| Introduction:Laryngeal cancer is one of the most common head and neck malignancies in Northeast China.The early symptoms of it are not obvious and most patients are diagnosed as middle and advanced stage accompanied by different degrees of invasion and lymphatic metastasis.As we known,the recurrence and metastasis of laryngeal cancer are important factors affecting the patient 5-year survival rate.At the same time the treatments of laryngeal cancer are still mainly on surgery and radiotherapy,lacking of diagnosis and prognosis of molecular markers.Therefore,in-depth study of the molecular mechanism of laryngeal cancer development will be conducive to discover them who have the potential clinical value.Tumor heterogeneity,one of the characteristics of malignant tumors,refers to the difference in tumor tissue composition and biological functions such as growth,migration and invasion ability as well as their susceptibility to drugs that due to the different cell expression profile,which is the main reason for tumor metastasis,recurrence and drug resistance.Therefore,analysis of tumor heterogeneity has potential clinical diagnostic value,and deeply study of the mechanism will provide a theoretical basis for the precise targeted therapy of tumors.There are two theories of tumor heterogeneity,namely the clonal selection and cancer stem cells.According to the theory of clonal selection,tumor heterogeneity is caused by the continuous mutation of single tumor cell population during development,and the population suitable for survival is retained.Cancer stem cell theory holds that tumors are composed of a small group of self-renewing cancer stem cells(CSC)and their heterogeneous clusters,thus tumors show heterogeneity.However,both theories support that tumor microenvironment(TME)plays an important role in the formation of Tumor heterogeneity.TME refers to the internal environment for the occurrence and development of tumor cells,including not only themselves,but also immune cells and stromal cells,as well as the intercellular microenvironment and infiltrated biomolecules in nearby areas.Recent years,individualized treatment based on tumor microenvironment has attracted more and more attention,especially the immunotherapy for lung cancer,ovarian cancer and pancreatic cancer whose quality of life and five-year survival rate have greatly benefited.However,little is known about the tumor microenvironment of laryngeal cancer.Mining the cell components,the interaction in the tumor microenvironment and the effects of laryngeal cancer cell in microenvironment is helpful to the discovery of biomarkers and potential therapeutic targets for the occurrence and development of laryngeal cancer.With the development of single-cell sequencing technology,researchers can study the biological process,the occurrence and development of diseases at the single-cell level.It has been widely used in tumor,developmental biology,clinical diagnosis,stem cell development and differentiation fields,and as the focus of current research.Single-cell sequencing on tumor can help us study tumor heterogeneity,microenvironment,track the metastasis and spread of cancer cells as well as understanding the evolution of drug resistance during treatment to provide new methods and ideas for clinical diagnosis,treatment and prognosis of malignant tumors.In this study,we conducted the following studies through the single cell sequencing of laryngeal cancer:1)analyzed the heterogeneity of laryngeal cancer and speculated the clonal evolution of malignant tumor cells;2)analyzed the differences in cell composition of each tissue were analyzed,and subsets closely related to the invasion and metastasis of laryngeal cancer were found;3)analyzed the copy number variations(CNV),malignant scores,transcription factors and signaling pathways regulating each subpopulation of epithelial cells to explore the mechanisms leading to laryngeal cancer cell heterogeneity;4)analyzed the function of immune cell subsets,the activated signaling pathways,developmental trajectories and key transcription factors were analyzed revealing the mechanism of immune escape of laryngeal cancer cells.Materials and methods:1.Experimental materials:human laryngeal cancer tissue(Tumor,T),border cancer tissue(Regions Tumor,R),normal epithelial tissue(Normal Tissue,N)and lymph node metastatic cancer tissue(Lymphoid Tissue,L)specimens,human laryngeal cancer Hep-2cell line.2.Experimental methods:preparation of single cell suspension,10×Genomics cell labeling and reverse transcription,c DNA library construction and amplification,second-generation sequencing,sequencing data preprocessing and normalization,cell cycle effect evaluation,multi-sample integration analysis,PCA dimensionality reduction,tSNE cell clustering and visualization,Infer CNV and sc Cancer analysis,Single R cell type annotation,pseudotime analysis,GSEA function and pathway enrichment analysis,SCENIC transcription factor analysis,immunofluorescence experiment,cells culture,gene transfection,Real-time PCR,Western Blot,chromatin immunoprecipitation,dual luciferase reporter gene activity detection,transwell experiment.Result:1.sc RNA-seq and analysis quality controlThe results of capillary electrophoresis experiments showed that the quality of the sample c DNA and c DNA library met the second-generation sequencing standards.The output of the Cell Ranger software shows that the quality control of single-cell RNA sequencing results is qualified,and the cell cycle genes has little effect on cell clustering and clustering,suggesting that the sequencing data can be analyzed.2.Analysis the heterogeneity of the laryngeal cancer microenvironmentThe tSNE cluster analysis results showed that there were 25 subgroups in laryngeal cancer tissues;Single R cell annotation and tissue immunofluorescence experiments showed that there are 7 groups including epithelial-derived cells,T lymphocytes,B lymphocytes,myeloid cells,NK cells,endothelial cells and fibroblasts;among them,the number of epithelial-derived cells and immune cells was large and included multiple cell clusters,while the number of stromal cells was few and included only one cluster.These results suggest that laryngeal cancer has complex heterogeneity,and the heterogeneity of epithelial-derived cells and immune cells were the main reasons for the heterogeneity of laryngeal carcinoma.3.The relationship between epithelial cell heterogeneity and laryngeal carcinoma invasion and metastasisThe re-clustering results of the epithelial-derived cell results showed that there ware 12subgroups of epithelial-derived cells.Among them,normal epithelial tissues were mainly composited with C0,C1 and C2 clusters,lymphoid tissues were mainly composited with C4,C5,C6,and C7.Tumors center tissues were mainly composited with C2,C6,C8,C9and C11 clusters,and the regions tumor tissues ware mainly composited with C0,C1,C3,C10,C4 and C5 clusters.The results of GSEA function enrichment and pseudotime analysis showed that there were 5 clusters(C0,C2,C7,C9 and C10)cytokeratosis was better,of which C2 and C9 were at the end of keratinization;2 clusters(C1 and C3)had significantly enhanced EMT function;3 clusters(C6,C8 and C11)had IFN-?response and neutrophil chemotaxis,and the they were in the early stage of development;2subgroups(C4 and C5)had strong proliferation ability and were at the end of proliferation and differentiation.These results suggested that C6,C8 and C11 subgroups were closely related to the invasion and metastasis of laryngeal cancer,and C4 and C5were related to the malignant proliferation of posterior cancer cells.4.The relationship between immune cell heterogeneity and laryngeal carcinoma invasion and metastasis4.1 The relationship between T/NK cell clusters and laryngeal carcinoma invasion and metastasisT/NK cell tSNE clustering and Single R annotation results showed that there were 11 cell clusters of T/NK cells,including 1 naive CD4~+T cell cluster,2 Tregs cell clusters,and 2CD4~+memory T cells clusters,2 CD8~+T cell clusters,1 NK cell cluster,1 CMP cells and 1??T cell cluster.Among them,tumor tissues infiltrated abundant T/NK cell clusters,mainly Tregs cells and??T cells;regions tumor tissues infiltrated Tregs cells,CD8+T cells and??T cells mainly;normal mucosal tissues existed CD8~+T cells,NK cells and CMP cells;CD4~+memory T cells,naive CD4~+T cells and Tregs cells are predominant in lymphatic metastatic cancer tissues.The results of GSEA functional enrichment analysis showed that the function cytotoxicity,antigen binding and chemotaxis and other functions were significantly activated in CD8~+T cells;translation-related functions were significantly activated in CD4~+memory T cells;TNF and its receptor,autophagy,and apoptosis were significantly activated in Tregs cells;cytotoxicity,antigen binding and exocrine functions were significantly activated in NK cells;redox reaction,chromosomal conformation changes,and cytotoxicity were significantly activated in??T cells.The results of pseudo-chronological analysis showed that the cells were at various stages of development in each cell subgroup.These results suggested that the laryngeal cancer tissue exhibited a state of T cell immunosuppression,the regions laryngeal cancer tissue was infiltrated with more immune-killing T cells,and the T cells in the lymphatic metastatic cancer tissue were in a naive state.4.2 The relationship between B cell clusters and laryngeal carcinoma invasion and metastasisThe results of tSNE clustering and Single R annotation showwd that there were 9 cell clusters of B cells,including 5 naive B cell clusters,2 plasma cell clusters,1 memory B cell cluster and 1 germinal center B cluster.Most of the B cells were located in the lymphoid tissues,and there is a small amount of infiltration in the tumor tissues.The results of GSEA function enrichment and pseudotime analysis showed that the germinal center B cell proliferation function was enhanced and was at the end of proliferation state;the unfolded protein response of some clusters of plasma cells were significantly activated and in the naive stage;memory B cells began to stimulate the antigen again response,but there were out a large number of proliferation and differentiation;naive B cell clusters were in different stages of immune response.These results suggested that part of B cells ware at an early stage of development,part of B cells had abnormal functions with little anti-tumor immunity.4.3 The relationship between myeloid cell clusters and laryngeal carcinoma invasion and metastasisThe results of tSNE clustering and Single R annotation showed that there were 7 cell clusters in myeloid cells,including 3 monocyte clusters,2 macrophage clusters,1neutrophil cluster and 1 dendritic cell cluster.T tissue had the highest proportion of macrophage subpopulation;R tissue had the highest proportion of neutrophil subpopulation;N tissue had the highest proportion of monocytes and macrophages;L tissue had the highest proportion of dendritic cell cluster,and a small part of neutrophils.The results of GSEA functional enrichment and pseudotime analysis showed that the macrophage C0 cluster and monocyte cluster the B cell proliferation regulation and differentiation function were significantly up-regulated,the T cell differentiation regulation function was significantly down-regulated,and the macrophage C3 cluster proliferated significantly up-regulated.Developmental trajectory was non-specific with multiple developmental stages;neutrophils promote angiogenesis and antibacterial immune response functions were significantly activated,and there were two states of early development and pro-inflammatory function differentiation states;dendritic cell the differentiation regulation function of T cells was significantly up-regulated,and most cells were in the early stages of development.5.Analysis of stromal cells functionThe results of GSEA functional enrichment analysis showed that fibroblast autophagy and proteasome-mediated protein degradation were significantly activated;endothelial cell autophagy,neutrophil function regulation and other functions were significantly activated.Fibroblasts had no tissue specificity;endothelial cells mainly existed in normal mucosal epithelial tissues.These results suggested that stromal cells had little effect on the invasion and metastasis of laryngeal cancer.6.Analysis of the communication network in laryngeal carcinomaThe results of cell-cell interaction analysis based on ligand-receptor results showed that epithelial-derived cells and fibroblasts were the most paired,followed by myeloid cells;the results of ligand-receptor specificity analysis showed that epithelial-derived cells and fibroblasts interactions were the most specific.These results suggested that epithelial-derived cells interacted closely with fibroblasts,while interacted less with T lymphocytes and B lymphocytes.7.Study on the formation mechanism of epithelial-derived cell heterogeneity7.1 Analysis of copy number variation of epithelial-derived cellsThe results of copy number variation analysis showed that the genes located at 3p,6q,7q,11q,and 12q in laryngeal cancer tissues were amplified,while the genes located at 3q,5p,5q,13p,and 13q were largely deleted;cancer regions tissues and cancer center tissues were similar,but the degree of gene amplification and deletion was reduced.The genes on chromosome 8 in the marginal cancer tissue was significantly amplified,and the chromosome 13 gene was obviously missing;there were also copy number variations in some chromosomal regions in normal tissues;in lymphoid tissues the genes copy number were extensive and irregular including amplification and deletion.The results of sc Cancer analysis showed that the malignant score of the epithelial-derived cell transcriptome had two peaks compared with the control.Most of the T and R groups were malignant cells,and the N group was mainly benign cells,which was consistent with the pathological tests results.These results suggested that the complex copy number variation of epithelial-derived cells in laryngeal cancer was one of the reasons for the formation of heterogeneity.7.2 Analysis of key pathways and transcription factors regulating epithelial cell heterogeneityGSEA pathway enrichment analysis results showed that NOTCH pathway played an important role in regulating cell invasion and metastasis;malignant cells that metastasize to lymphoid tissues mainly rely on oxidative phosphorylation for capacitation and proliferate vigorously;NF?B pathway regulated immune characteristics and metastasis in epithelial cells.Transcription factor enrichment analysis results show that transcription factors such as RAD21,BRCA1 and ETS1 played an important role in the proliferation of laryngeal cancer cells;transcription factors such as SOX2,SOX4,HOXC9,and YY1played an important role in the stemness and metastasis of laryngeal cancer cells;FOSL1and PITX1 played an important role in the keratinization process of laryngeal cancer cells.These results suggested that the EMT,IFN?response,NOTCH and NF?B pathways played an important role in the invasion and metastasis of laryngeal cancer.7.3 The influence and molecular mechanism of YY1/C-MYC/HMGB1 signal axis on the laryngeal carcinoma invasion and metastasisThe statistical analysis of the expression levels of YY1 and HMGB1 in head and neck squamous cell carcinoma in the TCGA database showed that both YY1 and HMGB1were significantly up-regulated in head and neck squamous cell carcinoma,and there was a positive correlation(P<0.05);their expression levels increased was closely related to lymph node metastasis of head and neck squamous cell carcinoma(P<0.05).The statistical results of downstream target genes of YY1 show that C-MYC is the direct downstream target gene of YY1;the expression levels were positively correlated in head and neck squamous cell carcinoma(P<0.05);knocking down YY1 in laryngeal cancer could significantly reduce C-MYC expression level(P<0.05).These results suggested that YY1 could transcriptionally activate the expression of C-MYC in laryngeal carcinoma and promoted the expression level of the C-MYC downstream gene HMGB1.Bioinformatics prediction results showed that HMGB1 was a downstream target gene of C-MYC;the expression levels were positively correlated in head and neck squamous cell carcinoma(P<0.05);in laryngeal cancer cells,overexpressed or knocking down C-MYC,the expression level of HMGB1 was significantly increased and decreased respectively(P<0.05);the double luciferase reporter gene activity test results showed that overexpression of C-MYC could significantly increase the promoter activity of HMGB1(P<0.05);Ch IP Experimental results showed that C-MYC could bind to the HMGB1promoter region.These results suggested that C-MYC cloud directly transcriptionally activate the expression of HMGB1.The results of transwell experiment showed that t overexpressed or knocking down C-MYC in laryngeal cancer cells significantly increased and decreased cell migration ability compared with the control group(P<0.05);knockdown HMGB1 decreased cell migration ability significantly(P<0.05);knockdown HMGB1 cloud significantly restore the promotion of C-MYC overexpression on cell migration.These results suggested that C-MYC cloud promote the migration of laryngeal cancer cells through HMGB1.8.Preliminary analysis of the formation mechanism of immune microenvironment heterogeneityThe results of T/NK cell GSEA pathway enrichment and transcription factor analysis showed that the TNF-beta signaling pathway played an important role in regulating cytotoxicity,and the Jak-STAT signaling pathway played an important role in regulating the activity of Treg cells.Transcription factors ETS1,TAF7,EOMES,etc.played an important role in CD8~+T cells and??T cells;REL,FOXP3,ETS1,etc.played an important role in Treg cells;and the transcribed factor scores in CD4~+memory T cells and naive CD4+T cells were are all low.The results of GSEA pathway enrichment analysis of B cell showed that glutathione metabolism was significantly up-regulated in naive B cells;plasma cell oxidative phosphorylation pathway was significantly down-regulated.Transcription factor analysis results showed that among naive B cells and memory B cells,FOXP1,HES1,ETS1,etc.scored higher(C0,C1,C2,C8),and MAFB,RELB,NFKB1,etc.scores higher in C3cluster;the scores of RAD21,REL,etc.in the germinal center B cells were relatively higher.The GSEA pathway enrichment analysis of myeloid cells showed that signal pathways such as oxidative phosphorylation and DNA replication in macrophages were significantly activated;TNF and MAPK signal pathways were significantly activated in monocytes;RIG-1-like receptors in neutrophils Significant activation of the pathway;significant activation of signal pathways such as RIG-1-like receptor pathway and T cell receptor in dendritic cells.Transcription factor analysis results showed that E2F1 and BRCA1 in macrophages promoted the proliferation of C3 subgroups,MAF,SPI1 and STAT1 promoted the phagocytic function of C0 subgroups;POU2F2,MEF2C and SPIB in monocytes(C2 and C7 clusters)promoted TNF and MAPK pathway activates and promoted the presentation of antigens to B cells;CREM,FOXO3,XBP1,KLF3 and BRCA1 promoted the phagocytic activity of the C5 subgroup.Conclusion:1.The microenvironment Laryngeal cancer is complex,which is mainly composed of 7types including epithelial-derived cells,T lymphocytes,B lymphocytes,myeloid cells,NK cells,endothelial cells and tumor-related fibroblasts.The heterogeneity of epithelial-derived cells and immune cells are the main reason for the heterogeneity of laryngeal cancer2.There are multiple cell clusters of epithelial-derived cells.Among them,C6,C8 and C11 subgroups are closely related to the invasion and metastasis of laryngeal cancer.C4and C5 are related to the malignant proliferation.The proliferation ability of tumor cells that metastasize to lymphoid tissue is significantly enhanced..3.The laryngeal cancer tissue show a state of immunosuppression;the marginal tissues of the laryngeal cancer show a state of inflammatory microenvironment;there are a large number of memory T cells and memory B cells in the lymphatic metastasis cancer tissue,but no immune response appeared.4.Epithelial-derived cells are closely related to fibroblasts and myeloid cells,and interact less with T lymphocytes and B lymphocytes.5.The complex copy number variation in epithelial-derived cells is one of the reasons for the formation of heterogeneity.6.A number of pathways including NOTCH and NF?B play an important role in laryngeal cancer progression.A number of transcription factors play an important role in laryngeal cancer heterogeneity.7.In laryngeal cancer,the transcription factor YY1 can transcriptionally activate the expression of C-MYC;C-MYC can directly transcriptionally activate the expression of HMGB1 and promote the migration of laryngeal cancer cells.8.A number of pathways play an important role in regulating laryngeal cancer immune microenvironment;and a number of transcription factors play an important role in immune cells heterogeneity. |
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