Volume 121, Issue 3, Pages 678-684 (September 2001) Claudin-4: A new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin  Patrick Michl, Malte Buchholz, Monika Rolke, Steffen Kunsch, Matthias Löhr, Bruce McClane, Shoichiro Tsukita, Gerhard Leder, Guido Adler, Thomas M. Gress  Gastroenterology  Volume 121, Issue 3, Pages 678-684 (September 2001) DOI: 10.1053/gast.2001.27124 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Overexpression of claudin-4 in pancreatic cancer tissues and colon carcinomas, as determined by Northern blot analysis. P1–P5, pancreatic adenocarcinomas, representative of 31 tested pancreatic adenocarcinomas; C1–C5, adenocarcinomas of the colon, representative of 12 tested colon cancers; PV, carcinoma of the papilla Vateri; G, gastric adenocarcinoma, representative of 3 tested gastric cancers; B, adenocarcinoma of the breast, representative of 4 tested breast cancer tissues; S, soft tissue sarcoma, representative of 2 tested sarcoma tissues; CP, chronic pancreatitis, representative of 9 tested chronic pancreatitis tissues; N, normal pancreas, representative of 9 tested normal pancreas tissues. (B) Expression of claudin-4 in human tissues; 44 of 61 tested tissues on a multiple tissue array are shown. 1, whole brain; 2, cerebral cortex; 3, cerebellum; 4, medulla oblongata; 5, putamen; 6, substantia nigra; 7, thalamus; 8, pituitary gland; 9, spinal cord; 10, heart; 11, aorta; 12, left atrium; 13, left ventricle; 14, esophagus; 15, stomach; 16, duodenum; 17, jejunum; 18, ileum; 19, ileocecum; 20, ascending colon; 21, transverse colon; 22, descending colon; 23, rectum; 24, kidney; 25, skeletal muscle; 26, spleen; 27, thymus; 28, peripheral blood leukocyte; 29, lymph node; 30, bone marrow; 31, trachea; 32, lung; 33, placenta; 34, bladder; 35, uterus; 36, prostate; 37, testis; 38, ovary; 39, liver; 40, pancreas; 41, adrenal gland; 42, thyroid gland; 43, salivary gland; 44, mammary gland. Signal intensities are expressed as arbitrary units. (C) Northern blot analysis of claudin-4 expression in various pancreatic cancer cell lines and HFL fibroblasts. Panc-1 cells were grown with or without TGF-β (10 ng/mL) for 24 hours under serum-free conditions. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunocytochemistry of claudin-4 in Capan-1 cells, grown (A) on chamber slides, (B) in a pancreatic adenocarcinoma tissue, and (C) in adjacent normal pancreatic tissue (original magnification 63×). Extracellular matrix is indicated by an asterisk, and tumor cells are indicated by an arrow. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of CPE on the pancreatic cancer cell lines Capan-1, Panc-1, and MiaPaca-2 and the fibroblast cell line HFL, as determined in trypan blue exclusion assays. The corresponding expression levels of claudin-4 RNA are shown below the bar graph. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Dose-dependent cytotoxic effect of CPE on Panc-1 cells, as determined in 86Rb-release assays. Results are expressed ± SEM and are representative of 3 independent experiments. *P < 0.05 compared with untreated cells. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Dose-dependent cytotoxic effect of CPE at different concentrations on Panc-1 cells transfected to overexpress TGF-β1 or mock-transfected Panc-1 cells, as determined in 86Rb-release assays. Results are expressed as mean ± SEM and are representative of 3 independent experiments. *P < 0.05 compared with TGF-β–transfected cells incubated with the same CPE concentration. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of CPE or NaCl 0.9% on the volume of Panc-1 nude mouse xenograft tumors, as determined on days 1 and 7. Results are representative of 2 independent experiments. In each experiment, xenograft tumors of 6 mice were injected with 2 μg CPE dissolved in 50 μL NaCL 0.9% and 6 xenograft tumors of control animals were injected with 50 μL NaCL 0.9% intratumorally on days 1, 2, 4, and 5. *P < 0.05 compared with tumor volume on day 1. Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Treatment of claudin-4–expressing Panc-1 xenografts and claudin-4–negative HT-1080 xenografts with CPE or NaCl 0.9%. Necrotic areas appear as bright, eosin-stained areas in the center of the H&E-stained tumor. Boxed inserts are shown at higher magnification (original magnification 20×). Gastroenterology 2001 121, 678-684DOI: (10.1053/gast.2001.27124) Copyright © 2001 American Gastroenterological Association Terms and Conditions