Shimonosono, M. | Search bioRxiv

bioRxiv2024

Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids

BackgroundEsophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbeccos Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. MethodsWe have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-{beta} receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. ResultsThe formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGF{beta} receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

cell biology↗

bioRxiv2023

ALDH2 dysfunction accelerates ESCC pathogenesis

The alcohol metabolite acetaldehyde is a potent human carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulates and causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent amongst East Asians. Chronic alcohol users with heterozygous ALDH2*2 display an increased risk for the development of esophageal squamous cell carcinoma (ESCC) and other alcohol-related cancers. However, how ALDH2 influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three dimensional organoids, and xenograft models. We find that alcohol exposure results in increased organoid formation and tumor growth concurrent with increased reactive oxygen species (ROS), increased DNA damage, and the enrichment of putative cancer stem cells (CSCs) characterized by high CD44 expression. Pharmacological activation of ALDH2 function by Alda-1 inhibits this phenotype, indicating that acetaldehyde is the primary driver of these changes. ALDH2 dysfunction also affects response to a commonly used chemotherapy for the treatment of ESCC. We find that Aldh2 dysfunction facilitated enrichment of CSCs following cisplatin-induced cell death and oxidative stress in murine organoids. Together, these data provide evidence that alcohol exposure, results in more aggressive tumors through enrichment of CSCs, which is augmented by ALDH2 dysfunction.

cancer biology↗