SOX2 regulates foregut squamous epithelial homeostasis and is lost during Barrett’s esophagus development
Ramon U. Jin, Yuanwei Xu, T. Mamie Lih, Yang-Zhe Huang, Toni M. Nittolo, Blake E. Sells, Olivia M. Dres, Jean S. Wang, Qing K. Li, Hui Zhang, Jason C. Mills
Ramon U. Jin, Yuanwei Xu, T. Mamie Lih, Yang-Zhe Huang, Toni M. Nittolo, Blake E. Sells, Olivia M. Dres, Jean S. Wang, Qing K. Li, Hui Zhang, Jason C. Mills
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Research Article Development Gastroenterology

SOX2 regulates foregut squamous epithelial homeostasis and is lost during Barrett’s esophagus development

Abstract

Esophageal adenocarcinoma is increasingly prevalent and is thought to arise from Barrett’s esophagus (BE), a metaplastic condition in which chronic acid and bile reflux transforms the esophageal squamous epithelium into a gastric-intestinal glandular mucosa. The molecular determinants driving this metaplasia are poorly understood. We developed a human BE organoid biobank that recapitulates BE’s molecular heterogeneity. Bulk and single-cell transcriptomics, supported by patient tissue analysis, revealed that BE differentiation reflects a balance between SOX2 (foregut/esophageal) and CDX2 (hindgut/intestinal) transcription factors. Using squamous-specific inducible Sox2-KO (Krt5CreER/+ Sox2Δ/Δ ROSA26tdTomato/+) mice, we observed increased basal proliferation, reduced squamous differentiation, and expanded metaplastic glands at the squamocolumnar junction, some tracing back to Krt5-expressing cells. CUT&RUN analysis showed SOX2 bound and promoted differentiation-associated targets (e.g., Krt13) and repressed proliferation-associated targets (e.g., Mki67). Thus, SOX2 is critical for foregut squamous epithelial differentiation, and its decreased expression is likely an initiating step in progression to BE and then to esophageal adenocarcinoma.

Authors

Ramon U. Jin, Yuanwei Xu, T. Mamie Lih, Yang-Zhe Huang, Toni M. Nittolo, Blake E. Sells, Olivia M. Dres, Jean S. Wang, Qing K. Li, Hui Zhang, Jason C. Mills

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Figure 4

Sox2Δ/Δ squamous organoids have increased proliferation and decreased maturation.

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Sox2Δ/Δ squamous organoids have increased proliferation and decreased m...
(A) Bright-field images of forestomach organoids from WT control and Sox2Δ/Δ mice. Right: tdTomato fluorescence indicates Cre activity and Sox2 deletion. Bottom: higher magnification of boxed regions. Scale bars: 3,000 μm. (B) Immunostaining of Sox2Δ/Δ squamous organoids shows SOX2 loss (green nuclear), tdTomato expression (red cytoplastic), increased Ki-67 (white nuclear), and reduced cytokeratin 13 (purple cytoplasmic). All scale bars: 100 μm. (C) Proliferation assay of squamous organoids tracked over 17 days; bright-field images shown. Scale bars: 1,000 μm. Below: total organoid area (mean ± SD, 3 wells/condition); AUC and 95% CI at day 17 shown. Two-tailed unpaired Student’s t test; P value indicated. (D) Transwell culture of squamous organoids under full media proliferation (left) and air-liquid interface (ALI) maturation conditions (right). H&E, SOX2 (brown nuclear), Ki-67 (purple nuclear, arrowheads), and cytokeratin 13 (red cytoplasmic) staining. Open arrowhead showing shed cell under ALI maturation conditions. Insets: higher magnification of boxed areas. Scale bars: 100 μm. (E) Transepithelial electric resistance (TEER) measurements (mean ± SD, 3 wells/condition) after 8 days in full media and 11 additional days under ALI conditions. AUC and 95% CI for full media and ALI conditions shown. Two-tailed unpaired Student’s t test; P values indicated. Images are representative of at least 3 independent experiments.