Bevacizumab Increases Viral Distribution in Human Anaplastic ...

Cancer Therapy: Preclinical

Bevacizumab Increases Viral Distribution in Human Anaplastic Thyroid Carcinoma Xenografts and Enhances the Effects of E1A-Defective Adenovirus dl922-947 Silvana Libertini,1 Irma Iacuzzo,1 Giuseppe Perruolo,1 Stefania Scala,3 Caterina Ierano',3 Renato Franco,4 Gunnel Hallden,5 and Giuseppe Portella1,2

Abstract

Purpose: Anaplastic thyroid carcinoma is a prime target for innovative therapy because it represents one of the most lethal human neoplasms and is refractory to conventional treatments such as chemotherapy and radiotherapy.We have evaluated a novel therapeutic approach based on the oncolytic replication-selective adenovirus dl922-947. Experimental Design: The antitumor efficacies of the E1ADCR2 (dl922-947) and DE1B55K (dl1520) mutants were compared in human thyroid anaplastic carcinoma cells in culture and in xenografts in vivo. To enhance the effects of dl922-947, anaplastic thyroid carcinoma tumor xenografts were treated with dl922-947 in combination with bevacizumab. Results: We showed that the efficacy of dl922-947 exceeded that of dl1520 in all tested anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, bevacizumab in combination with dl922-947 significantly reduced tumor growth compared with single treatments alone. Bevacizumab treatment significantly improved viral distribution in neoplastic tissues. Conclusions: Our data showed that dl922-947 had a higher oncolytic activity compared with dl1520 in anaplastic thyroid carcinoma cell lines and might represent a better option for virotherapy of anaplastic thyroid carcinoma. Moreover, bevacizumab increased the oncolytic effects of dl922-947 by enhancing viral distribution in tumors. The results described herein encourage the use of the dl922-947 virus in combination with bevacizumab.

Thyroid neoplasia comprises tumors with different molecular and clinical features, including well-differentiated follicular and papillary carcinomas and poorly differentiated and undifferentiated anaplastic carcinomas (1). Anaplastic thyroid carcinoma constitutes 1% to 7% of all thyroid cancer cases. It arises from thyroid follicular cells with morphologic features of malignant undifferentiated neoplasms

Authors’Affiliations: 1Dipartimento di Biologia e Patologia Cellulare e Molecolare; 2 Cattedra di Patologia Clinica Universita' Federico II; 3Clinical Immunology and 4 Pathology, National Cancer Institute, Fondazione ‘‘G. Pascale,’’ Naples, Italy; and 5 Cancer Research UK Molecular Oncology Unit, Barts and the London School of Medicine and Dentistry, London, United Kingdom Received 1/24/08; revised 4/21/08; accepted 4/30/08. Grant support: Associazione Italiana per la Ricerca sul Cancro; the Italian Ministry of Instruction, University and Research; and a grant from Fondazione Italiana per la Ricerca sul Cancro (S. Libertini). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: S. Libertini and I. Iacuzzo contributed equally to this work. Presented in part at the 31st Annual Meeting of EuropeanThyroid Association, September 2-6, 2006, Naples, Italy [Iacuzzo I, Libertini S, Fiorillo M, Pacelli R, Hallden G, Portella G. ‘‘An E1A mutant adenovirus (dl922-947) enhances the effects of paclitaxel, doxorubicin and radiation in human anaplastic thyroid carcinoma cell lines’’]. Requests for reprints: Giuseppe Portella, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta' di Medicina e Chirurgia, Universita' di Napoli Federico II, Via S. Pansini 5, 80131 Naples, Italy. Phone: 39-81-746-3056; Fax: 39-81-746-3037; E-mail: portella@ unina.it. F 2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-0200

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without features of thyroid differentiation (2). At odds with papillary and follicular carcinomas, which have a long-term favorable prognosis (3) with a high survival rate (>95%), patients affected by anaplastic thyroid carcinoma have a survival time of 2 to 6 months following diagnosis (4). The major impediment to successful control of the disease is the absence of active therapies (4); therefore, novel therapeutic approaches are required. Selectively replicating oncolytic viruses represent a novel therapeutic approach. Several viruses, including adenoviruses, have been engineered for selective targeting of neoplastic cells. The most common approach is the deletion of a viral gene whose product is necessary for its replication in normal cells but expendable in cancer cells (5). The first engineered replication-competent adenoviral mutant was dl1520 (Onyx015), with a deletion of the E1B-55K gene, making this mutant unable to inhibit p53 and apoptosis. Consequently, dl1520 could replicate in cancer cells lacking the functional p53 pathway but not in normal cells (6). The dl1520 mutant was predicted to replicate in most human cancers because the p53 pathway is nonfunctional in most human neoplasias. However, E1B-55K also mediates late viral RNA nuclear export, and the absence of this gene restricts viral replication to tumor cells able to complement the viral RNA export function (7). Despite these limitations, promising antitumor activity of dl1520 in combination with chemotherapy was shown in several clinical trials (8). Recently, results from a phase III clinical trial confirmed the ability of a similar oncolytic mutant (H101), with the E1B-55kD gene deleted, to

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increase the antitumor response rate of nasopharyngeal carcinoma in combination with cisplatin (9). The treatment of advanced or recurrent anaplastic thyroid carcinoma is complex because the tumor invades the trachea and causes death by asphyxiation. Clinical trials indicate that treatment with dl1520 could control locally aggressive neoplasms (8). We have previously shown that dl1520 is active against anaplastic thyroid carcinoma cell lines and tumor xenografts; however, high multiplicities of infection (MOI) of dl1520 were used (10, 11). These previous data indicate that oncolytic viruses may be developed for the treatment of anaplastic thyroid carcinoma. However, the limited efficacy of dl1520 we observed against anaplastic thyroid carcinoma cells highlights a need for oncolytic adenoviruses with higher replication efficiency and cytotoxicity. dl922-947 is a second-generation adenoviral mutant with a 24-bp deletion in the E1A conserved region 2 (E1A-CR2; ref. 12). This region normally interacts with host cell retinoblastoma protein and is necessary for binding and inactivation of the retinoblastoma protein family. The E1A-CR2 binding to retinoblastoma protein releases E2F, enabling S-phase entry and viral DNA replication. Consequently, the dl922-947 mutant is unable to induce progression from G1 into S phase of quiescent cells. The G1-S checkpoint is critical for cell growth progression and is lost in almost all cancer cells as a result of mutations or deletions of the retinoblastoma (RB) or CDKN2A genes and amplification or overexpression of cyclin D or CDK4 genes (13, 14). Cyclin D1 overexpression has been frequently observed in anaplastic thyroid carcinoma (15, 16) and the loss of RB-1 gene functions has a pivotal role in thyroid cells transformation (17). Furthermore, high expression levels of E2F have been observed in thyroid carcinomas, indicating alterations in the retinoblastoma pathway (18). It has previously been shown that, in a range of cancer cell lines not including anaplastic thyroid carcinoma cell lines, the in vitro efficacy of dl922-947 exceeded that of adenovirus 5 wild type (Ad5wt) and dl1520 (12, 19). In the present study, we evaluated the in vitro and in vivo effects of dl922-947 in anaplastic thyroid carcinoma cell lines, showing improved response rates and higher replication levels compared with dl1520. One of the major obstacles to successful clinical applications of therapeutic strategies based on replicating oncolytic viruses is the poor distribution of viral particles throughout the tumor mass. Studies in animal models with different oncolytic viruses have invariably shown a highly heterogeneous and incomplete dissemination of the virus (20, 21); similar data have been reported in clinical studies (22). The compromised vascular supply with necrotic areas, the distorted functional properties of tumor vessels, and the elevated tumor interstitial fluid pressure may contribute to an unequal viral distribution within the tumor and thereby reduce the uptake of the oncolytic virus by malignant cells (23, 24). Administration of monoclonal antibodies to vascular endothelial growth factor (VEGF) or VEGF receptor 2 has been shown to increase the uptake and efficacy of chemotherapy in experimental tumor models and in clinical studies (25). It has been suggested that vascular remodeling or normalization of tumor vessels after antiangiogenic treatment of solid tumors improves the delivery of chemotherapeutic drugs to tumor


tissue (26). It has previously been shown that the humanized anti-VEGF monoclonal antibody (bevacizumab) lowered tumor extracellular fluid volume and interstitial fluid pressure in xenograft tumors of human anaplastic thyroid carcinoma cells (27). Therefore, we decided to evaluate the effects of a combined treatment with bevacizumab and dl922-947 targeting anaplastic thyroid carcinoma tumor xenografts. We showed a significant reduction of tumor growth in response to the combination compared with each single agent treatment. Conversely, in vitro treatment of KAT-4 cells with bevacizumab plus dl922-947 did not show any effect on cell survival or viral replication. Our data show that dl922-947 has high replication efficiency in anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, the combined treatment with bevacizumab significantly improves the effects dl922-947 against anaplastic thyroid carcinoma tumor xenografts.

Materials and Methods Preparation of adenoviruses. dl1520 is a chimeric human adenovirus (Ad2-Ad5) with a deletion between nucleotides 2,496 and 3,323 and a stop codon (C to T transition in position 2022) in the E1B region (6). The dl922-947 adenoviral mutant has a 24-bp deletion in E1A-CR2 (12). AdGFP is a nonreplicating E1-deleted adenovirus encoding green fluorescent protein (GFP). Ad5wt is a nonmutant adenovirus used as a control. Viral stocks were expanded in the human embryonic kidney cell line HEK-293, purified, and stored, as previously reported (10, 11).Virus titer was determined by plaque-forming units (pfu) on HEK-293 cells. Cell lines. ARO and FRO human thyroid anaplastic carcinoma cell lines were kindly provided by Prof. J.A. Fagin (Memorial SloanKettering Cancer Center, New York, NY). KAT-4 cell line was obtained from Dr. Ain (University of Kentucky, Lexington, KY). Cells were grown in DMEM supplemented with 10% FCS and penicillin-streptomycin. All anaplastic thyroid carcinoma cell lines used have a nonfunctional p53 gene: in ARO and KAT-4 cells a 273 Arg!His substitution was observed, whereas in BHT-101 cells a 251 Ile!Thr substitution has been reported. FRO cells express very low levels of p53 (28, 29). Viability assay. For the evaluation of the cytoxic effects of the dl922-947 virus, cells (1 103) were seeded in 96-well plates. After 24 h, the medium was replaced with medium containing increasing concentrations of dl922-947, dl1520, or Ad5wt. Twelve days later, the cells were fixed with 50% trichloroacetic acid and stained with 0.4% sulforhodamine B in 1% acetic acid (30). The bound dye was solubilized in 100 AL of 10 mmol/L unbuffered Tris solution, and the absorbance was determined at 540 nm in a microplate reader (Bio-Rad). Quantitative PCR of adenoviral genomes and analysis of adenoviral gene expression. To quantify the amount of viral genomes, 1.5 105 cells were seeded in 12-well plates. After 24 h, cells were infected with dl922-947, dl1520, and Ad5wt at concentrations of 0.1, 1, and 100 pfu. At 48 h postinfection, cell supernatant was collected and viral DNA was extracted using a QIAamp DNA mini kit and quantified by real-time PCR using the primers 5¶-GCCACCGAGACGTACTTCAGCCTG-3¶ (upstream primer) and 5¶-TTGTACGAGTACGCGGTATCCT-3¶ (downstream primer) for the amplification of a 143-bp sequence of the viral hexon gene (from 99 to 242 bp). Viral titer was determined by plaque-forming units on HEK-293 cells, and a standard curve was constructed by assaying serial dilutions of dl1520, dl922-947, and Ad5wt viruses ranging from 0.1 to 100 pfu to quantify the input dose. To analyze the expression of adenoviral genes, 1.5 105 ARO and KAT-4 cells were seeded in 12-well plates and, after 24 h, infected with 10 pfu of dl 922-947 or dl 1520. Cells were harvested at

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dl922-947 and Bevacizumab

Fig. 1. Comparison of the cell killing activity and replication of dl922-947 and dl1520 in anaplastic thyroid carcinoma cell lines. A, cytoxic effects of the dl922-947 and dl1520 viruses were evaluated on ARO, FRO, and KAT-4 anaplastic thyroid carcinoma cell lines. Cells were seeded in 96-well plates and infected at different MOIs after 24 h. Cell survival was evaluated after 12 d. The Ad5wt adenovirus was used as a control. Points, mean percentages of the untreated cells from three different experiments; bars, SD. B, real-time PCR genome equivalent analysis. Anaplastic thyroid carcinoma cells were infected with dl922-947, dl1520, and Ad5wt at different MOIs (0.1, 1, and 100 pfu). At 48 h postinfection, cell medium was collected and viral DNA was extracted and quantified. dl922-947 ^ infected cells showed significant or highly significant differences in viral replication levels with respect to dl1520-infected cells. The difference between dl922-947 and Ad5wt replication levels was always al least significant. Columns, mean of three different experiments; bars, SD.

24 h postinfection; RNA was extracted with 1 mL Trizol (Invitrogen); and DNase treatment was done. One microgram of total RNA was reverse transcribed using a SuperScript II Reverse Transcriptase Kit (Invitrogen). Expression of E1A 13 S and Penton adenoviral genes was monitored using real-time PCR with the following specific primers: E1A 13 S forward, 5¶-AATGGCCGCCAGTCTTTT-3¶, and reverse, 5¶-ACACAGGACTGTAGACAA-3¶; Penton forward, 5¶-TAACCAGTCACAGTCGCAAG-3¶, and reverse, 5¶-CCCGCGCCTTAAACTTATT-3¶. As a control, the expression of b-actin gene was monitored using the following primers: forward, 5¶-GTCAGGCAGCTCGTAGCTCT-3¶, and reverse, 5¶TCGTGCGTGACATTAAGG AG-3¶. To calculate relative expression levels, the 2-DDCt method was used. Negative controls, samples without reverse transcriptase enzyme, or cDNA template was included in every PCR run, always resulting negative (data not shown). Western blot analysis. Proteins were extracted and subjected to SDSPAGE (10% polyacrylamide) under reducing conditions. Membranes were incubated with primary antibodies against cyclin A (1:500; Santa Cruz), p53 (1:1,000; Santa Cruz), or a-actin (1:500; A2103, Sigma) for 1 h at room temperature. AdGFP infection. KAT-4 cells were treated for 24 h with bevacizumab and then infected with AdGFP at varying MOIs. At 48 h postinfection, cells were trypsinized, washed, and resuspended in 300 AL of PBS and analyzed for GFP expression using a fluorescence-activated cell sorter cytometer (DakoCytomation) and Summit version 4.3 software (DAKO). Tumorigenicity assay. All experiments were done in 6-wk-old male athymic mice (Charles-River, Italy). To evaluate the effects of dl922-947 or dl1520 on anaplastic thyroid carcinoma xenografts, 60 athymic mice (for each cell line) were inoculated s.c. with 1 106 ARO or KAT-4 cells. When tumors were clearly detectable 20 d later, the animals were divided into three groups (20 animals per group) and tumor size was evaluated. Two respective groups received 1 108 pfu of dl922-947 or dl1520 twice per week intratumorally; one group was used as the control group. Tumor diameters were measured with calipers every other day until the animals were sacrificed. Tumor volumes (V) were calculated using the formula of rotational ellipsoid: V = (A B 2)/2 (A, axial diameter; B, rotational diameter). No mouse showed signs of wasting or other visible indications of toxicity. To evaluate the effects of dl922-947 in combination with bevacizumab, KAT-4 cells (1 106) were injected into the right flank of 80 athymic mice. After 40 d, tumor volume was evaluated and the animals


were divided into four groups (20 animals per group). Two groups received bevacizumab (5 mg/kg per mouse) i.p. on days 0 and 6, and on days 2 and 4, dl922-947 (1 107 pfu) was injected in the peritumoral area in a group treated with bevacizumab and in an untreated group. This treatment was repeated for 4 wk, and tumor diameters were measured. All mice were maintained at the Dipartimento di Biologia e Patologia Animal Facility. Animal experiments have been conducted in accordance with accepted standards of animal care and in accordance with the Italian regulations for the welfare of animals used in studies on experimental neoplasia, and the study was approved by our institutional committee on animal care. Tissue preparation, confocal microscopy, and immunohistochemistry. KAT-4 cells (1 106) were injected into the right flank of 20 athymic mice, and after 20 d, when tumors were clearly detectable, animals were randomized into two groups (10 animals per group). Bevacizumab (5 mg/kg per mouse) was administered i.p. only in one group at T = 0 and T = 5. AdGFP (1 106 pfu) was injected intratumorally in both groups at T = 6. After 2 d, animals were sacrificed, and tumors were excised, snap frozen in liquid nitrogen, and cut with a cryostat in thick sections for microscopic investigation. Sections were mounted onto untreated slides and coverslipped in slow-fade antifade (Molecular Probes) for microscopic investigation. Ten sections were sampled across the entire tumor mass to ensure they were representative of the distribution of GFP-positive signal into the tumor mass. For each slice, 10 images at high resolution were sampled from the periphery to the core of the section. Images were acquired using a ZeissLSM510 Meta argon-krypton laser scanning confocal microscope, with fluorescence excitation lines at 488 nm and emission filter BP505-550, 20 and 63 oil immersion objectives (Plan Apochromat; numerical aperture, 1.4; Zeiss), pixel depth of 12 bit, fixed box sizes of 512 512 pixels, and pinhole below 1 Airy unit. Four images from each optical section were averaged to improve the signal-to-noise ratio. The color scheme used was green for GFP-labeled structures. Tumor tissue sections obtained from formalin-fixed, paraffin-embedded samples of animals treated with bevacizumab or saline solution were stained by immunohistochemistry with antibody anti-CD31, endothelial-specific antigen (clone JC/70A, BioGenex; dilution 1:15), and microvessels density was evaluated according to the Weidner method (31). Briefly, tumor sections were scanned at low magnification (40) to identify the region of the section with the highest microvascular density (neovascular

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Fig. 2. Analysis of viral and cellular gene expression on viral infection. A, expression of adenoviral genes in anaplastic thyroid carcinoma ^ infected cells. ARO and KAT-4 cells were infected with 10 pfu of dl1520 or dl922-947 per cell. Expression levels of E1A 13 S or Penton genes were measured by real-time reverse transcription-PCR at 24 h postinfection. In both cell lines, dl922-947 infection induced higher expression levels of both viral genes. Columns, mean of three different experiments; bars, SD. B, analysis of cyclin A and p53 expression levels in anaplastic thyroid carcinoma cells infected with dl922-947 or dl1520. ARO and KAT-4 cells were infected with each virus at 50 pfu per cell; cells were harvested at 9, 12, and 24 h postinfection. In dl922-947 ^ infected cells, a strong reduction in cyclin A and p53 levels was observed starting from 12 h postinfection.

‘‘hotspot’’); this area was then counted at a magnification of 200 for the microvasculature highlighted by CD31. Quantitative real-time PCR of dl922-947 and dl1520 genomes in tumor xenografts. To evaluate the genome equivalent copies of dl922947 in animals treated with bevacizumab and dl922-947, KAT-4 cells (1 106) were injected into the right flank of 10 athymic mice. After 20 d, animals were divided into two groups with similar average tumor volume: a group received bevacizumab i.p. twice (at days 0 and 5), whereas saline solution was injected in the control group. At day 10, tumor volume was evaluated in both groups (control group, 124 F 52 AL; bevacizumab group, 120 F 57 AL), and dl922-947 was injected in the peritumoral area. After 48 h, animals of both groups were sacrificed; tumors were excised; DNA was extracted; and viral replication was evaluated by real-time PCR. DNA quality was analyzed by real-time PCR of b-actin gene. Statistical analysis. Comparisons among different treatment groups in the experiments in vivo were done by the ANOVA method and the Bonferroni post hoc test using the commercial software GraphPad Prism 4. For all the in vitro experiments, comparisons among groups were made with the ANOVA method and t test.

Results Analysis of dl922-947 cytopathic effects in human anaplastic thyroid carcinoma cell lines. First, we analyzed the effects of dl922-947 on anaplastic thyroid carcinoma-derived human cell lines and compared its activity to dl1520. All anaplastic thyroid carcinoma cell lines used in the study have a nonfunctional p53 gene, and alterations of the G1 phase progression inhibitor p16INK4a were observed in ARO and in FRO cell lines (32).


In all cell lines, dl922-947 showed a strong cell killing activity. ARO and KAT-4 cells were highly sensitive to dl922947 with respect to dl1520: at 0.0001 pfu per cell of dl922-947, about 90% of the cells were killed by the virus, whereas at the same MOI, dl1520 induced the death of about 30% for ARO cells and 10% for KAT-4 cells. In FRO cells, the same MOI of dl922-947 induced about 70% cell death. Interestingly, dl922-947 and dl1520 in FRO cells showed similar degrees of cytopathic effects (Fig. 1A). Consistent with previous results, the efficacy of dl922-947 exceeded that of Ad5wt (12, 19). To determine if the increased efficacy was related to a higher viral replication rate, we quantified changes in viral genomes by real-time PCR in anaplastic thyroid carcinoma cells infected for 48 hours with different MOIs of each virus. Analysis of genome equivalent copies in ARO and FRO cells showed highly significant (P < 0.01) or significant (P < 0.05) differences, respectively, at 1 and 100 pfu per cell. Conversely, in KAT-4 cells at 0.1 pfu per cell, a significant difference (P < 0.01) was observed (Fig. 1B). Next, we evaluated the expression of adenoviral genes in ARO and KAT-4 cells infected with dl922-947 or dl1520 for 24 hours. We selected two adenoviral genes whose expression is correlated with different stages of the adenoviral life cycle: E1A 13 S, representing genes transcribed from the immediate early region, and Penton, representing genes expressed from the late region. In both cell lines, dl922-947 infection induced higher expression levels of viral genes E1A 13 and Penton (Fig. 2A).

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Cyclin A, first expressed at the G1-S transition, is a major regulator of cell cycle progression, and it is required for S-phase entry and passage through G2 (33). In human fibroblasts, the cyclin A gene is a target for transcriptional activation by E1A, and the CR2 seems to be required for the activation of cyclin A gene expression in serum-starved fibroblasts (34). Accordingly, an E1A-deficient virus would fail to induce cyclin A expression, and conversely, a mutation of the E1B gene would induce an increase in cyclin A levels. Because the dl922-947 and dl1520 viruses have deletions in the E1A and E1B genes, respectively, the induction of cyclin A in ARO and KAT-4 cells was analyzed at different time points (hours) postinfection. Cells infected with the Ad5wt were used as a control. A strong reduction in cyclin A levels in dl922-947 – infected cells was observed starting from 12 hours postinfection (hpi), whereas no differences were observed in dl1520-infected cells (Fig. 2B). Similar data have previously been reported and were suggested to be due to the failure of dl1520 to shut down the translation of early viral mRNAs (7). To evaluate the activity of the E1B protein in ARO and KAT-4 cells on infection with dl922-947, dl1520, and Ad5wt, p53 levels were analyzed at different hours postinfection. No detectable p53 expression was observed in cells infected with Ad5wt or dl922-947, whereas in dl1520-infected cells, p53 was clearly detectable (Fig. 2B). Analysis of the antineoplastic effect of dl922-947 against anaplastic thyroid carcinoma tumor xenografts and comparison with dl1520 activity. To evaluate whether dl 922-947

inhibited the growth of ARO and KAT-4 tumor xenografts, 60 athymic mice for each cell line were inoculated s.c. with 1 106 cells (ARO or KAT-4). Twenty days later, 1 108 pfu of dl922-947 or dl1520 were injected intratumorally twice per week. Starting from day 10, a highly significant decrease (P < 0.01) in tumor volume was observed in dl922-947 – treated animals bearing ARO or KAT-4 xenografts compared with untreated or dl1520-treated animals (Fig. 3, top). From day 15, the dl1520-treated animals showed a significant reduction in tumor growth of ARO and KAT-4 xenografts. The inhibition in tumor growth was maintained until the end of the experiment. In 10% of the animals treated with dl922-947, a complete eradication of the tumor xenografts was observed (data not shown). None of the treated animals suffered from any clinically apparent side effects attributable to viral administration. Next, determination of genome equivalent copies in the ARO and KAT-4 xenografts by real-time PCR showed that the dl922947 – infected tumors had a higher viral DNA content than the corresponding dl1520-infected tumors (P < 0.01; Fig. 3, bottom). Effects of bevacizumab in combination with dl922-947 in vivo. It has been suggested that vascular remodeling or normalization of tumor vessels induced by antiangiogenic treatment could improve the delivery of drugs to tumor cells. Therefore, we analyzed the effects of bevacizumab in combination with dl922-947 in KAT-4 tumor xenografts. For this experiment, larger tumors were used because the vascular supply would more likely be compromised, leading to

Fig. 3. Tumor growth inhibition by dl922-947 and comparison with dl1520. A, tumor xenografts were induced by inoculating ARO or KAT-4 cells s.c. into athymic mice. After 20 d, animals were randomized and treated with dl922-947 or dl1520. The control group was injected with saline solution. dl922-947 induced a strong reduction in tumor growth, exceeding that of dl1520. From day 10, a highly significant decrease in tumor volume was observed in dl922-947 ^ treated animals with respect to the animals treated with dl1520. The differences in tumor growth were maintained until the end of the experiment. B, real-time PCR analysis of the genome equivalent copies. Animals bearing ARO or KAT-4 xenografts were injected in the peritumoral area with dl1520 or dl922-947 (5 107 pfu), and after 48 h, tumors were excised. DNA extraction was done starting from equal amounts of tumor (500 mg), and viral replication was evaluated by real-time PCR. Columns, mean of three different experiments; bars, SD.


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Fig. 4. Analysis of the effects of the bevacizumab in combination with dl922-947. KAT-4 cells were injected s.c. into athymic mice; animals were randomized after 40 d into four groups with similar tumor size; and two groups received bevacizumab i.p. every 5th day. dl922-947 was injected twice per week in a group treated with bevacizumab and in an untreated group. From day 17, a significant difference (P < 0.05) was observed between the control group and the group receiving the combined treatment. No significant differences were observed between the untreated control and dl922-947 treatment and between control and bevacizumab treatment. Furthermore, no statistically significant differences were observed between bevacizumab treatment and dl922-947 treatment. Tumor growth is expressed as a percentage of volume observed at T = 0.

the generation of hypoxic and necrotic areas. To better evaluate the effects of the combined treatment, a lower viral dose of dl922-947 (1 107 pfu) was used in these studies. Starting from T = 17, a significant difference (P < 0.05) was observed between the control group and the group receiving the combined treatment. From T = 20, the differences became highly significant (P < 0.01) up to the end of the treatment (Fig. 4). No statistically significant differences were observed between the bevacizumab treatment group and the control group up to the end of the experiment. Effects of bevacizumab in combination with dl922-947 in vitro. Next, we evaluated the effects of bevacizumab in combination with dl922-947 in vitro. KAT-4 cells were treated with bevacizumab for 48 hours before infection with dl922947. No significant differences were observed in cell survival and viral replication (Fig. 5A and B). Moreover, KAT-4 cells pretreated with bevacizumab for 48 hours and then infected with AdGFP at different MOIs showed no increase in GFP expression (Fig. 5C) or the number of positive cells (data not shown). These data suggest that the effects observed in vivo are not due to a direct activity of bevacizumab on KAT-4 cells or on viral uptake and replication. Viral distribution in bevacizumab-treated anaplastic thyroid carcinoma tumor xenografts. To evaluate the effects of bevacizumab on viral distribution, KAT-4 cells (1 106) were inoculated into 20 athymic mice, and after 20 days (T = 0), average tumor volumes were evaluated (83 F 32 AL) and animals were randomized into two groups. The groups received bevacizumab or saline solution, respectively, i.p. at T = 0 and T = 5. The average tumor volumes were evaluated at T = 6 (control group, 107 F 42 AL; bevacizumab group, 109 F 50 AL). AdGFP (1 106 pfu) was injected intratumorally in both groups. After 2 days, animals were sacrificed; tumors were excised; and GFP expression and distribution were evaluated by confocal microscopy analysis. In control tumors, a faint and localized fluorescence signal was observed, whereas in bevacizumab-treated tumors, a diffused and highly intense fluorescence signal was detected (Fig. 6A and B). Next, we evaluated the expression of adenoviral genes in ARO and KAT-4 cells infected with dl922-947 or dl1520 for 24 hours. We selected two adenoviral genes whose expression correlated with different stages of the adenoviral life cycle: E1A 13 S, representing genes transcribed from the immediate early


region, and Penton, representing genes expressed from the late region. Bevacizumab treatment induced a 5-fold increase in dl922-947 replication (Fig. 6C). The suppressive effect of bevacizumab on tumor angiogenesis was also evaluated, showing a decrease in microvessel density in bevacizumab-treated tumors (40/mmq) with respect to the untreated control (90/mmq), confirming that bevacizumab inhibits the neovascularization of anaplastic thyroid carcinoma xenografts.

Discussion Anaplastic thyroid carcinoma is associated with an extremely poor prognosis and leads to death in a very short time. The absence of active therapies is preventing the successful control of the disease (4); therefore, novel therapeutic approaches have been proposed. Gene therapy approaches based on viral vectors have been suggested for the treatment of anaplastic thyroid carcinoma. Different gene therapy strategies have been used (35); these include the combined transfer of the human cytokine Il-2 and HSV-tk (36). However, insufficient levels of gene expression often limited the therapeutic effects of replication-incompetent viruses. Replication-selective oncolytic viruses are being developed as a novel targeted form of anticancer treatment. Different viral vectors have been evaluated against anaplastic thyroid carcinoma cells, such as the oncolytic herpes virus NV1023 (37), the replication-competent vaccinia virus GLV-1h68 (38), and a conditionally replicative Wnt-a-catenin pathway – based adenovirus (39). The dl1520 mutant was the first replication-selective oncolytic virus described (6). Recently, results from a phase III clinical trial have shown the ability of H101, a mutant oncolytic adenovirus similar to dl1520, to increase the response rate of nasopharyngeal carcinoma in combination with cisplatin, confirming the potential clinical benefit of oncolytic adenoviruses (9). We have previously shown that dl1520 induces cell death of anaplastic thyroid carcinoma cell lines and also increases the effects of doxorubicin, paclitaxel (10), and radiotherapy (11). Recently, we have observed that lovastatin increases dl1520 cell killing activity against anaplastic thyroid carcinoma cells and tumor xenografts (40). However, high MOIs of dl1520 were used in these previous studies, highlighting the

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need for oncolytic adenoviruses with a higher replication activity. We decided to analyze the antitumor efficacy of the E1ACR2 – deleted oncolytic adenovirus dl922-947 in anaplastic thyroid carcinoma cell lines and tumor xenografts and compared its effects to dl1520, which has already been evaluated in a phase III clinical trial. In ARO and KAT-4 cell lines, dl922-947 induced a higher cytopathic effect compared with dl1520. The evaluation of viral replication in all anaplastic thyroid carcinoma cell lines confirmed a higher replication efficiency of the dl922-947 mutant. In FRO cells, the two mutant viruses showed similar levels of cell killing, although the dl922-947 mutant showed higher replication levels. It is possible that other mechanisms

besides viral replication are involved in the killing of FRO cells. In vivo, the dl922-947 mutant had superior efficacy in anaplastic thyroid carcinoma tumor xenografts, and a higher level of viral replication was observed in these tumors. Our data clearly showed that the E1A-CR2 mutant dl922-947 was more potent than the dl1520 mutant in anaplastic thyroid carcinoma cells in culture and in vivo. These observations suggest that the dl922-947 virus may have clinical potential in the treatment of anaplastic thyroid carcinoma. Anaplastic thyroid carcinoma is almost always fatal; death is attributable to upper airway obstruction and suffocation in half of patients and to a combination of local and distant disease in the remaining patients. Therefore, it is important to achieve

Fig. 5. In vitro effects of bevacizumab in combination with dl922-947. A, KAT-4 cells were treated with bevacizumab for 48 h before infection with dl922-947, and cell survival was evaluated after 12 d; no differences were observed. B, KAT-4 cells were treated with bevacizumab for 48 h before infection with dl922-947, and viral replication was evaluated by real-time PCR; no differences were observed. C, KAT-4 cells were pretreated with bevacizumab (48 h) and then infected with AdGFP at different MOIs. Bevacizumab treatment did not increase the GFP expression. Columns, mean of three different experiments; bars, SD.


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Fig. 6. Bevacizumab treatment enhances viral distribution and replication in anaplastic thyroid carcinoma xenografts. A, animals bearing KAT-4 xenografts were divided into two groups with similar tumor size. Animals received bevacizumab or saline solution i.p. at T = 0 and T = 5, respectively. AdGFP was injected intratumorally in both groups at T = 6. After 2 d, animals were sacrificed, and GFP expression and distribution were evaluated by confocal microscopy analysis. B, quantification of the digitized signal. C, genome equivalent copies of dl922-947 after combined treatment. Animals bearing KAT-4 xenografts were divided into two groups with similar average tumor size. One group received bevacizumab i.p. at days 0 and 5 and dl922-947 at day 10; the other group received saline solution and dl922-947 with the same schedule. After 2 d, animals were sacrificed; tumors were excised; and viral replication was evaluated by real-time PCR. Columns, mean of three different experiments; bars, SD.

local control of the disease. Accordingly, we chose to inject the dl922-947 mutant intratumorally because local administration of dl922-947 has been proved to be effective (19). An increasing number of oncolytic viruses are currently under clinical evaluation, and viruses derived from vaccinia, measles, mumps, and other strains have shown systemic efficacy following intratumoral injection or i.v. infusion (41). The therapy of disseminated diseases will benefit from these viruses and other novel mutants. However, it is also conceivable that oncolytic viruses without systemic efficacy still will be a useful tool for the treatment of locally aggressive diseases such as anaplastic thyroid carcinoma; moreover, local administration proved to be safe and to avoid toxic effects. Although previous results indicate that mutant oncolytic adenoviruses such as dl1520 and CV706 lack systemic efficacy (41), further studies are recommended to evaluate the efficacy and safety of dl922-947 after i.v. infusion. The successful application of therapeutic strategies based on replicating oncolytic viruses is limited by several obstacles such as preexisting immunity (42), emergence of virus-resistant cells, and incomplete viral dissemination (43). The latter represents the major obstacle contributing to virotherapy failure. Several factors contribute to poor viral distribution in the neoplastic tissues, such as the thickness of extracellular matrix, acidosis, and enhanced proteolytic activity (44). Hypoxia also contributes to reduce viral distribution by inducing the synthesis of angiogenic factors and leading to neoplastic angiogenesis (25). The abnormal vessel structure and properties and the absence of functioning lymphatic vessels in tumors increase the interstitial fluid pressure (44) interfering with viral spread and entry. Bevacizumab is a recombinant humanized monoclonal antibody that blocks the binding of VEGF to its receptor, and it has been tested in the clinic with promising results (25). Bevacizumab is effective mainly in combination with chemotherapeutic drugs (e.g., fluorouracil and irinotecan; ref. 25). It has been suggested that antiangiogenic agents potentiate chemotherapy by transiently ‘‘normalizing’’ the


abnormal tumor vasculature, making oxygen and drug delivery more efficient (44). Therefore, we hypothesized that bevacizumab could also improve viral distribution within anaplastic thyroid carcinoma tumor xenografts. It is important to note that blocking VEGF signaling reduces the interstitial fluid pressure in anaplastic thyroid carcinoma tumor xenografts (27). In the present study, we tested the effects of bevacizumab in combination with dl922-947 against anaplastic thyroid carcinoma tumor xenografts, showing that the combined treatment enhances the antineoplastic effects of the virus. Bevacizumab alone was not able to induce a statistically significant reduction of tumor growth, confirming a lack of direct antitumor activity against anaplastic thyroid carcinoma cells (27). This observation indicates that bevacizumab acts by enhancing dl922-947 oncolytic activity. It has previously been shown that bevacizumab, through the block of VEGF signaling, reduces the interstitial fluid pressure in anaplastic thyroid carcinoma (KAT-4) tumor xenografts (27). Therefore, it is possible that interstitial fluid pressure reduction allows a better diffusion of the locally administered dl922-947. This hypothesis is supported by the observation that bevacizumab pretreatment improves the dissemination of the reporter virus (AdGFP). It is worth noting that AdGFP injection was done in tumors with similar volume to avoid the possibility that the bevacizumab-treated group received higher viral load with respect to untreated animals. Furthermore, in bevacizumab-treated tumors, a more intense intracellular GFP staining was observed, suggesting an enhanced viral entry in neoplastic cells. This observation is in agreement with the increased viral replication observed in bevacizumab- and dl922-947 – treated tumors. It has been reported that bevacizumab induced a significant inhibition of tumor growth in an orthotopic model of anaplastic thyroid carcinoma (45). The discrepancy between our and previous data could be explained by taking into account that, in our study and in the study by Salnikov et al. (27), xenografts of KAT-4 cells of very large dimensions were

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used, whereas Prichard et al. (45) have used a different cell line (ARO) and an orthotopic model. Other strategies to enhance viral distribution into solid tumors have already been proposed, mostly based on pretreatment with proteolytic enzymes to alter extracellular matrix and facilitate virus dissemination (21, 46). Recently, it has been observed that matrix metalloproteinases 1 and 8, members of the matrix metalloproteinase enzymes, enhance viral delivery (47). These approaches could be useful, although a concern is represented by a potential increase of the metastatic spreading of neoplastic cells. Further studies are required to assess the feasibility of proteolytic enzyme treatment in the clinical setting. The antineoplastic and antiangiogenic effects and the clinical benefit of bevacizumab have already been shown, making the clinical use of a combined treatment of bevacizumab and oncolytic viruses feasible. Bevacizumab has also been used in combination with a nonreplicating adenovirus transducing the melanoma differentiation associated gene 7 (Ad MDa-7), showing the regression of lung tumors (48). However, the viral distribution in these tumors was not analyzed. Recently, in a rat glioma model, it has been shown that an angiostatic peptide (cRGD) enhanced antitumor efficacy

and viral replication of hrR3, a herpes simplex virus – derived oncolytic virus (49), confirming the potential benefit of viral treatment in combination with antiangiogenic agents. In summary, our data showed that dl922-947 has a higher oncolytic activity with respect to dl1520 and could represent a better option for virotherapy of anaplastic thyroid carcinoma. In conclusion, the results described herein could contribute to the development of new therapeutic protocols for the treatment of anaplastic thyroid carcinoma. Moreover, we have shown that bevacizumab increases the oncolytic effects of dl922-947 by increasing viral distribution. The results described encourage the use of the dl922-947 virus in combination with bevacizumab.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Acknowledgments We thank Dr. P. Formisano and Dr. C. Tang for the critical review of the manuscript, Dr. D.Viggiano for his help with confocal microscopy, and S. Sequino for his excellent technical assistance.

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