Extracellular Matrix Orchestration of Tissue Remodeling in the Chronically Inflamed Mouse Colon.

BACKGROUND & AIMS: Chronic inflammatory illnesses are debilitating and recurrent conditions associated with significant comorbidities, including an increased risk of developing cancer. Extensive tissue remodeling is a hallmark of such illnesses, and is both a consequence and a mediator of disease progression. Despite previous characterization of epithelial and stromal remodeling during inflammatory bowel disease, a complete understanding of its impact on disease progression is lacking. METHODS: A comprehensive proteomic pipeline using data-independent acquisition was applied to decellularized colon samples from the Muc2 knockout (Muc2KO) mouse model of colitis for an in-depth characterization of extracellular matrix remodeling. Unique proteomic profiles of the matrisomal landscape were extracted from prepathologic and overt colitis. Integration of proteomics and transcriptomics data sets extracted from the same murine model produced network maps describing the orchestrating role of matrisomal proteins in tissue remodeling during the progression of colitis. RESULTS: The in-depth proteomic workflow used here allowed the addition of 34 proteins to the known colon matrisomal signature. Protein signatures of prepathologic and pathologic colitic states were extracted, differentiating the 2 states by expression of small leucine-rich proteoglycans. We outlined the role of this class and other matrisomal proteins in tissue remodeling during colitis, as well as the potential for coordinated regulation of cell types by matrisomal ligands. CONCLUSIONS: Our work highlights a central role for matrisomal proteins in tissue remodeling during colitis and defines orchestrating nodes that can be exploited in the selection of therapeutic targets.

Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR.

Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas.

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3ß. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration.

BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.

A Diffusion-like Process Accommodates New Crypts During Clonal Expansion in Human Colonic Epithelium.

BACKGROUND & AIMS: Colorectal cancer (CRC) is thought to arise when the cumulative mutational burden within colonic crypts exceeds a certain threshold that leads to clonal expansion and ultimately neoplastic transformation. Therefore, quantification of the fixation and subsequent expansion of somatic mutations in normal epithelium is key to understanding colorectal cancer initiation. The aim of the present study was to determine how advantaged expansions can be accommodated in the human colon. METHODS: Immunohistochemistry was used to visualize loss of the cancer driver KDM6A in formalin-fixed paraffin-embedded (FFPE) normal human colonic epithelium. Combining microscopy with neural network-based image analysis, we determined the frequencies of KDM6A-mutant crypts and fission/fusion intermediates as well as the spatial distribution of clones. Mathematical modeling then defined the dynamics of their fixation and expansion. RESULTS: Interpretation of the age-related behavior of KDM6A-negative clones revealed significant competitive advantage in intracrypt dynamics as well as a 5-fold increase in crypt fission rate. This was not accompanied by an increase in crypt fusion. Mathematical modeling of crypt spacing identifies evidence for a crypt diffusion process. We define the threshold fission rate at which diffusion fails to accommodate new crypts, which can be exceeded by KRAS activating mutations. CONCLUSIONS: Advantaged gene mutations in KDM6A expand dramatically by crypt fission but not fusion. The crypt diffusion process enables accommodation of the additional crypts up to a threshold value, beyond which polyp growth may occur. The fission rate associated with KRAS mutations offers a potential explanation for KRAS-initiated polyps.

5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation.

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Correction: Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine.

[This corrects the article DOI: 10.1371/journal.pone.0004264.].

Quantifying Microsatellite Mutation Rates from Intestinal Stem Cell Dynamics in Msh2-Deficient Murine Epithelium.

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10-2) compared to wild-type epithelium (6.2 × 10-5).

Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts.

Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.

Bcl9 and Pygo synergise downstream of Apc to effect intestinal neoplasia in FAP mouse models.

Bcl9 and Pygo are Wnt enhanceosome components that effect ß-catenin-dependent transcription. Whether they mediate ß-catenin-dependent neoplasia is unclear. Here we assess their roles in intestinal tumourigenesis initiated by Apc loss-of-function (ApcMin), or by Apc1322T encoding a partially-functional Apc truncation commonly found in colorectal carcinomas. Intestinal deletion of Bcl9 extends disease-free survival in both models, and essentially cures Apc1322T mice of their neoplasia. Loss-of-Bcl9 synergises with loss-of-Pygo to shift gene expression within Apc-mutant adenomas from stem cell-like to differentiation along Notch-regulated secretory lineages. Bcl9 loss also promotes tumour retention in ApcMin mice, apparently via relocating nuclear ß-catenin to the cell surface, but this undesirable effect is not seen in Apc1322T mice whose Apc truncation retains partial function in regulating ß-catenin. Our results demonstrate a key role of the Wnt enhanceosome in ß-catenin-dependent intestinal tumourigenesis and reveal the potential of BCL9 as a therapeutic target during early stages of colorectal cancer.

Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer.

Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration.

The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism of, and requirement for, progenitor plasticity in mediating pathological response remain unknown. Here we show that phosphorylation of the transcription factor Atoh1 is required for both the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. As confirmed by lineage tracing, Atoh1+ cells (Atoh1(WT)CreERT2 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant Atoh1(9S/T-A)CreERT2 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis, Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenicity that affects overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium, and the balance between stem and progenitor fate is directly coordinated by ATOH1 multisite phosphorylation.

Itraconazole targets cell cycle heterogeneity in colorectal cancer.

Cellular dormancy and heterogeneity in cell cycle length provide important explanations for treatment failure after adjuvant therapy with S-phase cytotoxics in colorectal cancer (CRC), yet the molecular control of the dormant versus cycling state remains unknown. We sought to understand the molecular features of dormant CRC cells to facilitate rationale identification of compounds to target both dormant and cycling tumor cells. Unexpectedly, we demonstrate that dormant CRC cells are differentiated, yet retain clonogenic capacity. Mouse organoid drug screening identifies that itraconazole generates spheroid collapse and loss of dormancy. Human CRC cell dormancy and tumor growth can also be perturbed by itraconazole, which is found to inhibit Wnt signaling through noncanonical hedgehog signaling. Preclinical validation shows itraconazole to be effective in multiple assays through Wnt inhibition, causing both cycling and dormant cells to switch to global senescence. These data provide preclinical evidence to support an early phase trial of itraconazole in CRC.

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.

Metaplastic Cdx2-depleted cells can be very disruptive neighbors.

In this issue of JEM, Balbinot et al. (https://doi.org/10.1084/jem.20170934) describe an original mechanism where Cdx2 inactivation regulates intestinal metaplastic to neoplastic transition in a paracrine fashion. Surprisingly, the target cells are neighboring "normal" Cdx2-positive cells.

Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells.

Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.

Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny.

The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium.

Detection of colorectal dysplasia using fluorescently labelled lectins.

Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss dysplastic lesions. We tested here the ability of fluorescently labelled lectins to distinguish dysplasia from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue from the Apc(min) mouse and when applied to fixed human colorectal tissue sections. Wheat germ agglutinin (WGA) showed significantly decreased binding to adenomas in the mouse tissue and in sections of human colon from 47 patients. Changes in WGA binding to the human surface epithelium allowed regions containing normal epithelium (NE) or hyperplastic polyps (HP) to be distinguished from regions containing low-grade dysplasia (LGD), high-grade dysplasia (HGD) or carcinoma (C), with 81% sensitivity, 87% specificity and 93% positive predictive value (PPV). Helix pomatia agglutinin (HGA) distinguished epithelial regions containing NE from regions containing HP, LGD, HGD or C, with 89% sensitivity, 87% specificity and 97% PPV. The decreased binding of WGA and HPA to the luminal surface epithelium in human dysplasia suggests that these lectins may enable more sensitive detection of disease in the clinic using fluorescence colonoscopy.