Characterization of LGR5 stem cells in colorectal adenomas and carcinomas

Abstract

LGR5 is known to be a stem cell marker in the murine small intestine and colon, however the localization of LGR5 in human adenoma samples has not been examined in detail, and previous studies have been limited by the lack of specific antibodies. Here we used in situ hybridization to specifically examine LGR5 mRNA expression in a panel of human adenoma and carcinoma samples (n = 66). We found that a small number of cells express LGR5 at the base of normal colonic crypts. We then showed that conventional adenomas widely express high levels of LGR5, and there is no evidence of stereotypic cellular hierarchy. In contrast, serrated lesions display basal localization of LGR5, and the cellular hierarchy resembles that of a normal crypt. Moreover, ectopic crypts found in traditional serrated adenomas show basal LGR5 mRNA, indicating that they replicate the stem cell organization of normal crypts with the development of a cellular hierarchy. These data imply differences in the stem cell dynamics between the serrated and conventional pathways of colorectal carcinogenesis. Furthermore we noted high LGR5 expression in invading cells, with later development of a stem cell niche in adenocarcinomas of all stages.

Figure 1. Schematic diagram of the relationship between histological states of the classical and serrated pathways.

The upper half of the diagram represents the serrated pathway of colorectal carcinogenesis. Text in blue describes the genetic features of the serrated pathway (CIMP = CpG island methylator phenotype, MSI = microsatellite instability). The text in red describes some of the distinguishing features that are characteristic of the histological subtypes. The lower half of the diagram represents the classical pathway of carcinogenesis. Text in blue describes the genetic features of the classical pathway (CIN = chromosomal instability).

Figure 2. Stem cell architecture in human normal colon and hyperplastic polyps.

Representative H&E staining, in situ hybridization (LGR5; pink) and immunohistochemical staining (Ki67, KRT20, CD44; brown) in human colon samples, illustrating the normal stem cell architecture (A, site = ascending colon) and the maintenance of a stem cell niche in hyperplastic polyps (HPPs, B, site = descending colon). Black arrows indicate LGR5 positivity at the base of crypts. Scale bars represent 200 micron, scale bars of inserts represent 50 micron.

Figure 3. Stem cell architecture in human adenomas.

Representative H&E staining, in situ hybridization (LGR5; pink) and immunohistochemical staining (Ki67, KRT20, CD44; brown) in human colon samples, illustrating the absence of stem cell architecture in a conventional adenoma (A, site = transverse colon), and the maintenance of a relatively normal stem cell niche in a sessile serrated adenoma/polyp (SSA/P, B, site = caecum) and a traditional serrated adenoma (TSA, C, site = rectum). Black arrows indicate LGR5 positivity at the base of crypts. Scale bars represent 200 micron, scale bars of inserts represent 50 micron.

Figure 4. Diversity of LGR5 expression in conventional adenomas.

(A). Representative image of homogenous LGR5 expression throughout the adenomatous glands (site = transverse colon). (B). Representative image of heterogeneous LGR5 expression in a lesion (site = rectosigmoid). (C). Representative image of an adenomatous gland in which one branch expresses high LGR5, with the other half expressing low LGR5, suggestive of early dysregulation of the stem cell niche (site = rectosigmoid). (D). Representative images of LGR5 expression within an adenoma (site = rectosigmoid) that contains regions of high-grade dysplasia (HGD) and low-grade dysplasia (LGD). Scale bars in A-D represent 200 micron, scale bars in inserts represent 50 micron.

Figure 5. Expression of LGR5 in ectopic crypts.

Representative images showing expression of LGR5 and Ki67 in ectopic crypts of a traditional serrated adenoma (TSA, site = sigmoid colon). Black arrows identify LGR5 positivity at the base of ectopic crypts. Scale bars represent 100 micron, scale bars of inserts represent 20 micron.

Figure 6. Expression of LGR5 in invasion and adenocarcinoma.

(A). Representative images showing basal LGR5 expression in adenocarcinoma structures resembling crypts in an early stage cancer (pT1, site = sigmoid colon). (B). Representative images showing basal LGR5 expression in adenocarcinoma structures resembling crypts in a late stage cancer (pT3, site = sigmoid colon). (C). Representative images showing universally high LGR5 expression in an invasive cell population found within a conventional adenoma (site = sigmoid colon). Scale bars in A, B and C represent 500 micron, scale bars of inserts represent 100 micron.