MAPK antibodies (phospho-JNK, phospho-Erk1/2 and phospho-p38) were obtained from Cell Signaling Technology. S100A8 and S100A9 antibodies were purchased from Santa Cruz and the Bio-Plex Pro 27 Plex Human Cytokine, Chemokine and Growth Factor Assay was from Bio-Rad Laboratories Ltd., Hercules, CA, USA.
Pancreatic cancer cell lines; CFPAC-1 (ATCC: CRL-1918), Suit-2, BxPC-3 (ATCC: CRL-1687) and Panc-1 (ATCC: CRL-1469) were obtained from ATCC (Rockville, MD) and maintained in a humidified incubator at 37 °C in RPMI-1640 containing 10% FBS, 2 mm l-glutamine, 2500 IU/mL penicillin and 5 μg/mL streptomycin (all from Sigma, Poole, UK). All cell lines were authenticated using short tandem repeat profiling against international reference standards.
Isolation of human monocytes
Blood samples were obtained with written consent from two healthy volunteers under approved protocols at the Royal Liverpool University Hospital. Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) obtained from buffy coat preparations. Enriched monocyte fractions were isolated by positive selection using CD14-magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. Monocytes were eluted in 5 mL MACS buffer and the purity (> 90%) verified by FACS analysis using anti-CD14 conjugated FITC antibody or isotype control.
Stimulation of human monocytes
Freshly isolated monocytes were plated in 24-well plates in RPMI 1640 media supplemented with 10% FBS and stimulated with tumour cell-conditioned media from Panc-1, Suit-2 or BxPC-3 cell lines. Isolated monocytes were also treated with recombinant human growth factors (TGF-β, VEGF, IL-8 and FGF). After 24 h incubation, cell lysates were prepared using Tris/SDS lysis buffer and subjected to immuno-blotting assays.
Conditioned media preparation
Pancreatic cancer cells were counted and seeded in 6-well plates in RPMI media. After 24 h, cells were washed twice with PBS and incubated in serum free-RPMI and treated with 2 μg/mL of the recombinant proteins S100A8-GST, S100A9-GST or GST and incubated for an additional 24 h at 37 °C. For blocking experiments, cells were incubated with anti-RAGE antibody (R&D, Abingdon, UK) for 1 h prior to addition of recombinant proteins. For neutralising experiments, S100A8-GST and S100A9-GST proteins were incubated with respective neutralising antibodies (anti-S100A8 and anti-S100A9) for 1 h at 37 °C before treating cancer cells in culture. Cell supernatants were collected, centrifuged at 500x g for 5 min, to remove cellular debris, and then filtered through 0.2 μm filter units. Conditioned media were stored in -80 °C freezer until use.
Cytokines profiling assay
A Luminex assay (Bio-Plex Pro 27 Plex Human Cytokine kit) enabling the simultaneous measurement of 27 secreted cytokines in the conditioned media of pancreatic cancer cells was performed. Briefly, serially diluted standards and undiluted conditioned media (50 μL) were added to a microfilter plate containing antibody-coupled beads for each of the 27 analytes and incubated for 60 min with continuous shaking at room temperature. After washing steps, the biotinylated detection antibodies were added for 30 min with shaking. The microfilter plate was washed again, and Streptavidin-PE (50 μL) added and incubation continued at room temperature with shaking (900 rpm for 1 min followed by 300 rpm for 15 min). Assay buffer (125 μL) was added to each well of the microfilter plate before being read on a Bio-Plex 200 machine.
Total cell lysates were extraction by re-suspending cells in 100 mm Tris–HCl (pH 6.8) containing 2% w/v SDS and a protease inhibitor cocktail (Complete, Mini, EDTA-free protease inhibitors; Roche Applied Science, UK). Protein samples were quantified using a BCA protein assay kit (Perbio Science Ltd., Cramlington, UK) then subjected to SDS–PAGE. Separated proteins were transferred to hybond nitrocellulose membranes (Amersham Biosciences, Bucks, UK). Membranes were then blocked for 1 h with TBS containing 0.1% Tween-20 (TBS-T) and 5% milk (Bio-Rad Laboratories Ltd., Hemel Hempstead, Hertfordshire, UK) and then incubated overnight at 4 °C with antibodies specific for phospho-JNK, phospho-ERK1/2 and phospho-p38 (Cell Signaling Technology, Beverly,CA, USA) diluted 1:1000 in 5% BSA in TBST. The β-actin (clone AC-15, Sigma, Poole, UK) was used at a 1:10,000 dilution. Blots were washed with TBST and incubated for 1 h with horseradish-peroxidase (HRP)-conjugated secondary antibodies diluted 1:4000 in 5% skim milk in TBST. Bound HRP was visualised using the enhanced chemiluminescence kit (PerkinElmer Life Sciences, Bucks, UK).
NF-κB luciferase assay
Pancreatic cancer cells, Panc-1, were trypsinised, counted and transfected using nucleofector and nucleofector solution-R (Amaxa, UK) according to the manufacturer’s instructions. Cells were transfected with 3 μg of NF-κB plasmid or control plasmid from Clontech. 3 μg of GFP plasmid (Amaxa) was also used to monitor transfection efficiency. Cells were immediately removed from cuvettes, transferred into prepared 6-well plates and incubated in a humidified 37 °C/ 5% CO2 incubator. After 24 h of incubation, the cells were washed twice with PBS and incubated in serum free RPMI with or without 10 μg/mL of polymyxin-b, the latter used in order to monitor the stimulatory effect of bacterial LPS. Cells were treated with 2 μg/mL of either S100A8, S100A9, GST recombinant proteins or human TNF-α (50 ng/mL), as a positive control of NF-κB induction, and incubated for an additional 24 h. Luciferase activity was measured according to the Clontech kit recommendations (Clontech, Mountain View, CA, USA).
Statistical analyses were performed using student t-test. A p-value less than 0.05 was considered statistically significant.