Using Single Cell RNA-Seqeencing Approach To Analyze Single Cell Transcription Of Nasopharyngeal Carcinoma

Background:Nasopharyngeal carcinoma(NPC)is a kind of malignant tumor of epithelial origin,which occurs in the inner layer of the mucous membrane.The location of the disease is common in the pharyngeal recess and is a special type of head and neck cancer.Compared with other cancers,the incidence of nasopharyngeal cancer is lower,with unique geographical distribution and more complicated etiology.It’s generally considered that genetic susceptibility,infection of EB virus,and environmental factors were important causes of its incidence.The pathological features of nasopharyngeal carcinoma were mainly undifferentiated non-keratinizing carcinoma,and squamous cell carcinoma was rare.In the tissues of nasopharyngeal carcinoma,there was a large number of lymphocyte infiltration,also known as "lymphoid carcinoma".The incidence of nasopharyngeal carcinoma was hidden,and the tumor volume is usually small.Tissue samples obtained based on biopsy were few.Previous studies were based on cell populations,which were limited by small sampling volume and limited access to information.Infiltration of immune cells might cover the characteristics of nasopharyngeal carcinoma cells.Single-cell sequencing technology could sequence one single cell to obtain more information using a smaller amount of cells.At the same time,single-cell sequencing technology also helped to discover the population of cells that were masked by previous analysis methods because of the scarcity of the number.Therefore,single-cell transcriptome sequencing of nasopharyngeal carcinoma can help break through the bottlenecks of previous research and provide new ideas for further research.Objective:1.Optimize the protocol of nasopharyngeal carcinoma tissue dissociation;2.Describe the single cell transcription spectrum of nasopharyngeal carcinoma tissues and cell lines;3.Explore the heterogeneity of nasopharyngeal carcinoma tissues and cell lines;4.Describe the immune microenvironment of nasopharyngeal carcinoma tissue.Methods:1.Combining the method of mechanical dissociation and enzymatic dissociation to dissociate nasopharyngeal carcinoma biopsy tissue,obtain the single cell suspension of nasopharyngeal carcinoma,and evaluate the single cell activity ratio by trypan blue staining counting method;2.Construction of single cell cDNA library of nasopharyngeal carcinoma cell line and nasopharyngeal carcinoma tissue by Fluidigm-Cl single cell analysis system;3.Construct a single-cell cDNA library of nasopharyngeal carcinoma tissue by 10×Genomics system;4.Perform transcriptome sequencing through the illumina sequencing platform;5.Study the heterogeneity of nasopharyngeal cancer cell lines through principal component analysis and cluster analysis;6.Explore the subgroup of nasopharyngeal carcinoma tissue by t-SNE nonlinear cluster analysis;7.Characterize the transcriptome and immune cell subsets of nasopharyngeal carcinoma by bioinformatics analysis.Results:1.Optimize the dissociation procedure of nasopharyngeal carcinoma tissue to obtain an effective single cell suspensionWe adjusted the enzymolysis time and cell collection steps in the tissue dissociation procedure and found that using the Tumor Dissociation Kit(Human)kit,the enzymolysis system was based on DMEM or RPMI-1640 5mL,Enzyme H 150μL,enzyme R 75μL,enzyme A 25μL configuration.In the enzymatic hydrolysis process,the Meitianyi gentle MACS dissociator was used to mechanically dissociate three times.The enzymatic hydrolysis was incubated for 20 minutes,and the differential centrifugation was 300×g for 5 minutes;200xg for 5 minutes to obtain the final dissociation solution.Successfully obtained a single cell suspension with a high proportion of living cells,70%,to meet the needs of single cell sequencing.2.Successfully constructed a single cell cDNA library.The single-cell cDNA library of the nasopharyngeal carcinoma cell line was constructed by the Fluidigm-C1 single-cell gene analysis system,and the length of the obtained DNA fragment was 600-2000bp,the peak value was 1500-2000bp,and the quality of the cDNA library was qualified.3.There is cell heterogeneity in nasopharyngeal carcinoma cell linesSingle-cell transcriptome sequencing of the nasopharyngeal carcinoma cell line HK1-EBV by Fluidigm-C1 single-cell gene analysis system,principal component analysis and cluster analysis of the sequencing results,it was found that each cell line can be divided into 2-6 sub-cells Group,indicating that even nasopharyngeal carcinoma cell lines purified and cultured in vitro have cell heterogeneity.4.The infiltrating immune cells in nasopharyngeal carcinoma are mainly T cells.Single-cell sequencing of nasopharyngeal carcinoma tissues was carried out by Fluidigm-C1 single-cell gene analysis system and 10× Genomics system,and the cell types in nasopharyngeal carcinoma tissues were divided with the help of related gene markers.The immune cells infiltrated by nasopharyngeal carcinoma are mainly T cells,followed by B cells.Immune cells account for more than 80%of tissues.5.KI67 positive immune cell population was found in nasopharyngeal carcinomaThrough t-SNE analysis,we found that a subpopulation of cells in nasopharyngeal carcinoma is characterized by the positive gene KI67 associated with cell proliferation.Further analysis revealed that this subpopulation contains CD4+T cells,CD8+T cells,B cells and macrophages.Conclusion:1.We have established a method for single cell lysis of primary tumor tissue of nasopharyngeal carcinoma.2.For the first time,single cell transcriptome sequencing technology confirmed the existence of cell heterogeneity in nasopharyngeal carcinoma cell lines and primary tumor tissues.3.At the same time,the immune microenvironment of nasopharyngeal carcinoma is depicted,and a large number of immune cells are infiltrated,mainly T cells,followed by B cells.4.There are some immune cell populations in the nasopharyngeal carcinoma tumor microenvironment that have a tendency to proliferate.In summary,we optimized the dissociation process of nasopharyngeal carcinoma tissues and completed single-cell level transcription sequencing of nasopharyngeal carcinoma cell lines and primary tumor tissues.The analysis results suggest that the cell heterogeneity of nasopharyngeal carcinoma is widespread,and there is a large amount of immune cell infiltration in the tumor microenvironment,mainly T lymphocyte infiltration.Only a small part of the immune cells in nasopharyngeal carcinoma showed a tendency to proliferate.Through these studies,we further understand the complexity of nasopharyngeal carcinoma and provide a reference for further research.