Spatiotemporal Gene Expression Profiling In The Development Of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is a type of digestive system cancer with high incidence and mortality in the world.Previous studies have demonstrated that ESCC originates from epithelium and progresses through multiple stages under the combined effects of internal and external factors.The development of ESCC can be classified into five pathological stages according to the morphology and distribution of dysplastic cells,including normal,inflammation,low-grade intraepithelial neoplasia,high-grade intraepithelial neoplasia and invasive carcinoma.Early diagnosis and therapy of precancerous lesions can effectively interrupt their progression and reduce ESCC mortality,but there is still lack of comprehensive evaluation framework.Single-cell transcriptome studies have previously uncovered the dynamic gene regulation network of various cell types,identifying essential cells and molecules associated with ESCC development,therapy,and prognosis.However,the spatial distribution and interaction of crucial cells and molecules within the tissue remain unknown.Tissue is a complex ecosystem where each cell not only receives information from its surroundings but also has impacts on its neighbors.Therefore,it is urgent to elucidate the spatiotemporal gene expression alterations of specific cell types in esophageal multi-stage tissues for fully understanding the complicated tissue microenvironment and discovering potential molecular markers for ESCC detection,evaluation,and treatment.In this study,we have established a 4-NQO induced ESCC mouse model to mimic human esophageal carcinogenesis under external carcinogens and processed five pathological tissues for spatial transcriptome analysis using the Visium(10×Genomics)platform.We have revealed the gene expression characteristics of four canonical esophageal tissue regions that existed in all five pathological stages.Remarkably,we have identified a ESCC development region with high expression of tumor hallmark genes that emerged from inflammation and expanded during carcinogenesis.Combined with the single-cell transcriptome data of ESCC mouse model,we subsequently investigated the interactions between various cell types within each tissue region mediated by ligands and receptors.We found that EFNB1-EPHB4 interaction between mouse epithelial cell and epithelial cell enhanced and expanded from the basal layer to the superior layers as ESCC developed.It is noteworthy that human spatial and single-cell transcriptome data from multi-stage tissues also displayed the same spatiotemporal alterations of EFNB1-EPHB4 interaction between epithelial cell and epithelial cell during ESCC development.We further validated EFNB1-EPHB4 interaction through multiple immunofluorescence assays in mouse and human tissues as well as protein immunoprecipitation and cellular immunofluorescence staining in mouse ESCC primary cell MESCC and human KYSE30 cell line.Gene expression correlation analysis indicated that EFNB1 and EPHB4 related genes participate in epithelial cell proliferation,which are capable of regulating cell cycle and cell junction pathways.Upon knockdown and overexpression of EFNB1 and EPHB4 in mouse and human epithelial cells,cell viability assays and real-time fluorescence quantitative PCR analysis further showed that both EFNB1 and EPHB4 could promote epithelial cell proliferation by regulating cell cycle and cell junction pathways.In summary,this research investigated the spatiotemporal alterations of intercellular gene expression regulation in the tissue microenvironment during esophageal tumorigenesis.The EFNB1-EPHB4 interaction between epithelial cell and epithelial cell was determined to be enhanced and expanded from the basal layer as ESCC developed.This spatiotemporal regulation could promote cell proliferation in both mouse and human by regulating cell cycle and cell junction pathways to accelerate ESCC development.This study sheds new light on the significance of EFNB1-EPHB4 interaction between epithelial cell and epithelial cell during esophageal carcinogenesis and suggested potential new approaches for ESCC diagnosis and therapy targeting EFNB1 and EPHB4.