Single-cell Sequencing Revealed The Immunometabolic Heterogeneity In Non-small Cell Lung Cancer With COPD Overlapped

Part.1 Comprehensive Bioinformatics analysis reveals the impact of chronic obstructive pulmonary disease on lung cancer Background Lung cancer(LC)and chronic obstructive pulmonary disease(COPD)are both highmorbidity diseases and have a close relationship.Previous studies on the special population of COPD overlapping lung cancer mostly focused on the description of clinical features,lacking research at the cellular and molecular levels.In recent years,the use of bioinformatics methods to analyze the underlying biological information of diseases has become increasingly sophisticated.This study aims to use bioinformatics technology to comprehensively describe the cellular and molecular differences between COPD-overlapped lung cancer and lung cancer,to provide a molecular basis for the difference in clinical characteristics of COPD-overlapped lung cancer.Method The RNA sequencing data(count format),somatic gene mutation information,and clinical information of non-small cell lung cancer tissues were downloaded from The Cancer Genome Atlas(TCGA)database.All samples without accurate "fev1＿fvc＿ratio＿postbroncholiator" information were rejected.According to the lung function data,“fev1＿fvc＿ratio＿postbroncholiator<70” was defined as the COPDoverlapped lung cancer group.Lung adenocarcinoma and lung squamous cell carcinoma were separately grouped,and the differences in clinical features,genome mutation spectrum,and immune infiltration landscape between the two groups with and without chronic obstructive pulmonary disease overlap were compared,and differential expression analysis was performed.Then,the Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)pathway enrichment analysis was performed on the differentially expressed gene sets to explore their potential differences in biological processes and functional pathways.Finally,the WGCNA package of R software was used to perform weighted co-expression network analysis(WGCNA)on the screened differential gene expression profiles,and COPD-related module genes were selected and imported into the STRING database to construct a protein-protein interaction network(PPI)to screen out the core hub genes.Result After excluding the sample data without lung function-related indicators in the clinical information,we finally identified 83 lung adenocarcinoma samples(57 cases of simple lung adenocarcinoma,26 cases of lung adenocarcinoma combined with COPD)and 78 lung squamous cell carcinoma samples(37 cases of simple lung squamous cell carcinoma,41 cases of lung squamous cell carcinoma with COPD).NSCLC patients with and without COPD overlap didn’t differ significantly in clinical characteristics,including prognosis.COPD status did not lead to significant changes in the frequency of significant somatic mutations,however subtle changes occurred in mutation types.The two groups did not differ significantly in the vast majority of immune cell infiltration.In lung squamous cell carcinoma,the cyclooxygenase P450 pathway,the process of arachidonic acid metabolism,and the process of long-chain fatty acid metabolism in the combined COPD group and other cellular functional programs as well as tryptophan metabolism pathways,glycosaminoglycan degradation,and retinol metabolism metabolism-related pathways were up-regulated,while the regulation of chemotaxis,the negative regulation of apoptosis programs of endothelial cells and epithelial cells,ascorbic acid and aldonic acid metabolism,and PPAR signaling pathways were down-regulated.In lung adenocarcinoma,we also observed differences in the biological processes or signaling pathways involved in tumors with and without COPD.WGCNA and PPI analysis revealed that sodium channel α2(SCN2A),statherin(STATH),cytochrome P450 family members(CYP1A2and CYP1B1),γ-aminobutyric acid type A receptor(GABRA1),Claudin 17(CLDN17),and regulatory synaptic membrane exocytosis 1(RIMS1)may be potential key hub genes in COPD-overlapped squamous cell carcinoma.Vitamin D binding protein(GC)and ceruloplasmin(CP)may be potential key genes that influence the heterogeneity of COPD-overlapped lung adenocarcinoma.Conclusion The overlapping status of chronic obstructive pulmonary disease does not contribute significantly to the changes in clinical characteristics of lung cancer patients,nor does it directly affect the frequency and type of somatic mutation of lung cancer,but there are significant differences in the infiltration of immune cell populations in the local tumor microenvironment.Overlapping states of chronic obstructive pulmonary disease can alter multiple metabolic pathways and biological processes in the tumor microenvironment.In the lung squamous cell carcinoma group,SCN2 A,STATH,CYP1A2,CYP1B1,GABRA1,CLDN17,and RIMS1 may be potential COPD-related hub genes,and in the lung adenocarcinoma group,GC and CP may be potential COPD-related hub genes.Part.2Single-cell RNA-sequencing technology reveals tissue-specific immune signatures of COPD-overlapped lung cancer and COPD Background In a large number of studies on the carcinogenic mechanism of chronic obstructive pulmonary disease,chronic inflammation,DNA damage repair disorders,epithelialmesenchymal transition,and genetic susceptibility are all potential carcinogenic key pathways.However,there is still a lack of important research on the underlying mechanisms and key molecular pathways of COPD carcinogenesis.Single-cell sequencing technology is widely used in new species population identification,special pathogen screening,pathogen evolution paths,developmental biology,neuroscience,tumor heterogeneity research,and hematology identification.In this study,using single-cell RNA sequencing technology,we explored the key oncogenic pathways of chronic obstructive pulmonary disease and conducted in-depth studies on immune infiltration,cellular heterogeneity,and cellular metabolic profile remodeling under different conditions,to provide molecular-level insights for the identification,intervention,and treatment of special patient groups with chronic obstructive pulmonary disease and non-small cell lung cancer.Method Through clinical evaluation,we included three lung cancer patients with moderate or severe chronic obstructive pulmonary disease,pathologically suggestive of non-small cell lung cancer.Each patient was sampled with cancer tissue and COPD tissue far from the cancer nest,for a total of 6 samples.Subsequently,single-cell RNA sequencing analysis was performed on three COPD tissues and three lung cancer tissues overlapped with COPD,and cell composition maps were drawn under the two environments.Differential immune function and metabolic pathways were studied for the T cell population and macrophage population.Finally,single-cell RNA sequencing data of normal lung tissue,simple COPD disease tissue,simple lung cancer tissue,and overlapping lung cancer tissue were downloaded from the public database,and the previous results were verified.Result After data quality control,we found that most immune cell types were significantly enriched in the COPD group,and the LCCOPD group showed differential enrichment of T-regs and macrophage subtypes compared with the COPD group.FOXP3 m RNA expression was significantly increased in LCCOPD-related T-regs and positively correlated with the expression of CTLA-4 and PD-1,forming an immunosuppressive environment.Macrophages from different pathological settings show significant differences in cellular composition as well as different phenotypic dominance.COPDrelated macrophages are mainly M1 type,and the intensity of cell proliferation-related pathways is increased.LCCOPD-associated macrophages were dominated by M2-type and tumor-associated macrophages,and the chemotaxis of the immune population was gradually enhanced along the trajectory.Several metabolic and cytokine pathways were down-regulated in COPD tissues and up-regulated in the LCCOPD group,such as the SREBP pathway,IL2 signaling pathway,IFN-γsignaling pathway,glycolysis,glutamine metabolism,etc.related to different polarization processes.Based on the analysis of public single-cell datasets,we found that the COPD environment indirectly promotes carcinogenesis by accelerating the infiltration of T-regs and M2-type macrophages and creating an immunosuppressive environment in advance.Conclusion Our study provides an extensive cellular-level characterization of lung cancer overlapping COPD,a specific pathological type of lung cancer,and is the first to map the cellular composition of lung cancer in both COPD and COPD.Subsequent indepth analysis of T cell populations and macrophage populations under the two links revealed the association between COPD status and the formation of an immunosuppressive environment,thereby clarifying the role of COPD as a high-risk factor in the development of lung cancer.