Probes for LGR5

OBJECTIVE: Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer. DESIGN: 19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas. RESULTS: Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance. CONCLUSION: These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.

Abstract Aims Intestinal stem cell (ISC) markers such as LGR5, ASCL2, EPHB2 and OLFM4 and their clinical implications have been extensively studied in colorectal cancers (CRCs). However, little is known about their expression in precancerous lesions of CRCs. Here, we investigated the expression and distribution of ISC markers in serrated polyps and conventional adenomas. Methods and results RT-PCR analysis revealed that all ISC markers were significantly upregulated in conventional adenomas with low grade dysplasia (CALGs) compared with other lesions. RNA in situ hybridization confirmed that CALGs exhibited strong and diffuse expression of all ISC markers, which indicate a stem cell-like phenotype. However, normal colonic mucosa hyperplastic polyps and sessile serrated adenomas harbored LGR5+ cells that were confined to the crypt base and demonstrated an organized expression of ISC markers. Notably, in traditional serrated adenomas, expression of LGR5 and ASCL2 was localized to the ectopic crypts as in the normal crypts, but expression of EPHB2 and OLFM4 was distributed in a diffuse manner, which is suggestive of a progenitor-like features. Conclusions The expression and distribution profile of ISC markers possibly provides insights into the organization of stem and progenitor-like cells in each type of precancerous lesion of CRC

Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5+ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers-including OLFM4 and EPHB2-are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5+ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett's esophagus (BE)-which is histologically similar to intestinal metaplasia-exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5+ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease.

Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

Metaplasia and dysplasia in the corpus are reportedly derived from dedifferentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C-transcript expressing cells (PGC-transcript expressing cells) represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model.We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments, histological and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC-transcript expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC-transcript expressing cells with activated Kras, inactivated Apc and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice.Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC-transcript expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma, while Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma.The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.