Cancer-Stellate Cell Interactions Perpetuate the Hypoxia-Fibrosis Cycle in Pancreatic Ductal Adenocarcinoma1

Background and Aims Although both cancer and stellate cells (PSCs) secrete proangiogenic factors, pancreatic cancer is a scirrhous and hypoxic tumor. The impact of cancer-PSCs interactions on angiogenesis was analyzed. Methods Expression of periostin, CD31, and α-smooth muscle actin was assessed by immunohistochemistry. Human PSCs and cancer cells were cultivated under normoxia and hypoxia alone, or in coculture, to analyze the changes in their angiogenic and fibrogenic attributes, using enzyme-linked immunosorbent assay, immunoblot, and quantitative polymerase chain reaction analyses and growth of cultured endothelial cells in vitro. Results On the invasive front of the activated stroma, PSCs deposited a periostin-rich matrix around the capillaries in the periacinar spaces. Compared with the normal pancreas, there was a significant reduction in the microvessel density in chronic pancreatitis (five-fold, P < .001) and pancreatic cancer (four-fold, P < .01) tissues. In vitro, hypoxia increased PSCs' activity and doubled the secretion of periostin, type I collagen, fibronectin, and vascular endothelial growth factor (VEGF). Cancer cells induced VEGF secretion of PSCs (390 ± 60%, P < .001), whereas PSCs increased the endostatin production of cancer cells (210 ± 14%, P < .001) by matrix metalloproteinase-dependent cleavage. In vitro, PSCs increased the endothelial cell growth, whereas cancer cells alone, or their coculture with PSCs, suppressed it. Conclusions Although PSCs are the dominant producers of VEGF and increase endothelial cell growth in vitro, in the peritumoral stroma, they contribute to the fibrotic/hypoxic milieu through abnormal extracellular matrix deposition and by amplifying endostatin production of cancer cells

Syndecan-2 promotes perineural invasion and cooperates with K-ras to induce an invasive pancreatic cancer cell phenotype

Abstract Background We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells. Methods Syndecan-2 (SDC-2) expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. Results SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides) did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and - further downstream - phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. Conclusion SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.</p

AM251 induces changes in PSC phenotype.

<p>(A) Invasion assays demonstrated a trend toward increased invasiveness induced by AM251. (B) MMP-2 levels were unchanged by antagonist treatment. (C&D) AM251-treated pancreatic stellate cells were smaller and thinner, with a more stretched shape, an increased length, and a loss of intracellular fibres (control PSC: insets and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001701#pone-0001701-g008" target="_blank">Figure 8C&D</a>). Cells treated with AM630 (E&F) closely resembled the control PSC (see insets C&D and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001701#pone-0001701-g008" target="_blank">Figure 8C&D</a>). Original magnification: x 40 (C&E), x 80 (D&F). Nuclear stain: DAPI. White bars: control; black bars: AM251; grey bars: AM630. Data are shown as mean±SEM.</p

WIN de-activates pancreatic stellate cells.

<p>(A) IL-6 and MCP-1 secretion were significantly reduced by WIN (p = 0.001 and p = 0.0002, respectively), independent of TGFbeta (B; unchanged TGFbeta levels). The reduction in IL-6 and MCP-1 levels was partially reversed by a combination of the CB1-receptor and CB2-receptor antagonists AM251 and AM630 (A). While control PSC secreted significant amounts of fibronectin and collagen 1 (as seen by an intense signal at 220 and 190 kDa, respectively), treatment with WIN reduced the signal at the respective molecular weights (B; immunoblots of cell culture supernatants, pooled from three independent experiments). AlphaSMA levels were also suppressed by WIN (B; immunoblot of PSC cell lysates; gamma-tubulin: equal loading control). A combination of both antagonists AM251 and AM630 partially reversed the suppressive effects elicited by incubation with WIN (B, immunoblots of fibronectin, collagen 1 and alphaSMA). White bars: control; black bars: WIN55,212-2±AM251/AM630. Data are shown as mean±SEM.</p

Cannabinoid receptor 1 in human chronic pancreatitis tissues.

<p>CB1 immunohistochemistry: staining of tubular complexes (A) but immunonegativity of infiltrating mononuclear cells (B); various staining intensities of intrapancreatic nerves (C&D); pancreatic stellate cells in areas of fibrosis (E) or isolated and cultured in vitro (F). Original magnification: x20 (A, B, D), x40 (C), x80 (F). Insets: negative controls. Lower insets (A): CB1 in normal pancreas.</p

Effects of cannabinoid receptor antagonism on pancreatic stellate cells.

<p>(A) MTT assays after 48 hours' incubation of PSC with graded concentrations of CB1- and CB2-receptor antagonists AM251 and AM630 (p = n.s. for concentrations of 1.25, 2.5 and 5 µM). (B) LDH in cell culture supernatants and the fraction of apoptotic cells as judged by the GuavaNexin™ test. AM251 significantly increased IL-6 (C) but decreased MCP-1 secretion (D); AM630 had no effect on IL-6 (C) but induced a tendency towards increased MCP-1 levels (D). CB1-/2-receptor antagonism did not affect TGFbeta (E). Data are shown as mean±SEM (*, p<0.05).</p

Cannabinoid receptor blockade alters synthesis of ECM proteins.

<p>As assessed by immunoblot analysis of cell culture supernatants, AM251 and AM630 (at 2.5 µM) did not affect collagen and fibronection secretion. In PSC cell lysates, alphaSMA levels remain unchanged following CB1-receptor antagonism whereas AM630 induced increased alphaSMA protein levels. White bars: control; black bars: AM251; grey bars: AM630. Data are shown as mean±SEM.</p

Cannabinoid receptor 2 and endocannabinoid levels in chronic pancreatitis.

<p>Using an anti-CB2 antibody, tubular complexes were strongly stained (A). Invading mononuclear cells were also immunopositive (B), whereas intrapancreatic nerves were mostly unstained or only faintly positive (B). Pancreatic stellate cells were immunopositive for CB2 (C). The endocannabinoids anandamide (AEA) and 1+2-arachidonoylglycerol (1+2-AG) were lower in chronic pancreatitis (AEA: p = 0.14 and 1+2-AG: p = 0.0066; D). Cultured pancreatic stellate cells were faintly CB2-immunopositive (E). Original magnification: x20 (A, B), x40 (C), x80 (F). Insets: negative controls. Lower insets (A): CB2 in normal pancreas.</p

Cannabinoid receptor activation on PSC reduces growth, independent of apoptosis or necrosis.

<p>(A) MTT tests of WIN-treated PSC revealed dose-dependent inhibition of growth (p<0.05 at concentrations of 2.5 and 5 µM). (B&C) While AM251 and AM630 alone were not effective, pre-treatment with a combination of AM251 and AM630 blocked WIN-induced growth inhibition (D). The reduction in proliferated cells was neither due to necrosis (unchanged LDH levels) nor to apoptosis (constant low number of apoptotic cells in treated versus control PSC; E). Data are shown as mean±SEM (* p<0.05).</p