Genomic Characteristics And Multicellular Ecosystem Of Multiple Synchronous Lung Cancers(MSLCs)

Purpose: Precision clinical diagnosis and multicellular ecosystem analysis are the key to the treatment and prevention of multiple synchronous lung cancer(MSLC).However,it is still without a consensus solution in the clinical practice.In this study,we first performed next-generation whole-exome sequencing to gain further insights into the genetic alterations of MSLC and reveal the heterogeneity and evolutionary of MSLC.Second,single-cell sequencing technology was used to analyze the cellular composition and multicellular ecosystem of MSLC,especially to reveal the characteristics of immune cells.Finally,we established the prospective clinical cohort of MSLC.The precision diagnosis and dynamic evolution analysis of MSLC were conducted by whole-genome sequencing.Additionally,multicellular ecosystem and heterogeneity characteristics were revealed by combining single-cell sequencing analysis.Method: In this study,whole-exome sequencing and bioinformatics analysis were performed to reveal the mutation landscape and heterogeneity of MSLC.In vitro cell model and in vivo mouse model were used to verify the function and conservation of driver mutations in MSLC.Single-cell sequencing was used to analyze the cellular composition and multicellular ecosystem of MSLC.Based on the prospective clinical cohort of MSLC,whole-genome second-generation sequencing was used to precision diagnosis and dynamic evolution analysis of MSLC.Results: By whole-exome sequencing of four patients with MSLC,we identified driver mutations in every lesions of MSLC.The driver mutations in independent lesions of each individual have strong heterogeneity.However,functional studies show a strikingly conservative between driver mutations of different lesions.The driver mutations functionally converge on the MAPK and PI3K-AKT signaling pathways to drive the occurrence of tumor.Based on the single-cell RNA-seq of a MSLC case,we identified eight major cell types,including T cell,B cell,myeloid cell,NK cell,epithelial cell,endothelial cell,mast cell and fibroblast cell.The abundance of different cell types varies greatly.The cellular composition of MSLC is mainly composed of immune cells.Epithelial cells,including tumor cells,account for only 15% of the total number of cells in MSLC.Based on TCR sequencing,we found the tumor tissue exists specific T cell cloning amplification.Combining the TCR sequencing data of tumor tissue,paracancerous tissue and peripheral blood,we found that T cells in the tumor tissue MSLC come from lung tissue and peripheral lymphatic organs.Additionally,the most same T cell clones of different lesions in each MSLC patients were from the lung tissue.Conclusions: This study reveals the genomic heterogeneity and conservation of multiple lung cancer.Through the prospective clinical cohort of multiple lung cancer,we found that most lesions of MSLC have typical driving genes.The presence of a large number of immune cells and the amplification of specific T cell clones in MSLC reveal that the development of multiple lung cancer has been monitored by immune system.Together,out studies will hold important implications for the biological understanding of the multicellular ecosystem of MSLC and optimizing therapeutic strategies in MSLC.