Project Summary Barrett’s esophagus (BE) is a disease in which the squamous epithelium of the esophagus is replaced by a columnar intestinal epithelium (termed metaplasia), and is believed to affect 3-4 million people in the US. BE patients have a 30-125-fold greater risk of developing esophageal adenocarcinoma (EAC) via intermediate low- grade and high-grade dysplastic states, when compared to the general population. The pathogenesis of BE involves epigenetic/genomic aberrations (e.g. TP53 mutations), and an interplay with microenvironmental cues. Recent studies have demonstrated that ECM stiffening is associated with metaplasia (or BE)-dysplasia- adenocarcinoma sequence, and my novel data reveal that increased ECM stiffness, a critical biomechanical property, is characteristic of BE pathogenesis. This underscores a compelling need to understand mechanisms that foster BE metaplasia and dysplasia in the context of ECM stiffness. Therefore, this proposal will integrate my unique predoctoral training in biomaterial engineering with my new proposed career development training in epithelial cell and organoid biology to utilize an engineered HA-based hydrogel to elucidate the contribution of ECM stiffness in BE pathogenesis, and to identify matrix-activated therapeutic targets. This bioengineering approach will be combined with TP53 mutation (in the epithelium of BE) to understand BE pathogenesis. The overarching hypothesis of my proposal is that ECM stiffness modulation can recapitulate a dysplastic BE state, revealing novel underlying mechanisms of the disease. I will pursue this hypothesis through the following interrelated Specific Aims: Aim 1 focuses on establish a hydrogel platform to study the pro-dysplastic contribution of matrix properties in patient-derived BE organoids. Aim 2 focuses on in vitro identification and in vivo evaluation of therapeutic targets in stiff matrix-exposed patient-derived BE organoids. Aim 3 focuses on utilizing an hiPSC-derived platform of BE to elucidate the cooperation between intrinsic and extrinsic factors contributing to BE pathogenesis. This work is innovative because it uses an advanced bioengineering approach to elucidate the functional roles of ECM stiffness in human BE pathogenesis. Successful completion of this proposal will be both scientifically and clinically significant by defining the contribution of biomechanics to BE and laying a foundation for novel therapies to disrupt matrix stiffness in BE. The research proposal, training/career development/mentorship plans, advisory committee, RCR training, protected time for research, and institutional support will provide the foundation for me to transition to an eventual independent NIH funded tenure-track faculty investigator, who blends principles of basic biology, bioengineering, and translational medicine.

PROJECT NARRATIVE Barrett’s Esophagus (BE) is a prevalent disease in the US population. There are cell intrinsic (TP53 mutations) and cell extrinsic (extracellular matrix properties) mechanisms that foster BE pathogenesis. These mechanisms will be investigated in novel human induced pluripotent stem cell-derived organotypic raft and patient-derived 3D organoid culture systems to define how BE dysplasia occurs and to uncover translational therapeutic strategies.