JBMR

ORIGINAL ARTICLE

The Oncolytic Adenovirus D24‐RGD in Combination With Cisplatin Exerts a Potent Anti‐Osteosarcoma Activity Naiara 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 ~o‐García,2
Juan Fueyo,6 Candelaria Gomez‐Manzano,6 Wensceslao Torre,7 Fernando Lecanda,3 Ana Patin 1
and Marta M Alonso 1

Department of Department of 3 Department of 4 Department of 5 Department of 6 Department of 7 Department of 2

Medical Oncology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain Pediatrics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain Oncology, Center for Applied Medical Research (CIMA), Pamplona, Spain Orthopedics, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain Pathology, University of Texas (UT) MD Anderson Cancer Center, Madrid, Spain NeuroOncology, UT MD Anderson Cancer Center, Houston, TX, USA Thoracic Surgery, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain

ABSTRACT Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus D24‐RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that D24‐RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. 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 a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of D24‐RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of D24‐RGD for this devastating disease. © 2014 American Society for Bone and Mineral Research. KEY WORDS: OSTEOSARCOMA; ONCOLYTIC ADENOVIRUS; AUTOPHAGY

Introduction

O

steosarcoma is the most common malignant bone tumor in children and adolescents, and it is generally accepted that this neoplasm arises from primitive bone‐forming mesenchymal cells.(1) Most of osteosarcomas spontaneously occur during the first two decades of life, a period characterized by the rapid skeletal growth. The ontogeny of most osteosarcomas is characterized by alterations in the control of the cellular cycle, especially in the p53 and retinoblastoma tumor suppressor pathway.(2) Indeed, genetic studies have demonstrated that up to 80% of osteosarcomas harbor alterations in the RB1 gene or other events that result in RB1 inactivation.(3,4) Currently, standard treatment for high‐grade osteosarcoma includes preoperative and postoperative chemotherapy, and surgical

resection of the tumor while attempting to maintain maximum functionality. There are different clinical parameters associated with prognosis, but the most determinant are the development of metastasis and chemotherapy resistance. In spite of multimodal treatment, the survival fluctuates between 50% and 65%. Moreover, 20% of tumors have already metastasized at the time of diagnosis or they will during tumor treatment(1,5); the development of lung and/or bone metastasis is the most adverse prognostic factor in osteosarcoma. Thus, there is an urgent need for new targeted therapies to improve clinical outcome for metastatic patients. Oncolytic adenoviruses, designed for tumor‐ selective replication and destruction of cancer cells, represent a promising therapeutic strategy that could improve osteosarcoma prognosis.(6) Ad5‐D24‐RGD is a replication competent adenovirus that 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. Journal of Bone and Mineral Research, Vol. 29, No. 10, October 2014, pp 2287–2296 DOI: 10.1002/jbmr.2253 © 2014 American Society for Bone and Mineral Research

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for binding Rb protein), and displays enhanced infectivity due to the addition of an RGD‐4C motif in the fiber HI loop.(7,8) The RGD motif is able to interact with anb3 and anb5 integrins, which are widely expressed in neoplastic cells, including osteosarcoma cells.(9) Recent studies showed that replication‐competent oncolytic adenoviruses cause autophagic programmed cell death in tumor cells.(10) In autophagic cell death, unlike apoptotic cell death, caspases are not activated and neither DNA degradation nor nuclear fragmentation is apparent. Instead, autophagic cell death is characterized by degradation of the Golgi apparatus, polyribosomes, and endoplasmic reticulum before nuclear destruction; these organelles are preserved in apoptosis.(11,12) Therefore, it is possible that cells that are resistant to apoptosis, such as osteosarcoma cells, could be susceptible to autophagic death. One of the major obstacles to adenovirus systemic administration is the lack of antitumor effect owing to the clearance of the virus through the immune system.(13) In order to enhance the adenoviral therapeutic effect and to minimize opportunities for resistant cancer cells to emerge, combinations of drugs and adenovirus have shown promising synergistic anticancer effects.(14,15) In this study, we evaluated the therapeutic effects of D24‐RGD alone or in combination with standard therapy (cisplatin, doxorubicin, and methotrexate) in novel orthotopic and metastatic osteosarcoma models. Our results showed that treatment of osteosarcoma cells with D24‐RGD alone or with cisplatin induced a synergistic effect in vitro, mediated by autophagy. More importantly, this treatment was effective in xenograft 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 were developed at the University Clinic of Navarra, as described.(16) The 143B cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cell lines were maintained in a‐Minimum Essential Medium (a‐MEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C.

Adenovirus construction and infection Construction of D24‐RGD and viral infection have been described.(7,8)

Cell viability assay 531MII, 588M, 595M, and 678R cells were seeded at a density of 1  103 cells per well in 96‐well plates; the next day, cells were infected with D‐24‐RGD at MOIs of 0.1, 1, 5, 10, 25, 75, and 100, or with ultraviolet‐inactivated virus (UVi; 100 MOIs). In addition, cells were treated with doxorubicin, cisplatin, and methotrexate at concentrations ranging from 1  108 M to 1  103 M and where indicated cells were treated with 3‐Methyladenine (3‐MA; Sigma‐ Aldrich, St. Louis, MO, USA). Cell viability was assessed 7 days later 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 causing 50% of cells to be affected; ie, 50% survival). Chemotherapy and virus were then added in combinations, with each dose in each experiment plated in triplicate and each experiment performed

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three times. After fitting the combined dose‐response curve from a single representative experiment to a Chou‐Talalay line, Chou‐ Talalay combination indices (CIs) were calculated. Levels of interaction are defined as follows: CI > 1.1 indicates antagonism, CI between 0.9 and 1.1 indicates additive effect, CI < 0.9 indicates synergy.(19) A mean CI was calculated from data points with fraction affected (FA) >0.5. The FA range used to calculate the average CI values in the combination experiments did not include CI values of FA 650 nm) fluorescence emission from 1  104 cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur (Becton Dickinson [BD], San Jose, CA, USA) using the BD CellQuest software.

Immunoblotting For immunoblotting assays, samples at the same conditions and concentrations as described in the quantification of acidic vesicular organelles were subjected to SDS‐Tris‐glycine gel electrophoresis. Membranes were incubated with the following antibodies: E1A and actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fiber (NeoMarkers, Fremont, CA, USA), LC3 (Cell Signaling, Danvers, MA, USA), and a‐tubulin (Sigma‐Aldrich). The membranes were developed with Amersham ECL western blotting detection reagent (GE Healthcare, Pittsburgh, PA, USA).

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

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

Journal of Bone and Mineral Research

531MII osteosarcoma cells (5  105) were engrafted by injection through the tibial plateau in the primary spongiosa of both tibias of nude mice (5 mice per group; two legs) (Taconic Farms, Inc.). Twenty days after injection, animals were randomized to two groups (controls without treatment and D24‐RGD). D24‐RGD (3.8  107 plaque‐forming units [pfu]/animal) was administered intratibial 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 animals were used per each group). Seven days later animals were arbitrarily randomized to four groups (controls without treatment, D24‐RGD, cisplatin, and combination treatment D24‐RGD/cisplatin). D24‐RGD (2.5  108 pfu/animal) was intravenously administered in the tail vein, twice during the protocol. Cisplatin was given by intraperitoneal injection at 2 mg/kg  2 days per week until the end of the experimental period. Animals were sacrificed at day 40. Animals were weighed every week for the duration of the treatments.

PET analyses At the end of the experimental procedure in the orthotopic model, the effect on tumor activity was measured by positron emission tomography (PET) with the radiotracer 18 fluorodeoxyglucose (18F‐FDG). For PET procedure mice were fasted overnight but allowed to drink water ad libitum. The day of the study mice were anesthetized with 2% isoflurane in 100% O2 gas and 18F‐FDG (17.7  2.6 MBq in 80–100 mL) was injected in the tail vein. To avoid radiotracer uptake in the hindlimb muscle, 18 F‐FDG uptake was performed under continuous anesthesia for 50 minutes. PET imaging was performed in a dedicated small‐ animal Philips Mosaic tomograph (Cleveland, OH, USA), with 2‐mm resolution, 11.9‐cm axial field of view (FOV), and 12.8‐cm transaxial FOV. Anesthetized mice were placed horizontally on the PET scanner bed to perform a static acquisition (sinogram) of 15 minutes. Images were reconstructed using the 3D Ramla algorithm (a true 3D reconstruction) with two iterations and a relaxation parameter of 0.024 into a 128  128 matrix with a 1‐mm voxel size applying dead time, decay, random, and scattering corrections. For the assessment of 18F‐FDG uptake, all studies were exported and analyzed using the PMOD software (PMOD Technologies Ltd., Adliswil, Switzerland). Regions of interest (ROIs) were drawn on coronal 1‐mm‐thick small‐animal PET images on consecutive slices including entire hindlimbs. Finally, maximum standardized uptake value (SUVmax) was calculated using the formula SUV ¼ [tissue activity concentration (Bq/cm3)/injected dose (Bq)]  body weight (g).

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

Immunohistochemical analysis The paraffin‐embedded sections of the mice legs, lungs, and liver were immunostained for antibodies specific for adenoviral mouse‐hexon (Chemicon International, Inc., Temecula, CA, USA) and vimentin clone V9 (IS30; Dako Denmark A/S, Glostrup, Denmark), following conventional procedures. For immunohistochemical staining, Vectastain ABC kits (Vector Laboratories Inc., Burlingame, CA, USA) were used according the manufacturer’s instructions.

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Statistical analysis For the in vitro experiments, statistical analyses were performed using a two‐tailed Student’s t test. Data are expressed as mean  SD. These tests were used throughout the work. The SPSS 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 in vitro In order to determine whether oncolytic adenoviruses are a suitable therapy for pediatric osteosarcoma we evaluated the infection ability of a replication deficient DE1A‐RGD‐GFP adenovirus in four primary metastatic osteosarcoma cell lines (531MII, 588M, 595M, and 678R). We infected these cell lines with MOIs ranging from 10 to 100 and the amount of green cells was analyzed using flow cytometry. Our results showed that at 50 MOIs, 75% of the cells were infected in all cell lines tested (Fig. 1A). These data suggest that osteosarcoma cell lines are susceptible 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 proteins in our model (Fig. 1B; data not shown). Importantly, the virus was able to efficiently replicate in all the cell lines tested (Fig. 1C). For the 678R cell line the replication efficiency of D24‐RGD was lower and correlated with a lower infection capability (Fig. 1A). Next, we proceeded to evaluate the antitumoral effect of D24‐RGD in the same panel of osteosarcoma cell lines. The MTT assays showed that D24‐RGD induced cell death in a dose‐dependent manner. The virus displayed a potent antitumoral effect with IC50s ranging from 21 MOIs in the most sensitive cell line (588M) to 57.5 MOIs in the most resistant (678R) (Fig. 1D, Table 1). Together, these data suggest that in vitro D24‐RGD is able to replicate in and kill primary pediatric osteosarcoma cell lines.

Therapeutic effect of D24‐RGD in an orthotopic osteosarcoma 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 the treatment, we performed X‐ray analyses at days 21 and 53, and PET right before euthanasia (day 60). PET analyses revealed a remarkable decrease in the size of tumors treated with D24‐RGD compared to those of control‐treated mice (Fig. 2A, B; p ¼ 0.03). Pathological examination of hematoxylin and eosin (H&E) slides showed that in the control mice the tumors had grown such that they had even crossed the epiphysis of the tibias, giving place to transarticular 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 with D24‐RGD, as shown by staining against the adenoviral protein hexon (Fig. 2C).

The combination of D24‐RGD with chemotherapy enhanced the cytotoxic effect in osteosarcoma cell lines Next, we aimed to increase the antitumoral effect of D24‐RGD by combination with osteosarcoma standard chemotherapy; cisplatin, doxorubicin, and methotrexate (MTX). First, we determined the

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Fig. 1. D24‐RGD exerts a potent oncolytic effect in pediatric osteosarcoma cell lines. (A) Flow cytometry analyses of infectivity in osteosarcoma metastatic cell lines. The indicated cells lines were infected with a replication deficient construct expressing a modified fiber knob (AdGFP‐RGD). Data are shown as relative percentage (mean  SD) of GFP‐positive cells scored among 10.000 cells per treatment group. (B) Viral protein expression in osteosarcoma cell lines infected with D24‐RGD measured by western blot. (C) Quantification of D24‐RGD replication in the indicated cell lines. Viral titers were determined 3 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) per milliliter. 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 as the percentage (mean  SD) of cells alive after infection with D24‐RGD at the indicated multiplicities of infection (MOIs) relative to cells infected with UV‐inactivated D24‐RGD (control, equal to 100%).

IC50s of these drugs in our panel of cell lines (Fig. 3A, Supporting Fig. 1A, Supporting Table 1). As expected, cisplatin and doxorubicin displayed lower IC50s than MTX. To eliminate the possibility that the drugs could negatively interact with the adenovirus cycle we first performed Western blot analysis to evaluate the expression of the adenovirus late protein fiber in osteosarcoma cell lines concomitantly treated with the drugs and the virus (Fig. 3B, Supporting Fig. 1B). Expression levels of fiber were very similar in samples treated with D24‐RGD alone or in combination with either MTX or cisplatin. Strikingly, we did not detect fiber expression in cells that were treated with the virus in combination with doxorubicin. Further analyses using TCID50 assays confirmed the absence of D24‐RGD in the presence of doxorubicin (Fig. 3C). These data suggest that doxorubicin interferes with viral replication and thus is unsuitable to use in combination with D24‐RGD. In addition, our results uncovered MTX and cisplatin as candidates for a combination therapy with D24‐RGD. Because cisplatin displayed the best IC50s, we next quantified the antitumoral effect of cisplatin/D24‐RGD. Combination treatment resulted in a decrease of the cisplatin IC50 dose ranging from 0.5 to 1.0 logarithm (Fig. 3D, Supporting Fig. 2A, Table 1). Combination index showed that cisplatin/D24‐RGD had

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

Combination of D24‐RGD and cisplatin induces autophagy Next, we sought to elucidate the mechanism of action behind the synergy between D24‐RGD/cisplatin. Because it has been reported that cisplatin causes G2 cell‐cycle arrest,(23) first we analyzed the cell‐cycle profile of 531MII cells treated with cisplatin (1 mM) alone or in combination with D24‐RGD (10 MOI) (Supporting Fig. 3A). Treatment with cisplatin resulted in an arrest of 531MII cells in G2 (55.5%  2.7%, 72 hours; p < 0.001). As expected, treatment with D24‐RGD induced an increase of cells in S phase (61.3%  3.7% virus alone and 38.9%  4.1% virus plus cisplatin; p ¼ 0.01). However, the addition of D24‐RGD was sufficient to override the drug‐induced cell‐cycle arrest and at this time point the samples treated with virus alone or in combination with cisplatin showed more than 60% of the cells in S phase. These results suggest that the adenovirus mediates the abrogation of cell‐cycle arrest.

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Table 1. IC50 at 5 Days of the Different Drugs in Osteosarcomas Cell Lines Cell lines 531MII 588M 595M 678R

D24RGD (MOI)

CIS (mM)

D24RGD (5 MOIs)/CIS (mM)

D24RGD (10 MOIs)/CIS (mM)

32.4 23.3 40.1 57.5

1.15 5.35 7.69 1.1

0.43 0.38 0.88 0.76

0.09 0.09 0.41 0.08

IC50 ¼ 50% inhibitory concentration; MOI ¼ multiplicity of infection; CIS ¼ cisplatin.

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

combination treatment induced a process of autophagy with an increased in the number of vesicles and autophagosomes. In addition, we could observe the assembly of viral progeny. Importantly, treatment with the autophagy inhibitor 3‐MA resulted in a significant attenuation of the viral anti‐sarcoma effect (p ¼ 0.02 and p ¼ 0.01 for 3 mM and 5 mM 3‐MA, respectively, when compared with the effect of the virus alone) (Fig. 4D). Western blot analysis demonstrated that treatment with 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 when autophagy is induced. Treatment with the virus induced a decrease in the levels of p62 and an increase in the lipidation of LC3. This lipidation was attenuated after treatment with 3‐MA at the highest concentration (5 mM) (Fig. 4E). Moreover, addition of

Fig. 2. D24‐RGD anti‐sarcoma effect in orthotopic osteosarcoma model with the 531MII cell line. Tumors were developed by orthotopic injection of 500,000 531MII cells in the tibial tuberosity of female nude mice and 60 days later were sacrificed. (A) Representative images of X‐ray, PET analyses, and histologic sections (H&E) of mice mock‐treated (Control) or treated with D24‐RGD (3.8  107 plaque‐forming units [pfu]/animal). (B) Quantification of tumor burden by PET with the radiotracer 18 fluorodeoxyglucose (18F‐FDG). Maximum standardized uptake value (SUVmax) was calculated using the formula SUV ¼ [tissue activity concentration (Bq/cm3)/injected dose (Bq)]  body weight (g). Represented are the mean  SD, SUV values of the tumors of all animals 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).

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Fig. 3. Characterization of the anticancer effect of the combination of standard osteosarcoma chemotherapy and D24‐RGD. (A) Examination of cell viability after treatment with either cisplatin (CIS), doxorubicin (DOX), or methotrexate (MTX). 531MII cells were seeded at a density of 1  103 cells per well in 96‐well plates. The next day, the cells were treated with the indicated drugs at a concentration ranging from 0 to 1  103 mmol/L. Cell viability was assessed 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 infected with D24‐RGD (10 MOIs) alone or in combination with doxorubicin, cisplatin or MTX (1, 1 or 100 mmol/L respectively). Cells were harvested 72 hours after the infection. Actin is shown as a loading control (representative immunoblot). (C) Quantification of the replication phenotype of D24‐RGD in combination with chemotherapy. 531MII cell line was plated and treated with D24‐RGD (10 MOIs) alone or in combination with the indicated chemotherapy (1, 1, or 100 mmol/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 with D24‐RGD. 531MII cells were seeded at a density of 1  103 cells per well in 96‐well plates and the next day were infected with D24‐RGD at an MOI of 5, or 10 or with UVi‐D24‐RGD at 10 MOI. Where indicated, cells were treated with cisplatin at a concentration ranging from 0 to 1  103 mmol/L. Cell viability was assessed 5 days later using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. IC50 is the median‐effect dose (the dose causing 50% of cells to be affected; ie, 50% survival).

3‐MA (5 mM) to the combination treatment again resulted in a significant increase in the cell viability (p < 0.01; Fig. 4F). This increase in viability corresponded with a decrease in the viral proteins fiber and E1A and in an attenuation of the conversion of LC3I to LC3II (Fig. 4G). These data suggest that the autophagic inhibitor 3‐MA interferes with the viral cycle and thus dampen its anti‐sarcoma effect. Together, our results indicate that the combination treatment triggers autophagy in osteosarcoma cell lines.

Anti‐sarcoma effect of cisplatin in combination with D24‐RGD in two metastatic sarcoma models in vivo We next evaluated the antitumor effect of D24‐RGD in combination with cisplatin in two different models of osteosarcoma lung metastasis. We used the primary cell line 531MII because the tumors in the lungs resemble the patient’s metastasis and the 143B cell line because it is a very aggressive and already proven model. Animals were treated twice with an intravenous injection of 2.5  108 pfu/animal of D24‐RGD and/or cisplatin (2 mg/kg twice per week). The end point was to evaluate

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tumor burden, but in addition to assess whether the virus was able to reach the tumor metastasis after systemic administration and, if so, to examine the replication capability of the virus. Finally, we wanted to assess overall treatment toxicity for these animals. The phenotype of 531MII and 143B lung metastasis is depicted in Fig. 5A, C, respectively. Importantly, the group of animals treated with virus plus cisplatin showed a significant reduction in tumor burden compared with single agent treatment in both models (p ¼ 0.007 and p ¼ 0.02, for 531MII and 143B, respectively; Fig. 5B, D). Next, we asked whether the virus was able to replicate in the tumor in the presence of cisplatin. Of importance, both animals treated with D24‐RGD alone or in combination with cisplatin showed hexon expression, indicating that first, the virus was able to reach the tumor metastasis after systemic administration and, second, that the viral replication capability was not compromised in the presence of cisplatin (Fig. 5E). In addition, pathological examination of the mice livers treated with either D24‐RGD alone or in combination did not show signs of toxicity such as steatosis or cirrhosis and we only could detect residual hexon staining (Fig. 5F). Mice were weighed every week for the duration of the treatment and we

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Fig. 4. Combination of D24‐RGD with cisplatin induces autophagic cell death in pediatric osteosarcoma cell lines. (A) Acidic vesicle quantification in 531MII cells treated with cisplatin (1 mmol/L) and/or D24‐RGD (10 MOIs). Cells were stained with acridine orange (1 mg/mL) 72 hours after treatment and then subjected to flow cytometric analysis. Shown is the media and standard deviation of three experiment. (B) Western blot analyses of viral and autophagic proteins. Proteins were extracted from osteosarcoma cultures at 72 hours after treatment with D24‐RGD (10 MOI) and/or with cisplatin (1 mmol/L). The level of expression of tubulin is shown as the loading control. Densitometry analyses were performed and indicated in the blot. They represent the media and standard deviation of three independent experiments. Beclin levels have been calculated normalizing with tubulin levels meanwhile LC3 lipidation represents the ratio between LC3I and LC3II. The immunoblot shown is representative of three independent experiments. (C) Representative electron micrographs 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 autophagic multivacuolar bodies in the cytoplasm and (iv) the cluster of the progenies of D24‐RGD. Representative images from 20 cells are shown. (D) Examination of cell viability after treatment with either D24‐RGD (10 MOI) alone or in combination with 3‐MA (3 or 5 mM). 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 at 72 hours after treatment with D24‐RGD (10 MOI) and/or 3‐MA (3 or 5 mM). The level of expression of GRB2 is shown as the loading control. The immunoblot shown is representative of three independent experiments. The autophagic positive control is 531MII cells previously starved for 48 hours. (F) Examination of cell viability after treatment with either CIS (1 mmol/L), D24‐RGD (10 MOI) or CIS/D24‐RGD alone or in combination with 3‐MA (5 mM). Cell viability was assessed 3 days later using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. (G) Western blot analyses of viral and autophagic proteins in the presence of 3‐MA. Proteins were extracted from osteosarcoma cultures at 72 hours after the indicated treatments with or without 3‐MA (5 mM). The level of expression of GRB2 is shown as the loading control. The immunoblot shown is representative of three independent experiments.

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Fig. 5. Antitumor effect of D24‐RGD alone or in combination with cisplatin in two lung metastatic osteosarcoma models. (A) Metastatic lesions were developed by endovenous injection of 2  106 531MII cells in the tail vein of female nude mice. Animals were randomized as þ/controls (no treatment), cisplatin (2 mg/kg  2 days/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) 400 representative H&E photomicrographs of 531MII lung metastases; (c) 200 and (d) 400 vimentin (V9 antigen) immunohistochemistry showing the presence of multiple metastatic implants in the lungs of the mice. (B) Quantification of metastatic tumor burden (531MII) in the mice lungs after treatment. Bar representation of the tumor volume relative to the total lung surface (Wilcoxon test). The values represent mean tumor volumes of all the animals within a group and measures of the tumor areas were drawn automatically with an in‐house program. (C) Metastatic lesions were developed by endovenous injection of 1  106 143B cells in the tail vein of female nude mice. Animals were randomized as described in A. (a) 100 magnification of a lung metastasis; (b) 200 magnification of the previous lesion showing the intratumoral necrosis probably a result of the huge size of the metastatic mass; (c) 200 magnification showing a tumor trapping a
blood vessel and a

bronchioles; (d) 400 magnification of a metastatic lesion showing the typical appearance of the tumors, with atypical cells and mitosis. (D) Quantification of metastatic tumor burden (143B) in the mice lungs after combination treatments. Bar representation of the tumor volume relative to the total lung surface (Wilcoxon test). The values represent mean tumor volumes of all the animals within a group and measures of the tumor areas were drawn by hand. (E) Hexon immunostaining of the lungs of the animals treated with the indicated therapies. Representative photomicrographs of control and treated animals (magnification, 100). (F) Hexon immunostaining 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 the treatment. Mice from the different groups were weighted every week during treatment. Data is shown as the median  SD within each group in each time point.

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did not observe a significant weight loss (Fig. 5G). Together, these data indicate that the combination treatment is effective against osteosarcoma lung metastasis and that the systemic administration could be a feasible strategy to deliver the virus to the metastases without significant associated toxicities.

Discussion In this study we demonstrated that the adenovirus D24‐RGD alone or coadministered with cisplatin causes a potent antitumor effect mediated by autophagic cell death in vitro and, more importantly, in relevant preclinical models of primary and lung metastatic pediatric osteosarcoma. A previous study showed that D24‐RGD was effective against osteosarcoma in vivo and in vitro.(9,14,23) However, in our work we went on to further characterize the antitumoral effect D24‐RGD alone or in combination with cisplatin in vitro and in vivo. In addition, we used for the first time an orthotopic osteosarcoma animal model. The intratibial model better recapitulates the osteosarcoma phenotype and the physical barriers that the virus would confront in a clinical scenario. Our results underscore the adenovirus capability to induce a potent antitumoral effect, even in the presence of a dense extracellular matrix. Moreover, this virus has been evaluated in two clinical phase I/II studies for patients with gynecologic malignancies(24) and in patients with recurrent gliomas (Dr. Fueyo; personal communication, Brain Tumor Center, UT MD Anderson Cancer Center, Houston, TX, USA). In both studies D24‐RGD not only proved to be safe but also displayed an anticancer effect. These results support their use for other malignancies. Oncolytic viruses offer attractive therapeutic options for the multimodal management of different neoplasms, especially in cases of local recurrence, chemoresistance, or in the presence of lung metastases. Modified viruses could be a valid option for the use in combinatorial treatment in addition to standard chemotherapy. Furthermore, with the emergence of new tumor markers for chemoresistance, the response to current treatments could be anticipated. Thus, the subset of tumors potentially refractory to conventional chemotherapy could be subjected to a combinatorial viral treatment to improve chemosensitivity. In fact, there are several ongoing preclinical studies that used a variety of oncolytic viruses,(25–28) including adenovirus,(29) to target osteosarcoma with promising results. Because combination with chemotherapy could allow for a better viral replication, lower drug concentration and dissemination in larger tumor, we investigated the effect of D24‐RGD with standard chemotherapy. Aberrant D24‐RGD replication has been reported in the presence of doxorubicin, depending on the cell line.(14) In this line, our results showed that in all osteosarcoma cell lines tested, combination of these two agents resulted in an impaired adenoviral replication. In contrast, cisplatin and virus not only showed a potent antitumor effect in vitro 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 and oncolysis.(11,30,31) In addition, autophagy has been proposed as a survival mechanism against anticancer‐therapy–induced distress.(32–35) In many instances, upon treatment with chemotherapeutic agents, autophagy is triggered to aid in the removal of damaged proteins and organelles, thereby conferring stress tolerance, and sustaining viability under adverse conditions.34,35 In our model, our results point toward the first option being the autophagy that might be aiding in viral replication and thus

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promoting the anti‐sarcoma effect. In fact, this increase in cell autophagy could be the reason for efficacy improvement between cisplatin and D24‐RGD. Indeed, our data showed that the combination of both agents increases autophagy as indicated by the increase in beclin expression levels, LC3 lipidation and the electron microscopy micrographs. Moreover, the addition of the autophagy inhibitor 3‐MA to the combination treatment resulted in a significant reduction of the antitumor effect. These results suggest that the antitumoral effect exerted by the combination treatment was mediated by autophagic cell death and not by a prosurvival action of autophagy. One important concern in our research design was that viral systemic administration would lead to viral clearance and hepatotoxicity. However, at the dosage tested, not only we did not detect associated toxicities (degeneration of liver tissue, and severe weight loss(36); Fig. 5F, G), but importantly, we observed viral replication and a significant tumor burden reduction in the lungs. These data illustrates the potential ability of the virus to reach the metastasis through systemic administration while maintaining a safe toxicity profile. Together, our data underscore the potential of D24‐RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of D24‐RGD for this devastating disease.

Disclosures All authors state that they have no conflicts of interest.

Acknowledgments This work was supported by the European Union (Marie Curie IRG270459 to MMA); Spanish Ministry of Health (PI10/00399 to MMA; PI10/01580 to AP‐G), Spanish Ministry of Science and Innovation (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 the development and maintenance of cell lines and the animal model; GT analyzed and generated the immunohistochemistry data; data collection: JF, CG‐M,MSJ, LS, and WT; data analysis: MMA, AP‐G, FL, NM‐V, and GT; drafting the manuscript: MMA and AP‐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 take responsibility for the integrity of the data analysis.

References 1. Gorlick R, Anderson P, Andrulis I, et al. Biology of childhood osteogenic sarcoma and potential targets for therapeutic development: 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 retinoblastoma locus in osteosarcomas. Diagn Mol Pathol. 1996;5:214–9. 4. Feugeas O, Guriec N, Babin‐Boilletot A, et al. Loss of heterozygosity of the 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 Bone Tumor Committee. Children’s Oncology Group’s 2013 blueprint for research: bone tumors. Pediatr Blood Cancer. 2013;60:1009–15.

D24‐RGD WITH CISPLATIN EXERTS A POTENT ANTI‐OSTEOSARCOMA ACTIVITY

2295

6. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nat Biotechnol. 2000;18:723–7. 7. Fueyo J, Alemany R, Gomez‐Manzano C, et al. Preclinical characterization of the antiglioma activity of a tropism‐enhanced adenovirus targeted to the retinoblastoma pathway. J Natl Cancer Inst. 2003;95:652–60. 8. Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res. 2001;7:120–6. 9. Witlox AM, Van Beusechem VW, Molenaar B, et al. Conditionally replicative adenovirus with tropism expanded towards integrins inhibits osteosarcoma tumor growth in vitro and in vivo. Clin Cancer Res. 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 basic apoptotic machinery‐independent mechanism that resembles necrosis‐like programmed cell death. J Virol. 2004;78:12243–51. 11. Ito H, Aoki H, Kuhnel F, et al. Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. J Natl Cancer Inst. 2006;98:625–36. 12. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–34. 13. Nakashima H, Kaur B, Chiocca EA. Directing systemic oncolytic viral delivery to tumors via carrier cells. Cytokine Growth Factor Rev. 2010;21:119–26. 14. Graat HC, Witlox MA, Schagen FH, et al. Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus 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 by silencing the O6‐methylguanine‐DNA methyltransferase promoter. Cancer Res. 2007;67:11499–504. 16. Patino‐Garcia A, Zalacain M, Folio C, et al. Profiling of chemonaive osteosarcoma and paired‐normal cells identifies EBF2 as a mediator of osteoprotegerin 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 Immunol Methods. 1983;65:55–63. 18. Chou TC, Talalay P. Generalized equations for the analysis of inhibitions of Michaelis‐Menten and higher‐order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors. Eur J 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. Adv Enzyme 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 chemotherapy with antimetabolites. Pharmacol Ther. 2000;87:227–53.

2296

MARTINEZ‐VELEZ ET AL.

21. Gomez‐Manzano C, Fueyo J, Kyritsis AP, et al. Characterization of p53 and 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 of autophagy in temozolomide‐induced cytotoxicity for malignant glioma cells. Cell Death Differ. 2004;11:448–57. 23. Graat HC, van Beusechem VW, Schagen FH, et al. Intravenous administration 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 a tropism‐modified conditionally replicative adenovirus for recurrent malignant gynecologic diseases. Clin Cancer Res. 2010; 16:5277–87. 25. Morton CL, Houghton PJ, Kolb EA, et al. Initial testing of the replication competent Seneca Valley virus (NTX‐010) by the pediatric preclinical testing program. Pediatr Blood Cancer. 2010; 55:295–303. 26. Li G, Kawashima H, Ogose A, et al. Efficient virotherapy for osteosarcoma by telomerase‐specific oncolytic adenovirus. J Cancer Res Clin Oncol. 2011;137:1037–51. 27. Currier MA, Eshun FK, Sholl A, et al. VEGF blockade enables oncolytic cancer 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 oncolytic herpes virus but enhances therapeutic efficacy when given after virotherapy. 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 tissue sarcomas. 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 and autophagy‐triggered caspase activity. J Virol. 2011;85:4720–9. 32. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013;368:1845–6. 33. Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov. 2012;11:709–30. 34. Mathew R, White E. Autophagy, stress, and cancer metabolism: what doesn’t kill you makes you stronger. Cold Spring Harb Symp Quant Biol. 2011;76:389–96. 35. White E, DiPaola RS. The double‐edged sword of autophagy modulation in cancer. Clin Cancer Res. 2009;15:5308–16. 36. Duncan SJ, Gordon FC, Gregory DW, et al. Infection of mouse liver by human adenovirus type 5. J Gen Virol. 1978;40:45–61.

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