Supplemental methods and material
Reagents and transfections
We purchased SiRNAs from RiboBio (Guangzhou, China). We purchased human IL-18 over-expressing, knock-down and negative-control lentiviruses, and mouse IL-18 knock-down and negative control lentiviruses from Genechem (Shanghai, China). All transfections were carried out according to manufacturers’ instructions. We also bought recombinant mouse IL-18 (rmIL-18, Prospec, Israel), BAY11-7082 (Selleckchem, USA), recombinant human IL-18 (rhIL-18, Prospec) and curcumin (Sigma-Aldrich, USA).
Human Cytokine Array
A customized Human Cytokine Array was purchased from Raybiotech; the experiment was performed by a commercial service at KangChen Bio-Tech (Shanghai, China).
Hematoxylin and eosin staining and immunohistochemistry
We stained 4-μm paraffin-embedded sections of transplanted tumor tissue with hematoxylin and eosin (HE) and examined them with an Olympus BX51 microscope. Six randomly selected fields of each section were examined by two pathology specialists.
For IHC of IL-18, 4-μm paraffin-embedded tissues microarray sections were incubated overnight at 4°C with primary antibodies against human IL-18 (Medical & Biological Laboratories Co, Nagoya, Japan; 1:50) and then washed and incubated for 30 minutes with biotinylated goat anti-rabbit IgG. This antibody detects predominantly the mature form of IL-18. Sections were washed thoroughly and stained with diaminobenzidine. Images were captured using an Aperio ImageScope system. Each sample was examined by two pathology specialists who were blinded to patient diagnoses and outcomes. Staining intensity and positive cell proportions were recorded.
For IHC of IL-18, PCNA and Ki-67, 4-μm paraffin-embedded sections were incubated overnight at 4°C with primary antibodies against IL-18 (Medical & Biological Laboratories Co; 1:100), PCNA (Santa Cruz; 1:100), and Ki67 (Santa Cruz; 1:100) and then washed and incubated for 30 minutes with biotinylated goat anti-rabbit IgG. The sections were washed thoroughly, stained with diaminobenzidine, and examined by an Olympus BX51 microscope. Six randomly selected fields of each section were examined by two pathology specialists.
ELISA
Concentrations of IL-18 were measured with an ELISA kit (Medical & Biological Laboratories Co.), using the manufacturer’s protocol, which predominantly detects the mature IL-18 form (proform detection: ~0.7%). Mouse IFN-γ concentrations were measured using an ELISA kit (Boster, China) according to the manufacturer’s protocol.
CCK-8 assay
Cells were seeded in 96-well culture plates (2×104 cells/100μL/well) and grownin the incubator. We then added10μL of CCK-8 (Dojindo, Japan) solution to each well of the plate. Plates were incubated for 1 hour in the incubator and monitored the absorbance of the 450 nm valueusing a microplate reader.
Western blot analysis
Polyvinylidene difluoride (PVDF) membranes containing electrophoretically separated proteins from human primary pancreatic cancer cells or subcutaneous xenografts were separately incubated with the following antibodies: rabbit anti-IL-18 (Medical & Biological Laboratories Co; 1:500), rabbit anti-NF-κB (CST; 1:200), rabbit anti-p-NF-κB (CST; 1:200), rabbit anti-IKKα (CST; 1:200), rabbit anti-IKKβ (CST; 1:200), rabbit anti-p-IKKα/β (CST; 1:200), rabbit anti-p-IκBα (CST; 1:200), rabbit anti-IκBα (CST; 1:200), rabbit anti-MMP-3 (Boster; 1:200), rabbit anti-MMP-9 (Boster; 1:200), rabbit anti-c-myc (Boster; 1:200) antibody and rabbit anti-cyclin-D1 (Boster; 1:200). They were then incubated with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (CST; 1:2,000) and visualized by enhanced chemiluminescence (Boster).
Immunofluorescence
MIA-PaCa-2 and PANC-1 cells grown on cover slips in six-well plates were incubated overnight at 4°C with primary antibody to NF-κB (CST, 1:50), and then for 45 min at 37°C with FITC-conjugated goat anti-rabbit antibody (Boster). Slides were counterstained with DAPI to show cell nuclei and analyzed with an Olympus BX51 microscope.
RNA preparation and real-time PCR
Total RNA was extracted from human primary pancreatic cancer cells using a TRIzol kit (Invitrogen) according to the manufacturer's instructions. A 2-μg aliquot of each total RNA was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Kit (TransGen, China). Quantitative real-time PCR was performed using a SYBR Green Realtime PCR Master Mix (TransGen, China) according to the manufacturer’s instructions.
Supplementary table 1. Primer sequences for PCR.
Primers used in real-time PCR / Sequences (Forward and Reverse)Actin / F / CCTGGCACCCAGCACAAT
R / GGGCCGGACTCGTCATAC
MMP-1 / F / TGTCAGGGGAGATCATCGGGA
R / CTTCTCCCCGAATCGTAGTTATAGC
MMP-2 / F / GTGGATGCCGCCTTTAACTGG
R / GAAGAAGTAGCTGTGACCGCCG
MMP-3 / F / CCCAAATGCAAAGAAAGTGACAC
R / CAGCACAGGCAGGAGAAAACG
MMP-7 / F / GGGAACAGGCTCAGGACTATCTC
R / CAACATCTGGCACTCCACATCTG
MMP-8 / F / GCACTAACTTGACCTACAGGATTCG
R / CTGGTGAAGATGAGAGGTGATGC
MMP-9 / F / GGTTCGACGTGAAGGCGCAG
R / GCACTGCAGGATGTCATAGGTCAC
MMP-10 / F / CATTCAGTCTCTCTACGGACCTCC
R / ATTCAGGTTCAGGGTTCCAGTG
MMP-13 / F / TGGAATTAAGGAGCATGGCGAC
R / TTGCCGGTGTAGGTGTAGATAGG
cyclin-D1 / F / CCGTCCATGCGGAAGATC
R / ATG-GCCAGCGGGAAGAC
c-myc / F / ATCACAGCCCTCACTCAC
R / ACAGATTCCACAAGGTGC
Flow cytometry
Cells were dissociated, washed twice with BD PharMingen Stain Buffer (BD Bioscience, USA), resuspended with BD PharMingen Stain Buffer, antibodies added to the cells, incubated for 30 min at 4°C, and subjected to flow cytometry analysis (FACSAria, BD Immunocytometry Systems, USA).
We used the following antibodies in these assays: FITC Hamster anti-Mouse CD3e (BD), APC-Cy™7 Hamster anti-Mouse NK1.1 (BD), PE Rat anti-Mouse CD4 (BD), APC Rat anti-Mouse CD8a (BD), APC Rat anti-Mouse CD8a (BD), PE Rat anti-Mouse CD19 (BD), FITC Hamster anti-Mouse CD11c (BD), percp Rat anti-Mouse CD45R/B220 (BD), anti-mouse F4/80 (eBioscience, USA), PE-Cy™7 Mouse anti-Human CD11b/Mac-1 (BD), APC Rat anti-Mouse CD86 (BD), FITC Rat anti-Mouse CD49b (BD), APC-Cy™7 Rat anti-Mouse CD25 (BD), APC Rat anti-Mouse CD11b (BD), PE mouse anti-Human CD19 (BD), APC mouse anti-Human CD19 (BD), PE mouse anti-Human CD38 (BD), PE mouse anti-Human CD80 (BD), FITC mouse anti-Human CD3 (BD), APC mouse anti-Human CD44 (BD), PE-Cy™5 Mouse anti-Human CD8 (BD), PE mouse anti-Human CD1d (BD), PE mouse anti-Human CD8 (BD), PE mouse anti-Human CD56 (BD), FITC mouse anti-Human CD5 (BD), PE mouse anti-Human CD8 (BD), Anti-Mouse CD25 PE (eBioscience, USA), and Anti-Mouse CD11b PE (eBioscience).
Transwell assay
Cell invasion and migration were assessed using 24-well Corning Costar inserts with 8-μm pores. Upper surfaces of each insert were coated with Matrigel (BD) diluted 1:8, for 6 hours in an incubator. Cells (2×106) were added to upper chambers and incubated at 37°C, with migration assessed at 12 hours and invasion at 24 hours. Non-migrating and non-invading cells were removed with cotton buds from the top chambers. Cells remaining in bottom chambers were fixed with 100% methanol, stained with 1% crystal violet in 2% ethanol, and quantified visually in nine random fields using bright-field optics. Values for triplicate experiments are reported as mean ± SD of cell numbers.
Wound healing assay
Cells (5×106 per well) were cultured in six-well plates for 24 h. Cell layers were subsequently scratched with sterile plastic tips, washed with PBS twice, cultured for 24 h with medium containing 1% FBS, and photographed under an Olympus BX51 microscope.
Cytotoxicity experiments
Cytotoxic activities of T cells and NK cells were measured by a conventional 4-hour Chromium-51 (51Cr) release assay. Effector cells were seeded into round-bottom 96-well plates in 100 μL/well and at appropriate concentrations. PANC-1 and MIA-PaCa-2 cells were used as targets. Tumor targets (1×106 cells/mL) were incubated with 100 μCi sodium chromate (Perkin Elmer, USA) for 60 min at 37°C, washed, and incubated for an additional 30 min to allow spontaneous 51Cr release. They were then added to the effector cells at the indicated effector-to-target (E:T) ratios, and incubated for 18 h at 37°C in CO2 incubators. Supernatants were then transferred to LumaPlate-96 wells (Perkin Elmer). The plate dried down and counted on a Packard TopCount NXT. All cultures were performed in triplicate. Cytotoxicity percentages were calculated as: 100 × ([test 51Cr release] – [spontaneous 51Cr release]) / ([maximum 51Cr release] – [spontaneous 51Cr release]), where maximum and spontaneous counts were calculated from triplicates of target cells incubated with 2% Triton X (Sigma-Aldrich, USA) and in plain medium, respectively.
Dual-luciferase assay
PANC-1 cells were transiently transfected with reporter vectors with the rhIL-18 treatment. Cells were collected for luciferase assays after 48 hours’ cultivation, using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol.
Chromatin immunoprecipitation assay
The ChIP Assay Kit was bought from Merck Millipore and used according to the manufacturer’s protocol.
Supplementary table 2. Primer sequences for PCR.
Primers used in ChIP / Sequences (Forward and Reverse)c-myc promoter / F / AGGCGCGCGTAGTTAATTCA
R / TCGCATTATAAAGGGCCGGT
cyclin-D1 promoter / F / TGTCAGGGGAGATCATCGGGA
R / CTTCTCCCCGAATCGTAGTTATAGC
MMP-3 promoter / F / TCCAGTTTTCTCCTCTACCAAGAC
R / TTGCTTTCATCCAAATGGCAGCAG
MMP-9 promoter / F / GCCATGTCTGCTGTTTTCTAGAGG
R / CACACTCCAGGCTCTGTCCTCTTT