Mucinous neoplasia and high grade dysplasia induced by DSS.

<p>(A) <i>Smad3/Rag-DKO</i> DSS cycles. A cecal-colic multicystic mass (arrow) is present adjacent to the pancreas (asterisk). (B) <i>Smad3/Rag-DKO</i> 1.5%DSS. Multiple serosal masses are indicated on the mid to distal colon. (C) <i>Smad^−/−</i> DSS cycles. A large multicystic mesenteric mass. (D) <i>Smad^−/−</i> DSS cycles. Expansile mass in (A). Pancreas (asterisk) and mesenteric lymph node (upper right). Box region presented in (G). (E) Herniated proliferative mucosa (<b>H</b>) with compression the tunica muscularis (<b>TM</b>) with preservation of the submucosa (arrowhead). At left are invasive angular glands, dissecting mucin in the TM with focal penetration of the serosa (<b>S</b>) and intraperitoneal mucus (asterisk). (F) <i>Smad^−/−</i> DSS cycles. Mesenteric implant of mucinous cysts (asterisk) with mucin-producing epithelial lined glands (arrowheads). Note abscess (<b>A</b>) and mesenteric lymph node (<b>ML</b>). (G) Boxed region in (D). Note dissecting lakes of mucin and isolated epithelium and epithelial rafts within the cysts. (H) Higher magnification of asterisked region in (F). (I) <i>Smad3^+/−</i>3%DSS. Within mesenteric cysts are free large round cells (arrowhead) and clumps of basophilic cells (arrow) and rare signet rings (inset, Smad3−/− DSS cycles) (J) Foci of high grade dysplasia with in a hyperplastic polyp.</p

Characterization of Dextran Sodium Sulfate-Induced Inflammation and Colonic Tumorigenesis in <i>Smad3</i>^−/− Mice with Dysregulated TGFβ

<div><p>There are few mouse models that adequately mimic large bowel cancer in humans or the gastrointestinal inflammation which frequently precedes it. Dextran sodium sulphate (DSS)-induces colitis in many animal models and has been used in combination with the carcinogen azoxymethane (AOM) to induce cancer in mice. <i>Smad3</i>^−/− mice are deficient in the transforming growth factor beta (TGFβ) signaling molecule, <i>SMAD3</i>, resulting in dysregulation of the cellular pathway most commonly affected in human colorectal cancer, and develop inflammation-associated colon cancer. Previous studies have shown a requirement for a bacterial trigger for the colitis and colon cancer phenotype in <i>Smad3^−/−</i> mice. Studies presented here in <i>Smad3^−/−</i> mice detail disease induction with DSS, without the use of AOM, and show a) <i>Smad3</i>^−/− mice develop a spectrum of lesions ranging from acute and chronic colitis, crypt herniation, repair, dysplasia, adenomatous polyps, disseminated peritoneal adenomucinosis, adenocarcinoma, mucinous adenocarcinoma (MAC) and squamous metaplasia; b) the colon lesions have variable galactin-3 (Mac2) staining c) increased DSS concentration and duration of exposure leads to increased severity of colonic lesions; d) heterozygosity of <i>SMAD3</i> does not confer increased susceptibility to DSS-induced disease and e) disease is partially controlled by the presence of T and B cells as <i>Smad3</i>^−/−<i>Rag2</i>^−/− double knock out (DKO) mice develop a more severe disease phenotype. DSS-induced disease in <i>Smad3</i>^−/− mice may be a useful animal model to study not only inflammation-driven MAC but other human diseases such as colitis cystica profunda (CCP) and pseudomyxomatous peritonei (PMP).</p></div

Grading of DSS IBD.

*<p>These are scored for each section of large bowel (cecum, proximal colon, mid colon and distal colon) and summed for total IBD score.</p>**<p>Two extent scores are included in the IBD score. Extent 1 =  % of intestine affected in any manner; Extent 2 = % percent of intestine affected by the most severe score.</p

Squamous metaplasia and dysplasia in distal colons of DSS-treated <i>Smad3^−/−</i> mice.

<p><i>Smad3^+/−</i>, <i>Smad3^−/−</i> and WT mice were treated with either 1.5% DSS for one or 9 cycles. Experimental endpoint was 17 weeks. (A) Squamous cell metaplasia was scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079182#pone-0079182-t003" target="_blank">Table 3</a>. Pairwise comparisons between DSS-treated groups were via Mann-Whitney test. B) The same region of the distal colon as in (A) was scored for squamous cell dysplasia. No high grade (grade 3 or 4) dysplasia was detected. Incidence of dysplasia was compared via Fisher exact test. <i>*P</i>≤<i>0.05, **P</i>≤<i>0.01, ***P</i>≤<i>0.001, ****P</i>≤<i>0.0001</i>.</p

Dysplasia and invasive neoplasia scoring schema.

a<p>dysplasia score is assigned 0–4 for each section of large bowel, summed dysplasia are the sum of the scores in the individual segments (cecum, proximal colon, mid colon, distal colon).</p>b<p>number of invasive tumors is multiplied by size weight in each segment of the large bowel (cecum, proximal colon, mid colon, distal colon). Invasive tumor score is the sum of these values across the bowel).</p>c<p>diagnostic criteria for hyperplasia, adenoma, carcinoma and to distinguish invasion vs. pseudoinvasion (herniation) after Boivin et al 2003.</p

Patterns of galectin-3 expression.

<p><i>Smad3^−/−</i>3% DSS (A–C). Sections of formalin-fixed paraffin-embedded colon from a mouse with regions of normal proximal colon (A) mucosal adenomatous hyperplasia (B) and mucinous adenocarcinoma (C and D) immunohistologically stained for galectin-3. (A) In normal proximal colonic mucosa, galectin-3 expression is restricted to well-differentiated apical colonocytes with strong nuclear and lesser cytoplasmic signal. (B) Within adenomas there is loss of signal in the proliferative cells (arrows, mitotic figures) and staining cytoplasmic signal with indistinct to absent nuclear signal. (C) Within invasive crypts deep within the tunica muscularis (see A) there is loss of signal with gradual increase in signal as cells migrate into the peritoneal cavity and presumably differentiate to a mucinous phenotype. (D) <i>Smad3/Rag-DKO</i>, 1.5% DSS Strong cytoplasmic signal is present in the neoplastic epithelium lining mucinous peritoneal cysts and in free floating cells within the mucin pools (<b>P</b>).</p

Differential staining patterns between macrophage and epithelial cell markers.

<p>Representative images of formalin-fixed paraffin-embedded mucinous adenocarcinoma from a <i>Smad3/Rag-DKO</i> 1.5% DSS animal stained for galectin-3 (A), the macrophage marker F4/80 (B) and wide spectrum cytokeratin (C). (A) Galectin-3 staining is variable in the neoplastic epithelium with loss of signal in the invasive and less differentiated glands within the muscular tunics (MT). There is increased cytoplasmic signal in the epithelium lining the peritoneal mucinous lesions (P indicates peritoneal cavity). Note that activated macrophages express galectin-3 and F4/80 (B), whereas only the colonic epithelium is positive for cytokeratins (C).</p

Chronic typhlocolitis and associated secondary lesions induced by DSS.

<p>(A–C) Examples of chronic lesions. (A) <i>Smad3^−/−</i> DSS cycles. Cecum (Ce) and colon (PC, proximal; MC, mid; DC, distal; A, anus). The arrow and arrow head delineate region presented histologically in (D). (B) <i>Smad3/Rag-DKO</i> 1.5% DSS. A large multicystic mass is accompanied by retention of fecal pellets (arrow) consistent with partial obstruction. (C) <i>Smad3^−/−</i> DSS cycles. Multifocal mucin-filled cystic masses (arrows) are present. Ill-formed fecal pellet is present in the mid colon and the distal colon is thickened and opaque. (D–F) Subgross histological sections. (D) Proximal colon see (A). The gross appearance is due to an intussception (<b>I</b>) into the dilated distal lumen (<b>L</b>). (E) <i>Smad3^+/−</i>3% DSS. Distal colon and anus (<b>A</b>). The longitudinal mucosal folds of the distal colon (arrowheads) and the transition from glandular to metaplastic mucosa (asterisk) are indicated. (F) <i>Smad3^−/−</i> DSS cycles. Multifocal mucinous serosal and mesenteric masses (asterisks). The distal colon (<b>DC</b>) and pancreas (<b>P</b>) are indicated. (G) Chronic severe proliferative and lymphohistiocytic colitis with cryptitis and crypt abscesses. (H) Higher magnification of asterisked region in (E). Inset: Squamous regions may have areas of prominent cornification (right side) and non-cornified areas (left side). Arrow indicates direction of the anus. (I) <i>Smad3^−/−</i>3%DSS.The spectrum of chronic mucosal lesions includes moderate proliferative lymphohistiocytic colitis (arrow head) and atypical glands (*) that are not associated with active inflammation or ulceration.</p

Distal squamous cell metaplasia score^*.

*<p>Squamous cell metaplasia score was generated by adding the metaplasia score and two extent scores.</p>**<p>Extent is for the distal half of the distal colon where longitudinal mucosal folds start. Two extent scores are included in the total squamous metaplasia score. Extent 1 =  % of distal colon affected in any manner; Extent 2 = % percent of distal colon affected by the most severe score.</p>***<p>The highest grade dysplasia noted in the <i>Smad3</i>^−/− studies was 2 (low grade) but the higher scores are included here for completion.</p

Histopathology scores of DSS-treated <i>Smad3^−/−</i> and <i>Smad3/Rag-DKO</i> mice treated with different doses of DSS.

<p><i>Smad3^+/−</i>, <i>Smad3^−/−</i> and <i>Smad3/Rag-DKO</i> (DKO) mice were treated with either a single DSS cycle or 9 cycles of DSS. Experimental endpoint was 17 weeks. A) IBD score, B) Invasion score, C) Summed dysplasia score and (D) Distribution score (as described in materials and methods) are shown for individuals in each treatment group. Negative control groups were all statistically different (significance not shown in figure) from their respective DSS-treated group except for untreated water vs. 1.5% DSS <i>Smad3^−/−</i> in (B). <i>*P</i>≤<i>0.05, **P</i>≤<i>0.01.</i></p