| Objective:This study aims to investigate the characteristics of the tumor microenvironment and the role of tumor heterogeneity in cervical squamous cell carcinoma under different immune phenotypes.We will explore the mechanisms underlying the formation of specific immune phenotypes in different tumor states and their distribution and prognostic value at the individual level.Methods:This study used single-cell sequencing to analyze 14 cervical squamous cell carcinoma tumor tissues and 3 normal cervical tissues,and determined the degree of tumor immune infiltration through immunohistochemistry.In addition,by combining single-cell analysis and spatial transcriptomics,the study explored the characteristics of the tumor microenvironment under different immune phenotypes and the role of tumor heterogeneity.The mechanism was verified through cell and animal models.Moreover,based on the established cervical squamous cell carcinoma multi-omics cohort(94 samples),the individual tumor status was evaluated using transcriptome data,and the prognostic differences of tumor status were assessed in the treatment cohort.Results:Through the analysis of squamous epithelial cells,8 meta-programs(MP)were discovered,including 2 epithelial-related tumor states,namely MP6 and MP7.MP6 has keratinization-related genes and is expressed higher in rejection/desert-type tumors,while MP7 has immune response and glandular epithelium-related genes and is expressed higher in infiltrative-type tumors.The peritumoral TGFβ pathway activity is enhanced in the keratinizing epithelial-type tumor,and there is also a TGFβ-activated subpopulation of fibroblasts,i.e.CAF-MMP1,which is located close to this type of tumor.Interference with FABP5 affects the expression of MP6-related genes in cells and enhances the degree of immune infiltration in animal models of tumor.Normal fibroblasts are induced by MP6 tumors to transform into CAF-MMP11.The microenvironment of the MP7 tumor contains rich cell types and interactions,and the addition of IFNγ can upregulate the MP7 tumor state.MP-related genes are expressed consistently at the protein and phosphorylated protein levels.Squamous cell carcinoma patients who lean towards the MP7 state have a longer survival time after receiving immunotherapy.Conclusions:Cervical squamous cell carcinoma has two tumor states that are closely related to the degree of tumor immune infiltration,namely,Epi-KRT and Epi-Immune tumor states.The Epi-KRT state induces CAF to form an immune-rejecting environment through the TGFβ pathway.Both tumor states can be reversed.The distribution of Epi-KRT and EpiImmune tumor states among patients is universal and related to immune infiltration,and can serve as a prognostic indicator for immunotherapy.Part Ⅰ:Study of Heterogeneity in Cervical Squamous Cell CancerObjective:Different tumor immune phenotypes(including immune-infiltrated,excluded,and desert types)are crucial factors determining the response to tumor immunotherapy.Tumor heterogeneity and plasticity,as fundamental characteristics of tumors,play important roles in regulating the tumor immune microenvironment.This study aims to explore the tumor microenvironment characteristics of cervical squamous cell carcinoma under different immune phenotypes and the role of tumor heterogeneity in it.Methods:This study used single-cell sequencing on 14 cervical squamous cell carcinoma tumor tissues and 3 normal cervical tissues.The degree of tumor immune infiltration was determined by immunohistochemistry.Single-cell analysis was conducted to investigate the characteristics of the tumor microenvironment under different immune phenotypes and the role of tumor heterogeneity in it.Spatial transcriptomic data was used to validate the singlecell analysis.Results:Compared to excluded/desert type tumors,infiltrated tumors contain more exhausted T cells,which exhibit both cytotoxic and exhaustion features.In the analysis of squamous epithelial cells,eight meta-programs(MP)were identified,including two epithelial-related tumor states,MP6 and MP7.MP6 has genes related to keratinization,and its score is expressed higher in excluded/desert type tumors.MP7 has genes related to immune response and glandular epithelium,and its score is expressed higher in infiltrated tumors.Tumors in the MP6 state have fewer immune cells in their spatial microenvironment than tumors in the MP7 state,according to spatial chips..Conclusions:There are two tumor states in cervical squamous cell carcinoma that are closely related to the degree of tumor immune infiltration.Part Ⅱ:Study of Epi-KRT and Epi-Immune Cervical Squamous Cell CancerObjective:This study aims to explore the mechanisms by which different tumor states form specific immune phenotypes,elucidate the role of heterogeneous tumor states in the formation of tumor immune phenotypes,and identify potential targets for reversing immune phenotypes.Methods:This study used single-cell transcriptomics and spatial transcriptomics to conduct functional analysis on two tumor states.Through single-cell subpopulation analysis and spatial communication analysis,we investigated the reasons for the formation of specific immune phenotypes by different tumor states,which were validated through in vivo and in vitro models.Results:The Epi-KRT tumor exhibits stronger keratinization function,and there is receptor interaction represented by DSC2-DSG2 within the tumor.The TGFβ pathway activity is increased in the periphery of Epi-KRT tumor,and there is also a TGFβ-activated fibroblast subset,CAF-MMP1.The spatial location of Epi-KRT tumor and CAF-MMP1 is close.Interfering with FABP5 affects the expression of Epi-KRT-related genes in cells,leading to an enhanced immune infiltration in animal models of tumor growth.Normal fibroblasts are induced by Epi-KRT tumor to develop into CAF-MMP1.The Epi-Immune tumor microenvironment contains abundant cell types and interactions,and the addition of IFNy has an up-regulating effect on the Epi-Immune tumor state..Conclusions:Epi-KRT tumor state induces a tumor microenvironment that promotes immune exclusion via the TGFβ pathway.The tumor states can be reversed.Part Ⅲ:Multi-omics Cohort Study of Cervical Squamous Cell CancerObjective:The established cervical squamous cell carcinoma multi-omics cohort was used to assess the distribution of Epi-KRT and Epi-Immune tumor states at the individual level,and to explore the prognostic value of tumor status in the immunotherapy cohort.Methods:This study evaluated individual tumor status based on transcriptomic data by scoring the Epi-KRT state relative to the Epi-Immune state(Epi-KRT minus to Epi-Immune,K2I),and assessed the degree of immune infiltration in tumors through xCell and immunohistochemistry experiments.Correlation analysis was performed between K2I scores,immune infiltration,and proteomic and phosphoproteomic data.The prognostic differences between populations with high and low K2I scores were evaluated in the treatment cohort.Results:K2I scores were correlated with immune infiltration,and tumor-state-associated genes showed consistent expression at the proteomic and phosphoproteomic levels.Squamous cell carcinoma patients with low K2I scores had longer survival times under immunotherapy.Conclusions:The distribution of Epi-KRT and Epi-Immune tumor states among patients is universal and related to immune infiltration,and can serve as a prognostic indicator for immunotherapy. |
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