Volume 126, Issue 1, Pages 196-207 (January 2004) Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co- receptor gp130  Louise M. Judd, Barbara M. Alderman, Meegan Howlett, Arthur Shulkes, Chris Dow, Jill Moverley, Diane Grail, Brendan J. Jenkins, Matthias Ernst, Andrew S. Giraud  Gastroenterology  Volume 126, Issue 1, Pages 196-207 (January 2004) DOI: 10.1053/j.gastro.2003.10.066

Figure 1 Age-dependent changes in the stomachs of the mutant mice. Mice were killed at the ages specified, stomachs were removed, and pinned out for macroscopic examination. (A) Forty-week-old gp130WT/WT; (B–E) gp130757F/F, respective ages 9, 17, 26, and 48 weeks; and (F) 17-week-old gp130WT/757F. Arrow indicates site of gastric ulceration. Total stomach area (G) and adenoma area (H) were calculated and represented as a function of age of the mice. ∗Indicates that the mean value is significantly different (P < 0.05) from the age-matched control. ∗∗Indicates that the mean value is significantly different (P < 0.05) from the 0–6-week-old mice of the same genotype. (Original magnification 2×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 2 Morphologic analysis of the gastric mucosa in gp130757F/F mice demonstrating hyperplastic, inflammatory, and dysplastic changes. Sections of stomach from 6-week-old (A and B) and 40-week-old (C and D) gp130WT/WT (A and C) and gp130757F/F (B and D) mice were stained with H & E. The mucosa of gp130757F/F mice was severely hyperplastic demonstrating branching tortuous glands, with inflammatory foci associated with dysplasia (marked with asterisk) invading the muscularis mucosa (D and H). Numerous mitotic (E, white arrow) and apoptotic profiles (E, black arrow) were observed. (F) A proportion of the blood vessels contained thrombotic clots. (G) Numerous epithelial cells demonstrate nuclear atypia as well as significant nuclear stratification. (Original magnification: A–D, 50×; E, 250×; and F–H, 150×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 3 Immunohistochemical detection of PCNA reactive cells in wild-type antrum (A) gp130757F/F (B) tumor showing extensive proliferation in surface epithelium and atrophic glands. (C and D) Focal and variable proliferation of epithelial cells in well-developed tumors. (Original magnification: A, 36×; and B–D, 40×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 7 Metaplasia in antral tumors of the gp130757F/F mouse. Two types of metaplastic cells are present in mucosal glands: cells that are weakly PAS/Alcian blue/TFF2 positive but have a well-defined brush border (arrows in A–D) and cells with a foamy appearance (SPEM-like) that are strongly PAS/Alcian blue/TFF2 positive and intrinsic factor negative (asterisk in A–D). Near consecutive sections from a 40-week-old gp130757F/F tumor stained with H & E (A); PAS/Alcian blue (B); anti-TFF2 antiserum No. 6.4 (C); anti-intrinsic factor antiserum (D) (inset shows control intrinsic factor positive fundic chief cells). (Original magnification 350×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 4 Characterization of parietal and gastrin (G) cell populations in the stomachs of the gp130757F/F and gp130WT/WT mice. Paraffin fixed sections from gp130WT/WT (A and D) or gp130757F/F (B and E) stomachs were incubated with antibodies specific for either the β subunit of the H/K-ATPase (A and B), or gastrin (D and E) to detect parietal or G cells, respectively. The reactivity of the various primary antibodies was as described in the Material and Methods section. Immunoreactive cells were quantitated microscopically and expressed as a number of cells per length of gastric mucosa (C and F). ∗Indicates that the mean value is significantly different (P < 0.05) from the age-matched control. ∗∗Indicates that the mean value is significantly different (P < 0.05) from that of 6-week-old mice of the same genotype. (Original magnification 50×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 5 Analysis of tissue gastrin and gastric pH in gp130WT/WT and gp130757F/F mice were tested at different ages as described in the Materials and Methods (A) section. Steady-state gastrin production was addressed by Northern analysis of mRNA in stomach extracts using a specific radioactively labeled cDNA probe hybridized to total RNA, and the intensity of specific bands was quantified by densitometry and standardized to rL32 signal. (B) Gastrin protein in tissue extracts measured by radio-immunoassay. (C) pH from gastric washouts of fasted wild-type (shaded boxes) or gp130757F/F mice (solid circles) were compared at different developmental ages. ∗Indicates that the mean value is significantly different (P < 0.05) from age-matched controls. ∗∗Indicates that the mean value is significantly different (P < 0.05) from that of 6-week-old mice of the same genotype. (Original magnification 50×.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 6 Expression of trefoil 1 and 2 and Reg I peptides in stomachs of wild-type and gp130757F/F mice. Stomach sections of 40-week-old gp130WT/WT (A, C, and E) and gp130757F/F (B, D, and F) mice were hybridized with DIG-labeled cDNA probes specific for Reg I (A and B), TFF1 (C and D) or TFF2 (E and F). The sequence of probes was as described in the Materials and Methods section. Reg I and TFF1 production were also addressed by measuring mRNA by Northern analysis (G and H). Specific radioactively labeled cDNA probes were hybridized to total RNA samples, and the intensity of specific bands was quantitated by densitometry and standardized to rL32 signal. ∗Indicates that mean value is significantly different (P < 0.05) from age-matched controls. ∗∗Indicates that the mean value is significantly different (P < 0.05) from 4–6-week-old mice of the same genotype. (Original magnifications ×50.) Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)

Figure 8 Expression of growth factors in stomachs of gp130757F/F and gp130757WT/WT mice. mRNA levels of (A) epidermal growth factor-receptor (EGFr), (B) heparin binding-epidermal growth factor (HB-EGF), and (C) transforming growth factor-α (TGF-α) were measured semi-quantitatively by RT-PCR. Intensity of the bands was quantified by densitometry and standardized to the housekeeping gene GAPDH. ∗Indicates that the mean value is significantly different (P < 0.05) from age-matched controls. Gastroenterology 2004 126, 196-207DOI: (10.1053/j.gastro.2003.10.066)