TLR4 signaling modulates pancreatic carcinogenesis. (A–E) 4-wk-old p48Cre;KrasG12D mice were treated with saline or LPS and sacrificed at 4 wk. (A and B) Pancreata were stained with H&E, Trichrome, and CD45 (A) and weighed (B). (C–E) The presence of graded PanIN lesions (C), fibrotic area (D), and leukocytic infiltrate (E) were quantified by examining 10 high-powered fields (HPFs) per pancreas (n = 6 mice/group; ***, P < 0.001). (F and G) 4-wk-old p48Cre;KrasG12D mice were treated with saline, caerulein, or caerulein + TLR4 inhibitor. Representative H&E-stained sections are shown, and the number of dysplastic ducts per HPF was calculated (n = 5 mice/group; ***, P < 0.001). (H) Live pancreatic mononuclear cells from 6-mo-old p48Cre;KrasG12D or WT mice were gated and costained for CD45, CD11c, CD3, F480, B220, Gr1, and TLR4. Median fluorescence for TLR4 is shown for specific cellular subsets. Data are representative of experiments repeated three times. (I) Sections of normal human pancreas (n = 3) and human pancreatic cancer (n = 19) were stained for TLR4. Representative images are shown, and data were quantified (***, P < 0.001). (J) Pancreatic duct fluid was harvested at the time of surgical resection from four patients with pancreatic cancer and two patients with benign endocrine tumors and tested for TLR4 ligand levels on HEK-Blue reporter cells (***, P < 0.001). (K) Pancreatic ductal fluid from two patients with pancreatic carcinoma was harvested at the time of operative duct transection and analyzed for HMGB-1 and S100A9 expression by Western blotting. Error bars indicate standard error of the mean.
