Notch1 mutation drives clonal expansion in normal esophageal epithelium but impairs tumor growth

NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age, but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations may promote clonal expansion but impede carcinogenesis. Here we test this hypothesis. Visualizing and sequencing NOTCH1 mutant clones in aging normal human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild type cells, an effect enhanced by loss of the second allele. Notch1 loss alters transcription but has minimal effects on epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. We conclude that Notch1 mutations in normal epithelium are beneficial as wild type Notch1 promotes tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.

In vitro models of the human esophagus reveal ancestrally diverse response to injury

European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines.

Histological changes in the human esophagus following triamcinolone injection to prevent esophageal stricture after endoscopic submucosal dissection

Background Locoregional steroid injection prevents post-endoscopic submucosal dissection (ESD) esophageal stricture, but histological changes that occur following steroid injection in the human esophagus are unclear. This study investigated the histopathological characteristics caused by locoregional triamcinolone acetonide (TA) injection using human esophagectomy specimens. Methods From January 2014 to December 2019, among 297 patients (373 lesions) who underwent esophageal ESD, 13 patients who underwent additional esophagectomy after ESD were examined. Seven patients (TA group) with wide excisions were injected with TA after ESD and another six patients (Non-TA group) with smaller tumors were not injected with TA. The clinical background of these patients and histopathological features of ESD ulcer scar obtained from esophagectomy specimens were retrospectively investigated. Results The circumferential rate of ESD excision was more than three-quarters in all cases in the TA group, whereas it was less than three-quarters in the Non-TA group. No other statistical difference in the clinical background was found between the two groups. The subepithelial fibrous tissue of the ESD ulcer scar in the TA group was significantly thinner than that in the Non-TA group (P < 0.05). There was no significant difference in the thickness of the regenerated epithelium and muscularis propria layer of the ESD ulcer scar. Conclusions Histological finding of thinning of the subepithelial fibrous tissue of ESD ulcer scar in the human esophagus after TA injection was obtained. This suggests that TA suppresses the proliferation of the fibrous tissue of the subepithelial layer to help prevent esophageal stricture after widespread ESD in the human esophagus.

Novel Ex Vivo Model to Examine the Mechanism and Relationship of Esophageal Microbiota and Disease

Rates of esophageal cancer have increased over the last 40 years. Recent clinical research has identified correlations between the esophageal microbiome and disease. However, mechanisms of action have been difficult to elucidate performing human experimentation. We propose an ex vivo model, which mimics the esophagus and is ideal for mechanistic studies on the esophageal microbiome and resultant transcriptome. To determine the microbiome and transcriptome profile of the human distal esophagus, the microbiome was assessed in 74 patients and the transcriptome profile was assessed in 37 patients with and without Barrett’s esophagus. Thereafter, an ex vivo model of the esophagus was created using an air–liquid interfaced (ALI) design. This design created a sterile apical surface and a nutrient-rich basal surface. An epithelial layer was grown on the apical surface. A normal microbiome and Barrett’s microbiome was harvested and created from patients during endoscopic examination of the esophagus. There was a distinct microbiome in patients with Barrett’s esophagus. The ex vivo model was successfully created with a squamous epithelial layer on the apical surface of the ex vivo system. Using this ex vivo model, multiple normal esophageal and Barrett’s esophageal cell lines will be created and used for experimentation. Each microbiome will be inoculated onto the sterile apical surface of each cell line. The resultant microbiome and transcriptome profile on each surface will be measured and compared to results in the human esophagus to determine the mechanism of the microbiome interaction.

Lysophosphatidic acid mediated PI3K/AKT activation contributed to esophageal squamous cell cancer progression

Lysophosphatidic acid (LPA) and its G-protein-coupled receptors (Lpar1~Lpar6) mediate a plethora of activities associated with cancer growth and progression. However, there is no systematic study about whether and how LPA promotes esophageal squamous cell carcinoma (ESCC). Here we show that autotaxin (ATX), a primary LPA-producing enzyme, is highly expressed in ESCC, and overexpressed ATX is associated with the poor outcome of ESCC patients. Meanwhile, the expression of Lpar1 was much higher in ESCC cells compared with Het-1A (human esophagus normal epithelial cells). Functional experiments showed that LPA remarkably increased the proliferation and migration of ESCC cells. Furthermore, Lpar1 knockdown abolished the effect of LPA on ESCC cell proliferation and migration. Mechanistic studies revealed that LPA promoted ESCC cell lines proliferation and migration through PI3K/AKT pathway. Treatment of KYSE30 cell xenografts with Lpar1 inhibitor BMS-986020 significantly repressed tumor growth. Our results shed light on the important role of LPA in ESCC, and Lpar1 might be a potential treatment target for ESCC.

Submucosal gland neoplasms of the esophagus: an update and review

Submucosal glands (SMGs) present throughout human esophagus with clusters at either the upper third or lower third of the organ. SMGs tend to atrophy with age, and neoplasms arising in these glands are rare. In order to bring convenience to diagnosis, we summarize the histopathologic characteristics of all esophageal submucosal gland tumors (SGTs). Due to the morphological similarity, the nomenclature of salivary tumors is adopted for SGTs. However, there is great confusion about the definition and histogenesis of these tumors, especially the malignant subtypes. In the literature, esophageal mucoepidermoid carcinoma and adenoid cystic carcinoma usually adjoin the surface squamous epithelium and coexist with intraepithelial neoplasia or invasive squamous cell carcinoma (SCC). In addition, the typical gene alterations of salivary tumors have not been reported in these SGTs. Therefore, we propose to apply stringent diagnostic criteria to esophageal SGTs so as to exclude mimickers that are SCCs with various degree of SMG differentiation.

Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium

Background The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. Methods We established a novel stepwise protocol with transwell culture and an air–liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. Results We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. Conclusion We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

Lubiprostone protects esophageal mucosa from acid injury in porcine esophagus

Esophageal injury from acid exposure related to gastroesophageal reflux disease is a common problem and a risk factor for development of Barrett’s esophagus and esophageal adenocarcinoma. Our previous work highlights the benefits of using porcine esophagus to study human esophageal disease because of the similarities between porcine and human esophagus. In particular, esophageal submucosal glands (ESMGs) are present in human esophagus and proximal porcine esophagus but not in rodent esophagus. Although CFTR is expressed in the ducts of ESMGs, very little is known about CFTR and alternate anion channels, including ClC-2, in the setting of acid-related esophageal injury. After finding evidence of CFTR and ClC-2 in the basal layers of the squamous epithelium, and in the ducts of the ESMGs, we developed an ex vivo porcine model of esophageal acid injury. In this model, esophageal tissue was placed in Ussing chambers to determine the effect of pretreatment with the ClC-2 agonist lubiprostone on tissue damage related to acid exposure. Pretreatment with lubiprostone significantly reduced the level of acid injury and significantly augmented the recovery of the injured tissue ( P < 0.05). Evaluation of the interepithelial tight junctions showed well-defined membrane localization of occludin in lubiprostone-treated injured tissues. Pretreatment of tissues with the Na+-K+-2Cl− cotransporter inhibitor bumetanide blocked lubiprostone-induced increases in short-circuit current and inhibited the reparative effect of lubiprostone. Furthermore, inhibition of ClC-2 with ZnCl2 blocked the effects of lubiprostone. We conclude that ClC-2 contributes to esophageal protection from acid exposure, potentially offering a new therapeutic target. NEW & NOTEWORTHY This research is the first to describe the presence of anion channels ClC-2 and CFTR localized to the basal epithelia of porcine esophageal mucosa and the esophageal submucosal glands. In the setting of ex vivo acid exposure, the ClC-2 agonist lubiprostone reduced acid-related injury and enhanced recovery of the epithelial barrier. This work may ultimately provide an alternate mechanism for treating gastroesophageal reflux disease.