The BSR cells (Harbin Veterinary Research Institute) for virus rescue, and the human gastric adenocarcinoma cell lines SGC-7901 and AGS were purchased from the Cancer Cell Repository (Shanghai Cell Bank, Shanghai, China; 2010-02-20) and the American Type Culture Collection (ATCC; Rockville, MD, USA). Gastric adenocarcinoma cells SGC-7901 and AGS cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO₂ incubator. Cell culture reagents were acquired from Gibco (Grand Island, NY, USA). When the cells reached 50–70% confluence, they were infected with rL-hIFN-λ1 or NDV for further study. The NDV LaSota strain and the recombiant viruses were propagated and titrated in 9-day-old SPF embryonated chicken eggs. The NDV strain LaSota, SPF eggs and anti-NDV serum was provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Human PBMCs were isolated from peripheral blood of volunteer donor with density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Cells were collected and washed twice in RPMI-1640 medium, and then were resuspended at a final density of 10⁶/ml in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) both from Invitrogen. Con-A stimulations were performed in 24-well plates using 1×10⁶ whole PBMCs with 5 µg/ml Con-A (Sigma-Aldrich) in a final volume of 2 ml.

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tet-razolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Hoechst 33342 were obtained from Sigma-Aldrich. All PCR primers were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Total RNA from viral or tumour cells were extracted with TRIzol reagent (Invitrogen). Anti-cleaved caspase-3 was from ImmunoWay Biotechnology (Newark, DE, USA). Anti-IFN-λ1 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-IFNλ-R1 was from R&D Systems, Inc. (Minneapolis, MN, USA). HRP-conjugated goat anti-rabbit, HRP-conjugated goat anti-mouse and FITC-conjugated goat anti-mouse antibodies were purchased from CWBio (Shanghai, China). HRP-conjugated goat anti-rabbit, HRP-conjugated goat anti-mouse, and FITC-conjugated goat anti-mouse antibodies were purchased from CWBio. HRP-conjugated rabbit anti-chicken antibody was from EarthOx Life Sciences (Millbrae, CA, USA), and Cy3-conjugated rabbit anti-chicken antibody was purchased from KPL, Inc. (Gaithersburg, MD, USA). The polyvinylidene difluoride (PVDF) membrane and Luminata were provided by Millipore (Billerica, MA, USA). Trypsin, and EDTA-2Na were offered by Gibco. A mitochondrial transmembrane potential analysis kit was from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). All other supplies for cell culture were obtained from Corning Costar (Corning, NY, USA).

To construct a full-length recombinant genomic cDNA. The cDNA of the IFN-λ1 genes was amplified from a previous study (25). The primers for amplification of human IFN-λ1 gene are shown in (Table I). These primers included the gene start and termination sequences of the NDV genome and the Kozak sequence. The amplified human IFN-λ1 gene was inserted into plasmid pBluescriptII Ks(+) then tested with nucleotide sequence analysis, the newly recombinant plasmid was named as pBS. IFN-λ1. The amplified human IFN-λ1 gene was digested by PmeI and then inserted into the NDV genome cDNA through a unique PmeI site in the P-M intergenic region at nucleotide position 3165 of the NDV genome (8) to build the new recombinant plasmid named pBRN.hIFN-λ1 (Fig. 2).

The rescue of recombinant NDV viruses from cloned cDNA was as follows: BSR cells infected with A modified virus expressing T7 RNA polymerase at a multiplicity of infection (MOI) of 1.0 and then transfected with pBowing in a six-well plate with pBRN.hIFN-λ1 together with 3 helper plasmids [pBS-NP (0.4 µg), pBS-P (0.2 µg), and pBS-L (0.2 µg)]. After 16 h of incubation at 37°C, the medium was replaced with 2 ml Opti-MEM (Invitrogen) containing 0.5 µg of TPCK-trypsin and the cells were incubated for another 3 days at 37°C. The supernatant was then inoculated into the allantoic cavities of 9-day-old embryonated SPF eggs. After 72 h of incubation at 37°C, the allantoic fluid was harvested and the virus was identified by hemagglutination assay using 1% chicken red blood cells. The resultant recombinant viruses were designated as rL-hIFN-λ1.

Virus growth was determined both in cell culture and embryonated chicken eggs. Confluent monolayers of SGC cells were infected with rl, rL-hIFN-λ1 in 96-well plates at a MOI of 1.0. Every 12-h post-infection, the cellular monolayers of SGC cells were harvested by freeze-thawing three times. Virus titers were determined as 50% tissue culture-infective dose (TCID₅₀). Two hundred microliters of the recombinant virus dilution containing 10⁴ TCID₅₀ per ml was inoculated into the allantoic cavity of 9-day-old embryonated chicken eggs. After three days, the allantoic fluids of inoculated eggs were harvested and clarified by centrifugation at 1,000 × g for 10 min. Supernatants were used for titration as described above.

Tumour cells were cultured in 24-well plates for 24 h as previously described, infected with rL-hIFN-λ1 at a MOI of 1.0, treated with NDV and phosphate-buffered saline (PBS) as controls. Then, at 24-h post-infection, the cells were fixed in 4% paraformaldehyde at 4°C overnight, followed by immunofluorescence staining with antibodies against NDV, IFN-λ1 and Hoechst 33342 staining. These stained SGC cells were monitored using immunofluorescence microscopy.

To measure the proliferation level, the survial of cells was assessed with MTT with six replicates for NDV, rL-hIFN-λ1 and PBS. At 12, 24, 36, 48, 60 and 72-h post-infection, cell survival was determined by incubating the cells with MTT. Absorbance at 490 nm was determined with ELISA microplate readers (BioTek Instruments, Inc., Winooski, VT, USA).

Total RNA of virus or tumor cells was extracted from the cultured cells using TRIzol reagent. First-strand cDNA synthesis was performed using oligo(dT) primers and M-MLV reverse transcriptase. The primer set for the IFN-λ1 gene and the IFNLR1 gene were shown in (Table I). All the procedures were based on manufacturer's instructions.

Cells were cultured in 24-well plates with slides and 10% FBS DMEM at 37°C with 5% CO₂. Then, the SGC cells were infected with rL-hIFN-λ1 and NDV within the logarithmic growth phase and fixed in 4% paraformaldehyde. After fixing, the cells were stained according to the manufacturer's instructions. The slides were observed and imaged under an optical microscope. The apoptotic index (AI) was calculated as the number of apoptotic cells/(the number of apoptotic cells + the number of non-apoptotic cells) × 100%.

Gastric adenocarcinoma cells in six-well plates were infected with NDV or rL-hIFN-λ1 virus at MOI of 1.0. Then the supernatant was harvested at 0, 6, 12, 24, 36 and 48-h post-infection. The expression levels of IFN-λ1 in the supernatant were measured by means of enzyme immune assay as described by human IFN-λ1 platinum kit (eBioscience, San Diego, CA, USA). The OD450 of the samples were determined and plotted as a standard curve. The standard curve was generated by serially diluting the stock enzyme IFN-λ1 in dilution buffer supplied by the kit.

To measure the cytokine protein, cytokine levels within supernatants was measured with ELISA. Antibodies for IFN-λ1, and IFN-γ were purchased from eBioscience and for IL-13 from R&D Systems, Inc. Manufacturer's instructions were followed. Standards and culture supernatants were plated in triplicate.

SGC and AGS cells with or without treatment were lysed in RIPA lysis buffer with a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc.). The protein concentrations were measured using a BCA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). An equal amount of protein from each sample was loaded onto a 10% polyacrylamide gel and separated by electrophoresis. Then, the proteins were transferred to PVDF membrane (Millipore, Temecula, CA, USA). The membranes were blocked for 1 h in 5% BSA, incubated with primary antibodies against specific proteins (i.e., caspase-3 for apoptosis, anti-NDV for infection, anti-hIFN-λl for expression of recombiant gene, anti-IFNLR1 for detection of IFNλ-l receptor components and β-actin as a control) and then incubated with HRP-conjugated secondary antibodies. The protein bands were scanned using a Typhoon 9400 variable mode imager (Amersham Biosciences, Buckinghamshire, UK) and detected by Pierce ECL Plus substrate (Thermo Fisher Scientific, Inc.).

Annexin V/propidium iodide (PI) double-staining was used to detect membrane events according to the manufacturer's instructions (Biotech, Beijing, China). Flow cytometric analysis of labeled cells was performed using a FACSort flow cytometer (FACSCalibur) and data were analyzed using CellQuest software (both from BD Biosciences, Franklin Lakes, NJ, USA). The cytogram of the four quadrants in the figure was used to distinguish normal (Annexin V⁻/PI⁺), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic cells (Annexin V⁻/PI⁺). The sum of early and late apoptosis was presented as total apoptosis. Finally, staining of the cells was observed with a fluorescence microscope. All experiments were carried out in triplicate.

Purified virus particles were bound to 200-mesh Formvar carbon-coated nickel grids (Electron Microscopy Science, Hatfield, PA, USA). For immunolabeling, grids were blocked in PBS containing 2% globulin-free BSA (Sigma-Aldrich) and incubated with chicken anti-NDV polyclonal IgG. Grids were then washed in blocking solution and incubated in goat anti-chicken IgG conjugated to 10-nm gold beads (Sigma-Aldrich). The grids received a final wash, followed by negative staining with 1% phosphotungstic acid. They were examined under a model H7500 transmission electron microscope (Hitachi High Technologies America, Inc., Schaumburg, IL, USA) at 80 kV. Images were obtained by using an XR100 digital camera system (Advanced Microscopy Techniques, Danvers, MA, USA).

The data comparisons were performed using one-way analysis of variance (ANOVA) in SPSS v17.0 software. p<0.05 and p<0.01 were considered statistically significant. All experiments were repeated at least three times.

Recombinant NDV expressing the human IFN-λ1 was generated by inserting the IFN-λ1 gene in the genomic cDNA of the NDV LaSota strain (Fig. 1A). For virus recovery, the heterogeneous gene carrying plasmid was transferred into 9–11 days old SPF embryonated chicken eggs for efficient virus propagation. After incubation for 5 days, the allantoic fluids were harvested and analyzed in a rapid plate HA test using chicken erythrocytes and observed by electron microscopy (Harbin Veterinary Research Institute) (Fig. 1B). The obtained viruses were named as rL-hIFN-λ1. The presence and accuracy of the inserted IFN-λ1 gene, the sequences from rL-hIFN-λ1 genome were confirmed by nucleotide sequence analysis (data not shown), the sequences contained whole IFN-λ1 genome were cloned by RT-PCR (Fig. 1C) by primers NDV-PMEI (Table I) set above and blow the PMEI set on NDV. The results showed they represented the correct sequences successfully inserted into the NDV genome, and mutation did not occur in rL-hIFN-λ1.

To compare the growth characteristics of the recombinant viruses rL-hIFN-λ1 and NDV, viral titers of the supernatants collected from infected SGC cells at 0, 24, 36, 48 and 72 h were determined by TCID₅₀. The growth properties of rL-hIFN-λ1 in the SGC cells were similar to those of the parental virus NDV (Fig. 2A). The result demonstrated that the recombinant viruses had mostly retained the growth characteristics of the parental virus in the SGC cells.

The allantoic cavities of 10-day-old embryonated SPF chicken eggs were inoculated with 10⁴ TCID₅₀ of rL-hIFN-λ1. The allantoic fluid was harvested after 4-day incubation. The viral titers was determined in duplicate by end-point titration. The results of the replication of rL-hIFN-λ1 indicated that the amount of the recombinant virus remains similar after 10 passages (Fig. 2B). After 10 passages, the presence of the human IFN-λ1 gene was confirmed by RT-PCR (Fig. 2C) and nucleotide sequence analysis (data not shown).

To determine the expression of IFN-λ receptor specific receptor IFNLR1 in human gastric cancer cell lines. We used RT-PCR assays and western blot analysis to analyze mRNA and protein expression of IFNLR1, respectively. The IFNLR1 was expressed in all examined gastric cancer cell lines, while the expression level of mRNA or protein was divided vividly among cell lines (Fig. 3), the SGC and AGS gastric cells expressed more IFNLR1 compared with other gastric tumor cells. For this reason we chose these two cell lines for our further study. The IFN-λ receptor IFNLR1 expressed in gastric cancer cell lines showed potential as a target for IFN-λ1 antitumor therapy. We found that PBMCs (human peripheral blood mononuclear cells) also expressed IFNLR1, which allowed us to study the antitumor immune response of rL-hIFN-λ1.

To define whether the IFN-λ1 gene integrated in the NDV genome, the infected gastric tumor cell lines SGC were tested for production of the protein IFN-λ1. The presence of IFN-λ1 genes in the viral genome and expression in virus-infected cells were confirmed as positive for all passage generations using indirect immunofluorescence (Fig. 4A) and western blot analysis (Fig. 4B). In the indirect immunofluorescence the IFN-λ1 protein (green) was highly expressed in the rL-hIFN-λ1-infected group, while the NDV protein (red) was expressed in both the rL-hIFN-λ1-infected group and NDV-infected group. Neither IFN-λ1 nor NDV protein was expressed in the control group.

To define whether the IFN-λ1 gene integrated in the NDV genome leads to the production of biologically active IFN-λ1 in supernatants of rL-hIFN-λ1-infected gastric tumor cells, we measured the quantitation of IFN-λ1 by ELISA. The supernatants of tumour cells were harvested 24, 48, 72 and 96 h after the infection. The IFN-λ1 expression reached the highest level at 72 h to 223 ng/ml in the supernatants of rL-hIFN-λ1-infected SGC cells, whereas the SGC cells infected with NDV did not release any IFN-λ1 into the supernatants (Fig 4C).

To elucidate the proliferation changes of rL-hIFN-λ1-infected gastric adenocarcinoma cells, MTT assay was utilized to portray dose-response curves. After 24 h of rL-hIFN-λ1 or NDV infection, the gastric tumour cells were assessed by MTT assay. OD of MTT in the rL-hIFN-λ1-infected group was weaker compared with that in the NDV-infected group, suggested that the rL-hIFN-λ1-infected group had a greater inhibition ratio. Moreover, the inhibition ratio increased over the dose after infection (Fig. 5).

The recombinant virus rL-hIFN-λ1 was designed for cancer therapeutics, and apoptosis was measured in gastric carcinoma cell lines.

TUNEL assay kit was used for monitoring apoptosis. The results revealed that the number of apoptotic cells and the AI was much higher in the rL-hIFN-λ1 compared with NDV group, the negative control group showed much less apoptosis (p<0.01) (Fig. 6A).

Furthermore, the levels of cleaved caspase-3, a key protein to indicate apoptosis, were measured by western blot analysis at 24 h after virus infecting tumour cell lines. The expression levels of cleaved caspase-3 proteins were upregulated in the rL-hIFN-λ1 group compared with the other NDV group and the negative control group (Fig. 6B).

Flow cytometry analysis showed that early apoptosis of cells was higher in the rL-hIFN-λ1-infecting group than that in NDV group, early apoptosis was not detected in the control group (Fig. 6C).

Th1/Th2 balance is confirmed to play an important role in tumor growth, the high expression of Th1 response and reduction is capable of inducing antitumor immunity to prevent cancer cell lines in preclinical study (14,27) and gastric cancer in clinical research (28). To determine whether rL-hIFN-λ1 could elevate the secretion of chemokines of tumor microenvironment to promote antitumor immune responses, SGC cells were infected with rL-hIFN-λ1 or NDV, after cultured for 72 h, supernatants were collected to stimulate PBMCs for 5 days, then the production as quantified of T-cell cytokines IFN-γ (representative of Th1 responses) and IL-13 (representative of Th2 responses) by ELISA everyday. Our studies revealed that the Th1 cytokine IFN-γ increased significantly and the Th2 cytokine IL-13 was highly decreased in the rL-hIFN-λ1 group, as compared with NDV and control groups (p<0.01) (Fig. 7A). To confirm whether the Th1/Th2 immune response change was related to cytokine IFN-λ1 or the NDV viral, anti-IFN-λ1 or anti-NDV was added during the cell culture with the former supernatants, then cytokine levels were tested after PBMCs cultured for 5 days. The Th1 response was enhanced, and the Th2 response inhibited much more in the anti-IFN-λ1 group than in the anti-NDV group, NDV group and control group showed no significant change. The results demonstrated that the tumor microenvironment cytokine skewed towards a Th1 response duo to the high dose of IFN-λ1 expressed by the rL-hIFN-λ1-infected tumour cells (Fig. 7B).