| Background and aimsEsophageal carcinoma(EC)is a malignant tumor that originates from the epithelium or glands of the esophageal mucosa.In clinical practice,progressive dysphagia is the most typical symptom,and its incidence accounts for the eighth highest incidence of malignant tumors in the world.It is a serious threat to the health of all mankind.According to histological types,esophageal cancer can be divided into two types: esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).Adenocarcinoma is the most common type of esophageal cancer in Western countries,accounting for more than half of all new cases.China has a high incidence of esophageal cancer and squamous cell carcinoma accounts for about 90%of the total number of esophageal cancers in the country.ESCC has a high incidence and a poor prognosis and the five-year survival rate is only 30.3%.It is a common cause of death in malignant tumors in China.In the development of esophageal cancer,the interaction between the tumor microenvironment and tumor cells plays an important role,and affects the therapeutic effect of ESCC.The evolution of tumor cells and the abnormality of immune cells are particularly important,which involves the interaction of factors including stroma,blood vessels,lymphatic vessels,immune cells,and so on.The changes in this complex immune microenvironment are closely related to the occurrence and development of ESCC,patient survival,and the effectiveness of immunotherapy.Meanwhile,ESCC cells contain abundant tumor antigens,including tumor-associated antigens and neoantigens.Therefore,exploring the expression profile of various cells within tumor tissues and identifying biomarkers that recognize various cells will not only facilitate the early detection of ESCC but also provide targets for individualized cancer immunotherapy.The traditional methods have their limitations in the research of the tumor immune microenvironment.The single-cell transcriptome sequencing technologies that have emerged in recent years can conduct in-depth exploration of esophageal cancer tumor cells and tumor microenvironment,and identify different cell types.Through the expression profiles of different cell types,scientists can further search for sub types of cells and molecular biomarkers related to survival.Meanwhile,using this technology,individualized T-cell receptors(TCRs)sequences that may be used for future immunotherapy can be selected by V(D)J sequencing of activated T cells.In this study,we analyzed clinical ESCC tumor samples by single-cell sequencing,explored the formation mechanism of ESCC tumors.The results of analysis helped us understand ESCC complex microenvironment,providing new directions for the discovery of predictive biomarkers of ESCC and potential novel effective strategies for immunotherapy.MethodsWe first optimized collagenase digestion method to obtain high-quality single cells from esophageal cancer tumor tissues.Then we used this pre-optimized method to prepare single-cell suspensions of tumor tissues from two clinical ESCC patients,and perform a series of quality controls to ensure that cells can be used for subsequent single-cell sequencing.According to the characteristics of tumor microenvironment and the needs of subsequent data analysis,the obtained single cells were sorted into CD45 positive cell gruop and CD45 negative cell group by the immunomagnetic and immunoadsorption cell separation techniques,and then these sorted single cells were captured by the 10×genomics system and the sequencing library was constructed to perform the single-cell transcriptome sequencing.After data screening and standardization,Cell Ranger software is used to process the data,the cells are grouped by dimensionality reduction clustering to determine the cell types in the tumor microenvironment,and the differentially expressed genes of each cell types are analyzed in the meantime.After the cell types were determined,we carried out further sub type analysis of epithelial cells based on the characteristics of the two patient samples,specifically studied the tumor heterogeneity of different patients,and analyzed epithelial-mesenchymal transition(EMT)biomarkers in these tumor epithelial cells.Combined with the clinical patient data of ESCC in the TCGA database,the relationship between the differentially expressed genes in different sub typess and the survival rate of patients were analyzed.Subsequently,Hematoxylin and eosin(H&E)staining and multicolor immunofluorescence experiments were performed on paraffin sections of tumor tissues and para-carcinoma tissues to verify the tumor cell types and tumor microenvironment changes found by single-cell sequencing.Results(1)Using the optimized digestion method(with the concentration of 0.2 mg/ml type I collagenase and 0.2 mg/ml type IV collagenase mixed digestion method,digested at room temperature for 40 minutes),we can get single-cell viability above70% for the sorted both CD45 positive and negative group;which is also verified by flow cytometry technology following magnetic beads sorting.(2)The landscape of ESCC microenvironment was successfully drawn,which contains more than 12 cell types including nine immune cells(T cells,B cells,NK cells,and other cell types)and three non-immune cells(epithelial cells,endothelial cells and fibroblasts).The total number and proportion of different cell types were calculated in the different groups.(3)After selected ESCC clinical sample from the TCGA database,we analyzed the relationship of differentially expressed genes of the epithelial sub type with the overall patient survival.A sub type of epithelial cells with endothelial characteristics(the differentially expressed top genes in this group of cells were PECAM1,RAMP2,FLT1,and other genes)was significantly correlated with the survival of ESCC patients.(4)Epithelial-mesenchymal transition(EMT)-related genes of epithelial cells in CD45 negative cells were analyzed and identified EMT marker genes in the ESCC samples which include low expression markers of epithelial cells,such as E-cadherin/CDH1 and laminin(LAMA2)and high expression markers of mesenchymal cells,such as vimentin/VIM and fibronectin/FN1.(5)Through the classification of the CD45 positive cell population and the analysis of its V(D)J sequencing results,multiple CD8-positive T lymphocytes with the same TCR activation were found.ConclusionWe performed single-cell transcriptome sequencing on the obtained CD45-positive and CD45 negative cells to clarify the components of the ESCC microenvironment.The marker genes of each cell cluster were determined and distinguished.Through the analysis of epithelial cells in the tumor microenvironment,combined with related survival analysis,a set of key transition marker cell populations related to the carcinogenic evolution of epithelial cells are determined.We speculate that these cells play a role in the occurrence and evolution of esophageal cancer.Analysis of CD45 positive cells in the tumor microenvironment revealed multiple CD8 positive T lymphocytes with the same TCR sequence.We speculate that these cells may be activated by the specific antigen of ESCC and may perform potential immunotherapy effects in the respective patients. |
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