Volume 138, Issue 3, Pages 1003-1011.e5 (March 2010) Stat3 Is a Negative Regulator of Intestinal Tumor Progression in ApcMin Mice  Monica Musteanu, Leander Blaas, Markus Mair, Michaela Schlederer, Martin Bilban, Stefanie Tauber, Harald Esterbauer, Mathias Mueller, Emilio Casanova, Lukas Kenner, Valeria Poli, Robert Eferl  Gastroenterology  Volume 138, Issue 3, Pages 1003-1011.e5 (March 2010) DOI: 10.1053/j.gastro.2009.11.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Signal transducer and activator of transcription 3 (Stat3) is activated in intestinal tumors of ApcMin/+ mice. H&E staining and immunohistochemistry for Tyr-705-phosphorylated Stat3 (P-Stat3) revealed Stat3 activation (nuclear signals) in tumor cells (arrows) and stromal cells (arrowheads) of ApcMin/+ adenomas. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Loss of signal transducer and activator of transcription 3 (Stat3) affects tumor initiation in ApcMin/+ mice but enhances tumor growth at later stages of tumor development. (A) Analysis of tumor multiplicity (tumors/cm2 gut) and tumor size (mean tumor area) at early stages of tumor development revealed reduced number of adenomas upon loss of Stat3 in male and female mice. Male mice 1.5 months old (7 per genotype) and female mice 4 months old (5 per genotype) were used. (B) Analysis of tumor multiplicity (tumors/cm2 gut) and tumor size (mean tumor area) at later stages of tumor development revealed a significantly increased tumor size upon loss of Stat3 in male and female mice. Four-month-old male mice (n = 10 for Stat3flox/floxApcMin/+; n = 11 for Stat3ΔIECApcMin/+) and 7-month-old female mice (5 per genotype) were used. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Loss of signal transducer and activator of transcription 3 (Stat3) enhances intestinal tumor progression and invasiveness in ApcMin/+ mice. (A) H&E staining of carcinomas in 4-month-old male mice (upper panel) and 7-month-old female mice (middle panel). Upper left: a Stat3flox/floxApcMin/+ carcinoma invading the muscularis (arrow). Upper right: a Stat3ΔIECApcMin/+ carcinoma invading an intramural vein (arrow). Middle left: a Stat3flox/floxApcMin/+ carcinoma invading the muscularis (arrow). Middle right: a Stat3ΔIECApcMin/+ carcinoma infiltrating into the parenchyma of the pancreas (arrows). Immunohistochemical staining for Tyr-705-phosphorylated Stat3 (lower panel) confirmed loss of Stat3 in tumors of Stat3ΔIECApcMin/+ mice. Phosphorylated Stat3 was detected in nuclei of tumor cells (arrow) and in stromal cells (arrowhead) of Stat3flox/floxApcMin/+ mice. In contrast, Stat3ΔIECApcMin/+ tumors showed only signals in stromal cells (arrowheads). (B) Histopathological grading of intestinal tumors in male (left graphs) and female mice (right graphs) revealed an increased percentage of carcinomas upon loss of Stat3 (the percentage was calculated as follows: number of carcinomas per mouse/total tumor number per mouse ×100). 165 and 149 tumors identified in 4-month-old male Stat3flox/floxApcMin/+ (n = 11) and Stat3ΔIECApcMin/+ mice (n = 10), respectively, were used for analysis. The 110 and 69 tumors identified in 7-month-old female Stat3flox/floxApcMin/+ (n = 5) and Stat3ΔIECApcMin/+ (n = 5) mice, respectively, were used for analysis. Si, small intestine; nd, not detectable. (C) Kaplan-Meier plot showing premature lethality of male Stat3ΔIECApcMin/+ mice (n = 11) when compared to Stat3flox/floxApcMin/+ sex-matched littermates (n = 15). Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Increased proliferation in tumors of Stat3ΔIECApcMin/+ mice. (A) Immunohistochemistry for BrdU incorporation revealed a significant increase of proliferation in tumors of Stat3ΔIECApcMin/+ mice when compared to Stat3flox/floxApcMin/+ mice. (B, C) The apoptotic index, as assessed by TUNEL-staining (B) or staining for cleaved caspase 3 (C), was similar in Stat3ΔIECApcMin/+ mice and Stat3flox/floxApcMin/+ mice. The bars represent average numbers of positive cells ± standard error of mean in ≥12 tumors (n = 3 male mice per genotype). Positive cells or nuclei are indicated by arrows. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Increased levels of cyclin D1 in tumors of Stat3ΔIECApcMin/+ mice. (A) Real-time polymerase chain reaction analysis of signal transducer and activator of transcription 3 (Stat3) target genes demonstrating increased levels of cyclin D1 mRNA in intestinal epithelial cells (IEC) and tumors of Stat3ΔIECApcMin/+ mice when compared to Stat3flox/floxApcMin/+ mice (bars represent data ± standard error of mean from 3 animals per group; tumors were collected and individually pooled for RNA isolation from 4-month-old male mice). (B) Immunohistochemistry for cyclin D1 in tumors revealed stronger nuclear signals (arrows) in Stat3ΔIECApcMin/+ mice when compared to Stat3flox/floxApcMin/+ mice. (C) Up-regulation of cyclin D1 in tumors of Stat3ΔIECApcMin/+ mice was confirmed by Western blot analysis. Tumors were collected and individually pooled from 4-month-old male Stat3flox/floxApcMin/+ and Stat3ΔIECApcMin/+ mice. HSC70 expression was used as loading control. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Reduced levels of SMAD4 and CEACAM1 in tumors of Stat3ΔIECApcMin/+ mice. (A) Real-time polymerase chain reaction analysis demonstrating reduced levels of smad4 and ceacam1 mRNA in intestinal epithelial cells (IEC) and tumors of Stat3ΔIECApcMin/+ mice when compared to Stat3flox/floxApcMin/+ mice (bars represent data ± standard error of mean from 3 animals per group; tumors were collected and individually pooled from 4-month-old male mice). (B) Western blot analysis revealed reduced CEACAM1 protein expression in IEC and tumors of Stat3ΔIECApcMin/+ mice. HSC70 expression was used as loading control. (C) Immunohistochemistry for CEACAM1 (upper panel) confirmed reduced protein expression in Stat3-deficient tumors (arrow demonstrates CEACAM1 expression on apical membranes of Stat3flox/floxApcMin/+ tumor cells). Immunohistochemistry for β-catenin (lower panel) demonstrating up-regulation in Stat3flox/floxApcMin/+, and Stat3ΔIECApcMin/+ tumors (arrows demonstrate nuclear signals in tumor cells). (D) Quantitation of immunohistochemically stained sections by HistoQuest software demonstrated a stronger mean nuclear signal intensity for β-catenin in tumors of Stat3ΔIECApcMin/+ mice (left curves) (n = 8 tumors per genotype). Correspondingly, mean cytoplasmic staining intensity for β-catenin was reduced in tumors of Stat3ΔIECApcMin/+ mice (right curves). Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Generation and characterization of mice with conditional deletion of signal transducer and activator of transcription 3 (Stat3) in the intestinal epithelium. Exons 12 to 14 comprising the DNA binding domain of Stat3 were deleted using Villin-cre transgenic mice. (A) Southern blot analysis demonstrating Stat3 deletion in intestinal epithelial cells. fl/fl: tissues of mice with floxed Stat3 alleles; ΔIEC: tissues of mice with floxed Stat3 alleles harbouring the Villin-cre transgene. IEC, intestinal epithelial cells; kid., kidney; sto., stomach; spl., spleen; thy., thymus; bra., brain; fl, band for floxed Stat3; Δ: band for deleted Stat3. (B) Western blot analysis for total Stat3 and HSC70 (loading control) demonstrating loss of Stat3 protein in isolated epithelial cells from small intestine (si) and colon (c) of Stat3ΔIEC mice. (C) H&E-staining, Alcian blue-staining and immunohistochemistry for Lysozyme and Synaptophysin demonstrated the presence of enterocytes, goblet cells, paneth cells, and enteroendocrine cells in the small intestine of Stat3ΔIEC mice, respectively. Ki67-staining revealed proliferating cells in the crypts of Stat3ΔIEC mice. Corresponding cell types are indicated by arrowheads. (D) Quantitation of goblet cells, paneth cells and enteroendocrine cells demonstrating normal intestinal cell differentiation in Stat3ΔIEC mice. Positively stained cells for Alcian blue, Lysozyme, and Synaptophysin were quantified. No significant differences were identified in duodenum, jejunum, ileum and colon of Stat3ΔIEC mice when compared to Stat3flox/flox mice. Detailed quantitation is shown for goblet cells. The bars represent average numbers of positively stained cells ± standard error of mean in ≥12 crypts or villi (n = 3 mice per genotype). Si, small intestine; duod., duodenum; jejun., jejunum. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Loss of signal transducer and activator of transcription 3 (Stat3) enhances intestinal tumor size in ApcMin/+ mice. (A) Analysis of tumor multiplicity (tumors/mouse) in the duodenum, ileum, jejunum, and colon at late stages of tumor development. A sex-specific increase in tumor number was observed in the colon of male Stat3ΔIECApcMin/+ mice. (B) Analysis of tumor size (mean tumor area) at later stages of tumor development revealed a significantly increased tumor size in the jejunum and colon of male Stat3ΔIECApcMin/+ mice and the jejunum of female Stat3ΔIECApcMin/+ mice. The 165 and 149 tumors identified in 4-month-old male Stat3flox/floxApcMin/+ (n = 11) and Stat3ΔIECApcMin/+ mice (n = 10), respectively, were used for analysis. The 110 and 69 tumors identified in 7-month-old female Stat3flox/floxApcMin/+ (n = 5) and Stat3ΔIECApcMin/+ (n = 5) mice, respectively, were used for analysis. Si, small intestine; duod., duodenum; jejun., jejunum. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 H&E-stained sections of invasive carcinomas that developed in female Stat3ΔIECApcMin/+ mice. Frequently, a desmoplastic reaction was observed (arrowheads) and most tumors showed deep invasion into the muscularis propria (arrows). Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Reduced amount of activated signal transducer and activator of transcription 3 (Stat3) in the invasive region of Stat3flox/floxApcMin/+ carcinomas. (A) Immunohistochemical staining for Tyr-705-phosphorylated Stat3 in 4 carcinomas of Stat3flox/floxApcMin/+ mice. The demarcated lines in the uppermost low-magnification image confine regions in the main tumor (T) and invasive front (arrowheads). Note the reduced staining intensity in the invasive region when compared to the main tumor. The invasive regions in carcinomas are indicated by arrowheads in the high-magnification images below. (B) Scattergrams with quantitation of Tyr-705-phosphorylated Stat3 in the invasive region and main tumor using HistoQuest software (TissueGnostics GmbH). Regions of interest for the invasive front and main tumor were selected as indicated in (A) and nuclear intensity of phosphorylated Stat3 was quantified. The percentage of P-Stat3–positive nuclei is indicated in the right upper quadrant of the scattergrams. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 Vascularization, number of T cells, and number of macrophages was not significantly different between Stat3flox/floxApcMin/+, and Stat3ΔIECApcMin/+ tumors. Tumor vascularization was determined by immunohistochemistry for van Willebrand Factor (vWF) and quantified with ImageJ software. Presence of T cells and macrophages was quantified by CD3- and F4/80-immunohistochemistry, respectively. The bars represent average area of vessels or numbers of positive cells ± standard error of mean in ≥12 tumors (n = 3 mice per genotype). Positive vessels or cells are indicated by arrows. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 Comparable numbers of activated T cells in Stat3flox/floxApcMin/+ and Stat3ΔIECApcMin/+ tumors. T-cell activation was evaluated by co-immunofluorescence staining for CD3 (green) and Granzyme B (red). Cell nuclei are stained in blue (DAPI). Note that tumors (arrowheads) can be identified under the fluorescence microscope by the presence of double-positive (yellow) T-cells (arrows). 2.5×: low magnification; 40×: high magnification. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 7 Immunohistochemical staining revealed no major differences in protein levels of c-Myc, epidermal growth factor receptor, and E-cadherin in Stat3flox/floxApcMin/+, and Stat3ΔIECApcMin/+ tumors. Up-regulation of c-Myc in tumor tissue is indicated by arrows in the uppermost low-magnification panel (10×). The high-magnification images (40×) demonstrate nuclear staining in tumor tissue for c-Myc, characteristic dotted staining for epidermal growth factor receptor on the apical membrane and membrane staining for E-cadherin. Positive signals are indicated by arrows. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 8 Compensatory up-regulation of related signal transducer and activator of transcription (Stat) family members upon ablation of Stat3). (A) Real-time polymerase chain reaction analysis of Stat genes in intestinal epithelial cells (IEC) and tumors of Stat3ΔIECApcMin/+ mice, and Stat3flox/floxApcMin/+ mice. (B) Real-time polymerase chain reaction analysis of suppressor of cytokine signaling genes in intestinal epithelial cells (IEC) and tumors of Stat3ΔIECApcMin/+ mice and Stat3flox/floxApcMin/+ mice. Bars represent data ± standard error of mean from 3 animals per group; tumors were collected and individually pooled for RNA isolation from 4-month-old male mice. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 9 Reduced expression of CEACAM1 in signal transducer and activator of transcription 3 (Stat3)-deficient tumors of female mice (assessed by immunohistochemistry). The arrow demonstrates CEACAM1 expression on apical membranes of Stat3flox/floxApcMin/+ tumor cells. Gastroenterology 2010 138, 1003-1011.e5DOI: (10.1053/j.gastro.2009.11.049) Copyright © 2010 AGA Institute Terms and Conditions