Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201607296
ISSN1757-4684
AutoresKyle Potts, Chad R. Irwin, Nicole A. Favis, Desmond Pink, Krista M. Vincent, John D. Lewis, Ronald B. Moore, Mary Hitt, David H. Evans,
Tópico(s)Herpesvirus Infections and Treatments
ResumoResearch Article13 March 2017Open Access Transparent process Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti-tumor immunity with superior safety in bladder cancer models Kyle G Potts Kyle G Potts Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Chad R Irwin Chad R Irwin Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Nicole A Favis Nicole A Favis Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Desmond B Pink Desmond B Pink Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Krista M Vincent Krista M Vincent Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Anatomy & Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, ON, Canada Search for more papers by this author John D Lewis John D Lewis orcid.org/0000-0002-7734-1204 Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ronald B Moore Ronald B Moore Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Mary M Hitt Mary M Hitt Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author David H Evans Corresponding Author David H Evans [email protected] orcid.org/0000-0001-5871-299X Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Kyle G Potts Kyle G Potts Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Chad R Irwin Chad R Irwin Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Nicole A Favis Nicole A Favis Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Desmond B Pink Desmond B Pink Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Krista M Vincent Krista M Vincent Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Anatomy & Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, ON, Canada Search for more papers by this author John D Lewis John D Lewis orcid.org/0000-0002-7734-1204 Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ronald B Moore Ronald B Moore Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Mary M Hitt Mary M Hitt Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author David H Evans Corresponding Author David H Evans [email protected] orcid.org/0000-0001-5871-299X Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Author Information Kyle G Potts1,2,3, Chad R Irwin2,3,4, Nicole A Favis2,3,4, Desmond B Pink1,3, Krista M Vincent1,3,5, John D Lewis1,3, Ronald B Moore1,3,6, Mary M Hitt1,2,3,‡ and David H Evans *,2,3,4,‡ 1Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 2Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 3Cancer Research Institute of Northern Alberta (CRINA), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 4Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Anatomy & Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, ON, Canada 6Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 780 492-7997; E-mail: [email protected] EMBO Mol Med (2017)9:638-654https://doi.org/10.15252/emmm.201607296 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high-grade disease, Bacillus Calmette–Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell-cycle-regulated small subunit of ribonucleotide reductase (RRM2). The F4L-deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor-selective replication and cell killing. These F4L-deleted VACVs replicated selectively in immune-competent rat AY-27 and xenografted human RT112-luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY-27 tumors by VACV treatment developed anti-tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T-lymphocyte assays. Finally, F4L-deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L-deleted VACVs, with application as a promising therapy for patients with BCG-refractory cancers and immune dysregulation. Synopsis Vaccinia virus (VACV) mutated to render it incapable of synthesizing deoxynucleoside triphosphates (dNTPs) is a safe, highly selective, and potentially superior oncolytic agent in animal models for bladder cancer. F4L-deleted VACVs are highly attenuated in normal tissues and selectively replicate in, and kill bladder cancer cells that express elevated RRM2 levels. F4L-deleted VACVs replicate selectively in immune-competent rat AY-27 and xenografted human RT112-luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. Rats cured of AY-27 tumors by VACV treatment developed long-lasting anti-tumor immunity. F4L-deleted VACVs replicate in primary human bladder cancer explants and BCG-resistant cell lines. Introduction Over 90% of cases of bladder cancer are subtyped as urothelial cell carcinomas. When first diagnosed, about 80% of these cases are classified as non-muscle-invasive bladder cancer (NMIBC) [reviewed in (Anastasiadis & de Reijke, 2012; Potts et al, 2012; Delwar et al, 2016)], but unfortunately up to 80% of these patients will experience a recurrence within 5 years of initial treatment (van Rhijn et al, 2009). Current standards of care for low-risk patients include surgery and intravesical chemotherapy (Shen et al, 2008). The high-grade (Ta, T1, or carcinoma in situ) tumors are most likely to recur, and treatment for these patients includes surgery often followed by intravesical therapy with Bacillus Calmette–Guérin (BCG) (Shen et al, 2008). The side effects of BCG treatment include a risk of infection, cystitis, and prostatitis, and it can be hazardous for immunocompromised patients (Lamm et al, 1992). Moreover, about 30% of patients fail BCG therapy leaving cystectomy as the next common treatment option (Zlotta et al, 2009). How BCG works is poorly understood and it may simply be a pro-inflammatory agent (Redelman-Sidi et al, 2014). There is no evidence to suggest that BCG generates protective anti-tumor immunity and this may partly explain the high rate of treatment failure (Biot et al, 2012). In fact, there has been very little improvement in the treatment of high-grade NMIBC in the last 10–20 years and recurrence after BCG therapy is still one of the most significant problems in the management of bladder cancer (Downs et al, 2015). This highlights the urgent need for safer and more reliable bladder-sparing approaches. Oncolytic viruses are intended to replicate selectively in, and kill, cancer cells while sparing normal tissues [reviewed in (Potts et al, 2012; Russell et al, 2012; Kaufman et al, 2015)]. Some of the many cell pathways that affect virus replication are those that regulate cell proliferation and DNA replication, processes that are critically dependent upon deoxynucleoside triphosphate (dNTP) production (Aye et al, 2015). The rate-limiting step in dNTP biosynthesis is the de novo reduction of ribonucleoside diphosphates (rNDPs) to deoxyribonucleoside diphosphates (dNDPs) by the enzyme ribonucleoside diphosphate reductase (RNR) (Nordlund & Reichard, 2006). Since DNA virus replication requires dNTPs, this requirement for RNR activity provides an important biological feature that can be used to target DNA viruses to cancer cells. Many viruses exhibit oncolytic properties and a modified herpesvirus, Talimogene laherparepvec (T-Vec), recently received US clinical approval (Andtbacka et al, 2015). Vaccinia virus (VACV) has also been studied extensively as an oncolytic agent, with JX-594 (Pexa-Vec) having completed multiple phase II trials (Hwang et al, 2011; Heo et al, 2013; Cripe et al, 2015; Park et al, 2015). VACV is a large double-stranded DNA virus that efficiently infects many different cell types and encodes many of the proteins required for robust replication in normal cells (McFadden, 2005). These proteins include thymidine kinase (TK; the J2R gene product) and both large (RRM1; I4L gene product) and small (RRM2; F4L gene product) subunits of the heterodimeric RNR complex (Slabaugh et al, 1988). The virus-encoded components of RNR complex with each other and can form chimeras with cellular homologs (Hendricks & Mathews, 1998; Gammon et al, 2010). Most oncolytic VACVs reported to date encode mutations in J2R and little research has been conducted to determine whether mutating the RNR genes (Fend et al, 2016) might also produce advantageous oncolytic properties. The F4L gene is an important determinant of VACV virulence and viruses lacking F4L (∆F4L) are attenuated in vivo whereas ∆I4L mutants are not (Gammon et al, 2010). The fact that cellular RRM2 is cell-cycle-regulated whereas RRM1 is constitutively expressed can perhaps explain this observation (Eriksson et al, 1984) and leads to the prediction that a ∆F4L virus should replicate selectively in dividing cancer cells. This complementation-based strategy might be especially useful for treating more aggressive bladder cancers since increased levels of cellular RRM2 predict a poorer prognosis (Morikawa et al, 2010). Here we describe pre-clinical studies showing that VACV can be used safely as an intravesical treatment for NMIBC. We find that F4L-deleted VACVs retain much of their cytotoxicity and replication proficiency in bladder cancer cells. F4L-deleted VACVs also safely and effectively clear bladder tumors in animal models and induce a durable anti-tumor immunity. These findings highlight the potential for using a F4L-deleted VACV in treating bladder cancer, especially in patients who have failed BCG treatment or are immunosuppressed. Results Growth of VACV ΔF4L and ΔJ2R mutants in vitro Homologous recombination was used to disrupt the VACV (strain Western Reserve) F4L and J2R loci as shown in Fig EV1. Thirteen out of fifteen bladder cancer cell lines grown under high serum conditions (10%) supported robust virus replication, exceptions being UM-UC3-luc and UM-UC9 cells (Figs 1A and EV2). Under low serum conditions (0.1%), the wild-type (WT) and ∆J2R VACV grew as well as was seen in 10% serum. Although viruses lacking F4L also replicated efficiently in most of the cancer cell lines under low serum conditions, the ∆F4L VACVs grew poorly in 253J and AY-27 cells (Fig 1B). Most importantly, compared to WT, growth of ∆F4L∆J2R and ∆F4L VACVs in low serum was reduced > 4,000-fold in the NKC (normal epithelial kidney) line and > 250-fold in N60 (normal fibroblast) cell line, whereas the growth of ∆J2R VACV was only marginally reduced compared to WT in the NKC and N60 cells under the same low serum conditions (Figs 1 and EV2). Click here to expand this figure. Figure EV1. Genomic map of vaccinia virus constructsViruses were generated from the VACV Western Reserve strain. Viral thymidine kinase is encoded by the J2R gene. Subunit 2 of viral ribonucleotide reductase is encoded by the F4L gene. neo, neomycin gene; gusA, β-glucuronidase gene; lacZ, β-galactosidase gene; ITR, inverted terminal repeat; TKL, viral thymidine kinase gene left homology; TKR, viral thymidine kinase gene right homology; and WT, wild-type. Download figure Download PowerPoint Figure 1. ∆F4L∆J2R VACV retains much of the replication proficiency and cytotoxicity of WT VACV in bladder cancer cells A, B. Growth curves for the indicated VACV strains in subconfluent human bladder cancer cell lines, a rat bladder cancer cell line (AY-27), and a normal human skin fibroblast line (N60). The cells were infected with 0.03 PFU/cell. (A) Panel of cells grown under normal serum conditions. (B) Panel of cells grown under low (0.1%) serum conditions. Cultures were harvested at the indicated times and titered on BSC-40 cells. C, D. Survival of cell lines infected in vitro with the indicated VACV strains. Subconfluent cells were infected at the indicated multiplicities of infection (in PFU/cell). Uninfected cells were used as control. (C) Panel of cells grown under normal serum conditions. (D) Panel of cells grown under low (0.1%) serum conditions. The cells were incubated with resazurin to assess viability 3 days post-infection relative to uninfected control cells. Data information: Mean ± SEM is shown. For (A) and (B), data represent at least two independent lysates titered in duplicate. For (C) and (D) n ≥ 3. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. ∆F4L∆J2R VACV retains much of the replication proficiency of wild-type VACV in bladder cancer cellsGrowth curves for the indicated VACV mutants or WT VACV. Subconfluent cells were infected at a multiplicity of infection of 0.03 PFU/cell. Cultures were harvested at the indicated times and titered on BSC-40 cells. Normal human kidney epithelial cells grown under normal serum conditions (left) and 0.1% FBS (right). Panel of human bladder cancer cell lines (exception: MB49-luc, murine urothelial carcinoma) cultured in vitro with 10% FBS. Data information: Mean ± SEM is shown and data represent at least two independent lysates titered in duplicate. Download figure Download PowerPoint The effect of VACV on cell survival was determined using a resazurin-based viability assay. Similar to growth of virus under high serum conditions (Fig 1A), no dramatic difference in the efficiency of virus-mediated cell killing was seen among the different viruses under high serum conditions (Figs 1C and EV3). However, under low serum conditions, both N60 normal skin fibroblasts and NKC epithelial kidney cells became relatively resistant to ∆F4L and ∆F4L∆J2R VACV killing. Interestingly, in low serum conditions, 253J and AY-27 cancer cells were still highly susceptible to killing by ∆F4L∆J2R VACV (Fig 1D), even though virus replication was attenuated. This was a specific property of the ∆F4L∆J2R virus; 253J and AY-27 cells were still relatively resistant to the ∆F4L VACV. Finally, both N60 and NKC cells grown in 0.1% fetal bovine serum (FBS) showed a low proportion of cells in S-phase whereas the proportion of RT112-luc cells in S-phase remained high (Fig EV4), suggesting that proliferation status under our low serum growth conditions may mimic the proliferation status of normal and tumor tissues in vivo. These data indicate that the mutant VACVs, in particular ∆F4L∆J2R VACV, retained much of the cytotoxic capabilities and replication proficiency of WT virus in bladder cancer cells but do not replicate in non-dividing cells. Click here to expand this figure. Figure EV3. ∆F4L∆J2R VACV retains much of the cytotoxicity of wild-type VACV in bladder cancer cellsSurvival of cell lines infected in vitro with the indicated VACV strains. Subconfluent cells were infected at the indicated multiplicities of infection (in PFU/cell). Uninfected cells were used as control. Normal human kidney epithelial cells grown under normal serum conditions (left) and 0.1% FBS (right). Panel of human bladder cancer cell lines (exception: MB49-luc, murine urothelial carcinoma) cultured in vitro with 10% FBS. The cells were incubated with resazurin to assess viability 3 days post-infection relative to uninfected control cells. Uninfected cells were used as control. Data information: Mean ± SEM is shown and n ≥ 3. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Non-tumorigenic cells have reduced S-phase population when grown under lower serum conditionsCell cycle analyses of indicated cell lines. Indicated cells lines were grown in media supplemented with either 10% FBS or 0.1% FBS for 48 h, and then cell cycle distribution was monitored by flow cytometry after PI staining. Red traces indicate cells grown in 10% FBS and blue traces indicate cells grown in 0.1% FBS. Analysis of cell cycle phase distribution. Download figure Download PowerPoint Nucleotide biosynthetic proteins are elevated in bladder cancer cells One might expect that ∆F4L and/or ∆J2R strains would depend upon complementation from cellular RRM2 and TK1, respectively, to provide the dNTPs required for virus replication. Some limited data suggested that ∆F4L VACVs do grow better in cells expressing higher levels of RRM2 (Gammon et al, 2010). To examine this matter in more detail, the levels of proteins catalyzing nucleotide biosynthesis were quantified in a panel of human bladder cancer cell lines and in normal N60 fibroblasts under 10 and 0.1% serum conditions (Fig 2A and Appendix Fig S1). Western blots showed a general elevation in the levels of cellular RRM1, RRM2, and TK1 in cancer cell lines compared to normal cells (Appendix Fig S1). The abundance of the DNA damage-inducible form of R2, p53R2, did not significantly differ between the cancer cell lines and in normal skin fibroblasts. Figure 2. Elevated levels of proteins catalyzing nucleotide biosynthesis in bladder cancer cell lines and primary human tumor lysates Western blot showing RRM1, RRM2, p53R2, and TK1 expression in human bladder cancer cell lines and N60 normal human fibroblasts. β-tubulin is shown as a loading control. siRNA depletion of RRM2 in HeLa cells 3 days post-transfection as determined by Western blot analysis. Growth of the indicated VACV strains in subconfluent HeLa cells. The cells were treated for 24 h with a scrambled control siRNA ("Scram") or an RRM2-targeted siRNA and then infected with the indicated viruses at 0.03 PFU/cell. The cultures were harvested 2 days later and titered on BSC-40 cells. Western blot showing RRM1, RRM2, p53R2, and TK1 expression levels in human primary tumor tissues and adjacent normal urothelium. β-tubulin is shown as a loading control. Analysis of RRM1, RRM2, and TK1 expression levels from publicly available patient bladder cancer microarray data (NMIBC: non-muscle-invasive bladder cancer; MIBC: muscle-invasive bladder cancer). Data points denote log2-transformed MAS5.0 normalized values. The box limits represent the upper and lower quartiles. The median is marked by the horizontal line inside the box. The whiskers extend to the highest and lowest observed values. Western blot showing RRM1, RRM2, and TK1 expression in rat AY-27 bladder tumor tissue and the indicated normal tissues. β-tubulin and Ponceau S staining are shown as loading controls. In all Western blots, equal amounts of total protein (30 μg) were assayed. Data information: Mean ± SEM is shown. For (C) n = 4 and significance was determined by multiple t-test. Microarray data in (E) were analyzed using RStudio (v0.98.501) and significance analysis was performed using a one-way ANOVA followed by Tukey's HSD. Western blots are representative of at least two or three independent experiments. Download figure Download PowerPoint To demonstrate F4L-deleted VACVs' dependence on cellular RRM2, we tested whether knockdown of RRM2 in HeLa cells would prevent VACV replication. Efficient knockdown was achieved following transfection with RRM2-specific siRNAs, as confirmed by Western blot analysis (Fig 2B). The cells were then infected with the different VACVs and virus yield measured by plaque assay (Fig 2C). There was a significant reduction in ∆F4L and ∆F4L∆J2R VACV replication in cells with RRM2 knockdown, while WT or ∆J2R VACV replication was unaffected. We also examined expression of nucleotide metabolism proteins in samples isolated from primary human bladder tumors and from normal bladder urothelium. Western blot analysis showed elevated expression of both RRM1 and RRM2 in the tumor tissues relative to the normal urothelium (Fig 2D). Additionally, TK1 was only detectable in the tumor lysates, with two of these showing high expression levels, and only minimal expression in the remaining lysates. As in cultured cells, p53R2 expression was not specifically associated with tumors. These observations were generally corroborated by gene expression data obtained from primary tumor samples previously analyzed by Sanchez-Carbayo et al (2006). Reanalysis of these data showed that RRM2 and TK1 expression were significantly increased in both NMIBC and muscle-invasive bladder cancers (MIBC) when compared to the normal urothelium (Fig 2E). In contrast, RRM1 was only significantly over-expressed in MIBC. The expression level of these same proteins was also measured in different tissues recovered from an orthotopic rat AY-27 bladder cancer model (Fig 2F). We detected high RRM2 expression in tumor tissue as well as normal bladder. RRM1 did not appear elevated in tumors compared to normal tissues, and very little TK1 expression was detected in any of the tissues. VACVs encoding F4L and J2R mutations safely clear human bladder tumor xenografts The safety and oncolytic activity of the mutant VACVs was tested in xenograft models of human bladder cancer. These models were established by subcutaneous or orthotopic implantation of luciferase-expressing human RT112 cells (RT112-luc) in Balb/c immune-deficient mice. In the first study, we injected three doses of virus, each comprising 106 PFU of ∆J2R, ∆F4L, ∆F4L∆J2R, or UV-inactivated VACV as a control, directly into subcutaneous RT112-luc tumors (Fig 3A). An mCherry signal, indicative of virus replication, was detected in all mice before the third live virus injection (Appendix Fig S2) and all animals treated with live virus showed significantly prolonged survival compared to those treated with UV-inactivated VACV (Fig 3B). Both ∆F4L and ∆F4L∆J2R VACV significantly increased survival compared to animals treated with the ∆J2R strain (P = 0.015 and P = 0.001, respectively). Tumor growth was controlled in all animals treated with live viruses as determined by caliper measurements (Fig 3C), and by luciferase detection (Fig 3F and G, and Appendix Fig S3). Figure 3. ∆F4L∆J2R VACV safely and effectively clears subcutaneous human RT112-luc xenografted tumors Experimental scheme. Balb/c nude mice were injected with 2 × 106 RT112-luc cells in the left flank at day zero. Then, 106 PFU of UV-inactivated, ∆J2R, ∆F4L, or ∆F4L∆J2R VACV were injected into the tumors on days 10, 13, and 16 post-implantation. Overall survival of immunocompromised mice bearing RT112-luc flank tumors following treatment with the indicated viruses (n = 10 per group). Growth of individual virus-treated RT112-luc tumors. Legend as in (B). Analysis of individual animal body weights plotted as mean change in body weight relative to day 10. Legend as in (B). VACV titers in tissues taken from animals euthanized due to toxicity (note: only mice that had detectable (4/10) virus as determined by plaque assay are shown). Quantification of average luminescence (an indication of live tumor cells) from bladder tumors corresponding to (B). Area under the curve (AUC) calculation from the data in (F) (n = 5 per group). Data information: Mean ± SEM is shown. Animal survival was analyzed by log-rank (Mantel–Cox) test in (B). One-way ANOVA followed by Tukey's multiple comparison test was used in (G). For luciferase quantification in (F) and AUG calculations in (G) n = 5 representative animals. Download figure Download PowerPoint The ∆J2R virus showed strong anti-tumor activity, but this was only achieved with significant toxicity in Balb/c immune-deficient mice. Seven of ten ∆J2R VACV-treated mice were euthanized due to excessive weight loss (Fig 3D). The mice euthaniz
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