Probes for LGR5

Cancer associated fibroblasts (CAFs) are the dominant cell population in the cancer stroma. Gremlin 1 (GREM1), an antagonist of the bone morphogenetic protein pathway, is expressed by CAFs in a variety of human cancers. However, its biological significance for cancer patients is largely unknown. We applied RNA in situ hybridization (ISH) to evaluate the prognostic value of stromal GREM1 expression in a large cohort of 670 colorectal cancers (CRCs). Overall GREM1 expression in CRCs was lower than that of the matched normal mucosa, and GREM1 expression had a strong positive correlation with BMI1 and inverse correlations with EPHB2 and OLFM4. RNA ISH localized the GREM expression to smooth muscle cells of the muscularis mucosa, fibroblasts around crypt bases and in the submucosal space of a normal colon. In various colon polyps, epithelial GREM1 expression was exclusively observed in traditional serrated adenomas. In total, 44% of CRCs were positive for stromal GREM1, which was associated with decreased lymphovascular invasion, a lower cancer stage, and nuclear β-catenin staining. Stromal GREM1 was significantly associated with improved recurrence-free and overall survival, although it was not found to be an independent prognostic marker in multivariate analyses. In addition, for locally advanced stage II and III CRCs, it was associated with better, stage-independent clinical outcomes. In summary, CRCs are frequently accompanied by GERM1-expressing fibroblasts, which are closely associated with low lymphovascular invasion and a better prognosis, suggesting stromal GREM1 as a potential biomarker and possible candidate for targeted therapy in the treatment of CRCs.

Abstract

BACKGROUND:

Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures.

RESULTS:

Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research.

CONCLUSIONS:

We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Abstract

BACKGROUND & AIMS:

The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated.

METHODS:

We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5 mRNA-positive cells, irradiated, or given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues we analyzed the fate of progeny that expressed Bhlha15 mRNA. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using the TCGA dataset.

RESULTS:

Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors (EPs). Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with EPs, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors, towards that of ISCs. Bhlha15+ EPs with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells following dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon.

CONCLUSION:

In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation, in addition to traditional ISCs.