Colossal Crypts Bordering Colon Adenomas in ApcMin Mice Express Full-Length Apc

Abstract

Enlarged but nondysplastic crypts are frequently observed at the margins of colon tumors , forming what has been called a transitional epithelium. It is now thought that this is a reactive state and not a preneoplastic condition as previously suggested. We have used the mouse familial adenomatous polyposis model , Apc Min , to study these abnormal adenoma-associated crypts. We report that these nondysplastic crypts are enormous (as much as 10 times normal length) and branch more frequently than normal crypts. They express wild-type Apc protein and display the wild-type Apc allele. We conclude that the colossal crypts at adenoma mar- It has long been recognized that the colonic epithelium at the margins of adenocarcinomas, although not neoplastic, is not normal. The crypts in this transitional region are enlarged and often branched. 1-5 However, as a similar state is observed at the margins of an array of lesions, it is now believed that it represents a reactive state and not a preneoplastic condition 6,7 and has since been largely ignored. We have noticed in studies of a mouse model of familial adenomatous polyposis 8 that although the dysplastic epithelium forming the bulk of adenomas is Apc negative, there is often a layer of Apc-positive nondysplastic crypts underlying the tumor. 9 These Apc-positive crypts are often cut in cross section when the tumor is sectioned orthogonally to the surface. This observation raised questions about the relationship of the Apc-positive crypts to the overlying tumor and to the surrounding normal epithelium. We show here that they represent greatly elongated crypts, with normal Apc gene function, that extend under the tumor from the margins. Materials and Methods Isolation of Intact Epithelium and Scanning Electron MicroscopyFifteen week-old Apc Min mice (Jackson Laboratories, Bar Harbor, ME) were used. Intact epithelium was isolated, from small segments of colon each containing an adenoma, by intraventricular perfusion of 30 mmol/L EDTA in PBS followed by vibration into 4% paraformaldehyde in PBS. 10 After fixation for 30 minutes on ice, the isolated epithelium was washed in PBS and then rinsed and stored in methanol at Ϫ20°C. The colon adenomas and adjacent mucosa were dissected from the vibrated segments and fixed for 30 minutes in 2.5% glutaraldehyde in PBS. The adenomas were then processed for scanning electron microscopy (SEM) by post-fixing in 1% osmium tetroxide in 0.1 mol/L cacodylate HCl buffer, pH 7.4, and then dehydrated through a graded series of ethanol, critical-point dried, 11 and sputter coated with gold palladium. ImmunofluorescenceColossal crypts were microdissected from the isolated epithelium, rinsed in 1% Nonidet P-40 (NP40) in PBS, freeze-thawed at Ϫ20°C to increase permeability, rinsed in 1% NP40, and blocked at room temperature for 1 hour in PBS containing 10% newborn calf serum and 1% NP40. The crypts were incubated overnight at 4°C in rabbit polyclonal antibodies specific for the carboxy terminus of Apc protein (Santa Cruz Biotechnology, Santa Cruz, C...

E-cadherin Is Critical for Collective Sheet Migration and Is Regulated by the Chemokine CXCL12 Protein During Restitution

“…Initial morphological characterization of the intestine revealed increased crypt branching, a finding similar to that previously noted in N-cadherin mutant animals (49), p120catenin-deficient mice (50), and APC(Min) mice (51). Subsequent work using an inducible Cre mouse indicates that targeted deletion of .…”

Polyclonality of familial murine adenomas: Analyses of mouse chimeras with low tumor multiplicity suggest short-range interactions

“…Alternatively, loss of Apc͞APC in one epithelial clone may induce the expression of growth factors that affect mitotic rates in neighboring clones. The normal intestinal epithelium adjacent to tumors is often hyperplastic (25). Thus, the multiple hits in Apc͞APC that occur in polyclonal familial adenomas in the mammalian intestine may not be independent of one another.…”

The role of TGF-β and Wnt signaling in gastrointestinal stem cells and cancer

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