Volume 115, Issue 5, Pages 1254-1262 (November 1998) Malignant transformation of duct-like cells originating from acini in transforming growth factor α transgenic mice  Martin Wagner, Hardi Lührs, Günther Klöppel, Guido Adler, Roland M. Schmid  Gastroenterology  Volume 115, Issue 5, Pages 1254-1262 (November 1998) DOI: 10.1016/S0016-5085(98)70098-8 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Development of tubular complexes in TGF-α transgenic mice. Tissue sections were stained with H&E. (A) Early changes at day 14 include mild interlobular fibrosis (arrows) and decreased acinar cell height (asterisks). (B) At day 28, fibrosis (arrows) and altered acinar morphology (asterisks) is visible. (C) After 180 days, pancreatic fibrosis and typical tubular complexes (asterisks) can be seen next to normal acinar cells (arrowhead). At a higher magnification, the transition from acinar toward ductal morphology is evident. The inserts represent pancreas sections of littermate controls at the same age. A, B, and C: original magnification 50×; D: 250×. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of acinar and ductal markers in the pancreas of TGF-α transgenic mice. Sections were incubated with the respective antibodies or stained for carbohydrase activity. (A) Staining for amylase and acinar-1 in 180-day-old transgenic mice (TGF-α) compared with littermate controls (WT). (B) Carbonic anhydrase activity in tubular complexes of TGF-α and in controls (WT). Duct-1 expression is strongly expressed in tubular complexes. In control mice (WT) it is restricted to the ducts. (C) Staining for cytokeratin 8 (TROMA1) and 18 (TROMA2) in TGF-α transgenic (TGF-α) animals and littermate controls (WT). Original magnification 50×. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of the EGFR and TGF-α in the pancreas of TGF-α transgenic mice and control animals. In wild-type animals, (A) the EGFR is only expressed in ductal cells, (B) whereas cells within tubular complexes express high levels of EGFR. (C) Western analysis indicates an increase of the EGFR at days 28 and 180. (D) Slot blot analysis with 10 μg of total RNA shows a constant high level of TGF-α expression in the pancreas of 0 to 180-day-old TGF-α transgenic animals. The 185 ribosomal probe served as control. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of the EGFR and TGF-α in the pancreas of TGF-α transgenic mice and control animals. In wild-type animals, (A) the EGFR is only expressed in ductal cells, (B) whereas cells within tubular complexes express high levels of EGFR. (C) Western analysis indicates an increase of the EGFR at days 28 and 180. (D) Slot blot analysis with 10 μg of total RNA shows a constant high level of TGF-α expression in the pancreas of 0 to 180-day-old TGF-α transgenic animals. The 185 ribosomal probe served as control. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of the EGFR and TGF-α in the pancreas of TGF-α transgenic mice and control animals. In wild-type animals, (A) the EGFR is only expressed in ductal cells, (B) whereas cells within tubular complexes express high levels of EGFR. (C) Western analysis indicates an increase of the EGFR at days 28 and 180. (D) Slot blot analysis with 10 μg of total RNA shows a constant high level of TGF-α expression in the pancreas of 0 to 180-day-old TGF-α transgenic animals. The 185 ribosomal probe served as control. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Dysplastic changes and tumors in the pancreas of TGF-α transgenic mice. (A) Highly proliferating tubular complexes are clustered and surrounded by delicate connective tissue (animal 195; original magnification 50×). (B) The higher magnification (animal 190; original magnification 100×) reveals irregular cellular morphology. This includes pleomorphic nuclei with abnormal chromatin patterns and frequent mitotic figures in ductal structures. (C) Large cysts are lined with flat epithelium (animal 333; original magnification 50×). (D) In some animals we found intracystic proliferations characterized by a multilayer epithelium and intracystic, papillary projections with a varying degree of nuclear anaplasia (animal 194; original magnification 50×). (E) Tumors display a papillary to cystic phenotype surrounded by dense connective tissue (animal 316; original magnification 12.5×). (F) A higher magnification reveals the origin of the tumors from large cysts. Tumors invade the connective tissue and show a marked nuclear anaplasia (animal 315; original magnification 100×). Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Characterization of tumors with acinar and ductal markers. (A) Sections were incubated with antibodies as indicated or stained for carbonic anhydrase activity. Compared with normal pancreatic tissue, tumor sections display reduced immunoreactivity for amylase and acinar-1, whereas carbonic anhydrase activity and reactivity for duct-1 was increased. (B) The expression of cytokeratin 8 and 18 remained unchanged within tubular complexes. Tumors overexpressed the EGFR. Original magnification 50×. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Expression of p53 in the pancreas of TGF-α transgenic mice and control animals. Sections were incubated with polyclonal antiserum to p53. Tumors and tubular complexes show enhanced nuclear staining for p53 compared with wild-type pancreas and the remaining normal acinar cells in TGF-α transgenic mice. Original magnification 250×. Gastroenterology 1998 115, 1254-1262DOI: (10.1016/S0016-5085(98)70098-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions