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ORIGINAL ARTICLE Different responses of human pancreatic adenocarcinoma cell lines to oncolytic Newcastle disease virus infection PRA Buijs1, CHJ van Eijck1, LJ Hofland2, RAM Fouchier3 and BG van den Hoogen3 Newcastle disease virus (NDV) is a naturally occurring oncolytic virus with clinically proven efficacy against several human tumor types. Selective replication in and killing of tumor cells by NDV is thought to occur because of differences in innate immune responses between normal and tumor cells. In our effort to develop oncolytic virotherapy with NDV for patients with pancreatic cancer, we evaluated the responses to NDV infection and interferon (IFN) treatment of 11 different established human pancreatic adenocarcinoma cell lines (HPACs). Here we show that all HPACs were susceptible to NDV. However, this NDV infection resulted in different replication kinetics and cytotoxic effects. Better replication resulted in more cytotoxicity. No correlation was observed between defects in the IFN pathways and NDV replication or NDV-induced cytotoxicity. IFN production by HPACs after NDV infection differed substantially. Pretreatment of HPACs with IFN resulted in diminished NDV replication and decreased the cytotoxic effects in most HPACs. These findings suggest that not all HPACs have functional defects in the innate immune pathways, possibly resulting in resistance to oncolytic virus treatment. These data support the rationale for designing recombinant oncolytic NDVs with optimized virulence that should likely contain an antagonist of the IFN pathways. Cancer Gene Therapy (2014) 21, 24–30; doi:10.1038/cgt.2013.78; published online 3 January 2014 Keywords: pancreatic adenocarcinoma; oncolytic virotherapy; Newcastle disease virus; innate immunity INTRODUCTION An estimated 277 000 new cases of pancreatic adenocarcinoma arise worldwide yearly, and 266 000 patients die of this disease.1 Radical surgery is the only potential curative treatment option to date. However, even for the 10–15% of patients with resectable disease, the prognosis is poor: median survival after resection is 24 months and 5-year survival is 20%.2 For patients with locally advanced or metastatic pancreatic cancer, the outlook is even grimmer: with palliative chemotherapy, median survival is just 6 months.3 Development of novel treatment options for pancreatic cancer is of crucial importance. The local or systemic administration of oncolytic viruses such as Newcastle disease virus (NDV) to cancer patients is a promising treatment strategy with encouraging results in a variety of tumor types.4–7 NDV is a replication-competent oncolytic virus belonging to the family Paramyxoviridae with a natural avian host range. The virus has a replication cycle confined to the cell cytoplasm without integration or recombination. NDV strains are categorized in three different groups based on the severity of disease they cause in birds: lentogenic (avirulent), mesogenic (intermediate virulent) and velogenic (virulent). Although NDV can cause severe and lethal disease in poultry, infection is relatively asymptomatic in humans.8 NDV selectively replicates in and destroys tumor cells while sparing normal cells, presumably because of defective interferon (IFN) signaling pathways in tumor cells.9–13 Infection of normal cells leads to IFN production that inhibits viral replication. The tumor-specific replication and subsequent induced cytolysis, and the inability of the virus to spread among healthy cells, make NDV a safe cancer therapeutic agent. The IFN produced by normal cells surrounding the tumor cells upon NDV infection, besides having antiviral effects, might exert an antiproliferative effect against human pancreatic adenocarcinoma cell lines (HPACs).14 Several naturally occurring NDV strains have shown antitumor activity without major side effects in phase I–II clinical trials.7,8,15,16 However, the success of these therapies was only marginal, likely because of the IFN sensitivity or overattenuation of the virus. In addition, some tumor cells are still capable of mounting effective antiviral responses that may, at least partially, explain the observed resistance of some tumors to oncolytic NDV therapy.17,18 The generation of recombinant viruses has provided opportunities to improve the efficacy of oncolytic NDV virotherapy.17–23 Pancreatic tumors are heterogeneous in cell composition, and information is lacking on defects in IFN pathways in these cells. Our ultimate goal is to generate an optimized recombinant NDV with destroying capacity of all different cell types of pancreatic tumors. For a rational design, we evaluated the susceptibility of 11 HPACs for NDV, response to this infection with regard to IFN production and cytotoxicity, and response to IFN treatment. MATERIALS AND METHODS Cell lines and culture conditions The human pancreatic adenocarcinoma cell lines were obtained from the American Type Culture Collection (Wesel, Germany) and authentication was performed using Short Tandem Repeat profiling.24 Cells were used not more than 25 passages after thawing. PANC-1, MIA PaCa-2, BxPC-3, Hs 700T, Hs 766T, CFPAC, SU.86.86, AsPC-1, Capan-1, Capan-2 and HPAF-II were cultured in RPMI-1640 medium supplemented with 100Uml� 1 penicillin, 100Uml� 1 streptomycin, 2mM L-glutamine (PSG) and 5% or 1Department of Surgery, Erasmus MC, Rotterdam, The Netherlands; 2Department of Internal Medicine, Division of Endocrinology, Erasmus MC, Rotterdam, The Netherlands and 3Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands. Correspondence: Dr BG van den Hoogen, Department of Viroscience, Erasmus MC, s-Gravendijkwal 230, Room Ee-1767, 3015 CE Rotterdam, The Netherlands. E-mail: b.vandenhoogen@erasmusmc.nl Received 26 September 2013; revised 15 November 2013; accepted 23 November 2013; published online 3 January 2014 Cancer Gene Therapy (2014) 21, 24–30 & 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt 10% HyClone characterized fetal bovine serum (FBS HC; Thermo Fischer Scientific, Breda, The Netherlands). The non-neoplastic human lung fibroblast cell line MRC-5 was cultured in Dulbecco’s modified Eagle’s medium supplemented with PSG and 10% FBS HC. The 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with PSG, 0.5mgml� 1 geneticin, 1mM sodium pyruvate, 1% non-essential amino acids and 10% FBS HC. Vero clone 118 cells25 were cultured in Iscove’s modified Dulbecco’s medium supplemented with PSG and 10% FBS HC. All cells were kept at 37 1C with 5% CO2 in a humidified incubator. Periodically, cells were tested and confirmed to be mycoplasma free. All media and supplements were purchased from GIBCO (Life Technologies, Bleiswijk, The Netherlands). Newcastle disease virus All infection experiments were performed with lentogenic wild-type NDV isolated from a cloacal swab of a wild female mallard taken on 17 October 2002 in Lekkerkerk, The Netherlands. The virus was cultured in embryo- nated chicken eggs using standard techniques. The F cleavage site was sequenced and the deduced amino acid sequence was found to be 112GRQGRL117, confirming this to be a lentogenic NDV. All infection experi- ments were performed in the presence of reduced concentration of FBS HC (3%), without the addition of trypsin. Virus stocks were titrated by end point dilution assay in Vero clone 118 cells. To read out infection, cells were stained with chicken polyclonal anti-NDV antibody and rabbit-FITC labeled anti-chicken IgG antibody (1:2000 and 1:1000 dilution respectively; both from Abcam, Cambridge, UK) at 72h after inoculation. Viral titers were calcu- lated using the method of Reed and Muench.26 In infection experiments, the term MOI refers to multiplicity of infection. An MOI of 1 equals one 50% tissue culture infective dose per cell. Low, medium and high MOIs are defined in this article as an MOI of 0.01, 0.1 and 1, respectively. Replication curves 1.5� 106 cells in T25 flasks (Corning, Amsterdam, The Netherlands) were inoculated with NDV at low MOI. After 1 h of incubation, cells were washed three times with phosphate-buffered saline, and fresh infection medium was added. At time points 0, 6, 12, 24, 48 and 72 h after washing, duplicates of 100ml supernatant were collected, mixed with 100ml 50% (w/v) sucrose and frozen at � 80 1C. Samples were titrated in quad- ruplicate as described before. Cytotoxicity assay Quadruplicates of 2� 104 cells per well in 96-well plates (Corning) were either mock inoculated or inoculated with NDV at different MOIs (low– medium–high). After 48 h, 100ml fresh medium was added. At time points 0, 24, 48, 72, 96 and 120 h after inoculation, cells were washed once with phosphate-buffered saline and lysed by incubation with 100ml 0.9% Triton X for 45min at 37 1C. Lactate dehydrogenase in 50ml of lysate sample was assayed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Leiden, The Netherlands) following the manufacturer’s instructions. Cell viability is presented as percentage absorption (450 nm, Tecan Infinite F200, Giessen, The Netherlands) of inoculated versus mock-inoculated cells that were considered to be 100% viable. Although measurements were performed at multiple time points, we only show data from time point 5 days after inoculation, as this represented differences between observed cytotoxicity the best. IFN measurement with luciferase bioassay To determine the presence of type I IFN in the supernatants of infected cells, a bioassay using a plasmid with the IFN-stimulated response element (ISRE) fused to the firefly luciferase gene (pISRE-Luc; Agilent Technologies, Amstelveen, The Netherlands) as reporter was used. At 24 h after inocu- lation, supernatants of (mock-) infected cells were collected and stored at � 20 1C. This time point was chosen to be able to measure IFN production without possible interference from decreasing cell viability and/or secondary IFN signaling loops. The 293T cells were transfected using the calcium phosphate method27 with pISRE-Luc as reporter plasmid and Simian virus 40-Renilla-luciferase (pRL-SV40; Promega) as internal control. The next day, the collected supernatants were treated with ultraviolet to inactivate virus present in the supernatant and 300ml was placed on the transfected 293T cells, together with 200ml fresh 293T medium. After 24 h, luminescent signals were generated using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions and detected with a Tecan Infinite F200 microplate reader. Renilla luciferase signals were used to normalize firefly luciferase signals to correct for 293T cell number and transfection efficiency. IFN produced by NDV-infected cells is presented as fold change as compared with mock-infected cells. Recombinant human IFN-b-1a (Merck Calbiochem, Darmstadt, Germany) was used as a positive control. NDV staining and fluorescence-activated cell sorting (FACS) analysis Cells were harvested by trypsinization and subsequently fixed and permeabilized using Cytofix/Cytoperm following the manufacturer’s instructions (BD, Breda, The Netherlands). Next, cells were incubated on ice for 1 h with chicken polyclonal anti-NDV antibody (1:800 dilution), washed and subsequently incubated on ice for another hour with rabbit- FITC labeled anti-chicken IgG antibody (1:400 dilution). Cells were washed and fixed in 2% paraformaldehyde. FITC signals were detected using a BD FACS Calibur cytometer and FACS data were analyzed using BD CellQuest Pro software. RNA isolation and quantitative real-time PCR RNA was isolated using the High Pure RNA Isolation kit (Roche, Woerden, The Netherlands) following the manufacturer’s instructions. RNA was reverse transcribed using a two-step protocol. First, 22 ml (of a total of 50ml) of eluted RNA was mixed with 2ml random primers (500mgml� 1; Promega), 2 ml dNTPs (10mM) and 1 ml RNAse inhibitor (40Uml� 1; Promega). This mix was heated for 5min at 65 1C and immediately put on ice. Next, 8ml FS buffer (5� ), 2 ml DTT (0.1 M), 2 ml Superscript III RT (200Uml� 1; Life Technologies) and 1ml RNAse inhibitor (40U ml� 1) was added. This mix was incubated for 5min at 25 1C and for 1 h at 60 1C to synthesize complementary DNA. Quantitative real-time PCR was performed using 4 ml of complementary DNA in an ABI PRISM 7000 Sequence Detection System (Life Technologies) using TaqMan gene expression assays for ISG56, OAS1 and Mx1 (Hs00356631, Hs00973637 and Hs00895608; all from Life Technologies). b-Actin was used as household gene (forward primer 30- GGCATCCACGAAACTACCTT-50 , reverse primer 30-AGCACTGTGTTGGCGTA- CAG-50 , probe 30-ATCATGAAGTGTGACGTGGACATCCG-50). Results are pre- sented as fold change of treated samples versus control samples (triplicates), calculated using the 2�DDCT method. 28 Statistical analysis Continuous data were compared between the groups using the Mann– Whitney U-test. Correlations between continuous data were calculated using Spearman’s rank correlation coefficient (rho). The P-values of o0.05 were considered statistically significant. RESULTS For a rational design of recombinant NDVs with improved oncolytic efficiency acting against all different sorts of pancreatic tumor cells, we evaluated the responses to NDV for 11 different HPACs with regard to cytotoxic effects and innate immune responses: PANC-1, MIA PaCa-2, BxPC-3, Hs 700T, Hs 766T, CFPAC, SU.86.86, AsPC-1, Capan-1, Capan-2 and HPAF-II. HPACs differ in susceptibility to NDV replication and subsequent cytotoxic effects To assess the susceptibility of the 11 HPACs to NDV replication and NDV-induced cytolysis, multicycle replication kinetic studies with wild-type lentogenic NDV were conducted. Cells were inoculated at low MOI and virus titers in the culture supernatant were determined at different time points after inoculation. MRC-5, a normal human fibroblast cell line known to be susceptible to NDV infection, was taken as control. Based on NDV replication capacity in the different HPACs, the cells were categorized into three groups (Table 1 and Figure 1): supporting high replication (peak titer41.0� 105: Capan-1, HPAF- II and SU.86.86), supporting low to medium replication (peak titer 1.0� 104–1.0� 105: Capan-2, BxPC-3, AsPC-1, PANC-1 and CFPAC) and supporting minimal replication (peak titer o1.0� 104: Hs 766T, Hs 700T and MIA PaCa-2). Oncolytic NDV for treatment of pancreatic cancer PRA Buijs et al 25 & 2014 Nature America, Inc. Cancer Gene Therapy (2014), 24 – 30 The differences observed in NDV replication were reflected in the induced cytotoxicity (Figure 2). In general, cells with high virus replication showed cytotoxicity already at low MOI (SU.86.86, Capan-1, HPAF-II), whereas cells displaying low to medium virus replication required inoculation at medium MOI to kill most cells. Inverse correlation was observed between peak viral titers and percentage cell survival at low and medium MOI (Spearman’s rho � 0.77 and � 0.74, respectively). MIA PaCa-2 and Hs 700T did not support virus replication and displayed cytotoxic effects only at high MOI. These findings indicate that NDV replication is an important factor to cause cytotoxicity in HPACs. HPACs differ in IFN production and signaling pathways For most HPACs, variation of NDV replication corresponded with differences in cytotoxic effects induced by the NDV infection. The observed variation in replication might result from differences in innate immune responses between the HPACs, as was described in earlier studies in other cell lines.10–13 To investigate differences in the IFN production pathway, cells were inoculated with NDV at high MOI to obtain high percentages of infected cells and 24 h later IFN content in the supernatant was determined (Figure 3a). Out of 11 HPACs, 6 (SU.86.86, Capan-1, Capan-2, BxPC-3, AsPC-1, MIA PaCa-2) failed to induce substantial IFN production upon NDV infection. This lack of IFN production was not because of low percentage of infected cells, as all HPACs displayed 468% infected cells by FACS analysis upon inoculation with this high MOI. The insensitivity of MIA PaCa-2 cells to NDV infection and induced cytotoxic effects could not be explained by IFN production as these cells did not produce IFN upon NDV infection. Interestingly, five HPACs (HPAF-II, CFPAC, PANC-1, Hs 766T and Hs 700T) did induce IFN production upon NDV infection, as did control MRC-5 fibroblasts. This is not an unusual finding as IFN production by tumor cells has been demonstrated before.11,13,19 No correlation was found between virus replica- tion or cytotoxic effects of NDV and IFN production in these cells (Spearman’s rho � 0.27; � 0.08; � 0.13 and � 0.32 for replication; cytotoxicity at low, medium and high MOI, respectively). This is best demonstrated by the HPAF-II cell line that produces relatively large amounts of IFN, yet is highly susceptible to NDV. The tumor selectivity of NDV has also been shown to be because of defects in the IFN signaling pathway.11 To test for defects in the Janus kinase/signal transducer and activator of transcription signaling pathway, cells were either mock treated or treated with 1000 IUml� 1 of recombinant human IFN-b. After 24 h, expression of mRNA of three important IFN-stimulated genes (ISGs; ISG56, 20–50-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance 1 (Mx1)) was determined (Figure 3b). As expected, all three tested ISGs in normal human lung fibroblast MRC-5 cells were upregulated upon IFN treatment, although these cells also produced IFN upon NDV infection. Of the five IFN-producing HPACs (HPAF-II, CFPAC, PANC-1, Hs 766T and Hs 700T), four displayed a diminished upregulation as compared with MRC-5 fibroblasts for at least one of the mRNAs. Although lower than MRC-5 control, they still displayed at least a tenfold upregulation of at least one of the assayed ISG-mRNAs that might still be biologically significant. For Hs 700T cells, which demon- strated production of IFN upon NDV infection, two out of three tested ISGs were highly upregulated upon IFN treatment. Thus, Hs 700T cells produce IFN upon NDV infection, and a mostly intact IFN signaling pathway in these cells results in expression of antiviral genes. Failure to upregulate OAS1 mRNA suggests that Hs 700T cells have a mutation in or deletion of this antiviral gene, but nevertheless they were still protected from infection. In summary, most of the tested HPACs showed a retained ability to respond to type I IFN, and the significance of the lower magnitude of response in some HPACs is unclear. Furthermore, no statistical correlation (tested with Spearman’s rank) was found between virus replication or cytotoxic effects of NDV and ISG-mRNA upregulation. IFN-b pretreatment can hamper NDV replication and protect from NDV-induced cytotoxicity in most HPACs To study whether the observed differences in IFN signaling also reflected a functional effect on replication of NDV in these cells, multicycle replication curves for NDV were generated in HPACs pretreated with IFN-b. As shown in Figure 4a, pretreatment of the HPACs with IFN-b decreased virus replication in most cells to levels comparable to resistant HPACs, and replication was delayed in Capan-1 and SU.86.86 cells by 24 h (Figure 4b). IFN-b pretreatment also decreased NDV-induced cytotoxic effects in most cells (Figure 5), whereas IFN-b did not induce measurable cytotoxicity by itself after 24 h (data not shown). Table 1. Virus yield obtained after inoculation of MRC-5 (normal fibroblast) and 11 different HPACs with NDV Cell line Peak titer (TCID50 per ml) MRC-5 1.0� 104 Capan-1 3.4� 106 HPAF-II 1.6� 106 SU.86.86 1.7� 105 Capan-2 6.6� 104 BxPC-3 5.5� 104 AsPC-1 3.1� 104 PANC-1 1.3� 104 CFPAC 1.2� 104 Hs 766T 4.6� 103 Hs 700T 2.8� 103 MIA PaCa-2 2.7� 103 Abbreviations: HPAC, human pancreatic adenocarcinoma cell line; NDV, Newcastle disease virus; TCID50, 50% tissue culture infective dose. Figure 1.