| Objective: Lung cancer,which adenocarcinoma is the most common subtype,is the malignant tumor with the highest mortality in China.Although the multidisciplinary diagnosis and treatment of lung cancer have made great progress in recent years,a considerable number of patients are already in advanced stages or have distant metastases when they are diagnosed.The survival time of lung adenocarcinoma patients with EGFR,ALK,or ROS1 gene mutations was significantly prolonged by targeted therapy.However,more than 30% of the unknown driving mutation genes were not detected,and targeted drug resistance and non-sensitive gene mutations are still difficult challenges in clinical practice.The dilemma of clinical treatment of lung cancer is not only the occult of early symptoms but also the heterogeneity of lung cancer,which leads to the difficulty of the unified treatment plan.Revealing the molecular differences between lung cancer patients can explore the causes of cancer and provide significant guidance for the key molecular targets of targeted or immunotherapy of lung cancer.The evolution of lung adenocarcinoma underwent a multi-stage process including gene mutation,molecular expression abnormality,atypical adenomatous hyperplasia(AAH),adenocarcinoma in situ(AIS),minimally invasive adenocarcinoma(MIA),and invasive adenocarcinoma(IA).Previously study found that some key signaling pathways are activated in the progression of mice lung adenoma to lung adenocarcinoma There are also reports found the frequency of TP53,KRAS,EGFR, and other driver genes mutation in the process of lung adenocarcinoma progression is significantly increased in MIA.However,there is still a lack of systematic research in terms of the evolution of human lung adenocarcinoma,especially the early evolution mechanism of lung cancer and tumor heterogeneity are still not clear.The study of tumor genome characteristics based on sequencing technology provides an effective basis for the early diagnosis and treatment of lung cancer.Single-cell sequencing can analyze the specific cell population at the single-cell level,and the technology has been successfully applied to identify cell types and understand the complex subpopulations in organs,tissues,and various cancers.This study focused on the evolution of lung adenocarcinoma,using single cell RNA sequencing technology to describe the transcriptome characteristics from atypical adenomatous hyperplasia to invasive adenocarcinoma,and combining with bioinformatics analysis to construct a personalized regulatory network of lung adenocarcinoma evolution,so as to explore the changes of the tumor microenvironment and key molecular targets in the progression of lung adenocarcinoma.It would be better for the early diagnosis of patients with lung cancer and provide the theoretical basis for reasonable intervention.Materials and Methods: According to the inclusion and exclusion criteria,a total of 52 samples of tumor and normal lung tissues from patients with lung adenocarcinoma were collected in the department of thoracic surgery.All cancer samples included AAH,AIS,MIA,and IA pathological types,which were identified according to 2015 WHO classification standard.The clinical characteristics of these participants,including age,gender,pathology,and tumor stage,were recorded at recruitment.The experiment was divided into two parts as follows: Chapter I: Characteristics of stromal cells in the tumor microenvironment.The samples were prepared into single-cell suspension,and the single-cell c DNA libraries were obtained by 10 x Genomics platform,then sequenced by Novaseq-6000.The raw data were mapped to the human genome(build hg19)using Cell Ranger software,then the sequence was de-duplicated and identified to output the gene expression matrices and create the Seurat object.The low-quality cells were filtered,and the data were normalized.The annotations of cell identity on major clusters were defined by the expression of known marker genes.The subclusters of endothelial cells,fibroblasts,lymphocytes,and myeloid cells in tumor tissues and normal tissues were identified.We used Gene Set Enrichment Analysis(GSEA)and cell trajectory analysis to analyze the composition and gene expression differences of different stromal cell types in the two tissues.Chapter II: Characteristic analysis of epithelial cells.The epithelial cells from the tumor and normal lung tissue were re-clustered to identify cell types.The malignant epithelial cells were distinguished by the infer CNV method,and the origin of lung adenocarcinoma was defined,then verified by protein immunofluorescence staining.We selected cell clusters that closely resemble those of AT2 and AT2-like cancer cells and then tracked the gene expression changes along the trajectory from non-malignant tissues to AAH,AIS,MIA,and eventually IA.We identified differentially expressed genes that exhibited dynamic expression over the pseudotime axis,then ordered these genes along the temporal pseudotime and reconstructed a diffusion map.We constructed the gene regulatory network in normal lung cells and cancer cells using the big SCale2 algorithm.And we mapped the cell-cell interaction and receptor-ligand pair between all major cell types using Cell Phone DB.To verify the bioinformatic analysis results,we used the protein immunofluorescence staining,flow cytometry,RNA in situ hybridization,and quantitative PCR analysis.Results: Chapter I: 1.A total of 52 freshly resected lung specimens of four subtypes(3 AAH,5 AIS,9 MIA,and 17 IA)were collected from 25 patients,along with 18 adjacent normal lung tissues from a distal region within the same lobe,which served as controls.We characterized the transcriptome of 268,471 cells from samples,with an average of about 5000 single cells per sample and a median of 966 genes detected per cell.2.Endothelial cells proliferated actively in lung adenocarcinoma,and stalk-like cells trail behind the tip cells during angiogenic sprouting,forming the stalk of the sprout,were enriched in AAH stage.GSEA results showed that MYC_TARGETS_V1 was the hallmark gene set with the highest enrichment score in tumor endothelium,while the INFLAMMATORY_RESPONSE gene set was low expression indicated that the immune response activity was decreased.3.Fibroblast-like cells and myofibroblasts were the main types of cancer-associated fibroblasts and increased gradually with the evolution of lung adenocarcinoma.Smooth muscle cells were observed in IA stage tumors and a few normal tissues.GSEA results showed that EPITHELIAL_MESENCHIMAL_TRANSITION was the hallmark gene set with the highest enrichment score.4.The number of NK cells in tumor decreased gradually with the progression of the lung adenocarcinoma suggested that the innate immune function weakened.CD8+ T cells were enriched in IA stage,while Treg immunosuppressive cells were significantly increased in AAH to IA stage,indicating that the tumor microenvironment developed towards an immunosuppressive direction.5.Alveolar macrophages showed a decreasing trend in the MIA and IA stages,and tumor-associated macrophages significantly increased in the IA stage.Alveolar macrophages transformed into tumor-associated macrophages weakened the inhibitory effect of inflammation,and enhanced the ability of tumor angiogenesis,migration,invasion,and metastasis.Chapter II: 1.The gene expression characteristics of AT2-like cells and AT2 cells were similar,and infer CNV results showed that the AT2-like cluster were malignant cells with obvious chromosomal variation in IA stage.During the evolution from AAH to IA stage,the expression of the AT2 cell marker gene SFTPC decreased gradually,and the same results were observed in the mouse lung adenocarcinoma model,demonstrating that AT2 cells are the origin of adenocarcinoma.2.Cell trajectory analysis showed that lung adenocarcinoma progressed according to the pathways of AAH,AIS,MIA,and IA,and the expressions of WIF1,LAMP3,MUC1,AQP5,and other genes were gradually down-regulated;the expressions of MT-ND4,MT-ND2,and JUN were up-regulated approximately in MIA stage;the expressions of VIM,S100A4,VEGF,MDK,SOX4,LYZ,TIMP1 and other genes were up-regulated in the late pathological stage.3.The results of gene regulatory network analysis showed that the up-regulated genes in cancer cells were mainly related to energy metabolism,ribosome synthesis,and MHC-II molecular expression,which suggests that these activities are essential during adenocarcinoma progression.4.During the evolution of lung adenocarcinoma,AT2 cells lost characteristic deletion in the lung epithelial lineage and dedifferentiated into a stem-like state.The stem-like genes FOSB,JUN,IF127,and S100A4 were gradually up-regulated,and the expression level of Wnt signal regulator Lgr5 was increased.Meanwhile,the EMT process was gradually enhanced.5.AT2 cell interacted specifically with the myeloid cell subset by LGALS9 receptor, but AT2-like cells tightly interacted with myeloid cells via ANXA1,FN1 and MDK.6.Plasma mi RNA-10 a and β-hydroxybutyric acid levels were significantly increased in early pathological stages(AIS and MIA),and the differences were statistically significant.MDK and TIMP1 were similar with CEA and highly expressed in plasma of patients with advanced lung adenocarcinoma.Conclusion: 1.The progression of lung adenocarcinoma is closely related to stromal cells.Endothelial cells,fibroblasts,and immune cells promote tumor angiogenesis and form an immunosuppressive microenvironment,which is conducive to the invasion and progression of cancer cells.2.AT2 cells underwent dedifferentiation to a stem-like state and further initiate tumor progression.AT2-like cells with the characteristics of AT2 cells were first observed in the AAH stage and gradually progressed to AIS,MIA,and IA.Meanwhile,the loss of AT2 feature became more and more prominent,so it can be inferred that AT2 cells are the original cells of human lung adenocarcinoma.3.Wnt signaling pathway is activated during the evolution of lung adenocarcinoma.The progression of AT2 cells from AAH to IA is accompanied by the high and low expression of different genes at different stages.LAMP3,MUC1,FOSB,JUN,IF127,S100A4,MDK,SOX4,LYZ,and other genes may be the key molecular targets in the evolution of lung adenocarcinoma.4.AT2-like cells express high levels of ANXA1,MDK,FN1,and C5AR1 genes,then interact with immune cells through ligand-receptor complexes,which may become new targets for immunotherapy of lung adenocarcinoma.5.Ribosome synthesis and energy metabolism genes are highly expressed in lung adenocarcinoma.Plasma markers mi RNA-10 a and β-hydroxybutyric acid may become sensitive indicators for the diagnosis of early lung cancer.MDK and TIMP1 can help us to monitor the progress of advanced lung cancer. |
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