Dissecting Dynamic Alterations Of Epithelial And Stromal Environment In Multi-stage Progression Of Esophagus Cancer By Single-cell Transcriptomic Analysis

Esophageal squamous-cell carcinoma(ESCC)is one of the most frequently occurring gastrointestinal malignancies in China,with a highest incidence in the world.It is believed that the development of ESCC is a multi-stage,progressive process.With the stimulation of genetic and environmental factors,normal esophageal epithelial cells gradually develop into precancerous lesions,and then finally become invasive carcinoma.Epithelial cell hyperplasia is an early stage in the esophageal carcinogenesis.Clinically,the pathology of esophageal cancer is mainly divided into two types:esophageal squamous cell carcinoma and esophageal adenocarcinoma.The histological changes in esophageal carcinogenesis are from normal squamous epithelial,esophagitis,low-grade intraepithelial neoplasia(LGIN),high-grade intraepithelial neoplasia(HGIN)cancer in situ to final invasive ESCC.In this process,esophagitis,low-grade and high-grade intraepithelial neoplasia are precancerous lesion stages.It has been well known that components in tumor microenvironment,including immune cells,stromal cells,and some non-cellular substances such as the extracellular matrix,play important roles in cancer development and cancer cells can make a microenvironment favoriting their evolution.The advent of single-cell RNA sequencing and advance in bioinformatics has enabled a more comprehensive and refined dissection of the components in highly complex tumor microenvironment,which has resolved some long-standing problem for tumor development.However,up to date,most studies on deciphering ESCC microecosystems used late-stage tumor samples and no studies reporting the analysis of dynamic molecular changes in the multi-step ESCC tumorigenesis.In the present study,we recruited 14 patients with ESCC in Linzhou hospital,Henan Province and collected surgically removed normal(n=8),inflammatory(n=10),lowgrade intraepithelial neoplasia(n=6),high-grade intraepithelial neoplasia(n=9),and ESCC(n=14)samples for single-cell transcriptome sequencing to dissect the dynamic molecular alterations and interactions across the various different stages during ESCC development.We have found that the HGIN is a critical stage for ESCC development driven by expression alterations of specific gene programs.We have found that epithelial cells downregulate the mucosal defense and differentiation program but upregulate the antioxidation and proliferation programs from HGIN to ESCC stage,which cooperatively promote the malignant phenotype of precancerous cells.We have analyzed the distribution of endothelial cell subtypes at different pathological stages and found that the infiltration of tumor associated endothelial cells(TECs)is an event that distinguishes ESCC from other precancerous lesions.By analysis of the heterogeneity of stromal microenvironment in ESCC,we have identified 4 major fibroblast subtypes and found that the fraction of cancer-associated fibroblasts(CAFs)and normal membrane fibroblasts(NMFs)changed oppositely across the advance of the lesions.The accumulation of CAFs is a late event in ESCC development.Analysis of the interaction between CAFs and epithelial cells expressing different gene programs showed that CAFs may interact with epithelial cells through CD74-MIF,EGFR-MIF and HLA-C-FAM3C to promote the malignant phenotypes of precancerous cells.We have also found that the mucosal program expressed by epithelial cells may negatively regulate CAF formation.The epithelial mucosal defense program was downregulated beginning from HGIN,which enhanced the activation and angiogenesis of CAF,further promoting ESCC progression.Taken together,these results shed light for the first time on how epithelial cells and stromal cells progress and interact with each other at single-cell level in the development of ESCC.