Top-down morphogenesis of colorectal tumors

Abstract

One of the fundamental tenets of oncology is that tumors arise from stem cells. In the colon, stem cells are thought to reside at the base of crypts. In the early stages of tumorigenesis, however, dysplastic cells are routinely found at the luminal surface of the crypts whereas the cells at the bases of these same crypts appear morphologically normal. To understand this discrepancy, we evaluated the molecular characteristics of cells isolated from the bases and orifices of the same crypts in small colorectal adenomas. We found that the dysplastic cells at the tops of the crypts often exhibited genetic alterations of adenomatous polyposis coli (APC) and neoplasia-associated patterns of gene expression. In contrast, cells located at the base of these same crypts did not contain such alterations and were not clonally related to the contiguous transformed cells above them. These results imply that development of adenomatous polyps proceeds through a top-down mechanism. Genetically altered cells in the superficial portions of the mucosae spread laterally and downward to form new crypts that first connect to preexisting normal crypts and eventually replace them.

Figure 1

(A) Hematoxylin and eosin stained section of a typical small adenomatous polyp. Dysplastic epithelium is located superficially in the crypts and is contiguous with the underlying histologically normal epithelium at the base. (B) Note the abrupt transition between dysplastic and normal-appearing epithelial cells at the mid-point of this crypt. (C) Adjacent normal colonic mucosae for comparison.

Figure 2

Proliferative activity assessed with the Ki-67 antibody. Ki-67 immunoreactivity is broadly distributed throughout the dysplastic epithelium at the top of the crypts (A and B). Within the normal-appearing epithelium at the bottom of the crypts, only the basal and surrounding cell layers are labeled (A and B), similar to the pattern observed in normal colonic epithelium unassociated with neoplasia (C).

Figure 3

β-Catenin expression in small adenomas. β-catenin is highly expressed and distributed throughout the dysplastic epithelium at the top of the crypts, where it is found in the nucleus as well as in the cytoplasm and plasma membrane (A and B). In contrast, staining is less intense in the normal-appearing epithelium at the bottom of the crypts, and staining is confined to the membranes (A and B). The staining pattern at the bottom of the adenomatous crypts is similar to that observed in normal colonic epithelium unassociated with neoplasia (C).

Figure 4

Sequential probability ratio test analysis of LOH. Digital SNP analysis by using polymorphic markers within the APC gene was performed on the microdissected components of the adenomas. The resultant microdissected fractions consisted of dysplastic cells (D) from the tops of the crypts and underlying normal epithelium (U) from the same crypts. The x axis represents the total number of alleles counted, whereas the y axis represents the observed proportion of the two alleles. Samples whose observed allelic proportions was above curve 2 were interpreted to have undergone LOH, whereas samples whose observed allelic proportion was below curve 1 were interpreted as being in allelic balance. ▴, The allelic proportion of an individual DNA sample from the indicated crypt component, with the numbers corresponding to the adenomas listed in Table 1.

Figure 5

Dysplastic epithelial cells harbor APC mutations. Examples of DNA sequencing gels of PCR products derived from microdissected components of the adenomas are shown. The microdissected fractions consisted of dysplastic cells (D) from the tops of the crypts and underlying normal epithelium (U) from the same crypts. The numbers refer to the adenomas listed in Table 1.

Figure 6

Models of morphogenesis of sporadic adenomatous polyps. (A) Transformation of a single epithelial cell occurs at the base of the crypt (arrow) by virtue of APC inactivation. The transformed cell proliferates and passively migrates upward as a result of routine epithelial turnover. Once the transformed cells reach the superficial portion of the mucosae, they continue to proliferate and migrate and begin to populate the superficial mucosae of the adjacent crypts. The adenomatous epithelium thereby confronts the normal epithelium of the adjacent crypts, pushing the latter downward and gradually replacing it from top-to-bottom. (B) Similar to A except that the initial transformation event occurs in an epithelial cell in the intercryptal zone lying between crypt orifices (arrow). (C) En face view of surface, indicating spread of intercryptal dysplastic epithelial cell to adjacent crypts.