Background
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and a leading cause of cancer deaths worldwide [
1]. Despite advances in our understanding of the pathophysiology of PDAC, there have been limited improvements in clinical outcome. This is in part due to the presence of a highly reactive stroma that promotes carcinogenesis, confers resistance to conventional therapy [
2‐
5] and increases PDAC aggressiveness [
6]. The tumour stroma comprises extracellular matrix and a variety of cell types including cancer-associated fibroblasts, stellate cells, endothelial cells and inflammatory cells such as macrophages and monocytes [
7,
8]. The latter secrete the potent chemokines S100A8 and S100A9 which are low molecular proteins of S100 family of EF-hand calcium binding proteins. Both proteins are constitutively expressed in cells of myeloid origin such as monocytes and neutrophils [
9]. Once secreted in the extracellular space, S100A8/A9 act as chemo-attractants recruiting further inflammatory cells and creating an inflammatory microenvironment that promotes tumour development [
10,
11]. In-vivo and in-vitro experiments have shown a strong link between the levels of S100A8/S100A9 and several disorders such as cystic fibrosis, rheumatoid arthritis, atherosclerosis and cardiovascular disease [
11‐
14]. Secreted S100A8/S100A9 proteins have also been implicated in cancer growth [
15,
16] and in the establishment of a favourable environment for metastasis by promoting the migration of monocytes and tumor cells to metastatic sites [
17,
18]. This process involves vascular endothelial growth factor-A (VEGF-A) and TGF-ß-SMAD4 signalling pathways and both toll-like receptor-4 (TLR-4) [
19], and the receptor for advanced glycation end-products (RAGE) [
20]. However little is known about S100A8 and S100A9-mediated cross talk between stromal monocytes and pancreatic cancer cells. In the current study, we show that exposing monocytes to pancreatic tumour cell-derived conditioned media increased the expression of both S100A8 and S100A9. Secreted S100A8/A9 from monocytes, in turn, led to secretion of several cytokines from pancreatic tumour cells. This was mediated by RAGE and was associated with phosphorylation of Erk1/2, p38 kinase and activation of the nuclear factor kappa-light-chain-enhancer of activated B cells pathway (NF-κB).
Methods
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.
Cell culture
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.
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.
Immunoblotting analysis
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 analysis
Statistical analyses were performed using student t-test. A p-value less than 0.05 was considered statistically significant.
Discussion
The complex interaction between pancreatic tumour cells and surrounding immune cells is increasingly understood to be vital in sustaining tumour growth and progression [
23,
24]. Stromal cells potentiate tumour development through the secretion of a complex network of autocrine and paracrine factors including cytokines, proteases and growth factors [
25,
26].
We previously showed that the stromal compartment of colorectal and pancreatic cancers contain CD14
+ monocytic cells expressing S100A8 and S100A9 proteins [
21]. Moreover, we observed that Smad4-negative pancreatic and colorectal tumours contain fewer stromal S100A8-positive monocytes than their Smad4-positive counterparts [
21,
27], suggesting a regulated relationship between cancer cells and surrounding monocytic cells. No such relationship was observed for S100A9-positive monocytes, indicating a distinction between S100A8- and S100A9-expressing tumour-associated monocytes. We also reported that exogenously added S100A8 and S100A9 proteins enhanced the migration and proliferation of colorectal and pancreatic cancer cells in culture [
15]. In the current study, we found that S100A8 and S100A9 proteins influenced cytokine secretion from pancreatic cancer cells in overlapping but also distinct ways. Whilst both recombinant S100A8-GST and S100A9-GST proteins induced increased secretion of the pro-inflammatory cytokines IL-8 and TNF-α and the growth factor FGF, only S100A8-GST induced PDGF secretion. This again highlights a distinction between these two closely related S100 proteins.
S100A8/A9 are reported to stimulate cells through binding to the cellular receptor RAGE, a primary receptor which, when activated, is associated with amplified inflammatory conditions and tumour progression [
27]. Here we found that the secretion of TNF-α and FGF was mediated via the RAGE receptor, whilst the RAGE receptor was not required for IL-8 and PDGF secretion. S100A8/A9 signaling from receptors other than the RAGE receptor has previously been reported, including the binding of S100A8/A9 to Toll-like receptor-4 receptor and the newly discovered EMMPRIN receptor that exclusively binds to S100A9 but not S100A8 [
28,
29].
Previous studies have elucidated a role for S100A8/A9 in preparing a pre-metastatic niche in distant organs through activation of serum amyloid A3 and toll-like receptor 4 (TLR4) [
17,
19]. For patients with pancreatic cancer, release of S100A8/A9 proteins from myeloid cells increases CD33
+CD14
−HLA-DR
− myeloid-derived suppressor cells [
30]. Our observation that media conditioned from pancreatic cancer cells, as well as the individual cytokines TGF-β and TNF-α, induced the expression of S100A8 and S100A9 proteins in HL-60 cells and primary monocytes supports the concept of a paracrine feedback loop, whereby cancer cells induce monocytic S100A8/A9 expression through cytokine secretion and the S100 proteins in turn induce cytokine secretion. Secretion of TGF-β by pancreatic cancer cells induces extra-cellular matrix (ECM) formation and enhances fibrosis [
31]. Both TGF-β and TNF-α have been associated with increased cancer cell proliferation and motility. Additionally, both TGF-β and TNF-α participate in the creation of inflammatory environments that enhance tumorigenesis and immunosuppressive activity blocking the host’s anti-tumour response [
32]. Induction of S100A8 and S100A9 expression in response to primary lung tumor cell-derived soluble factors VEGF-A, TGF-β and TNF-α in myeloid cells prior to tumor metastasis has been reported [
17].
Binding of S100A8 and S100A9 to RAGE receptor activates a cascade of downstream signalling pathways [
27]. We report here a RAGE-dependent increase in levels of phosphor-Erk1/Erk2 and phospho-p38 phosphorylation following S100A8 and S100A9 treatment of pancreatic cancer cells, although we did not examine the basal levels of these proteins. Furthermore, the S100 proteins activated the NF-κB pathway through RAGE. Activation of the MAPK/NF-κB signaling axis is known to enhance tumor growth, survival, and migration [
33]. Moreover, NF-κB activation is also critical in establishing an inflammatory microenvironment which in turn sets the stage for cancer progression [
33].
In summary, our data suggest the existence of paracrine feedback loop between stroma-associated S100A8/A9-secreting cells and pancreatic tumour cells. This feedback loop may affect tumour progression. Hence, targeting S100A8/A9 represents a potential therapeutic option to curtail the aggressive nature of pancreatic cancer.
Acknowledgements
The authors would like to acknowledge King Abdullah International Medical Research Center (KAIMRC) for their financial support to cover the publication fees.