Single-cell RNA Sequencing Reveals Phenotypic Diversity Of Cells In The Tumor Microenvironment Of Lung Adenocarcinoma And The Prognostic Significance Of Tumor-infiltrating Plasma Cells

Objectives:Purpose: Lung cancer has surpassed breast,prostate,colorectal,and brain cancers globally combined,and is the most common cause of cancer-related deaths worldwide.Lung adenocarcinoma is the most common subtype of lung cancer in the world;however,overall survival of lung cancer patients remains disappointing regardless of subtype.The tumor microenvironment has long been a key and central part of tumor research,and is important for understanding the processes of tumorigenesis,progression,and metastasis,as well as for tumor diagnosis,prevention,and prognosis.The tumor microenvironment is distinctly heterogeneous,and traditional sequencing technologies only target the mixed state of many tissue samples,ignoring the possible existence of multiple different cells within the samples.On the other hand,single-cell RNA sequencing technology addresses this bottleneck by providing information on the status of individual cells within a tissue.It allows identification of different cell subpopulations at the single cell level and precise quantitative analysis of gene expression profiles of different cell types,thus revealing the complexity of molecular composition and the differences between them.Lung adenocarcinoma cell heterogeneity is a key factor in tumor survival and prognosis,but the number of samples from single-cell studies of lung adenocarcinoma is limited and there is a lack of corresponding clinical prognostic information.In contrast,bulk transcription analysis provides clinical information for a large cohort.Thus,the combination of single-cell and bulk transcriptome analysis can characterize tumor heterogeneity and determine the association of the immune landscape with clinical outcomes.Methods:We downloaded single-cell RNA sequencing data from eight LUAD samples from public databases,analyzed and organized the sequencing data,and identified 16 cell types,including B cells,plasma cells,endothelial cells,mast cells,CD8+ T cells,CD4+ T cells,Treg,depleted T cells,epithelial cells,dendritic cells,macrophages,dendritic cells(DCs),p DCs,monocytes,and fibroblasts,etc.A Complete Transcriptome Atlas of Lung Adenocarcinoma Single Cells was constructed.Then,based on the intratumoral heterogeneity of lung adenocarcinoma,the composition of each cell type in the lung adenocarcinoma bulk transcriptome RNA-seq cohort was imputed based on single-cell transcriptome analysis by using the bulk RNA-seq data from 503 LUAD samples of The Cancer Genome Atlas(TCGA)for deconvolution,and the deconvolution analysis was used to quantitatively estimate the composition of each tumor,and to explore the cell lineage status of lung adenocarcinoma.Meanwhile,the relationship between tumor cell lineage and tumor development and patient prognosis was investigated,and clinically significant cell subtypes in the LUAD microenvironment were further identified.We also identify the core genes of relevant subpopulations that play a role in the tumor microenvironment by exploring the genetic characteristics of the relevant subpopulations and demonstrate the role of relevant cell subtypes and genes in LUAD prognosis through clinical data collection and immunohistochemistry.Results:Higher plasma cell infiltration in the tumor microenvironment was associated with better survival of lung adenocarcinoma.Further characterization of plasma cell subpopulations revealed an active role of tumor-infiltrating plasma cells in tumor immunity.High-throughput data screening revealed an important role of plasma cell-specific expression of TNFRSF17 in lung adenocarcinoma prognosis.In addition,we showed by immunohistochemistry combined with clinical follow-up data that higher tumor plasma cell infiltration and high plasma cell-specific expression of TNFRSF17 were associated with better survival of lung adenocarcinoma.The differential expression of TNFRSF17 was also verified in paraffin sections by immunohistochemistry.It indicates that TNFRSF17 may play a key role in the potential mechanism of plasma cell prognostic contribution to LUAD.In summary,this study used single-cell transcriptome sequencing to demonstrate the transcriptome profile of lung adenocarcinoma at single-cell resolution and used a deconvolution approach to combine single-cell sequencing data with bulk transcriptome data,thus revealing the relationship between cell subtypes and tumor clinical features and clarifying the prognostically important cell subtypes in the tumor microenvironment and the core genes that play this role,which provides new ideas for the precision treatment of lung adenocarcinoma.Conclusion: 1.Phenotypic diversity of cells in the lung adenocarcinoma microenvironment.2.High plasma cell infiltration in the microenvironment is associated with better survival rates.3.TNFRSF17 may play a role in the potential mechanism of plasma cell prognostic contribution to LUAD.