Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-ter... Full description

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APCKO) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APCKO organoids by overexpression of Wnt target genes and nuclear-translocated β-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/β-catenin signaling activation in BE neoplastic transformation. This system can be used to study other ‘driver’ pathway alterations in BE-associated neoplasia, avoiding signaling noise present in immortalized or cancer-derived cell lines. •A long-term expansion Wnt-activated human BE organoid model has been established using CRISPR-Cas9 combined with a niche-based selection system.•Compared to wild-type BE organoids, Wnt-activated BE organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, overexpression of Ki67, c-myc and cyclin D1, and enhanced protrusive migration ability, which disrupted reorganization and integrity of the basement membrane.•This CRISPR-Cas9 editing human-derived organoid model recapitulates the role of activated Wnt signaling during BE neoplastic transformation.