The Oncolytic Adenovirus Δ24-RGD in Combination with Cisplatin Exerts a Potent Anti-Osteosarcoma Activity

The Oncolytic Adenovirus D24‐RGD in Combination WithCisplatin Exerts a Potent Anti‐Osteosarcoma ActivityNaiara Martinez‐Velez,1,2 Enric Xipell,1 Patricia Jauregui,1 Marta Zalacain,2 Lucía Marrodan,2

Carolina Zandueta,3 Beatriz Vera,1 Leire Urquiza,1 Luis Sierrasesúmaga,2 Mikel San Julián,4 Gemma Toledo,5

Juan Fueyo,6 Candelaria Gomez‐Manzano,6 Wensceslao Torre,7 Fernando Lecanda,3 Ana Pati~no‐García,2�

and Marta M Alonso1�1Department of Medical Oncology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain2Department of Pediatrics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain3Department of Oncology, Center for Applied Medical Research (CIMA), Pamplona, Spain4Department of Orthopedics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain5Department of Pathology, University of Texas (UT) MD Anderson Cancer Center, Madrid, Spain6Department of NeuroOncology, UT MD Anderson Cancer Center, Houston, TX, USA7Department of Thoracic Surgery, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain

ABSTRACTOsteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack ofresponse to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatmentstrategies that overcome both of these problems. Our purposewas to elucidate whether the use of the oncolytic adenovirusD24‐RGDalone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our resultsshowed that D24‐RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition ofcisplatin. D24‐RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin.Of importance, administration of D24‐RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in asignificant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential ofD24‐RGD to become a realistic therapeutic option for primary andmetastatic pediatric osteosarcoma. Moreover, this study warrants afuture clinical trial to evaluate the safety and efficacy of D24‐RGD for this devastating disease. © 2014 American Society for Bone andMineral Research.

KEY WORDS: OSTEOSARCOMA; ONCOLYTIC ADENOVIRUS; AUTOPHAGY

Introduction

Osteosarcoma is the most commonmalignant bone tumor inchildren and adolescents, and it is generally accepted that

this neoplasm arises from primitive bone‐forming mesenchymalcells.(1) Most of osteosarcomas spontaneously occur during thefirst two decades of life, a period characterized by the rapidskeletal growth. The ontogeny of most osteosarcomas ischaracterized by alterations in the control of the cellular cycle,especially in the p53 and retinoblastoma tumor suppressorpathway.(2) Indeed, genetic studies have demonstrated that upto 80% of osteosarcomas harbor alterations in the RB1 geneor other events that result in RB1 inactivation.(3,4) Currently,standard treatment for high‐grade osteosarcoma includespreoperative and postoperative chemotherapy, and surgical

resection of the tumor while attempting to maintain maximumfunctionality. There are different clinical parameters associatedwith prognosis, but the most determinant are the development ofmetastasis and chemotherapy resistance. In spite of multimodaltreatment, the survival fluctuates between 50% and 65%.Moreover, 20% of tumors have already metastasized at thetime of diagnosis or they will during tumor treatment(1,5); thedevelopment of lung and/or bone metastasis is the most adverseprognostic factor in osteosarcoma. Thus, there is an urgent needfor new targeted therapies to improve clinical outcome formetastatic patients. Oncolytic adenoviruses, designed for tumor‐selective replication and destruction of cancer cells, represent apromising therapeutic strategy that could improve osteosarcomaprognosis.(6) Ad5‐D24‐RGD is a replication competent adenovirusthat harbors a 24–base pair deletion in the E1A region (responsible

Received in original form December 17, 2013; revised form April 1, 2014; accepted April 10, 2014. Accepted manuscript online April 16, 2014.Address correspondence to: Marta M Alonso, PhD, Department of Medical Oncology, Clínica Universidad de Navarra, CIMA Building, Avd. Pio XII, 55 Pamplona,Spain. E‐mail: [email protected]�AP‐G and MMA contributed equally to this work as senior authors.Additional Supporting Information may be found in the online version of this article.

ORIGINAL ARTICLE JJBMR

Journal of Bone and Mineral Research, Vol. 29, No. 10, October 2014, pp 2287–2296DOI: 10.1002/jbmr.2253© 2014 American Society for Bone and Mineral Research

2287

for binding Rb protein), and displays enhanced infectivity due tothe addition of an RGD‐4C motif in the fiber HI loop.(7,8) The RGDmotif is able to interact with anb3 and anb5 integrins, which arewidely expressed in neoplastic cells, including osteosarcomacells.(9) Recent studies showed that replication‐competent onco-lytic adenoviruses cause autophagic programmed cell death intumor cells.(10) In autophagic cell death, unlike apoptotic cell death,caspases are not activated and neither DNA degradation nornuclear fragmentation is apparent. Instead, autophagic cell deathis characterized by degradation of the Golgi apparatus, poly-ribosomes, and endoplasmic reticulumbefore nuclear destruction;these organelles are preserved in apoptosis.(11,12) Therefore, itis possible that cells that are resistant to apoptosis, such asosteosarcoma cells, could be susceptible to autophagic death.

One of the major obstacles to adenovirus systemic adminis-tration is the lack of antitumor effect owing to the clearance ofthe virus through the immune system.(13) In order to enhance theadenoviral therapeutic effect and to minimize opportunities forresistant cancer cells to emerge, combinations of drugs andadenovirus have shown promising synergistic anticancereffects.(14,15) In this study, we evaluated the therapeutic effectsof D24‐RGD alone or in combination with standard therapy(cisplatin, doxorubicin, and methotrexate) in novel orthotopicand metastatic osteosarcoma models. Our results showed thattreatment of osteosarcoma cells with D24‐RGD alone or withcisplatin induced a synergistic effect in vitro, mediated byautophagy. More importantly, this treatment was effective inxenograft osteosarcoma models at tibial and lung locations,markedly reducing tumor burden.

Materials and Methods

Cell lines and culture conditions

The sarcoma cell lines 531MII, 588M, 595M, and 678R weredeveloped at the University Clinic of Navarra, as described.(16)

The 143B cell line was obtained from the American Type CultureCollection (ATCC, Manassas, VA, USA). All the cell lines weremaintained in a‐Minimum Essential Medium (a‐MEM) supple-mentedwith 10% fetal bovine serum in a humidified atmospherecontaining 5% CO2 at 37°C.

Adenovirus construction and infection

Construction of D24‐RGD and viral infection have beendescribed.(7,8)

Cell viability assay

531MII, 588M, 595M, and 678R cells were seeded at a density of1� 103 cells per well in 96‐well plates; the next day, cells wereinfected with D‐24‐RGD at MOIs of 0.1, 1, 5, 10, 25, 75, and 100, orwith ultraviolet‐inactivated virus (UVi; 100 MOIs). In addition, cellswere treated with doxorubicin, cisplatin, and methotrexate atconcentrations ranging from1� 10�8M to 1� 10�3M andwhereindicated cells were treated with 3‐Methyladenine (3‐MA; Sigma‐Aldrich, St. Louis, MO, USA). Cell viability was assessed 7 dayslater using the MTT assay (Sigma‐Aldrich), as described.(17) Dose‐response curves were analyzed using CalcuSyn Software (Biosoft,Cambridge, UK). CalcuSyn fits the dose‐response curves to Chou‐Talalay lines.(18) IC50 is the median‐effect dose (the dose causing50% of cells to be affected; ie, 50% survival). Chemotherapy andvirus were then added in combinations, with each dose in eachexperiment plated in triplicate and each experiment performed

three times. After fitting the combined dose‐response curve froma single representative experiment to a Chou‐Talalay line, Chou‐Talalay combination indices (CIs) were calculated. Levels ofinteraction are defined as follows: CI> 1.1 indicates antagonism,CI between 0.9 and 1.1 indicates additive effect, CI< 0.9 indicatessynergy.(19) A mean CI was calculated from data points withfraction affected (FA) >0.5. The FA range used to calculate theaverage CI values in the combination experiments did not includeCI values of FA<0.5, whichwas considered as not relevant growthinhibition,(20) because one aims to achieve the maximal effect ofthe combination tested on cancer cells.

Viral replication assay

Osteosarcoma cells were seeded at a density of 1� 104 cells/wellin six‐well plates and 20 hours later were infected with theD24‐RGD at an MOI of 10 and treated with doxorubicin, cisplatin,and methotrexate at concentrations of 0.1mM, 1mM, and 10mM,respectively. Three days later, cells were collected and the tissueculture infection dose replication assay (TCID50) method wasused to determine the final viral titration, as described.(7)

Cell‐cycle analysis

Cell‐cycle phase distribution was analyzed by measuring DNAcontent, as described.(21)

Quantification of acidic vesicular organelles

Autophagy was assessed in the cell lines by quantification ofacidic vesicular organelles, as described.(22) Briefly, cells weretreated with D24‐RGD (10 MOI) and/or cisplatin (1mM). Threedays later cells were stained with acridine orange (1mg/mL,15 minutes) (Sigma‐Aldrich) and analyzed by flow cytometry.Green (510–530 nm) and red (>650 nm) fluorescence emissionfrom 1� 104 cells illuminated with blue (488 nm) excitation lightwas measured with a FACSCalibur (Becton Dickinson [BD], SanJose, CA, USA) using the BD CellQuest software.

Immunoblotting

For immunoblotting assays, samples at the same conditions andconcentrations as described in the quantification of acidicvesicular organelles were subjected to SDS‐Tris‐glycine gelelectrophoresis. Membranes were incubated with the followingantibodies: E1A and actin (Santa Cruz Biotechnology, SantaCruz, CA, USA), fiber (NeoMarkers, Fremont, CA, USA), LC3 (CellSignaling, Danvers, MA, USA), and a‐tubulin (Sigma‐Aldrich). Themembranes were developed with Amersham ECL westernblotting detection reagent (GE Healthcare, Pittsburgh, PA, USA).

Transmission electron microscopy

Transmission electronmicroscopy (TEM) was employed to detectautophagy. The treated samples were fixed in 2.5% (wt/vol)glutaraldehyde in 0.1M cacodylate buffer, pH 7.4, and then theywere maintained in fixative followed by dehydration through agraded series of ethanol and embedded in Epon. Ultrathinsections (65 nm) were cut and stained with uranyl acetate andReynold’s lead citrate. The sections were examined in a Jeol 1210transmission electron microscope (Jeol Ltd., Herts, UK).

Animal studies

Ethical approval was granted by the Animal Ethical Committee ofthe University of Navarra. For the orthotopic intratibial model,

2288 MARTINEZ‐VELEZ ET AL. Journal of Bone and Mineral Research

531MII osteosarcoma cells (5� 105) were engrafted by injectionthrough the tibial plateau in the primary spongiosa of both tibiasof nude mice (5 mice per group; two legs) (Taconic Farms, Inc.).Twenty days after injection, animals were randomized to twogroups (controls without treatment and D24‐RGD). D24‐RGD(3.8� 107 plaque‐forming units [pfu]/animal) was administeredintratibial once a week during the first 3 weeks of treatment.Animals were sacrificed at day 60. For the lung metastatic model,143B and 531MII cell lines (1� 106 and 2� 106 cells, respectively)were injected through the tail vein (10 animalswere usedper eachgroup). Seven days later animals were arbitrarily randomized tofour groups (controls without treatment, D24‐RGD, cisplatin, andcombination treatment D24‐RGD/cisplatin). D24‐RGD (2.5� 108

pfu/animal) was intravenously administered in the tail vein, twiceduring the protocol. Cisplatin was given by intraperitonealinjection at 2mg/kg� 2 days per week until the end of theexperimental period. Animals were sacrificed at day 40. Animalswere weighed every week for the duration of the treatments.

PET analyses

At the end of the experimental procedure in the orthotopicmodel, the effect on tumor activity was measured by positronemission tomography (PET) with the radiotracer 18 fluorodeox-yglucose (18F‐FDG). For PET procedure mice were fastedovernight but allowed to drink water ad libitum. The day ofthe study mice were anesthetized with 2% isoflurane in 100% O2

gas and 18F‐FDG (17.7� 2.6 MBq in 80–100mL) was injected inthe tail vein. To avoid radiotracer uptake in the hindlimb muscle,18F‐FDG uptake was performed under continuous anesthesia for50 minutes. PET imaging was performed in a dedicated small‐animal Philips Mosaic tomograph (Cleveland, OH, USA), with2‐mm resolution, 11.9‐cm axial field of view (FOV), and 12.8‐cmtransaxial FOV. Anesthetized mice were placed horizontally onthe PET scanner bed to perform a static acquisition (sinogram) of15 minutes. Images were reconstructed using the 3D Ramlaalgorithm (a true 3D reconstruction) with two iterations and arelaxation parameter of 0.024 into a 128� 128 matrix with a1‐mm voxel size applying dead time, decay, random, andscattering corrections. For the assessment of 18F‐FDG uptake, allstudies were exported and analyzed using the PMOD software(PMOD Technologies Ltd., Adliswil, Switzerland). Regions ofinterest (ROIs) were drawn on coronal 1‐mm‐thick small‐animalPET images on consecutive slices including entire hindlimbs.Finally, maximum standardized uptake value (SUVmax) wascalculated using the formula SUV¼ [tissue activity concentration(Bq/cm3)/injected dose (Bq)]�body weight (g).

X‐ray analyses

X‐ray radiography was performed for the mice bearingosteosarcoma orthotopic xenografts at days 21 and 53, withmice placed on the prone position on sensitive radiographic film(MIN‐R, Eastman Kodak).

Immunohistochemical analysis

The paraffin‐embedded sections of themice legs, lungs, and liverwere immunostained for antibodies specific for adenoviralmouse‐hexon (Chemicon International, Inc., Temecula, CA, USA)and vimentin clone V9 (IS30; Dako Denmark A/S, Glostrup,Denmark), following conventional procedures. For immunohis-tochemical staining, Vectastain ABC kits (Vector Laboratories Inc.,Burlingame, CA, USA) were used according the manufacturer’sinstructions.

Statistical analysis

For the in vitro experiments, statistical analyses were performedusing a two‐tailed Student’s t test. Data are expressed asmean� SD. These tests were used throughout the work. TheSPSS v15 (Statistical Package for The Social Sciences, Chicago, IL,USA) program was used for the statistical analysis.

Results

D24‐RGD exerts a potent anti‐osteosarcoma effect invitro

In order to determine whether oncolytic adenoviruses are asuitable therapy for pediatric osteosarcoma we evaluated theinfection ability of a replication deficient DE1A‐RGD‐GFPadenovirus in four primary metastatic osteosarcoma cell lines(531MII, 588M, 595M, and 678R). We infected these cell lines withMOIs ranging from 10 to 100 and the amount of green cells wasanalyzed using flow cytometry. Our results showed that at 50MOIs, 75% of the cells were infected in all cell lines tested(Fig. 1A). These data suggest that osteosarcoma cell lines aresusceptible to infection with a tropism‐modified adenovirus.Next, we assessed the expression of early (E1A) and late (fiber)viral proteins in the four cell lines previously infected with D24‐RGD. We observed a robust expression of E1A and fiber proteinsin our model (Fig. 1B; data not shown). Importantly, the virus wasable to efficiently replicate in all the cell lines tested (Fig. 1C). Forthe 678R cell line the replication efficiency ofD24‐RGDwas lowerand correlatedwith a lower infection capability (Fig. 1A). Next, weproceeded to evaluate the antitumoral effect of D24‐RGD in thesame panel of osteosarcoma cell lines. The MTT assays showedthat D24‐RGD induced cell death in a dose‐dependent manner.The virus displayed a potent antitumoral effect with IC50s rangingfrom 21MOIs in themost sensitive cell line (588M) to 57.5MOIs inthe most resistant (678R) (Fig. 1D, Table 1). Together, these datasuggest that in vitro D24‐RGD is able to replicate in and killprimary pediatric osteosarcoma cell lines.

Therapeutic effect of D24‐RGD in an orthotopicosteosarcoma animal model

In order to test the anti‐osteosarcoma effect of D24‐RGD in vivo,we engrafted the 531MII cell line in the tibias of nude mice.Twenty days after cell injection animals were either mock treated(control) or treated with D24‐RGD. To evaluate the efficacy of thetreatment, we performed X‐ray analyses at days 21 and 53, andPET right before euthanasia (day 60). PET analyses revealed aremarkable decrease in the size of tumors treated with D24‐RGDcompared to those of control‐treated mice (Fig. 2A, B; p¼ 0.03).Pathological examination of hematoxylin and eosin (H&E) slidesshowed that in the control mice the tumors had grown such thatthey had even crossed the epiphysis of the tibias, giving place totransarticular tumors in some mice. Tumors treated with D24‐RGD showed extensive areas of necrosis (Fig. 2A). Importantly,D24‐RGD replicated efficiently in the tibias of mice treated withD24‐RGD, as shown by staining against the adenoviral proteinhexon (Fig. 2C).

The combination of D24‐RGD with chemotherapyenhanced the cytotoxic effect in osteosarcoma cell lines

Next, we aimed to increase the antitumoral effect of D24‐RGD bycombination with osteosarcoma standard chemotherapy; cisplat-in, doxorubicin, andmethotrexate (MTX). First, we determined the

Journal of Bone and Mineral Research D24‐RGD WITH CISPLATIN EXERTS A POTENT ANTI‐OSTEOSARCOMA ACTIVITY 2289

IC50s of these drugs in our panel of cell lines (Fig. 3A, SupportingFig. 1A, Supporting Table 1). As expected, cisplatin anddoxorubicin displayed lower IC50s than MTX. To eliminate thepossibility that the drugs could negatively interact with theadenovirus cycle we first performed Western blot analysis toevaluate the expression of the adenovirus late protein fiber inosteosarcoma cell lines concomitantly treatedwith the drugs andthe virus (Fig. 3B, Supporting Fig. 1B). Expression levels of fiberwere very similar in samples treated with D24‐RGD alone or incombination with either MTX or cisplatin. Strikingly, we did notdetect fiber expression in cells that were treated with the virus incombination with doxorubicin. Further analyses using TCID50

assays confirmed the absence of D24‐RGD in the presence ofdoxorubicin (Fig. 3C). These data suggest that doxorubicininterferes with viral replication and thus is unsuitable to use incombination with D24‐RGD. In addition, our results uncoveredMTX and cisplatin as candidates for a combination therapy withD24‐RGD. Because cisplatin displayed the best IC50s, we nextquantified the antitumoral effect of cisplatin/D24‐RGD. Combi-nation treatment resulted in a decrease of the cisplatin IC50 doseranging from 0.5 to 1.0 logarithm (Fig. 3D, Supporting Fig. 2A,Table 1). Combination index showed that cisplatin/D24‐RGD had

a synergistic anti‐osteosarcoma effect in all the cell lines tested(Supporting Fig. 2B).

Combination of D24‐RGD and cisplatin inducesautophagy

Next, we sought to elucidate themechanismof action behind thesynergy between D24‐RGD/cisplatin. Because it has beenreported that cisplatin causes G2 cell‐cycle arrest,(23) first weanalyzed the cell‐cycle profile of 531MII cells treated withcisplatin (1mM) alone or in combination with D24‐RGD (10 MOI)(Supporting Fig. 3A). Treatment with cisplatin resulted in anarrest of 531MII cells in G2 (55.5%� 2.7%, 72 hours; p< 0.001).As expected, treatment with D24‐RGD induced an increase ofcells in S phase (61.3%� 3.7% virus alone and 38.9%� 4.1%virus plus cisplatin; p¼ 0.01). However, the addition of D24‐RGDwas sufficient to override the drug‐induced cell‐cycle arrest andat this time point the samples treated with virus alone or incombination with cisplatin showedmore than 60% of the cells inS phase. These results suggest that the adenovirus mediates theabrogation of cell‐cycle arrest.

Fig. 1. D24‐RGD exerts a potent oncolytic effect in pediatric osteosarcoma cell lines. (A) Flow cytometry analyses of infectivity in osteosarcomametastaticcell lines. The indicated cells lines were infected with a replication deficient construct expressing a modified fiber knob (AdGFP‐RGD). Data are shown asrelative percentage (mean� SD) of GFP‐positive cells scored among 10.000 cells per treatment group. (B) Viral protein expression in osteosarcoma celllines infected with D24‐RGD measured by western blot. (C) Quantification of D24‐RGD replication in the indicated cell lines. Viral titers were determined3 days after infection at an MOI of 10 by the tissue culture infection dose‐50 (IC50) method in 293 cells and expressed as plaque‐forming units (pfu) permilliliter. Data are shown as the mean� SD of three independent experiments. (D) Cell viability analyses of D24‐RGD infected osteosarcoma cell lines.Cell viability was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays 5 days after infection. Data are shown asthe percentage (mean� SD) of cells alive after infection with D24‐RGD at the indicated multiplicities of infection (MOIs) relative to cells infected withUV‐inactivated D24‐RGD (control, equal to 100%).


Because the analysis of the cell‐cycle in 531MII cells treatedwith D24‐RGD and cisplatin did not reveal a significant increasein the percentage of cells in subG0‐G1 we assessed whether thismultimodal treatment could induce autophagy.(12) Our resultsshowed that both agents alone increased the amount of acidicvesicles (18%� 7.6% and 28%� 7% for cisplatin and D24‐RGD,respectively). Importantly, the combination of D24‐RGD andcisplatin was significantly more effective at increasing the acidicvesicles than either treatment alone (68%� 7.5%; p< 0.01)(Fig. 4A, Supporting Fig. 3B). In addition, we examined theautophagy‐related biochemical marker beclin, and the conver-sion of LC3I to LC3II. Interestingly, Western blot analysis of 531MIIcells treatedwith the combination treatment showed an increasein the expression levels of beclin and in the lipidation of LC3I toLC3II (Fig. 4B). Moreover, TEM images (Fig. 4C) showed that

combination treatment induced a process of autophagy with anincreased in the number of vesicles and autophagosomes. Inaddition, we could observe the assembly of viral progeny.Importantly, treatment with the autophagy inhibitor 3‐MAresulted in a significant attenuation of the viral anti‐sarcomaeffect (p¼ 0.02 and p¼ 0.01 for 3mM and 5mM 3‐MA,respectively, when compared with the effect of the virus alone)(Fig. 4D). Western blot analysis demonstrated that treatmentwith 3‐MA resulted in a downregulation of the viral protein fiber,suggesting a less efficient viral replication. P62 is a ubiquitin‐binding scaffold protein the levels of which decrease whenautophagy is induced. Treatment with the virus induced adecrease in the levels of p62 and an increase in the lipidation ofLC3. This lipidation was attenuated after treatment with 3‐MA atthe highest concentration (5mM) (Fig. 4E). Moreover, addition of

Table 1. IC50 at 5 Days of the Different Drugs in Osteosarcomas Cell Lines

Cell lines D24RGD (MOI) CIS (mM) D24RGD (5 MOIs)/CIS (mM) D24RGD (10 MOIs)/CIS (mM)

531MII 32.4 1.15 0.43 0.09588M 23.3 5.35 0.38 0.09595M 40.1 7.69 0.88 0.41678R 57.5 1.1 0.76 0.08

IC50¼ 50% inhibitory concentration; MOI¼multiplicity of infection; CIS¼ cisplatin.

Fig. 2. D24‐RGD anti‐sarcoma effect in orthotopic osteosarcoma model with the 531MII cell line. Tumors were developed by orthotopic injection of500,000 531MII cells in the tibial tuberosity of female nude mice and 60 days later were sacrificed. (A) Representative images of X‐ray, PET analyses, andhistologic sections (H&E) ofmicemock‐treated (Control) or treatedwithD24‐RGD (3.8� 107 plaque‐forming units [pfu]/animal). (B) Quantification of tumorburden by PET with the radiotracer 18 fluorodeoxyglucose (18F‐FDG). Maximum standardized uptake value (SUVmax) was calculated using the formulaSUV¼ [tissue activity concentration (Bq/cm3)/injected dose (Bq)]�body weight (g). Represented are the mean� SD, SUV values of the tumors of allanimals in the same group (Wilcoxon test). (C) H&E and hexon immunostaining of the tibias of animals either control‐treated or infected with D24‐RGD.Representative photomicrographs of control and treated animals (magnification, �100).


3‐MA (5mM) to the combination treatment again resulted in asignificant increase in the cell viability (p< 0.01; Fig. 4F). Thisincrease in viability corresponded with a decrease in the viralproteins fiber and E1A and in an attenuation of the conversion ofLC3I to LC3II (Fig. 4G). These data suggest that the autophagicinhibitor 3‐MA interferes with the viral cycle and thus dampen itsanti‐sarcoma effect.

Together, our results indicate that the combination treatmenttriggers autophagy in osteosarcoma cell lines.

Anti‐sarcoma effect of cisplatin in combination withD24‐RGD in two metastatic sarcoma models in vivo

We next evaluated the antitumor effect of D24‐RGD incombination with cisplatin in two different models of osteosar-coma lung metastasis. We used the primary cell line 531MIIbecause the tumors in the lungs resemble the patient’smetastasis and the 143B cell line because it is a very aggressiveand already proven model. Animals were treated twice with anintravenous injection of 2.5� 108 pfu/animal of D24‐RGD and/orcisplatin (2mg/kg twice per week). The end point was to evaluate

tumor burden, but in addition to assess whether the virus wasable to reach the tumor metastasis after systemic administrationand, if so, to examine the replication capability of the virus.Finally, we wanted to assess overall treatment toxicity for theseanimals. The phenotype of 531MII and 143B lung metastasis isdepicted in Fig. 5A, C, respectively. Importantly, the group ofanimals treated with virus plus cisplatin showed a significantreduction in tumor burden compared with single agenttreatment in both models (p¼ 0.007 and p¼ 0.02, for 531MIIand 143B, respectively; Fig. 5B, D). Next, we asked whether thevirus was able to replicate in the tumor in the presence ofcisplatin. Of importance, both animals treated with D24‐RGDalone or in combination with cisplatin showed hexon expression,indicating that first, the virus was able to reach the tumormetastasis after systemic administration and, second, that theviral replication capability was not compromised in the presenceof cisplatin (Fig. 5E). In addition, pathological examination of themice livers treated with either D24‐RGD alone or in combinationdid not show signs of toxicity such as steatosis or cirrhosis andwe only could detect residual hexon staining (Fig. 5F). Mice wereweighed every week for the duration of the treatment and we

Fig. 3. Characterization of the anticancer effect of the combination of standard osteosarcoma chemotherapy and D24‐RGD. (A) Examination of cellviability after treatment with either cisplatin (CIS), doxorubicin (DOX), or methotrexate (MTX). 531MII cells were seeded at a density of 1� 103 cells per wellin 96‐well plates. The next day, the cells were treated with the indicated drugs at a concentration ranging from 0 to 1� 103mmol/L. Cell viability wasassessed 5 days later using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. (B) Expression of the late adenoviral gene fiber.531MII cells were plated and 24 hours later infectedwithD24‐RGD (10MOIs) alone or in combinationwith doxorubicin, cisplatin orMTX (1, 1 or 100mmol/Lrespectively). Cells were harvested 72 hours after the infection. Actin is shown as a loading control (representative immunoblot). (C) Quantification of thereplication phenotype of D24‐RGD in combination with chemotherapy. 531MII cell line was plated and treated with D24‐RGD (10 MOIs) alone or incombination with the indicated chemotherapy (1, 1, or 100mmol/L, respectively). Three days after infection, cell lysates were used to infect 293 cells.Viral titers were determined by the tissue culture infection dose‐50 (IC50) method. (D) Median‐effect doses (IC50) of cisplatin alone or in combination withD24‐RGD. 531MII cells were seeded at a density of 1� 103 cells per well in 96‐well plates and the next day were infectedwithD24‐RGD at anMOI of 5, or 10or with UVi‐D24‐RGD at 10 MOI. Where indicated, cells were treated with cisplatin at a concentration ranging from 0 to 1� 103mmol/L. Cell viability wasassessed 5 days later using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) assay. IC50 is themedian‐effect dose (the dose causing 50%of cells to be affected; ie, 50% survival).


Fig. 4. Combination of D24‐RGD with cisplatin induces autophagic cell death in pediatric osteosarcoma cell lines. (A) Acidic vesicle quantification in 531MIIcells treated with cisplatin (1mmol/L) and/or D24‐RGD (10 MOIs). Cells were stained with acridine orange (1mg/mL) 72 hours after treatment and thensubjected to flow cytometric analysis. Shown is the media and standard deviation of three experiment. (B) Western blot analyses of viral and autophagicproteins. Proteinswere extracted fromosteosarcoma cultures at 72 hours after treatmentwithD24‐RGD (10MOI) and/orwith cisplatin (1mmol/L). The level ofexpression of tubulin is shown as the loading control. Densitometry analyses were performed and indicated in the blot. They represent the media andstandard deviation of three independent experiments. Beclin levels have been calculated normalizing with tubulin levels meanwhile LC3 lipidationrepresents the ratio between LC3I and LC3II. The immunoblot shown is representative of three independent experiments. (C) Representative electronmicrographs showing the ultrastructure of the control (mock‐infected); (i) and combination treatment D24‐RGD/CIS (ii, iii, iv). Note the vacuoles in the virus‐infected cells but not in the untreated cells. Close‐ups of combination treatment cell illustrated in (iii) shows the complex autophagicmultivacuolar bodies inthe cytoplasmand (iv) the cluster of the progenies ofD24‐RGD. Representative images from20 cells are shown. (D) Examinationof cell viability after treatmentwith eitherD24‐RGD (10MOI) alone or in combinationwith 3‐MA (3 or 5mM). Cell viability was assessed 3 days later using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. (E) Western blot analyses of viral and autophagic proteins. Proteins were extracted from osteosarcoma cultures at72 hours after treatment with D24‐RGD (10 MOI) and/or 3‐MA (3 or 5mM). The level of expression of GRB2 is shown as the loading control. The immunoblotshown is representative of three independent experiments. The autophagic positive control is 531MII cells previously starved for 48 hours. (F) Examination ofcell viability after treatment with either CIS (1mmol/L), D24‐RGD (10 MOI) or CIS/D24‐RGD alone or in combination with 3‐MA (5mM). Cell viability wasassessed 3 days later using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. (G) Western blot analyses of viral and autophagicproteins in the presence of 3‐MA. Proteinswere extracted fromosteosarcoma cultures at 72 hours after the indicated treatmentswithorwithout 3‐MA(5mM).The level of expression of GRB2 is shown as the loading control. The immunoblot shown is representative of three independent experiments.


Fig. 5. Antitumor effect ofD24‐RGDaloneor in combinationwith cisplatin in two lungmetastatic osteosarcomamodels. (A)Metastatic lesionsweredevelopedbyendovenous injection of 2� 106 531MII cells in the tail vein of female nude mice. Animals were randomized asþ/controls (no treatment), cisplatin (2mg/kg� 2days/week� 4 weeks), D24‐RGD (2.5� 108 pfu/week� 3 weeks), or cisplatin/D24‐RGD, and at day 40 animals were euthanized. (a) �200 and (b) �400representative H&E photomicrographs of 531MII lungmetastases; (c)�200 and (d)�400 vimentin (V9 antigen) immunohistochemistry showing the presence ofmultiplemetastatic implants in the lungsof themice. (B)Quantificationofmetastatic tumor burden (531MII) in themice lungs after treatment. Bar representationofthe tumor volume relative to the total lung surface (Wilcoxon test). The values representmean tumor volumesof all the animalswithin agroup andmeasures of thetumor areas were drawn automatically with an in‐house program. (C) Metastatic lesions were developed by endovenous injection of 1� 106 143B cells in the tailvein of female nudemice. Animals were randomized as described in A. (a)�100magnification of a lungmetastasis; (b)�200magnification of the previous lesionshowing the intratumoral necrosis probably a result of the huge size of themetastaticmass; (c)�200magnification showing a tumor trapping a �blood vessel anda ��bronchioles; (d)�400magnification of a metastatic lesion showing the typical appearance of the tumors, with atypical cells and mitosis. (D) Quantification ofmetastatic tumorburden (143B) in themice lungs after combination treatments. Bar representationof the tumor volume relative to the total lung surface (Wilcoxontest). The values representmean tumor volumesof all the animalswithin agroupandmeasuresof the tumor areasweredrawnbyhand. (E) Hexon immunostainingof the lungs of the animals treatedwith the indicated therapies. Representative photomicrographs of control and treated animals (magnification,�100). (F) Hexonimmunostaining of the livers of animals treated with the indicated therapies. Representative photomicrographs of control and treated animals (magnification,upper panel �100 and lower panel �200). (G) Body weight plotting of animals treated with either single agent or combination during the duration of thetreatment. Mice from the different groups were weighted every week during treatment. Data is shown as themedian� SDwithin each group in each time point.


did not observe a significant weight loss (Fig. 5G). Together, thesedata indicate that the combination treatment is effective againstosteosarcoma lung metastasis and that the systemic administra-tion could be a feasible strategy to deliver the virus to themetastases without significant associated toxicities.

Discussion

In this study we demonstrated that the adenovirus D24‐RGDalone or coadministered with cisplatin causes a potent antitumoreffect mediated by autophagic cell death in vitro and, moreimportantly, in relevant preclinical models of primary and lungmetastatic pediatric osteosarcoma.A previous study showed that D24‐RGD was effective against

osteosarcoma in vivo and in vitro.(9,14,23) However, in our workwewent on to further characterize the antitumoral effect D24‐RGDalone or in combination with cisplatin in vitro and in vivo. Inaddition, we used for the first time an orthotopic osteosarcomaanimal model. The intratibial model better recapitulates theosteosarcoma phenotype and the physical barriers that the viruswould confront in a clinical scenario. Our results underscore theadenovirus capability to induce a potent antitumoral effect, evenin the presence of a dense extracellular matrix. Moreover, thisvirus has been evaluated in two clinical phase I/II studies forpatients with gynecologic malignancies(24) and in patients withrecurrent gliomas (Dr. Fueyo; personal communication, BrainTumor Center, UT MD Anderson Cancer Center, Houston, TX,USA). In both studies D24‐RGD not only proved to be safe butalso displayed an anticancer effect. These results support theiruse for other malignancies.Oncolytic viruses offer attractive therapeutic options for the

multimodal management of different neoplasms, especially incases of local recurrence, chemoresistance, or in the presence oflung metastases. Modified viruses could be a valid option forthe use in combinatorial treatment in addition to standardchemotherapy. Furthermore, with the emergence of new tumormarkers for chemoresistance, the response to current treatmentscould be anticipated. Thus, the subset of tumors potentiallyrefractory to conventional chemotherapy could be subjected to acombinatorial viral treatment to improve chemosensitivity. Infact, there are several ongoing preclinical studies that used avariety of oncolytic viruses,(25–28) including adenovirus,(29) totarget osteosarcoma with promising results.Because combination with chemotherapy could allow for a

better viral replication, lower drug concentration and dissemi-nation in larger tumor, we investigated the effect of D24‐RGDwith standard chemotherapy. Aberrant D24‐RGD replicationhas been reported in the presence of doxorubicin, dependingon the cell line.(14) In this line, our results showed that in allosteosarcoma cell lines tested, combination of these two agentsresulted in an impaired adenoviral replication. In contrast,cisplatin and virus not only showed a potent antitumor effect invitro but also in the two osteosarcoma lung metastatic models.Recent studies suggest than adenovirus have the ability to

induce autophagy, which may promote virus replication andoncolysis.(11,30,31) In addition, autophagy has been proposed as asurvival mechanism against anticancer‐therapy–induced dis-tress.(32–35) In many instances, upon treatment with chemother-apeutic agents, autophagy is triggered to aid in the removal ofdamaged proteins and organelles, thereby conferring stresstolerance, and sustaining viability under adverse conditions.34,35

In our model, our results point toward the first option being theautophagy that might be aiding in viral replication and thus

promoting the anti‐sarcoma effect. In fact, this increase in cellautophagy could be the reason for efficacy improvementbetween cisplatin and D24‐RGD. Indeed, our data showed thatthe combination of both agents increases autophagy asindicated by the increase in beclin expression levels, LC3lipidation and the electron microscopy micrographs. Moreover,the addition of the autophagy inhibitor 3‐MA to the combinationtreatment resulted in a significant reduction of the antitumoreffect. These results suggest that the antitumoral effect exertedby the combination treatment was mediated by autophagic celldeath and not by a prosurvival action of autophagy.

One important concern in our research design was that viralsystemic administration would lead to viral clearance andhepatotoxicity. However, at the dosage tested, not only we didnot detect associated toxicities (degeneration of liver tissue,and severe weight loss(36); Fig. 5F, G), but importantly, weobserved viral replication and a significant tumor burdenreduction in the lungs. These data illustrates the potentialability of the virus to reach the metastasis through systemicadministration while maintaining a safe toxicity profile.

Together, our data underscore the potential of D24‐RGD tobecome a realistic therapeutic option for primary and metastaticpediatric osteosarcoma. Moreover, this study warrants a futureclinical trial to evaluate the safety and efficacy of D24‐RGD forthis devastating disease.

Disclosures

All authors state that they have no conflicts of interest.

Acknowledgments

This work was supported by the European Union (Marie CurieIRG270459 to MMA); Spanish Ministry of Health (PI10/00399 toMMA; PI10/01580 to AP‐G), Spanish Ministry of Science andInnovation (Ramo´n y Cajal contract RYC‐2009‐05571 to MMA).

Authors’ roles: Study design: MMA and AP‐G; study conduct:NM‐V, EX, PJ, MZ, LM, CZ, LU, and BV were involved in thedevelopment and maintenance of cell lines and the animalmodel; GT analyzed and generated the immunohistochemistrydata; data collection: JF, CG‐M,MSJ, LS, and WT; data analysis:MMA, AP‐G, FL, NM‐V, and GT; drafting themanuscript: MMA andAP‐G; revising manuscript content: MMA, AP‐G, NM‐V, and FL;approving the final version of the manuscript: MMA, AP‐G, NM,FL, LS, MSJ, JF, CG‐M, WT, and GT; MMA and AP‐G takeresponsibility for the integrity of the data analysis.

References

1. Gorlick R, Anderson P, Andrulis I, et al. Biology of childhoodosteogenic sarcoma and potential targets for therapeutic develop-ment: meeting summary. Clin Cancer Res. 2003;9:5442–53.

2. Mitelman F. Recurrent chromosome aberrations in cancer. Mutat Res.2000;462:247–53.

3. Belchis DA, Meece CA, Benko FA, Rogan PK, Williams RA, Gocke CD.Loss of heterozygosity and microsatellite instability at the retinoblas-toma locus in osteosarcomas. Diagn Mol Pathol. 1996;5:214–9.

4. Feugeas O, Guriec N, Babin‐Boilletot A, et al. Loss of heterozygosity ofthe RB gene is a poor prognostic factor in patients with osteosarcoma.J Clin Oncol. 1996;14:467–72.

5. Gorlick R, Janeway K, Lessnick S, Randall RL, Marina N; COG BoneTumor Committee. Children’s Oncology Group’s 2013 blueprint forresearch: bone tumors. Pediatr Blood Cancer. 2013;60:1009–15.


6. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancertherapy. Nat Biotechnol. 2000;18:723–7.

7. Fueyo J, AlemanyR, Gomez‐ManzanoC, et al. Preclinical characterizationof the antiglioma activity of a tropism‐enhanced adenovirus targeted tothe retinoblastoma pathway. J Natl Cancer Inst. 2003;95:652–60.

8. Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R. Aconditionally replicative adenovirus with enhanced infectivity showsimproved oncolytic potency. Clin Cancer Res. 2001;7:120–6.

9. Witlox AM, Van Beusechem VW, Molenaar B, et al. Conditionallyreplicative adenovirus with tropism expanded towards integrinsinhibits osteosarcoma tumor growth in vitro and in vivo. Clin CancerRes. 2004;10:61–7.

10. Abou El Hassan MA, van der Meulen‐Muileman I, Abbas S, Kruyt FA.Conditionally replicating adenoviruses kill tumor cells via a basicapoptotic machinery‐independent mechanism that resemblesnecrosis‐like programmed cell death. J Virol. 2004;78:12243–51.

11. Ito H, Aoki H, Kuhnel F, et al. Autophagic cell death of malignantglioma cells induced by a conditionally replicating adenovirus. J NatlCancer Inst. 2006;98:625–36.

12. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy incancer development and response to therapy. Nat Rev Cancer.2005;5:726–34.

13. Nakashima H, Kaur B, Chiocca EA. Directing systemic oncolytic viraldelivery to tumors via carrier cells. Cytokine Growth Factor Rev.2010;21:119–26.

14. Graat HC, Witlox MA, Schagen FH, et al. Different susceptibility ofosteosarcoma cell lines and primary cells to treatment with oncolyticadenovirus and doxorubicin or cisplatin. Br J Cancer. 2006;94:1837–44.

15. Alonso MM, Gomez‐Manzano C, Bekele BN, Yung WK, Fueyo J.Adenovirus‐based strategies overcome temozolomide resistance bysilencing the O6‐methylguanine‐DNA methyltransferase promoter.Cancer Res. 2007;67:11499–504.

16. Patino‐Garcia A, Zalacain M, Folio C, et al. Profiling of chemonaiveosteosarcoma andpaired‐normal cells identifies EBF2 as amediator ofosteoprotegerin inhibition to tumor necrosis factor‐related apoptosis‐inducing ligand‐induced apoptosis. Clin Cancer Res. 2009;15:5082–91.

17. Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J ImmunolMethods. 1983;65:55–63.

18. Chou TC, Talalay P. Generalized equations for the analysis ofinhibitions of Michaelis‐Menten and higher‐order kinetic systemswith two or more mutually exclusive and nonexclusive inhibitors. EurJ Biochem. 1981;115:207–16.

19. Chou TC, Talalay P. Quantitative analysis of dose‐effect relationships:the combined effects of multiple drugs or enzyme inhibitors. AdvEnzyme Regul. 1984;22:27–55.

20. Peters GJ, van der Wilt CL, van Moorsel CJ, Kroep JR, Bergman AM,Ackland SP. Basis for effective combination cancer chemotherapywith antimetabolites. Pharmacol Ther. 2000;87:227–53.

21. Gomez‐Manzano C, Fueyo J, Kyritsis AP, et al. Characterization of p53and p21 functional interactions in glioma cells en route to apoptosis.J Natl Cancer Inst. 1997;89:1036–44.

22. Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S. Role ofautophagy in temozolomide‐induced cytotoxicity for malignantglioma cells. Cell Death Differ. 2004;11:448–57.

23. Graat HC, van Beusechem VW, Schagen FH, et al. Intravenousadministration of the conditionally replicative adenovirus Ad5‐Delta24RGD induces regression of osteosarcoma lung metastases.Mol Cancer. 2008 Jan 23;7:9.

24. Kimball KJ, Preuss MA, Barnes MN, et al. A phase I study of atropism‐modified conditionally replicative adenovirus for recur-rent malignant gynecologic diseases. Clin Cancer Res. 2010;16:5277–87.

25. Morton CL, Houghton PJ, Kolb EA, et al. Initial testing of thereplication competent Seneca Valley virus (NTX‐010) by thepediatric preclinical testing program. Pediatr Blood Cancer. 2010;55:295–303.

26. Li G, Kawashima H, Ogose A, et al. Efficient virotherapy forosteosarcoma by telomerase‐specific oncolytic adenovirus. J CancerRes Clin Oncol. 2011;137:1037–51.

27. Currier MA, Eshun FK, Sholl A, et al. VEGF blockade enables oncolyticcancer virotherapy in part by modulating intratumoral myeloid cells.Mol Ther. 2013;21:1014–23.

28. Eshun FK, Currier MA, Gillespie RA, Fitzpatrick JL, Baird WH, Cripe TP.VEGF blockade decreases the tumor uptake of systemic oncolyticherpes virus but enhances therapeutic efficacy when given aftervirotherapy. Gene Ther. 2010;17:922–9.

29. Sasaki T, Tazawa H, Hasei J, et al. Preclinical evaluation of telomerase‐specific oncolytic virotherapy for human bone and soft tissuesarcomas. Clin Cancer Res. 2011;17:1828–38.

30. Jiang H, White EJ, Gomez‐Manzano C, Fueyo J. Adenovirus’s last trick:you say lysis, we say autophagy. Autophagy. 2008;4(1):118–20.

31. Jiang H, White EJ, Rios‐Vicil CI, Xu J, Gomez‐Manzano C, Fueyo J.Human adenovirus type 5 induces cell lysis through autophagy andautophagy‐triggered caspase activity. J Virol. 2011;85:4720–9.

32. Choi AM, Ryter SW, Levine B. Autophagy in human health anddisease. N Engl J Med. 2013;368:1845–6.

33. Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as apotential therapeutic target for diverse diseases. Nat Rev DrugDiscov. 2012;11:709–30.

34. Mathew R, White E. Autophagy, stress, and cancer metabolism: whatdoesn’t kill you makes you stronger. Cold Spring Harb Symp QuantBiol. 2011;76:389–96.

35. White E, DiPaola RS. The double‐edged sword of autophagymodulation in cancer. Clin Cancer Res. 2009;15:5308–16.

36. Duncan SJ, Gordon FC, Gregory DW, et al. Infection of mouse liver byhuman adenovirus type 5. J Gen Virol. 1978;40:45–61.


Documents

The Oncolytic Adenovirus Δ24-RGD in Combination with Cisplatin Exerts a Potent Anti-Osteosarcoma Activity