Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Associates With Functional Alterations in Circulating microRNAs.

BACKGROUND AND AIMS: The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. METHODS: Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies. RESULTS: miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. CONCLUSIONS: These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.

Protein kinase A-mediated phosphorylation of naked cuticle homolog 2 stimulates cell-surface delivery of transforming growth factor-α for epidermal growth factor receptor transactivation.

The classic mode of G protein-coupled receptor (GPCR)-mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)-mediated cleavage of plasma membrane-anchored EGFR ligands. Herein, we show that the Gαs-activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E2 (PGE2 ) transactivate EGFR through increased cell-surface delivery of the EGFR ligand transforming growth factor-α (TGFα) in polarizing madin-darby canine kidney (MDCK) and Caco-2 cells. This is achieved by PKA-mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα-containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE2 rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser-223, a process that is facilitated by the molecular scaffold A-kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell-surface delivery of TGFα and increased EGFR activation. Thus, GPCR-triggered, PKA/AKAP12/NKD2-regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.

Murine Intrarectal Instillation of Purified Recombinant Clostridioides difficile Toxins Enables Mechanistic Studies of Pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474-4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Colon epithelial cell TGFß signaling modulates the expression of tight junction proteins and barrier function in mice.

Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGFß signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFß and intestinal barrier function. Here, we examine the role of TGFß signaling via SMAD4 in modulation of colon barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), Western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFß1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFß signaling, and TGFß-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared with healthy controls. Canonical TGFß signaling modulates the expression of tight junction proteins and barrier function in mouse colon.NEW & NOTEWORTHY We demonstrate that canonical TGFß family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.

p120-catenin controls contractility along the vertical axis of epithelial lateral membranes.

In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.

Inhibition of EGFR/ErbB does not protect against C. difficile toxin B.

Clostridioides difficile is a common cause of diarrhea and mortality, especially in immunosuppressed and hospitalized patients. C. difficile is a toxin-mediated disease, but the host cell receptors for C. difficile toxin B (TcdB) have only recently been revealed. Emerging data suggest TcdB interacts with receptor tyrosine kinases during infection. In particular, TcdB can elicit Epidermal Growth Factor Receptor (EGFR) transactivation in human colonic epithelial cells. The mechanisms for this function are not well understood, and the involvement of other receptors in the EGFR family of Erythroblastic Leukemia Viral Oncogene Homolog (ErbB) receptors remains unclear. Furthermore, in an siRNA-knockdown screen for protective genes involved with TcdB toxin pathogenesis, we show ErbB2 and ErbB3 loss resulted in increased cell viability. We hypothesize TcdB induces the transactivation of EGFR and/or ErbB receptors as a component of its cell-killing mechanism. Here, we show in vivo intrarectal instillation of TcdB in mice leads to phosphorylation of ErbB2 and ErbB3. However, immunohistochemical staining for phosphorylated ErbB2 and ErbB3 indicated no discernible difference between control and TcdB-treated mice for epithelial phospho-ErbB2 and phospho-ErbB3. Human colon cancer cell lines (HT29, Caco-2) exposed to TcdB were not protected by pre-treatment with lapatinib, an EGFR/ErbB2 inhibitor. Similarly, lapatinib pre-treatment failed to protect normal human colonoids from TcdB-induced cell death. Neutralizing antibodies against mouse EGFR failed to protect mice from TcdB intrarectal instillation as measured by edema, inflammatory infiltration, and epithelial injury. Our findings suggest TcdB-induced colonocyte cell death does not require EGFR/ErbB receptor tyrosine kinase activation.

Cecum axis (CecAx) preservation reveals physiological and pathological gradients in mouse gastrointestinal epithelium.

The mouse cecum has emerged as a model system for studying microbe-host interactions, immunoregulatory functions of the microbiome, and metabolic contributions of gut bacteria. Too often, the cecum is falsely considered as a uniform organ with an evenly distributed epithelium. We developed the cecum axis (CecAx) preservation method to show gradients in epithelial tissue architecture and cell types along the cecal ampulla-apex and mesentery-antimesentery axes. We used imaging mass spectrometry of metabolites and lipids to suggest functional differences along these axes. Using a model of Clostridioides difficile infection, we show how edema and inflammation are unequally concentrated along the mesenteric border. Finally, we show the similarly increased edema at the mesenteric border in two models of Salmonella enterica serovar Typhimurium infection as well as enrichment of goblet cells along the antimesenteric border. Our approach facilitates mouse cecum modeling with detailed attention to inherent structural and functional differences within this dynamic organ.

Distinct roles for interleukin-23 receptor signaling in regulatory T cells in sporadic and inflammation-associated carcinogenesis.

Introduction: The pro-inflammatory cytokine interleukin-23 (IL-23) has been implicated in colorectal cancer (CRC). Yet, the cell-specific contributions of IL-23 receptor (IL-23R) signaling in CRC remain unknown. One of the cell types that highly expresses IL-23R are colonic regulatory T cells (Treg cells). The aim of this study was to define the contribution of Treg cell-specific IL-23R signaling in sporadic and inflammation-associated CRC. Methods: In mice, the role of IL-23R in Treg cells in colitis-associated cancer (CAC) was investigated using azoxymethane/dextran sodium sulphate in wild-type Treg cell reporter mice (WT, Foxp3 YFP-iCre), and mice harboring a Treg cell-specific deletion of IL-23 (Il23r ΔTreg). The role of IL-23R signaling in Treg cells in sporadic CRC was examined utilizing orthotopic injection of the syngeneic colon cancer cell line MC-38 submucosally into the colon/rectum of mice. The function of macrophages was studied using clodronate. Finally, single-cell RNA-seq of a previously published dataset in human sporadic cancer was reanalyzed to corroborate these findings. Results: In CAC, Il23r ΔTreg mice had increased tumor size and increased dysplasia compared to WT mice that was associated with decreased tumor-infiltrating macrophages. In the sporadic cancer model, Il23r ΔTreg mice had increased survival and decreased tumor size compared to WT mice. Additionally, MC-38 tumors of Il23r ΔTreg mice exhibited a higher frequency of pro-inflammatory macrophages and IL-17 producing CD4+ T cells. The decreased tumor size in Il23r ΔTreg mice was macrophage-dependent. These data suggest that loss of IL-23R signaling in Treg cells permits IL-17 production by CD4+ T cells that in turn promotes pro-inflammatory macrophages to clear tumors. Finally, analysis of TCGA data and single-cell RNA-seq analysis of a previously published dataset in human sporadic cancer, revealed that IL23R was highly expressed in CRC compared to other cancers and specifically in tumor-associated Treg cells. Conclusion: Inflammation in colorectal carcinogenesis differs with respect to the contribution of IL-23R signaling in regulatory T cells.

An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Concomitant antibiotic and mercury resistance among gastrointestinal microflora of feral brook trout, Salvelinus fontinalis.

Twenty-nine bacterial isolates representing eight genera from the gastrointestinal tracts of feral brook trout Salvelinus fontinalis (Mitchell) demonstrated multiple maximal antibiotic resistances and concomitant broad-spectrum mercury (Hg) resistance. Equivalent viable plate counts on tryptic soy agar supplemented with either 0 or 25 µM HgCl(2) verified the ubiquity of mercury resistance in this microbial environment. Mercury levels in lake water samples measured 1.5 ng L(-1); mercury concentrations in fish filets ranged from 81.8 to 1,080 ng g(-1) and correlated with fish length. The presence of similar antibiotic and Hg resistance patterns in multiple genera of gastrointestinal microflora supports a growing body of research that multiple selective genes can be transferred horizontally in the presence of an unrelated individual selective pressure. We present data that bioaccumulation of non-point source Hg pollution could be a selective pressure to accumulate both antibiotic and Hg resistant bacteria.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms.

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development.

Monoclonal antibodies to DIPA: a novel binding partner of p120-catenin isoform 1.

The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for ß-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPA-specific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.

An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes.

The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a beta-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa beta-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.