Development of orthotopic pancreatic cancer model for observation of metastasis. (A) Development of orthotopic pancreatic cancer and gross inspection. 1. PANC-1–derived clones were orthotopically implanted in the parenchyma of the pancreatic head. 2. Luciferase activity imaging at two days following tumor cell injection confirming orthotopic implantation at the injection site without cell spillage. 3–8. Metastasis to the peritoneum, peritoneum and liver, liver, diaphragm and liver, mesenteric lymph node and intestinal wall, and lung, indicated by arrowheads (▲), were obvious macroscopically. (B) Histological analysis of metastatic lesions. Metastatic lesions of the spleen, liver, downward mucosa of the intestine, intestinal wall, lung, vein tumor thrombi (H&E staining, left: ×100 magnification; right: ×200 magnification, Δ indicates tumor). (C) Increased incidences of metastasis at different sites in MUC4/Y overexpression group compared to the control group (n = 8 per group). Comparisons between groups were tested with the chi-square test, *P <0.05.