Table 1.
Grading of DSS IBD.
Table 2.
Dysplasia and invasive neoplasia scoring schema.
Table 3.
Distal squamous cell metaplasia score*.
Figure 1.
Smad3−/− mice are more susceptible to DSS-induced colitis and cancer than Smad3+/− mice.
(A) Percent weight change of Smad3+/− and Smad3−/− mice treated with 3% DSS in drinking water for 7 days. Mice were euthanized when they reached endpoint criteria as outlined in Methods. week 1, n = 16 and n = 11; week2, n = 13 and n = 6; week3, n = 13 and n = 4; week 5 and after, n = 13 and n = 3 (Smad3+/− and Smad3−/−, respectively). (B) Survival analysis of mice treated with 3% DSS in (A). Endpoint 33 weeks. (C) Survival analysis (27 week endpoint) of Smad3+/− and Smad3−/− animals treated with 9 cycles of DSS. IBD score (D), Summed dysplasia score (E) and Invasion score (F) of same study as in (C). P-values (D–F) derived from a Mann-Whitney test.
Figure 2.
Histopathology scores of DSS-treated Smad3−/− and Smad3/Rag-DKO mice treated with different doses of DSS.
Smad3+/−, Smad3−/− and Smad3/Rag-DKO (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 Smad3−/− in (B). *P≤0.05, **P≤0.01.
Figure 3.
Characteristics of acute typhlocolitis induced by DSS.
(A) Smad3−/−1.5% DSS. Cecum (Ce) and colon (PC, proximal; MC, mid; DC, distal; A, anus). The cecum is contracted with darkly discolored contents. The colon lacks formed fecal pellets. The mid and distal colon are thickened and turgid. (B) Smad3+/−3% DSS. Subgross hematoxylin and eosin-stained section of DSS-exposed colon. The cecum has been removed. The mid and distal colon the luminal (L) contents are fluid with no formed fecal pellets. The submucosa (arrow) is markedly expanded by edema. (C) Smad3+/−3% DSS. Mid colon at the junction of erosive mucosal loss and effacement by inflammatory cells adjacent to intact mucosa (M). Note the muscularis mucosae (arrowheads) and the expansion of the submucosa (*) with inflammatory cells. Tunica muscularis as indicted (TM). (D) Smad3+/−3% DSS. Exudate adherent to ulcerated mucosa contains degenerate inflammatory cells, erythrocytes, fibrin and proteinaceous fluid with hazy coccoid bacterial colonies (arrow). (E) Smad3−/−3%DSS. Chronic-active proliferative and ulcerative colitis. The mucosa adjacent to an ulcer is proliferative with irregular and angular glands with loss of goblet cells, and increased mitotic figures. (F) Smad3−/−1.5% DSS. Glands adjacent to areas of active inflammation are classified as indefinite for dysplasia due to active inflammatory milieu. Dilated crypt filled with filamentous bacteria with a portion escaping into the adjacent lamina propria/submucosa (arrow).
Figure 4.
Chronic typhlocolitis and associated secondary lesions induced by DSS.
(A–C) Examples of chronic lesions. (A) Smad3−/− 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) Smad3/Rag-DKO 1.5% DSS. A large multicystic mass is accompanied by retention of fecal pellets (arrow) consistent with partial obstruction. (C) Smad3−/− 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 (I) into the dilated distal lumen (L). (E) Smad3+/−3% DSS. Distal colon and anus (A). The longitudinal mucosal folds of the distal colon (arrowheads) and the transition from glandular to metaplastic mucosa (asterisk) are indicated. (F) Smad3−/− DSS cycles. Multifocal mucinous serosal and mesenteric masses (asterisks). The distal colon (DC) and pancreas (P) 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) Smad3−/−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.
Figure 5.
Mucinous neoplasia and high grade dysplasia induced by DSS.
(A) Smad3/Rag-DKO DSS cycles. A cecal-colic multicystic mass (arrow) is present adjacent to the pancreas (asterisk). (B) Smad3/Rag-DKO 1.5%DSS. Multiple serosal masses are indicated on the mid to distal colon. (C) Smad−/− DSS cycles. A large multicystic mesenteric mass. (D) Smad−/− DSS cycles. Expansile mass in (A). Pancreas (asterisk) and mesenteric lymph node (upper right). Box region presented in (G). (E) Herniated proliferative mucosa (H) with compression the tunica muscularis (TM) with preservation of the submucosa (arrowhead). At left are invasive angular glands, dissecting mucin in the TM with focal penetration of the serosa (S) and intraperitoneal mucus (asterisk). (F) Smad−/− DSS cycles. Mesenteric implant of mucinous cysts (asterisk) with mucin-producing epithelial lined glands (arrowheads). Note abscess (A) and mesenteric lymph node (ML). (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) Smad3+/−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.
Figure 6.
Patterns of galectin-3 expression.
Smad3−/−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) Smad3/Rag-DKO, 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 (P).
Figure 7.
Differential staining patterns between macrophage and epithelial cell markers.
Representative images of formalin-fixed paraffin-embedded mucinous adenocarcinoma from a Smad3/Rag-DKO 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).
Figure 8.
Squamous metaplasia and dysplasia in distal colons of DSS-treated Smad3−/− mice.
Smad3+/−, Smad3−/− 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 Table 3. 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. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.