Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential

Background & Aims

Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis.

Methods

We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining.

Results

Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-ID^High) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID^Low). RNA sequencing suggested increased autophagy in Cyto-ID^High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-ID^Low and Cyto-ID^High cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-ID^High group. Ki67 expression was also increased in organoids generated by Cyto-ID^High cells, including in basal cells localized beyond the outermost cell layer.

Conclusions

Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.