Oncolytic virotherapy for osteosarcoma using midkine ... - Nature

Cancer Gene Therapy (2014) 21, 126–132 & 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt

ORIGINAL ARTICLE

Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses M Takagi-Kimura1, T Yamano2, M Tagawa3 and S Kubo1 Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma. Cancer Gene Therapy (2014) 21, 126–132; doi:10.1038/cgt.2014.7; published online 28 February 2014 Keywords: oncolytic virotherapy; fiber-modified adenovirus; midkine promoter; osteosarcoma

INTRODUCTION Osteosarcoma is the most common primary bone tumor, mainly occurring in young children and adolescents.1,2 It is an aggressive malignant tumor that typically metastasizes to the lungs at an early phase.1,3 Despite the increase in survival rates because of improvements in chemotherapy, patients presenting with pulmonary metastases at the time of diagnosis still have a poor prognosis, and novel therapeutic paradigms are urgently needed.1–3 An emerging technology with considerable promise as a novel cancer treatment is the use of oncolytic viruses that are capable of tumor-selective replication.4,5 The primary approach to achieve tumor-selective viral replication has been to replace endogenous viral regulatory sequences with a tumor-specific promoter, such as the midkine (Mdk) promoter. Mdk is a basic heparin-binding growth factor that is a developmentally important retinoic acidresponsive protein strongly induced during mid-gestation. Mdk expression in normal human adult tissues is limited;6–9 however, Mdk is overexpressed in various human cancers6,7,10–14 and implicated in cancer development because of its mitogenic effects,15,16 promotion of angiogenesis,17 and antiapoptotic,18,19 fibrinolytic20 and transforming21 activities. We recently developed MOA5 oncolytic vectors based on human adenoviruses serotype 5 (Ad5), in which viral replication was strictly regulated by the tumor-specific Mdk promoter.22 We demonstrated that MOA5 was highly effective in malignant mesothelioma models.22 Another critical factor for the success of oncolytic virotherapy is the viral infectivity of target cancer cells. This is mainly dependent on the physical binding of adenoviral fiber knobs to their specific cellular receptors. Ad5 is commonly used as a gene transfer vector,

and uses the coxsackievirus/adenovirus receptor (CAR) as its main cellular receptor.23–26 Although CAR is ubiquitously expressed in normal epithelial cells, its expression is downregulated in many tumor types.26,27 Previous studies27–29 suggest that tumor progression and aggressiveness correlates with suppressed CAR expression, with this limitation exacerbated in advanced cancers. Downregulation of CAR expression in tumors therefore represents a significant obstacle in the use of Ad5 vectors for oncolytic virotherapy. In contrast, adenovirus serotype 35 (Ad35) uses the complementregulating protein CD46 as its cellular receptor.30 CD46 is expressed at low levels on all nucleated cells, but its expression is upregulated in cancer cells.31–36 We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from Ad35; the resulting vector was designated MOA35. MOA35 has increased viral infectivity and confers enhanced transduction efficiency and antitumor efficacy in malignant mesothelioma cells, especially those in which CAR expression is downregulated.37 In this study, we investigated the feasibility of these two oncolytic adenoviruses as treatments for human osteosarcoma. We evaluated Mdk promoter activity and CAR/CD46 expression in multiple osteosarcoma cell lines. Subsequently, we tested and compared the antitumor efficacies of MOA5 and MOA35 in vitro and in vivo.

MATERIALS AND METHODS Cell lines and cell culture Normal human osteoblast cells and their specific media were purchased from Lonza Japan (NHOst, Tokyo, Japan). Human dermal fibroblasts and

1 Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2Department of Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan and 3Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan. Correspondence: Dr S Kubo, Department of Genetics, Hyogo College of Medicine, 1-1 Mukogawa-cho, Hyogo, Nishinomiya 663-8501, Japan. E-mail: [email protected] Received 5 November 2013; accepted 5 February 2014; published online 28 February 2014

Oncolytic virotherapy for osteosarcoma M Takagi-Kimura et al

127 their specific media were purchased from Cell Systems Corporation (Kirkland, WA, USA). The human embryonic kidney 293 (HEK293) cell lines (Microbix, Toronto, ON, Canada)38 were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). Human osteosarcoma cell lines, HOS, MG-63 and Saos-2, were purchased from the RIKEN BioResearch Center (Tsukuba, Ibaraki, Japan), while the MNNG-HOS and U2OS human osteosarcoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. All cells were incubated at 37 1C/5% CO2.

Mdk mRNA expression analysis Expression of Mdk mRNA in cell lines was analyzed using quantitative reverse transcription polymerase chain reaction (qPCR) assays. Total RNA was extracted from semiconfluent cell cultures grown in 10-cm dishes using ISOGEN RNA extraction solution (Nippon Gene, Tokyo, Japan), and then treated with DNase to remove genomic DNA contamination. The isolated RNA was then analyzed by qPCR for expression of Mdk and glyceraldehyde3-phosphate dehydrogenase RNA using the TaqMan One-Step RT-PCR Master Mix Reagent Kit (Applied Biosystems Japan, Tokyo, Japan), following the manufacturer’s instructions. The primers and TaqMan probe for Mdk (Hs00171064_m1) and glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1) were purchased from Applied Biosystems Japan (TaqMan gene expression assays). Briefly, assays were conducted in triplicate, with 40 ng of total RNA added to the reaction mixture (TaqMan One-step RT-PCR Master Mix Reagents) containing 18 pmol of each primer and 5 pmol of probe. Reactions were amplified for one cycle of 30 min at 48 1C and 10 min at 95 1C, followed by 50 cycles of 15 s at 95 1C and 1 min at 60 1C.

Virus infectivity assays To compare the infectivity of adenoviral vectors with the wild-type Ad5 or modified Ad35 fibers, we used replication-deficient adenoviruses (Ad843 and Ad844). Cells (1 105 cells per well) were seeded in 24-well plates and infected with serially diluted Ad843 or Ad844. At 24 h after infection, EGFP titers were determined by flow cytometry and compared between Ad843 and Ad844 (Ad844/Ad843), after normalizing to baseline values in normal human osteoblasts.

In vitro cytotoxicity assays To determine the oncolytic activity of each adenovirus in various cell lines, cells were cultured on 24-well plates (1 105 cells per well) and infected at a multiplicity of infection (MOI) of 0 (control), 0.1, 1, 10, 100 or 1000. Half the supernatant was replaced with fresh medium containing 4% fetal bovine serum every day. On day 7, cells were fixed with 10% buffered formalin containing 1% crystal violet for 30 min, followed by three washes in tap water and air-dried. For quantitative analysis of the cytocidal effect of each adenovirus, three sets of 96-well tissue-culture plates containing 5 103 cells per well were infected with MOA5 or MOA35 at MOIs of 0 (control), 0.1, 1, 10 or 100. Half of the supernatant was replaced with fresh medium containing 4% fetal bovine serum every other day. On days 2, 4, 6 and 8 after infection, viable cell numbers, of triplicate cultures, were determined using the Alamar blue method according to the manufacturer’s instructions (Alamar Biosciences, Sacramento, CA, USA). Briefly, 40 ml of Alamar blue was aseptically added to cultures, which were then returned to the incubator for 3 h. Fluorescence was measured using an ARVO X4 multilabel plate reader (Perkin-Elmer, Waltham, MA, USA) with an excitation wavelength of 544 nm and an emission wavelength of 590 nm. The percentage of viable cells was determined by calculating the fluorescence of viable cells compared with wells containing no viral vectors.

Assessment of Mdk promoter activity The relative luciferase activities of promoter constructs were quantified in cell lines transfected with a reporter plasmid containing the firefly luciferase gene driven by the Mdk or SV40 promoter, together with a Renilla luciferase-expressing plasmid (pRL-TK; Promega, Madison, WI, USA). Cells were lysed at 48 h after transfection and assayed for luciferase activity using the Dual-Glo Luciferase Assay System (Promega). Data are presented as the mean±s.d. calculated from triplicate assays.

CAR and CD46 expression profile analysis Cells were incubated with mouse monoclonal anti-human antibodies against CAR (ab9891) and CD46 (ab789; Abcam, Cambridge, UK). Following this, cells were incubated with an Alexa Fluor 488-conjugated goat antimouse immunoglobulin G secondary antibody (A11001; Molecular Probes, Eugene, OR, USA), and analyzed on a FACSCalibur flow cytometer with CellQuest software version 3.1f (Becton Dickinson Japan, Tokyo, Japan).

Subcutaneous human osteosarcoma xenograft models All animal experiments were conducted according to institutional guidelines as per an approved protocol. Human osteosarcoma xenografts were established in 6- to 7-week-old female BALB/c-nu/nu (nude) mice (Charles River Japan, Yokohama, Japan) through subcutaneous inoculation of 1 106 MG-63 cells into the right dorsal flank. When tumors reached a diameter of approximately 5 mm, mice were intratumorally injected with 100 ml of phosphate-buffered saline, or 100 ml of MOA5 or MOA35 (5 107 TU) diluted in phosphate-buffered saline on days 0 and 7 (n ¼ 10 mice per group). Mice were observed closely and tumors measured two times a week. Tumor volume was calculated using the following formula: a b2 0.5, where a and b were the large and small diameters, respectively.

Statistical analysis Results are presented as the mean±s.d. The statistical significance of differences was calculated using Student’s t-test, with a P-value o0.05 considered significant.

Vector plasmids and virus production The oncolytic adenovirus vector plasmids, pMOA5 and pMOA35, have been described previously as Ad888 (pAd5-Mdk-E1) and Ad889 (pAd5/F35-MdkE1), respectively.37 Each vector contains the Mdk-E1 expression cassette, and an enhanced green fluorescent protein (EGFP) marker gene driven by the human cytomegalovirus promoter. Similarly, for control replicationdefective adenoviruses, the Mdk-E1 cassette of MOA5 and MOA35 was replaced with the phosphoglycerate kinase-1 (PGK) promoter-driven bgalactosidase (b-gal) reporter gene to create Ad843 (pAd5-PGK-b-gal) and Ad844 (pAd5/F35-PGK-b-gal), respectively. This allowed for the genome of the four adenoviruses to be adjusted such that they were the same size.37 All vectors contained an independent cytomegalovirus promoter-driven EGFP marker gene in the adenoviral backbone to enable the monitoring of the presence and spread of the adenovirus. All vectors were propagated in HEK293 cells, purified by CsCl ultracentrifugation and dialyzed against 10 mM Tris-HCl buffer (pH 8.0) with 10% glycerol. The titers of the vectors were determined by conventional limiting dilution on HEK293 cells, and were found to have similar viral yields. The particle to plaque-forming unit ratios of these vectors ranged from 16.3 to 25.1. Titers were also determined by EGFP expression, using a FACSCalibur flow cytometer, as EGFP-transducing units (TU) per milliliter. & 2014 Nature America, Inc.

RESULTS Tumor-specific Mdk mRNA expression and Mdk promoter activities in human cell lines To evaluate whether oncolytic adenoviruses with the Mdk promoter could be used for targeting osteosarcomas, we analyzed the expression levels of Mdk in human osteosarcoma cell lines by qPCR. Mdk mRNA was expressed in the five osteosarcoma cell lines tested (HOS, MNNG-HOS, MG-63, Saos-2 and U2OS), but expression levels were low in osteoblasts and fibroblasts (Figure 1a). To analyze Mdk promoter activity, luciferase reporter assays were used. The Mdk promoter was found to be significantly activated in all of the five osteosarcoma cells lines examined (Figure 1b). The level of activation was equal to or greater than that in HEK293 cells, in which Mdk promoter activity is confirmed to be activated.22 These findings suggest that Mdk is a promising tumor-specific promoter for transcriptional targeting of osteosarcoma cells, and could be used effectively to regulate replication of oncolytic adenoviruses. Cancer Gene Therapy (2014), 126 – 132


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Figure 1. Evaluation of tumor-specific midkine (Mdk) promoter activities in vitro. (a) Relative Mdk mRNA levels in cell lines were determined by quantitative reverse transcription polymerase chain reaction (qPCR). Total RNA was extracted from normal osteoblasts, fibroblasts, human embryonic kidney 293 (HEK293) and osteosarcoma cell lines (HOS, MNNG-HOS, MG-63, Saos-2 and U2OS). Primers and probes were specific for Mdk and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was used as an endogenous control for normalization. (b) Mdk promoter activities in vitro. Relative luciferase activities in various cell lines transfected with a firefly luciferase-expressing reporter plasmid driven by the Mdk or SV40 promoter, together with a Renilla luciferase-expressing plasmid. Data were normalized to baseline values in osteoblast cells, and shown as the mean±s.d. calculated from triplicate assays.

Cell surface expression of CAR and CD46 CAR and CD46 expression in the human osteosarcoma cell lines was evaluated by flow cytometry (Figure 2). CAR expression was low in osteoblasts, MNNG-HOS and MG-63 cells, whereas the other cell lines exhibited higher levels of expression. In contrast, CD46 was highly expressed in all osteosarcoma cell lines except osteoblasts. These findings suggest that CD46, as opposed to CAR, as a cellular receptor for adenoviral vectors confers more effective transduction in MNNG-HOS and MG-63 osteosarcoma cells. Infectivity of adenoviruses To investigate whether modification of the fiber enhanced infectivity, we compared the infectivity of two replication-defective Ad5 vectors. Ad843 contains a wild-type Ad5 fiber, whereas Ad844 has the modified Ad5/F35 fiber in which the CAR-binding domain of the Ad5 fiber knob was replaced with the CD46-binding domain from Ad35 (Figure 3a). Of the cells expressing high levels of CAR, HOS cells were less efficiently infected (0.49±0.11-fold) with Ad844 (Ad5/F35 fiber) compared with Ad843 (Ad5 fiber), while Saos-2 and U2OS cells were infected at the same levels for both adenoviruses (Figure 3b). In contrast, MNNG-HOS and MG-63 cells exhibited low CAR expression levels, and were infected more efficiently with Ad844 compared with Ad843 (6.73±0.96- vs 7.80±0.65-fold, respectively). Our results confirm that replacement of the CAR-binding domain in the Ad5 fiber knob with the CD46-binding domain from Ad35 confers enhanced cellular susceptibility to Ad5-based adenovirus in osteosarcoma cell lines with low levels of CAR expression. Oncolytic efficiency of Mdk promoter-regulated oncolytic adenoviruses in vitro To investigate tumor-specific cytotoxicity, normal cells and the five osteosarcoma cell lines were infected with Ad843, Ad844, Cancer Gene Therapy (2014), 126 – 132

Figure 2. Cell surface expression of coxsackievirus/adenovirus receptor (CAR) and CD46 in human osteosarcoma cells. The expression of CAR (adenovirus serotype 5 (Ad5) receptor) and CD46 (Ad35 receptor) were evaluated in human cell lines by flow cytometry. Non-shaded, experimental groups; shaded, negative controls.

MOA5 or MOA35 at various MOIs. Both replication-defective adenoviruses, Ad843 and Ad844, showed no apparent cell death at an MOI up to 1000 in all cell lines tested (Figure 4). In contrast, both MOA5 and MOA35 caused dose-dependent cytolysis in all osteosarcoma cell lines, although sensitivity varied between cell lines (Figure 4). In cells expressing high levels of CAR, MOA5 caused cell death at a lower MOI than that for MOA35 (10 vs 100) in HOS cells. Both oncolytic adenoviruses caused comparable cell death in Saos-2 and U2OS cells. By contrast, in MNNG-HOS and MG-63 cells, MOA35 caused cell death at a lower MOI than that for MOA5 (10 vs 100). MOA35 did not appear to induce cell death in osteoblasts and fibroblasts in which the Mdk promoter was not activated. These findings are consistent with the levels of infectivity of the adenoviruses seen in these cell lines (Figure 3b). & 2014 Nature America, Inc.


129 Viral dose- and time-dependent oncolysis by Mdk promoter-regulated oncolytic adenoviruses in vitro The cytotoxicity of oncolytic adenoviruses was assayed by infecting cultures with MOA5 or MOA35 at various MOIs. MOA5 and MOA35 caused no significant cytocidal effect in osteoblast

Figure 3. Modification of the adenovirus fiber, and validation of adenoviruses with or without fiber modification. (a) Structure of adenovirus fibers. The wild-type adenovirus serotype 5 (Ad5) fiber, and Ad5/F35 modified fiber in which the coxsackievirus/adenovirus receptor (CAR)-binding domain of the Ad5 fiber knob was replaced with the CD46-binding domain from Ad35. (b) Infectivity of adenoviruses with the wild-type Ad5 fiber or the modified Ad5/ F35 fiber. Cell lines were infected with replication-defective adenoviruses (Ad843 with the Ad5 fiber, or Ad844 with the Ad5/ F35-modified fiber). At 24 h after infection, EGFP titers were determined by flow cytometry and compared. Data were normalized to baseline values in human embryonic kidney 293 (HEK293) cells, and shown as the mean±s.d. calculated from triplicates.

cells, but caused varying degrees of viral dose- and timedependent cytotoxicity in osteosarcoma cell lines (Figure 5). In the MNNG-HOS and MG-63 cells, MOA35 caused significantly more extensive cell death than MOA5. In contrast, MOA5 caused more extensive (HOS) or comparable (Saos-2 and U2OS) cell death compared with MOA35, which is consistent with the levels of infectivity of the adenoviruses and oncolytic efficiency seen in these cell lines (Figures 3 and 4).22 These findings show that the infectivity of adenoviruses with or without fiber modification critically determines the cytotoxicity of oncolytic adenoviruses in the osteosarcoma cells we investigated. Antitumor efficacy of recombinant adenoviruses in an osteosarcoma xenograft model We examined in vivo antitumor efficacy in a subcutaneous lowlevel CAR MG-63 xenograft model. Treatment with MOA5 did not cause a significant reduction in tumor volume (1109±221 mm3) by day 56 compared with phosphate-buffered saline treatment (1348±337 mm3) (Figure 6). In contrast, MOA35 treatment resulted in a significant reduction in tumor volume after day 28 (362±105 mm3 on day 56; Po0.01) compared with MOA5. These results indicate that fiber modification in Mdk promoter-regulated oncolytic adenoviruses enhances the antitumor efficiency of Mdktargeted virotherapy in vivo.

DISCUSSION The use of replication-competent viruses represents an emerging technology with the potential to achieve highly efficient gene transfer to tumors. As each successfully transduced tumor cell becomes a virus-producing cell, this could sustain further transduction events after the initial administration. Previously, we developed the oncolytic Ad5 vector, MOA5, in which viral replication was regulated by the tumor-specific Mdk promoter.22

Figure 4. Oncolytic efficiency of midkine Mdk promoter-regulated oncolytic adenoviruses in vitro. Normal human cells (osteoblasts and fibroblasts), and the five human osteosarcoma cell lines were infected with Ad843, Ad844, MOA5 or MOA35 at multiplicity of infection (MOIs) ranging from 0.1 to 1000. Crystal violet staining of viable cells was used to evaluate oncolytic activity at 7 days after infection. & 2014 Nature America, Inc.

Cancer Gene Therapy (2014), 126 – 132


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Figure 5. Viral dose- and time-dependent oncolysis by midkine (Mdk) promoter-regulated oncolytic adenoviruses in vitro. Cells were infected with MOA5 or MOA35, at multiplicities of infection from 0.1 to 100. On the indicated days, the number of surviving cells were determined using Alamar blue. Data shown are the mean±s.d. calculated from triplicates.

Figure 6. In vivo antitumor effects of oncolytic adenoviruses in an MG-63 osteosarcoma xenograft model. MG-63 tumors were subcutaneously grown in nude mice and injected with MOA5, MOA35 or phosphate-buffered saline (PBS). Tumor volumes were measured two times a week, and data shown are the mean±s.d. *Po0.05.

We subsequently developed MOA35, in which the CAR-binding domain of the Ad5 fiber knob from MOA5 was replaced with the CD46-binding domain of Ad35.37 In this study, we demonstrated the effective transduction, along with the in vitro and in vivo therapeutic efficacy, of these Mdk promoter-regulated oncolytic adenoviruses with or without fiber modification in human osteosarcoma models. To achieve tumor-selective viral replication of MOA5 or MOA35, Mdk promoter activity is vital. We found that Mdk promoter activity was significantly activated in human osteosarcoma cell lines (Figure 1b). However, the relative promoter activities in the osteosarcoma cells were approximately 6- to 15-fold higher than that in normal human osteoblast cells. Our findings from a Cancer Gene Therapy (2014), 126 – 132

previous study showed that Mdk promoter activity was highly activated in malignant mesothelioma cells, around 29- to 3013fold higher than that in normal mesothelial cells. Despite this small difference in Mdk activity between human osteosarcoma and normal cells, the Mdk promoter-regulated oncolytic adenoviruses caused tumor-specific cytotoxicity in human osteosarcoma cells without affecting normal cells (Figures 4 and 5), suggesting the involvement of anti-viral responses in normal cells.39 Our results indicate that the Mdk promoter confers tumor-selective transcriptional targeting in oncolytic adenoviruses, in particular for human osteosarcomas. Another critical factor for the replicative lytic spread of oncolytic viruses in solid tumors is viral infectivity of target tumor cells. This mainly depends on the physical binding of the adenoviral fiber knob to specific cellular receptors. We evaluated the expression levels of the CAR (Ad5 receptor) and CD46 (Ad35 receptor) receptors for adenoviruses in five human osteosarcoma cell lines. Very low levels of CAR expression were observed in MNNG-HOS and MG-63 cells, whereas the other osteosarcoma cell lines exhibited high expression levels. CD46 was highly expressed in all osteosarcoma cell lines. In HOS cells (high levels of CAR expression), MOA5 caused cell death more extensively than MOA35, while both oncolytic adenoviruses caused comparable levels of cell death in Saos-2 and U2OS cells (Figures 4 and 5). In the MNNG-HOS and MG-63 cells (low levels of CAR expression), MOA35 caused greater levels of cell death than MOA5. These findings are consistent with the expression profile of CAR/CD46 (Figure 2), and also with the level of infectivity for these adenoviruses in these cell lines (Figure 3b). Our results indicate that the evaluation of CAR/CD46 and Mdk expression in tumor specimens is recommended in clinical situations for selecting suitable oncolytic adenoviruses. MOA5 and MOA35 use different cellular receptors, allowing for tailored virotherapy based on CAR and CD46 receptor expression levels, and facilitating more effective treatments against cancers with upregulated Mdk expression. Three kinds of oncolytic adenoviruses have been reported for the treatment of human osteosarcoma. The first was the oncolytic adenovirus Ad5-D24RGD.40–43 Ad5-D24RGD contains a mutant E1A that cannot bind to the retinoblastoma protein, limiting replication of this virus in cancer cells with constitutively active & 2014 Nature America, Inc.


131 E2F. In addition, the virus is unable to replicate in non-dividing normal cells with functional retinoblastoma protein.44 Ad5-D24RGD also carries a cyclic Arg-Gly-Asp (RGD-4C) integrin-binding motif in its fiber knob domain chimeric fibers to circumvent CAR deficiency, and to enhance integrin binding for effective targeting of adenoviruses to osteosarcoma cells that express low levels of CAR and high levels of integrins.45 The second oncolytic adenovirus reported contains a human telomerase reverse transcriptase promoter and wild-type Ad5 fiber,46–48 or a modified fiber with the RGD integrin-binding motif in the fiber knob.49 The third virus is an oncolytic Ad5 vector without fiber modification and driven by a survivin promoter, which is activated in human osteosarcoma cells.50 Our study is the first to demonstrate tumor-selective cytotoxicity and antitumor effects of Mdk promoter-regulated oncolytic adenoviruses. To the best of our knowledge, we are also the first group to evaluate the effects of Ad35 fiber modification in adenoviruses on transduction and therapeutic efficacy in experimental human osteosarcoma models. To evaluate the in vivo therapeutic efficacy of MOA5 and MOA35 in low CAR-expressing MG-63 osteosarcoma cells, in the present study, we tested a reduced amount of the oncolytic adenoviruses for intratumoral administration in subcutaneously xenografted models. The reduced viral amount is based on our previous observations that low-dose administration (two injections of 5 107 TU; total 1 108 TU) serves to make the difference of therapeutic efficacy of MOA5 and MOA35 more evident in subcutaneous tumor models of low CAR-expressing MSTO-211 H mesothelioma cells,37 in which high-dose administration (two injections of 5 108 TU; total 1 109 TU) achieved complete tumor regression in combination with suicide gene therapy.22 Therefore, further enhancement of therapeutic efficacy of MOA5 and MOA35 would be expected by increasing the virus amount and/or by the combination of radiation and chemotherapy,51 particularly when the oncolytic viruses are armed with suicide genes or immunomodulatory genes.5,52 In conclusion, we identified that tumor-specific Mdk is highly useful in targeting oncolytic adenoviruses to osteosarcoma. In addition, we demonstrated that oncolytic effects of the Mdk promoter-regulated oncolytic adenoviruses that use different cellular receptors depend on the expression of the CAR and/or CD46 receptors. This indicates that the evaluation of CAR/CD46 and Mdk expression in tumor specimens should be conducted in clinical settings for selecting suitable oncolytic adenoviruses. Therefore, because MOA5 and MOA35 target different cellular receptors, this allows for individually tailored virotherapy against various cancers with upregulated Mdk expression, including human osteosarcoma. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank members of the Joint-Use Research Facilities from Hyogo College of Medicine for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25460484 and 23591973), and a Grant-in-Aid for Researchers from Hyogo College of Medicine.

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