| Background:Gastrointestinal stromal tumor(GIST)is the most common mesenchymal tumor of the gastrointestinal tract,with a domestic incidence of approximately 1 in 100,000-2in 100,000 and a significant rise in recent years.The treatment of GIST has been revolutionized by the emergence of the KIT inhibitor Imatinib.Currently,Imatinib is the standard first-line treatment for metastatic cases of GIST.The response rate to the second and third-line regimens sunitinib and regorafenib is poor and the clinical benefit is limited.Thus,the efficacy of drug therapy associated with GIST remains suboptimal,and resistance to targeted agents such as Imatinib in GIST is a key factor limiting further improvement in GIST prognosis.However,the molecular mechanisms underlying the generation and development of drug resistance in GIST are still unclear.Therefore,exploring the molecular mechanisms of GIST metastasis and drug resistance progression and finding effective biomarkers and therapeutic targets are of significant importance to improve the prognosis of GIST.Tumor-infiltrating immune cells are an essential component of the tumor microenvironment and are not only involved in regulating the local anti-tumor immune response,but are also the main target cells relevant for immunotherapy.A comprehensive analysis of tumor-infiltrating immune cells can help to understand the molecular mechanisms of tumor immune escape and provide new ideas for the development of therapeutic targets.Previous studies have identified a large number of tumor-infiltrating immune cells within the GIST microenvironment,mainly macrophages and T lymphocytes,as well as B lymphocytes,natural killer cells and dendritic cells.Also,it has been shown that the density of M2 macrophage infiltration is significantly higher in metastatic recurrent tumors than in the primary tumor.Therefore,an insight into the composition of the immune cells within the GIST microenvironment and their interactions with tumor cells is crucial to elucidate the mechanisms of GIST metastasis and drug resistance.Single cell RNA sequencing enables analysis of complex cellular composition and heterogeneity at the individual cell level,by identifying differential gene expression and epigenetic factors caused by single cell genomic mutations,as well as novel cell-specific markers and cell types.Single cell RNA sequencing technologies can provide greater insight into the complex interactions between the immune microenvironment and tumor cells,and more thoroughly characterize the tumor ecosystem.A comprehensive analysis of the immune microenvironment of GIST tumors using single cell RNA sequencing will help to better understand the molecular mechanisms of drug resistance in GIST and provide new ideas and strong evidence to support the precise diagnosis and treatment of GIST.Using 10x Genomics high-throughput single-cell RNA sequencing technology,we systematically resolve the immune and tumor cell profiles within the GIST immune microenvironment at the single-cell level,combined with multiple immunofluorescence staining to verify the cellular composition characteristics within the GIST immune microenvironment;meanwhile,we use the GIST public dataset to explore the immunophenotyping of GIST patients,combined with clinicopathological data,to identify key molecular markers and therapeutic targets for GIST,providing a scientific basis for precise diagnosis and treatment of drug resistance in GIST.Methods:Sixteen tissue samples from eight GIST patients were included in this study,including eight tumor tissues and eight matched normal tissues next to the tumor;single-cell RNA sequencing was performed on the 16 tissue samples using 10x Genomics technology;the raw single-cell RNA sequencing data were subjected to data splitting and cell barcoding using Cell Ranger software,and the raw sequencing data were compared with the GRCh38 human reference genome to generate the original gene-cell expression matrix;assess the quality of each cell based on the number of UMIs,genes and mitochondrial genes per cell,and filter out low quality cells;use"Double Finder"to predict and remove potential double or multiple Cell clustering and clustering of sequenced cells using unsupervised clustering methods and downscaling and visualization of results using t-SNE;cell type identification based on differential genes,known marker genes and copy number variation analysis to fully resolve the immune and tumor cell profiles of GIST;construction of a GIST public dataset(GSE136755)with The ss GSEA method was used to construct GIST-associated immunophenotypes(GICs)consisting of eight immune-associated genes and two stroma-associated gene sets;a clinical cohort of 64 primary GIST cases undergoing radical surgery at Zhongshan Hospital of Fudan University was used to validate the immune cells in the tumor microenvironment and to investigate the correlation between immune cell subsets and patients’clinicopathological factors and prognosis.Results:In this study,single-cell RNA sequencing was performed on clinical samples from16 GIST patients,and a total of 153,515 high-quality single-cell RNA data were obtained;various known immune cell subsets were present within the GIST tumor microenvironment,such as CD4~+T cells,CD~+8 T cells,NK cells,dendritic cells,macrophages,epithelial cells,B cells,etc.,indicating that the GIST tumor microenvironment The immune cell types within the GIST tumor microenvironment are diverse,complex and heterogeneous.The analysis of the number of immune cells in the tumor and the normal tissues adjacent to the tumor revealed that NK cells,DC cells and macrophages were highly infiltrated in the tumor compared to the normal tissues adjacent to the tumor;further analysis of the macrophage subpopulation revealed that macrophages were mainly present in the tumor,accounting for 87.7%of all cells;further analysis of the polarisation status of macrophages in the GIST tumor revealed that M2 type Further analysis of the polarisation status of macrophages in GIST tumors revealed that M2 macrophage markers such as CD163 and MRC1 were expressed on almost all macrophages,whereas M1 macrophage markers such as CD80,HLA-G and IL1R2 were weakly expressed on macrophages,suggesting that a large number of immunosuppressive M2 macrophages were infiltrating in GIST tumors;the expression of tumor infiltrating B lymphocytes(Tumor infiltrating B The clustering analysis of Tumor infiltrating B lymphocyte(TIB)revealed the presence of four TIB cell subtypes in GIST tumor tissue:Follicular＿B cells,GC＿B＿cells,Ig G＿Plasma＿cells,and Ig A＿Plasma＿cells.Plasma＿cells);a significantly higher number of Ig G＿Plasma＿cells and a significantly lower infiltration of Ig A＿Plasma＿cells in tumor tissue compared to normal tissue adjacent to the tumor;a significantly higher ratio of Ig G/Ig A plasma cells in tumor tissue;Further analysis of transcriptomic data revealed that the TIB cell subpopulation influenced the formation of TLS in GIST tumors through the CCL5-CCR5 chemotaxis;transcriptomic data and the set of 8immune-related genes and 2 stroma-related genes were used to differentiate GIST into three immunophenotypes:GIC-C type showed"immune infiltration",GIC-B type showed"moderate immune infiltration",and GIC-A type showed"Immune desert".The immunofluorescence staining of CD3~+T cells and CD20~+B cells in GIST tumor tissues in our centre verified the existence of three subtypes of GICs in the GIST microenvironment,and the three immunophenotypes were significantly correlated with the tumor risk grade of GIST patients.Further Ig G immunohistochemical staining,combined with the clinical characteristics of the patients,revealed that the patients in the Ig G high expression group had better survival,suggesting that Ig G plasma cells may play an internal anti-tumor immune function in the GIST microenvironment.Conclusion:This study applied single-cell RNA sequencing technology to comprehensively resolve the immune and tumor cell profiles within the GIST tumor microenvironment at the single-cell level,demonstrating the high complexity and heterogeneity of cells within the GIST microenvironment,and revealing the dynamic changes and functional transformation profiles of various cell types within and adjacent to the tumor.Combined with transcriptomic data,the immunophenotyping associated with clinical risk factors in GIST patients was identified,and the impact of Ig G plasma cell infiltration on good prognosis was verified in a large clinical cohort,providing a new idea and solid foundation for individualized and precise medical treatment of GIST.Figure 47 Table 12 Reference 71... |
PDF Full Text Request