rNDV-B1/Fas tumor–treatedsamples were stained with polyclonal anti-active caspase 3 protein (red) and hematoxylin (blue) for counterstaining. Black squares note magnified areas.Black arrows and dot square note caspase 3–positive cells. Scale bar, 200 mm. D, analysis of myeloid populations within the tumor microenvironment. B16-F10 cellswere implanted in the flank of the posterior right leg of C57BL/6 mice. Starting on day 10 after tumor cell line injection, the animals were intratumorallytreated every 24 hourswith a total of three doses of 5� 106 PFU of rNDV-B1/Fas, rNDV-B1, or PBS for control mice. Twenty-four hours after the last dose, the tumorswere removed and specifically processed for the isolation and analysis by flow cytometry of the different myeloid cells lineages. Values for each of thepopulations were expressed as a percentage with respect to the total CD45-positive cells isolated from each tumor (� , P < 0.05; �� , P < 0.005).






treatment of such aggressive tumors, providing a local therapythat combines the specificity of viral infection of cancer cells andthe enhancement of the proapoptotic capacity of NDV.

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

Authors' ContributionsConception and design: S. Cuadrado-Castano, J. Ayllon, A. García-Sastre,E. VillarDevelopment of methodology: S. Cuadrado-Castano, J. Ayllon, M. Mansour,S. Tripathi, E. VillarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Cuadrado-Castano, J. Ayllon, M. Mansour, J. dela Iglesia-Vicente, S. JordanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Cuadrado-Castano, J. Ayllon, M. Mansour, J. de laIglesia-Vicente, S. Jordan, S. Tripathi, E. VillarWriting, review, and/or revision of the manuscript: S. Cuadrado-Castano,J. Ayllon, M. Mansour, A. García-Sastre, E. VillarAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Cuadrado-CastanoStudy supervision: A. García-Sastre, E. Villar

AcknowledgmentsThe authors thank Faustino Mollinedo and Consuelo Gajate (CIC, Sala-

manca, Spain) for providing pL430-Fas plasmid,Martin Perez-Andres (assistantprofessor, Department Medicine, Serv. Cytometry, CIC-Universidad de Sala-manca) for his cooperation, and Richard Cadagan and Osman Lizardo forexcellent technical assistance. Confocal laser scanning microscopy was per-formed at the Icahn School of Medicine at Mount Sinai-Microscopy SharedResource Facility.

Grant SupportThis study was financially supported by R01AI088770 from NIAID (to A.

García-Sastre) and PI08/1813 from the Spanish Fondo de InvestigacionesSanitarias (co-financed by FEDER funds from the EU; to E. Villar). S. Cua-drado-Castano was a predoctoral fellowship holder from the Spanish JCYL(cofinanced by European Social Funds; EDU/330/2008; 2008-2012). M. Man-sour was supported by NCI 5 T32 CA 78207-13 training grant.

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

Received October 27, 2014; revised February 18, 2015; accepted March 2,2015; published online March 11, 2015.

References1. Parks GD, Lamb RA. Paramyxoviridae: the viruses and their replication. In:

Fields BN, Knipe DM, Howley PM, editors. Fields virology. 5th ed. Phila-delphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2007.p. 48.

2. Cassel WA, Garrett RE. Newcastle disease virus as an antineoplastic agent.Cancer 1965;18:863–8.

3. Sinkovics JG,Horvath JC.Newcastle disease virus (NDV): brief history of itsoncolytic strains. J Clin Virol 2000;16:1–15.

4. Zamarin D, Palese P. Oncolytic Newcastle disease virus for cancer therapy:old challenges and new directions. Future Microbiol 2012;7:347–67.

5. ZamarinD,HolmgaardRB, Subudhi SK, Park JS,MansourM, Palese P, et al.Localized oncolytic virotherapy overcomes systemic tumor resistance toimmune checkpoint blockade immunotherapy. Sci Transl Med 2014;6:226ra32.

6. LamHY, Yeap SK, RasoliM,Omar AR, Yusoff K, Suraini AA, et al. Safety andclinical usage ofNewcastle disease virus in cancer therapy. J BiomedBiotech2011;2011:718710.

7. Peeters BP, de LeeuwOS, KochG, Gielkens AL. Rescue of Newcastle diseasevirus from cloned cDNA: evidence that cleavability of the fusion protein is amajor determinant for virulence. J Virol 1999;73:5001–9.

8. Nakaya T, Cros J, Park MS, Nakaya Y, Zheng H, Sagrera A, et al. Recom-binant Newcastle disease virus as a vaccine vector. J Virol 2001;75:11868–73.

9. Vigil A, ParkMS,Martinez O, ChuaMA, Xiao S, Cros JF, et al. Use of reversegenetics to enhance the oncolytic properties of Newcastle disease virus.Cancer Res 2007;67:8285–92.

10. Zhao H, Janke M, Fournier P, Schirrmacher V. Recombinant Newcastledisease virus expressing human interleukin-2 serves as a potential candi-date for tumor therapy. Virus Res 2008;136:75–80.

11. Vigil A, Martinez O, Chua MA, Garcia-Sastre A. Recombinant Newcastledisease virus as a vaccine vector for cancer therapy. Mol Ther 2008;16:1883–90.

12. ZamarinD, Vigil A, Kelly K, Garcia-Sastre A, Fong Y. Genetically engineeredNewcastle disease virus for malignant melanoma therapy. Gene Ther2009;16:796–804.

13. Puhler F, Willuda J, Puhlmann J, Mumberg D, Romer-Oberdorfer A, BeierR. Generation of a recombinant oncolytic Newcastle disease virus andexpression of a full IgG antibody from two transgenes. Gene Ther 2008;15:371–83.

14. Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, BlagosklonnyMV, et al. Molecular definitions of cell death subroutines: recommenda-tions of the Nomenclature Committee on Cell Death 2012. Cell DeathDiffer 2012;19:107–20.

15. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol2007;35:495–516.

16. Igney FH, Krammer PH. Death and anti-death: tumour resistance toapoptosis. Nat Rev Cancer 2002;2:277–88.

17. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancerand cancer therapy. Cancer 1994;73:2013–26.

18. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancergenetics and chemotherapy. Cell 2002;108:153–64.

19. Kaufmann SH, Vaux DL. Alterations in the apoptotic machinery andtheir potential role in anticancer drug resistance. Oncogene 2003;22:7414–30.

20. Fabian Z, Csatary CM, Szeberenyi J, Csatary LK. p53-independent endo-plasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virusstrain in tumor cell lines. J Virol 2007;81:2817–30.

21. Elankumaran S, Rockemann D, Samal SK. Newcastle disease virus exertsoncolysis by both intrinsic and extrinsic caspase-dependent pathways ofcell death. J Virol 2006;80:7522–34.

22. Ravindra PV, Tiwari AK, Ratta B, Chaturvedi U, Palia SK, Chauhan RS.Newcastle disease virus-induced cytopathic effect in infected cells is causedby apoptosis. Virus Res 2009;141:13–20.

23. MansourM, Palese P, ZamarinD.Oncolytic specificity ofNewcastle diseasevirus is mediated by selectivity for apoptosis-resistant cells. J Virol2011;85:6015–23.

24. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor super-families: integrating mammalian biology. Cell 2001;104:487–501.

25. Trauth BC, Klas C, Peters AM,Matzku S,Moller P, FalkW, et al.Monoclonalantibody-mediated tumor regression by induction of apoptosis. Science1989;245:301–5.

26. Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449–56.27. Yonehara S, Ishii A, YoneharaM. A cell-killingmonoclonal antibody (anti-

Fas) to a cell surface antigen co-downregulated with the receptor of tumornecrosis factor. J Exp Med 1989;169:1747–56.

28. Siegel RM, Chan FK, Chun HJ, Lenardo MJ. The multifaceted role of Fassignaling in immune cell homeostasis and autoimmunity. Nat Immunol2000;1:469–74.

29. Schneider P, Bodmer JL, Holler N,MattmannC, Scuderi P, Terskikh A, et al.Characterization of Fas (Apo-1, CD95)-Fas ligand interaction. J Biol Chem1997;272:18827–33.

30. Schutze S, Tchikov V, Schneider-Brachert W. Regulation of TNFR1 andCD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol2008;9:655–62.

31. Zhao Y, Sui X, Ren H. From procaspase-8 to caspase-8: revisiting structuralfunctions of caspase-8. J Cell Physiol 2010;225:316–20.






32. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al. TwoCD95 (APO-1/Fas) signaling pathways. Embo J 1998;17:1675–87.

33. Takahashi T, Tanaka M, Inazawa J, Abe T, Suda T, Nagata S. Human Fasligand: gene structure, chromosomal location and species specificity. IntImmunol 1994;6:1567–74.

34. Pardo J, Bosque A, Brehm R, Wallich R, Naval J, Mullbacher A, et al.Apoptotic pathways are selectively activated by granzyme A and/or gran-zyme B in CTL-mediated target cell lysis. J Cell Biol 2004;167:457–68.

35. Muschen M, Warskulat U, Beckmann MW. Defining CD95 as a tumorsuppressor gene. J Mol Med (Berl) 2000;78:312–25.

36. Debatin KM, Krammer PH. Death receptors in chemotherapy and cancer.Oncogene 2004;23:2950–66.

37. Israelow B, Narbus CM, Sourisseau M, Evans MJ. HepG2 cells mount aneffective antiviral interferon-lambda based innate immune response tohepatitis C virus infection. Hepatology 2014;60:1170–9.

38. Park MS, Garcia-Sastre A, Cros JF, Basler CF, Palese P. Newcastle diseasevirus V protein is a determinant of host range restriction. J Virol 2003;77:9522–32.

39. Livak KJ ST. Analysis of relative gene expression data using real-timequantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8.

40. Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, et al. SPOTS:signaling protein oligomeric transduction structures are early mediators ofdeath receptor-induced apoptosis at the plasma membrane. J Cell Biol2004;167:735–44.

41. Shinoura N, Ohashi M, Yoshida Y, Kirino T, Asai A, Hashimoto M, et al.Adenovirus-mediated overexpression of Fas induces apoptosis of gliomas.Cancer Gene Ther 2000;7:224–32.

42. Gajate C, Mollinedo F. Lipid rafts and Fas/CD95 signaling in cancerchemotherapy. Recent Pat Anticancer Drug Discov 2011;6:274–83.

43. Zhuang S, Kochevar IE. Ultraviolet A radiation induces rapid apoptosis ofhuman leukemia cells by Fas ligand-independent activation of the Fasdeath pathways. Photochem Photobiol 2003;78:61–7.

44. Delmas D, Rebe C, Lacour S, Filomenko R, Athias A, Gambert P, et al.Resveratrol-induced apoptosis is associated with Fas redistribution in therafts and the formation of a death-inducing signaling complex in coloncancer cells. J Biol Chem 2003;278:41482–90.

45. Legembre P, Beneteau M, Daburon S, Moreau JF, Taupin JL. Cutting edge:SDS-stable Fas microaggregates: an early event of Fas activation occurringwith agonistic anti-Fas antibody but not with Fas ligand. J Immunol2003;171:5659–62.

46. Lee KH, Feig C, Tchikov V, Schickel R, Hallas C, Schutze S, et al. The role ofreceptor internalization in CD95 signaling. EMBO J 2006;25:1009–23.

47. RavindraPV, Tiwari AK,Ratta B, BaisMV,ChaturvediU, Palia SK, et al. Timecourse of Newcastle disease virus-induced apoptotic pathways. Virus Res2009;144:350–4.

48. Shimizu M, Yoshimoto T, Sato M, Matsuzawa A, Takeda Y. Frequency andresistance of CD95 (Fas/Apo-1) gene-transfected tumor cells to CD95-mediated apoptosis by the elimination and methylation of integratedDNA. Int J Cancer 2006;119:585–92.

49. Soengas MS, Lowe SW. Apoptosis and melanoma chemoresistance. Onco-gene 2003;22:3138–51.

50. Caldwell SA, RyanMH,McDuffie E, Abrams SI. The Fas/Fas ligand pathwayis important for optimal tumor regression in a mouse model of CTLadoptive immunotherapy of experimental CMS4 lungmetastases. J Immu-nol 2003;171:2402–12.






2015;14:1247-1258. Published OnlineFirst March 11, 2015.Mol Cancer Ther Sara Cuadrado-Castano, Juan Ayllon, Mena Mansour, et al. ModelsVirus Promotes Tumor Remission in Syngeneic Murine Cancer Enhancement of the Proapoptotic Properties of Newcastle Disease

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Enhancement of the Proapoptotic Properties of Newcastle ... › content › molcanther › 14 › 5 › 1247.full.pdfpase 8 or InSolution Caspase 9 (EMD Millipore; Ref 218840 and 218841,