Abstract
Esophageal adenocarcinoma (EAC) is a highly heterogeneous disease, dominated by large-scale genomic rearrangements and copy number alterations. Such characteristics have hampered conventional target-directed drug discovery and personalized medicine strategies, contributing to poor outcomes for patients. We describe the application of a high-content Cell Painting assay to profile the phenotypic response of 19,555 compounds across a panel of six EAC cell lines and two tissue-matched control lines. We built an automated high-content image analysis pipeline to identify compounds that selectively modified the phenotype of EAC cell lines. We further trained a machine-learning model to predict the mechanism of action of EAC selective compounds using phenotypic fingerprints from a library of reference compounds. We identified a number of phenotypic clusters enriched with similar pharmacological classes, including methotrexate and three other antimetabolites that are highly selective for EAC cell lines. We further identify a small number of hits from our diverse chemical library that show potent and selective activity for EAC cell lines and that do not cluster with the reference library of compounds, indicating they may be selectively targeting novel esophageal cancer biology. Overall, our results demonstrate that our EAC phenotypic screening platform can identify existing pharmacologic classes and novel compounds with selective activity for EAC cell phenotypes.