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Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Author(s): Kelley S. Yan [1, 2]; Claudia Y. Janda [3]; Junlei Chang [1]; Grace X. Y. Zheng [4]; Kathryn A. Larkin [1]; Vincent C. Luca [3]; Luis A. Chia [1]; [...]

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration. [ABSTRACT FROM AUTHOR]

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase- like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies. [ABSTRACT FROM AUTHOR]

Esophageal injury is a risk factor for diseases such as Barrett's esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair. NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans., (Copyright © 2017 the American Physiological Society.)

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24(+) fraction that contained the majority of Lgr5-EGFP(+) putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24(+)EpCAM(+) fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies.

Intestinal stem cells (ISCs) have been studied for more than three decades; however, their isolation has remained a challenge. We hypothesized that, just as for stem cells of other tissues, one or more membrane markers would allow positive selection of ISCs by antibody-based sorting. To explore this hypothesis, microarray data of putative ISC fractions generated by side population sorting and laser capture microdissection were subjected to bioinformatic analysis to identify common membrane antigens. The microarray comparison suggested CD24 as a candidate surface marker, and immunohistochemistry showed expression of CD24 in epithelial cells of crypt bases. Flow cytometry of jejunal epithelial preparations revealed a CD24(+) CD45(-) fraction comprising ∼1% of the cells. Analysis with epithelial cell adhesion molecule and CD31 confirmed that the cell preparations were epithelial and without endothelial contamination. Cycling cells identified by prior injection with 5-ethynyl-2'-deoxyuridine were found predominantly in the CD24(lo) subfraction. Transcript analysis by real-time RT-PCR showed this subfraction to be enriched in the ISC markers leucine-rich-repeat-containing G-protein-coupled receptor 5 (40-fold) and Bmi1 (5-fold), but also enriched in lysozyme (10-fold). Flow cytometry with anti-lysozyme antibodies demonstrated that Paneth cells comprise ∼30% of the CD24(lo) subfraction. Additional flow analyses with leucine-rich-repeat-containing G-protein-coupled receptor 5-enhanced green fluorescent protein (EGFP) epithelium demonstrated colocalization of EGFP(hi) and CD24(lo). In contrast, CD24 cells were negative for the quiescent ISC marker doublecortin and CaM kinase-like-1. Culture of CD24(lo) cells in Matrigel generated organoid structures, which included all four epithelial lineages, thus giving functional evidence for the presence of ISCs. We conclude that the CD24(lo) fraction of jejunal epithelium is highly enriched with cycling ISCs. This isolation method should be useful to many investigators in the field to advance both the basic understanding of ISC biology and the therapeutic applications of ISCs.