Single-cell Transcriptomic Profiles Reveal The Interpatient And Intratumor Heterogeneity Of Lung Adenocarcinoma

BackgroundThe incidence and mortality of lung cancer in China have increased significantly due to the various factors,especially outdoor air pollution.The ability of diagnosis and treatment of lung cancer have been significantly improved in the past ten years,when the computed tomography(CT)was included in physical examination project.The development of surgical techniques,such as surgical options and the application of thoracoscope improved its precision and reduced the incidence of surgical complications.The application of targeted therapy,such as EGFR tyrosine kinase inhibitors significantly improved the survival of lung adenocarcinoma patients with EFGR mutations.However,lung cancer is still one of the leading causes of globally cancer-related death.Early screening has improved the diagnosis rate of early-stage lung cancer.However,there are still large number of patients on the middle and advanced stage,when they were diagnosed as lung cancer.For these patients,surgery could not be a radical method,therefore,radiotherapy,chemotherapy and targeted therapy have become the main treatments.However,there is a high probability tumor will resist to treatment using chemotherapy or targeted therapy,which was also called chemoresistance.The tumor cells with chemoresistance will gradually become the main subgroup within the tumor and eventually the whole tumor will resist to the therapy.Tumor heterogeneity is one of the important characteristics of malignant tumors,which can cause differences between tumor subgroups in abilities of proliferation,invasion,metastasis,drug sensitivity,and other aspects.Tumor heterogeneity is observed not only in intra-tumoral and inter-patient,but also in primary and metastatic tumors.The tumor cells within one patient also have different biological characteristics,including morphology,molecular subtypes,metastasis patterns,and different responses to treatments.Single-cell sequencing(SCS)technology,aimed at analyzing single cell level genetic information,including different cell subgroups,transcript,protein,and epigenetics expression spectrum information.Now,it widely used in neuroscience,the heterogeneity of tumor research and circulating tumor cells.Single-cell transcriptome sequencing aimed to reveal tumor heterogeneity at single-cell level,which was expected to provide new therapeutic targets and treatments for lung cancer patients.ObjectiveThis study analyzed 75 lung adenocarcinoma patients’ single-cell transcriptome data downloaded from GEO database(GSE123902,GSE131907),including 23 primary lung adenocarcinoma samples,13 brain metastases samples,1 sample of bone metastases,1 sample of adrenal metastasis,7 metastasis lymph nodes,5 pleural effusion samples,10 normal lymph nodes and 15 normal lung tissue samples.MethodsUsing R package Seurat to analyze single-cell sequencing data,and cell types were identified by R package SingleR.R package InferCNV was used to isolate tumor cells from normal epithelial cells.Pseudo-time trajectories analysis was performed using R package Monocle3.R package GSVA was used to analyze the differently expression of pathways between cell subgroups.KEGG and GO analysis were performed by Cytoscape software.Results1.Identification and isolation of tumor cells from samplesA total of 170831 cells were isolated by Seurat,including 80002 cells from primary tumors,35364 cells from brain metastases,and 55465 cells from normal adjacent tissues.SingleR package were used to identify epithelial cells and tumor cells.4014 normal epithelial cells were identified in adjacent tissues.In addition,Copy number variation(CNV)analysis was performed on all cells.It was found that CNVs were amplified or deleted in multiple chromosomes in tumor cells.2.Heterogeneity analysis in brain metastases and primary tumorsIntegrating 18 primary tumors and 10 brain metastases samples without preoperative chemotherapy,we compared differently expressed genes.Based on Kyoto Encyclopedia of Genes and Genomes(KEGG)database and the gene ontology(GO)database,we performed pathway analysis.The analysis results involved translation initiation,RNA transport,endoplasmic reticulum stress,exosomes,cell adhesion unfolded protein reaction and protein binding.Gene Set Variation Analysis(GSVA)found differences in EMT,MYC,glycolysis,oxidative phosphorylation pathway.3.Intra-tumor heterogeneity analysisTo explore the heterogeneity in one single sample,we selected brain metastasis sample GSM3516671 and advanced primary tumor sample GSM3516665.A total of 806 tumor cells were isolated in brain metastasis sample,and four cell subgroups were obtained by Seurat package.Monocle3 package was used for pseudo-time analysis.The pathway enrichment analysis in each cell subgroup was conducted by Cytoscape software.It was found that genes related to oxidative phosphorylation and prostaglandin metabolism were up-regulated in the first subgroup.In the second subgroup,multiple genes up-regulated associated with chemotherapy resistance.In the advanced primary tumor sample,the analysis showed that the second subgroup up-regulated genes associated with metastasis and EMT,which probably derived from alveolar type II epithelial cells.ConclusionUsing single-cell transcriptome sequencing technology,we can explore intra-tumoral and inter-patient heterogeneity in patients with lung adenocarcinoma.The results may explain the phenomenon that patients with same pathology subtype response different outcomes with the same treatment.It showed the importance of individual therapy and single-cell sequencing techniques,which have the capability to provide individualized treatment.In addition,it also provided technical support for exploring the mechanism of chemoresistance and helped to reformulate the appropriate treatment.