Microbiota metabolized Bile Acids accelerate Gastroesophageal Adenocarcinoma via FXR inhibition.

Background: The incidence of Barrett esophagus (BE) and Gastroesophageal Adenocarcinoma (GEAC) correlates with obesity and a diet rich in fat. Bile acids (BA) support fat digestion and undergo microbial metabolization in the gut. The farnesoid X receptor (FXR) is an important modulator of the BA homeostasis. The capacity of inhibiting cancer-related processes when activated, make FXR an appealing therapeutic target. In this work, we assess the role of diet on the microbiota-BA axis and evaluate the role of FXR in disease progression. Results: Here we show that high fat diet (HFD) accelerated tumorigenesis in L2-IL1B mice (BE- and GEAC- mouse model) while increasing BA levels and enriching gut microbiota that convert primary to secondary BA. While upregulated in BE, expression of FXR was downregulated in GEAC in mice and humans. In L2-IL1B mice, FXR knockout enhanced the dysplastic phenotype and increased Lgr5 progenitor cell numbers. Treatment of murine organoids and L2-IL1B mice with the FXR agonist obeticholic acid (OCA) deacelerated GEAC progression. Conclusion: We provide a novel concept of GEAC carcinogenesis being accelerated via the diet-microbiome-metabolome axis and FXR inhibition on progenitor cells. Further, FXR activation protected with OCA ameliorated the phenotype in vitro and in vivo, suggesting that FXR agonists have potential as differentiation therapy in GEAC prevention.

Telomere shortening accelerates tumor initiation in the L2-IL1B mouse model of Barrett esophagus and emerges as a possible biomarker.

Barrett's esophagus (BE) is a precursor of the esophageal adenocarcinoma (EAC). BE- development and its progression to cancer is associated with gastroesophageal reflux disease. However, there is currently no molecular risk prediction model that accurately identifies patients at high risk for EAC. Here, we investigated the impact of shortened telomeres in a mouse model for Barrett esophagus (L2-IL1B). The L2-IL1B mouse model is characterized by IL-1ß-mediated inflammation, which leads to a Barrett-like metaplasia in the transition zone between the squamous forestomach and glandular cardia/stomach. Telomere shortening was achieved by mTERC knockout. In the second generation (G2) of mTERC knockout L2-IL1B.mTERC-/- G2 mice exhibited telomere dysfunction with significantly shorter telomeres as measured by qFISH compared to L2-IL1B mice, correlating with stronger DNA damage in the form of phosphorylation of H2AX (γH2AX). Macroscopically, tumor area along the squamocolumnar junction (SCJ) was increased in L2-IL1B.mTERC-/- G2 mice, along with increased histopathological dysplasia. In vitro studies indicated increased organoid formation capacity in BE tissue from L2-IL1B.mTERC-/- G2 mice. In addition, pilot studies of human BE-, dysplasia- and EAC tissue samples confirmed that BE epithelial cells with or without dysplasia (LGD) had shorter telomeres compared to gastric cardia tissue. Of note, differentiated goblet cells retained longer telomeres than columnar lined BE epithelium. In conclusion, our studies suggest that shortened telomeres are functionally important for tumor development in a mouse model of BE and are associated with proliferating columnar epithelium in human BE. We propose that shortened telomeres should be evaluated further as a possible biomarker of cancer risk in BE patients.

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett's esophagus.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett's esophagus. METHODS: Six L2-IL1B mice with advanced stage of disease (12-16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12-14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. RESULTS: Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. CONCLUSIONS: This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett's esophagus. Further investigations are needed to assess its use in the clinical setting.

Targeted Hsp70 fluorescence molecular endoscopy detects dysplasia in Barrett's esophagus.

PURPOSE: The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. METHODS: Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. RESULTS: Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized  by human BE dysplastic patient-derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. CONCLUSION: In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

High-Fructose Diet Alters Intestinal Microbial Profile and Correlates with Early Tumorigenesis in a Mouse Model of Barrett's Esophagus.

Esophageal adenocarcinoma (EAC) is mostly prevalent in industrialized countries and has been associated with obesity, commonly linked with a diet rich in fat and refined sugars containing high fructose concentrations. In meta-organisms, dietary components are digested and metabolized by the host and its gut microbiota. Fructose has been shown to induce proliferation and cell growth in pancreas and colon cancer cell lines and also alter the gut microbiota. In a previous study with the L2-IL-1B mouse model, we showed that a high-fat diet (HFD) accelerated EAC progression from its precursor lesion Barrett's esophagus (BE) through changes in the gut microbiota. Aiming to investigate whether a high-fructose diet (HFrD) also alters the gut microbiota and favors EAC carcinogenesis, we assessed the effects of HFrD on the phenotype and intestinal microbial communities of L2-IL1B mice. Results showed a moderate acceleration in histologic disease progression, a mild effect on the systemic inflammatory response, metabolic changes in the host, and a shift in the composition, metabolism, and functionality of intestinal microbial communities. We conclude that HFrD alters the overall balance of the gut microbiota and induces an acceleration in EAC progression in a less pronounced manner than HFD.

Anti-inflammatory chemoprevention attenuates the phenotype in a mouse model of esophageal adenocarcinoma.

Barrett's esophagus (BE) is the main known precursor condition of esophageal adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is probably mediated by chronic esophageal inflammation, secondary to gastroesophageal reflux disease in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high-fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, whereas both NSAIDs were effective chemoprevention agents in the accelerated HFD-fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.

Notch signaling drives development of Barrett's metaplasia from Dclk1-positive epithelial tuft cells in the murine gastric mucosa.

Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC), but its cellular origin and mechanism of neoplastic progression remain unresolved. Notch signaling, which plays a key role in regulating intestinal stem cell maintenance, has been implicated in a number of cancers. The kinase Dclk1 labels epithelial post-mitotic tuft cells at the squamo-columnar junction (SCJ), and has also been proposed to contribute to epithelial tumor growth. Here, we find that genetic activation of intracellular Notch signaling in epithelial Dclk1-positive tuft cells resulted in the accelerated development of metaplasia and dysplasia in a mouse model of BE (pL2.Dclk1.N2IC mice). In contrast, genetic ablation of Notch receptor 2 in Dclk1-positive cells delayed BE progression (pL2.Dclk1.N2fl mice), and led to increased secretory cell differentiation. The accelerated BE progression in pL2.Dclk1.N2IC mice correlated with changes to the transcriptomic landscape, most notably for the activation of oncogenic, proliferative pathways in BE tissues, in contrast to upregulated Wnt signalling in pL2.Dclk1.N2fl mice. Collectively, our data show that Notch activation in Dclk1-positive tuft cells in the gastric cardia can contribute to BE development.

Elimination of NF-κB signaling in Vimentin+ stromal cells attenuates tumorigenesis in a mouse model of Barrett's Esophagus.

Chronic inflammation induces Barrett's Esophagus (BE) which can advance to esophageal adenocarcinoma. Elevated levels of interleukin (IL)-1b, IL-6 and IL-8 together with activated nuclear factor-kappaB (NF-κB), have been identified as important mediators of tumorigenesis. The inflammatory milieu apart from cancer cells and infiltrating immune cells contains myofibroblasts (MFs) that express aSMA and Vimentin. As we observed that increased NF-κB activation and inflammation correlates with increased MF recruitment and an accelerated phenotype we here analyze the role of NF-κB in MF during esophageal carcinogenesis in our L2-IL-1B mouse model. To analyze the effect of NF-κB signaling in MFs, we crossed L2-IL-1B mice to tamoxifen inducible Vim-Cre (Vim-CreTm) mice and floxed RelA (p65fl/fl) mice to specifically eliminate NF-κB signaling in MF (IL-1b.Vim-CreTm.p65fl/fl). The interaction of epithelial cells and stromal cells was further analyzed in mouse BE organoids and patient-derived human organoids. Histological scoring of IL-1b.Vim-CreTm.p65fl/fl mice showed a significantly attenuated phenotype compared with L2-IL-1B mice, with mild inflammation, decreased metaplasia and no dysplasia. This correlated with decreased proliferation and increased differentiation in cardia tissue of IL-1b.Vim-CreTm.p65fl/fl compared with L2-IL-1B mice. Distinct changes of cytokines and chemokines within the local microenvironment in IL-1b.Vim-CreTm.p65fl/fl mice reflected the histopathological abrogated phenotype. Co-cultured NF-κB inhibitor treated MF with mouse BE organoids demonstrated NF-κB-dependent growth and migration. MFs are essential to form an inflammatory and procarcinogenic microenvironment and NF-κB signaling in stromal cells emerges as an important driver of esophageal carcinogenesis. Our data suggest anti-inflammatory approaches as preventive strategies during surveillance of BE patients.

Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma.

BACKGROUND & AIMS: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis. METHODS: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5+ (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB. RESULTS: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5+ cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5+ cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5+ cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation. CONCLUSIONS: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.

BarrettNET-a prospective registry for risk estimation of patients with Barrett's esophagus to progress to adenocarcinoma.

Risk stratification in patients with Barrett's esophagus (BE) to prevent the development of esophageal adenocarcinoma (EAC) is an unsolved task. The incidence of EAC and BE is increasing and patients are still at unknown risk. BarrettNET is an ongoing multicenter prospective cohort study initiated to identify and validate molecular and clinical biomarkers that allow a more personalized surveillance strategy for patients with BE. For BarrettNET participants are recruited in 20 study centers throughout Germany, to be followed for progression to dysplasia (low-grade dysplasia or high-grade dysplasia) or EAC for >10 years. The study instruments comprise self-administered epidemiological information (containing data on demographics, lifestyle factors, and health), as well as biological specimens, i.e., blood-based samples, esophageal tissue biopsies, and feces and saliva samples. In follow-up visits according to the individual surveillance plan of the participants, sample collection is repeated. The standardized collection and processing of the specimen guarantee the highest sample quality. Via a mobile accessible database, the documentation of inclusion, epidemiological data, and pathological disease status are recorded subsequently. Currently the BarrettNET registry includes 560 participants (23.1% women and 76.9% men, aged 22-92 years) with a median follow-up of 951 days. Both the design and the size of BarrettNET offer the advantage of answering research questions regarding potential causes of disease progression from BE to EAC. Here all the integrated methods and materials of BarrettNET are presented and reviewed to introduce this valuable German registry.

High-Fat Diet Accelerates Carcinogenesis in a Mouse Model of Barrett's Esophagus via Interleukin 8 and Alterations to the Gut Microbiome.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Progression from BE to cancer is associated with obesity, possibly due to increased abdominal pressure and gastroesophageal reflux disease, although this pathogenic mechanism has not been proven. We investigated whether environmental or dietary factors associated with obesity contribute to the progression of BE to EAC in mice. METHODS: Tg(ED-L2-IL1RN/IL1B)#Tcw mice (a model of BE, called L2-IL1B mice) were fed a chow (control) or high-fat diet (HFD) or were crossbred with mice that express human interleukin (IL) 8 (L2-IL1B/IL8 mice). Esophageal tissues were collected and analyzed for gene expression profiles and by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. Organoids were established from BE tissue of mice and cultured with serum from lean or obese individuals or with neutrophils from L2-IL1B mice. Feces from mice were analyzed by 16s ribosomal RNA sequencing and compared to 16s sequencing data from patients with dysplasia or BE. L2-IL1B were mice raised in germ-free conditions. RESULTS: L2-IL1B mice fed an HFD developed esophageal dysplasia and tumors more rapidly than mice fed the control diet; the speed of tumor development was independent of body weight. The acceleration of dysplasia by the HFD in the L2-IL1B mice was associated with a shift in the gut microbiota and an increased ratio of neutrophils to natural killer cells in esophageal tissues compared with mice fed a control diet. We observed similar differences in the microbiomes from patients with BE that progressed to EAC vs patients with BE that did not develop into cancer. Tissues from dysplasias of L2-IL1B mice fed the HFD contained increased levels of cytokines that are produced in response to CXCL1 (the functional mouse homolog of IL8, also called KC). Serum from obese patients caused organoids from L2-IL1B/IL8 mice to produce IL8. BE tissues from L2-IL1B mice fed the HFD and from L2-IL1B/IL8 mice contained increased numbers of myeloid cells and cells expressing Cxcr2 and Lgr5 messenger RNAs (epithelial progenitors) compared with mice fed control diets. BE tissues from L2-IL1B mice raised in germ-free housing had fewer progenitor cells and developed less dysplasia than in L2-IL1 mice raised under standard conditions; exposure of fecal microbiota from L2-IL1B mice fed the HFD to L2-IL1B mice fed the control diet accelerated tumor development. CONCLUSIONS: In a mouse model of BE, we found that an HFD promoted dysplasia by altering the esophageal microenvironment and gut microbiome, thereby inducing inflammation and stem cell expansion, independent of obesity.

Origins of Metaplasia in the Esophagus: Is This a GE Junction Stem Cell Disease?

The incidence of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett's esophagus (BE) has been increasing steadily in the western world in recent decades. Understanding the cellular origins of BE and the conditions responsible for their malignant transformation would greatly facilitate risk assessment and identification of patients at risk of progression, but this topic remains a source of debate. Here, we review recent findings that have provided support for the gastroesophageal junction (GEJ) as the main source of stem cells that give rise to BE and EAC. These include both gastric cardia cells and transitional basal cells. Furthermore, we discuss the role of chronic injury and inflammation in a tumor microenvironment as a major factor in promoting stem cell expansion and proliferation as well as transformation of the GEJ-derived stem cells and progression to EAC. We conclude that there exists a large amount of empirical support for the GEJ as the likely source of BE stem cells. While BE seems to resemble a successful adaptation to esophageal damage, carcinogenesis appears as a consequence of natural selection at the level of GEJ stem cells, and later glands, that expand into the esophagus wherein the local ecology creates the selective landscape for cancer progression.