Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. We generated tamoxifen-inducible, squamous epithelial-specific (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. 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) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID). RNA sequencing suggested increased autophagy in Cyto-ID 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 and Cyto-ID cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-ID group. Ki67 expression was also increased in organoids generated by Cyto-ID cells, including in basal cells localized beyond the outermost cell layer. 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.

中文翻译：

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自噬有助于食管上皮的稳态，其中高自噬囊泡水平标志着基底细胞增殖有限且自我更新潜力增强

自噬在食管良性和恶性病理中发挥作用。在这里，我们的目标是定义自噬在食管上皮稳态中的作用。我们生成了他莫昔芬诱导的鳞状上皮特异性（自噬相关 7）条件敲除小鼠，以利用组织学和生化分析评估对食管稳态的影响以及对致癌物 4-硝基喹啉 1-氧化物 (4NQO) 的反应。我们根据自噬囊泡 (AV) 识别染料 Cyto-ID 的荧光对食管基底细胞进行荧光激活细胞分选，然后对这些细胞进行透射电子显微镜、图像流式细胞术、三维类器官测定、RNA 测序和细胞分析。周期分析。对三维类器官进行传代、单细胞 RNA 测序、细胞周期分析和免疫染色。鳞状上皮中的基因自噬抑制导致稳态条件下食管基底细胞的增殖增加，并且与用 4NQO 治疗的小鼠体重显着减轻相关，进一步显示上皮组织结构受到干扰。具有高 AV 水平 (Cyto-ID) 的食管基底细胞在初始铺板时表现出有限的类器官形成能力，但比具有低 AV 水平 (Cyto-ID) 的食管基底细胞传代效率更高。 RNA 测序表明 Cyto-ID 食管基底细胞的自噬增加，同时细胞周期进程减少，后者经细胞周期分析得到证实。对 Cyto-ID 和 Cyto-ID 细胞生成的三维类器官进行单细胞 RNA 测序，鉴定出 Cyto-ID 组中 3 个细胞群的扩增和 G2/M 相关基因的富集。在 Cyto-ID 细胞生成的类器官中，包括位于最外层细胞层之外的基底细胞中，Ki67 表达也有所增加。自噬有助于维持食管增殖分化梯度。 AV水平高的食管基底细胞增殖有限，并产生具有增强自我更新能力的三维类器官。