Single-Cell Transcriptomic Alterations In Multistep Development Of Esophageal Squamous-Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the most common upper gastrointestinal malignancies in China.The development of ESCC usually takes many years.Squamous cell dysplasia limited to the mucosa is recognized as intraepithelial neoplasia(IN),a precancerous lesion.Based on the cytological abnormality and proportion of dysplastic cells in mucosa,IN is divided into low-grade and high-grade.The low-grade intraepithelial neoplasia(LGIN)takes longer time to develop into more severe lesions,while the high-grade intraepithelial neoplasia(HGIN)takes shorter time to become invasive ESCC.Furthermore,patients with the same grade IN have different prognosis.Nowadays,there is no applicable strategy for assessing the outcome of patients bearing IN.Therefore,a deeper understanding of molecular etiology of ESCC development through multi-step precancerous lesions is of significance for illustrating underlying pattern of tumorigenesis and providing new markers for early diagnosis and treatment.Through the development of next-generation sequencing and high-resolution single-cell sequencing technologies,scientists have elucidated the genomic and transcriptomic variations in ESCC including driver genes and abnormal activated signaling pathways.Recent studies have profiled the single-cell transcriptomic landscape of the microenvironment in ESCC and found the widespread immunosuppression phenomenon in tumor,which suggests the importance of microenvironment during tumorigenesis.However,most of the researchers have focused on the comparison between tumor and non-tumor tissues,and few of them have analyzed the dynamic changes of cell types and molecules of precancerous lesions.In this study,we have collected samples from multiple regions of 14 ESCC specimens and obtained 14 cases ESCC,9 cases of HGIN,6 cases of LGIN,10 cases of inflammation(INF)and 8 cases of normal epithelia(NOR).We have performed single-cell transcriptomic sequencing(scRNA-seq,10× Genomic)on the 47 multistep tumorigenesis samples and captured 110,969 single cells.We have comprehensively profiled the transcriptomic landscape of microenvironment of multistep ESCC tumorigenesis,and revealed the dynamic expressional changes of immune cell types and cell-cell interactions during ESCC development.We have identified the extensive infiltration of tumor associated macrophages(TAM)and tolerable dendritic cells(tDC)in ESCC,which plays an important role in promoting tumor progression and immunosuppression.Moreover,epithelial cells can recruit TAM through the secretion of cytokines and this interaction signal is enhancing during tumorigenesis.During early stage of HGIN,CD8~+T cells have already experienced exhaustion,but the cytotoxic activity has not been up-regulated.CD4~+Treg cells have also intensified the effect of immunosuppression in the microenvironment,providing a mild surroundings for the development of dysplasia.Moreover,we have performed cell-cell interaction analysis and have found that various types of cells in the microenvironment can further recruit a large number of pro-inflammatory and immunosuppressive cells by expressing cytokines and chemokines to mediate cell-cell chemotaxis.Collectively,these results indicate that immune cells in the microenvironment provide a hotbed for the tumorigenesis ESCC.In summary,we have employed a high-throughput scRNA-seq analysis and depicted a complete cell atlas of the landscape in multistep ESCC development.We have characterized the transcriptomic features of immune cell clusters and elucidated the possible immune cell subtypes that function as tumor promoting and immunosuppressing.This study provides new understandings about how immune cells behave during ESCC development at early stage before invasive carcinoma,which offers valuable data to support early diagnosis and prevention of ESCC.