High Content Imaging of Barrett's-Associated High Grade Dysplasia Cells Following siRNA Library Screening Reveals Acid Responsive Regulators of Cellular Transitions.

ABSTRACT Background and Aims 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. Methods 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. Results 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. Conclusion 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.