Barrett’s oesophagus (BO), an intestinal-type metaplasia (IM), typically arising in conjunction with gastro-oesophageal reflux disease, is a prominent risk factor for the development of oesophageal adenocarcinoma (OAC). The molecular similarities between IM and normal intestinal tissues are ill-defined. Consequently, the contribution of intestine-enriched factors expressed within BO to oncogenesis is unclear. Herein, using transcriptomics we define the intestine-enriched genes expressed in meta-profiles of BO and OAC. Interestingly, 77% of the genes differentially expressed in a meta-profile of BO were similarly expressed in intestinal tissues. Furthermore, 85% of this intestine-like signature was maintained upon transition to OAC. Gene networking analysis of transcription factors within this signature revealed a network centred upon NR5A2, GATA6 and FOXA2, whose over-expression was determined in a cohort of BO and OAC patients. Simulated acid reflux was observed to induce the expression of both NR5A2 and GATA6. Using siRNA-mediated silencing and an NR5A2 antagonist we demonstrate that NR5A2-mediated cancer cell survival is facilitated through augmentation of GATA6 and anti-apoptotic factor BCL-XL levels. Abrogation of NR5A2-GATA6 expression in conjunction with BCL-XL co-silencing resulted in synergistically increased sensitivity to chemotherapeutics and photo-dynamic therapeutics. These findings characterize the intestine-like signature associated with IM which may have important consequences to adenocarcinogenesis.

Effective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett's esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation. This study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors. By using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway-associated genes supporting EAC cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor-activated signaling pathways (TYROBP-spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice. These data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

There is no doubt that chronic gastroesophageal reflux disease increases the risk of esophageal adenocarcinoma (EAC) by several fold (odds ratio, 6.4; 95% CI, 4.6–9.1), and some relationships between reflux disease–mediated inflammation and oncogenic processes have been explored; however, the precise interconnections between the immune response and genomic instabilities underlying these pathologic processes only now are emerging. Furthermore, the precise cell of origin of the precancerous stages associated with EAC development, Barrett’s esophagus, be it cardia resident or embryonic remnant, may shape our interpretation of the likely immune drivers. This review integrates the current collective knowledge of the immunology underlying EAC development and outlines a framework connecting proinflammatory pathways, such as those mediated by interleukin 1β, tumor necrosis factor α, leukemia inhibitory factor, interleukin 6, signal transduction and activator of transcription 3, nuclear factor-κB, cyclooxygenase-2, and transforming growth factor β, with oncogenic pathways in the gastroesophageal reflux disease–Barrett’s esophagus–EAC cancer sequence. Further defining these immune and molecular railroads may show a map of the routes taken by gastroesophageal cells on their journey toward EAC tumor phylogeny. The selective pressures applied by this immune-induced journey likely impact the phenotype and genotype of the resulting oncogenic destination and further exploration of lesser-defined immune drivers may be useful in future individualized therapies or enhanced selective application of recent immune-driven therapeutics.

Esophageal adenocarcinoma (EAC) develops from within Barrett's esophagus (BE) concomitant with gastro-esophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of the BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. siRNA library screening and high content imaging analysis outlined changes in BE-HGD (High grade dysplasia) and EAC cell morphologies following gene silencing. Gene expression microarray (GEM) data and low pH exposures studies modelling GERD-associated pulses (pH4.0, 10 Mins) and tumour microenvironments (pH6.0, constant) were utilised. Statistical analysis of siRNA screening data defined 207 genes (Z-score>2.0), in 12 distinct morphological clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes of this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1 and PRC1. Guided by GEM study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a non-overlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterised by differential rates of cell motility, pseudopodia formation and altered expression of the mesenchymal markers vimentin and E-cadherin. Collectively we have demonstrated that low pH microenvironments associated with GERD, and tumour invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonisation and invasion.

Classical organoid models of Barrett’s esophagus (BE) lack accessible luminal surfaces, tissue depth and tractable stem cell and immune cell components. Herein, we aimed to seed adult tissue-resident stem cells (ASC) with advanced organ-on-a-chip (OOAC) microfluidics to provide a platform upon which to study the immunobiology of BE and associated high grade dysplasia (HGD). ASCs from BE and HGD and gastroesophageal junction (GEJ) were obtained using methods analogous to ground-state stem cell culture and conditional reprogramming. ASC differentiation was achieved on Transwells and flexible OOAC with tissues examined by immunohistochemistry and single-cell RNA sequencing gene expression analysis. Monocytic cell line THP1 was utilized in transmigration experiments. Monolayered ASCs exhibited colony formation, self-renewal and air–liquid interface-mediated differentiation resulting in 3D furrow formation, mucus production and cell types reflective of tissue of origin-foveolar, goblet, enterocyte-like. Levels of intestinal marker Trefoil Factor 3 were abundant in BE tissue, significantly lower in HGD and absent in GEJ. The gastric marker Gastrokine1 was only expressed in HGD- and GEJ-differentiated ASCs. Comparatively, tissues derived from OOAC displayed significantly enhanced villus-like structure, height, and prolonged survival (<19 days) when compared to Transwell-differentiated equivalents. Single-cell RNAseq analysis detected populations representative of stem cells, transit amplifying stem cells, early and late enterocytes, goblet and enteroendocrine cells and CXCL8 positive immunomodulatory cells in both systems. However, significantly higher levels of proliferating cells, terminal enterocyte-like, Goblet cells, and enteroendocrine-like cells were observed in BE-OOAC systems. THP1 cells were tracked under flow conditions in BE-OOAC and their transmigration through the endothelial layer was evident only in the presence of organoid tissue above. These findings showed that OOACs offer more physiologically relevant environments, promote deeper cellular differentiation, and increased cellular tractability when seeded as ASCs rather than classical organoid aggregates.

GATA family transcription factors are somatically variable (SV) in esophageal adenocarcinomas (EAC) and inducible by simulated reflux. Our study examines the mechanisms whereby GATA family members, GATA4, GATA6 and the atypical TRPS-1, influence oncogenesis during the Barrett's esophagus (BE) metaplasia-dysplasia transition preceding EAC. RNAseq analyses of esophageal cell lines and lesion-derived adult stem cells (ASCs) in conjunction shRNA- or CRISPR-facilitated gene silencing, together with re-analysis of The Cancer Genome Atlas data (TCGA), spatial transcriptomics and organ-on-a-chip (OOAC) studies were employed. While a GERD history positively correlated with GATA4/6 SV and a columnar-associated gene signature (ANPEP/GATA4) in TCGA EAC cases, it negatively associated with a squamous lineage-linked signature (TP63/SOX15) containing TRPS1. In experimental data, opposing effects on regulators of squamous and columnar lineage identity were uncovered between TRPS1 and classical GATA factors (GATA4/6). Interrogation of this GATA "fulcrum" defined further genes (CGN, IL6R and GPRC5B) targeted for TRPS1-mediated suppression or GATA4/6 activation. A novel spatial transcriptomic signature of BE-associated high-grade-dysplasia (HGD) captured GATA fulcrum action through GPRC5B expression. Functionally, GPRC5B was found to be low-pH-responsive, to increase proliferative and colony formation rates, and when over-expressed facilitate a hyper proliferative HGD-like transformation of BE-ASCs. Using an OOAC platform, cellular overgrowth, reduced luminal villus structures, lower goblet cell numbers, and loss of intestine-associated marker gene expression (TFF3/MUC2), were observed following GPRC5B over-expression in BE-ASCs, mirroring HGD. This study identifies critical GATA factor-mediated processes underlying cellular phenotype in the BE-HGD-EAC transition and identifies GATA-inducible GPRC5B as a functional marker and possible driver of progression through HGD to EAC.