Single-cell RNA Sequencing Dissecting Cellular Heterogeneity And Dynamic Changes In Peritoneal Microenvironment During Gastric Cancer Peritoneal Metastasis

Background: Gastric cancer(GC)is one of the most common cancers and is the second most-diagnosed cancer and the second leading cause of cancer deaths in China,which is one of the malignant diseases that seriously threaten the health of Chinese residents.Although the clinical diagnosis and treatment strategies for GC have been greatly improved,the prognosis of GC is still poor and unsatisfactory.Peritoneal metastasis is the most common way of distant metastasis for GC,which is the main factor causing the poor prognosis of GC.Once peritoneal metastasis occurs,the overall survival time of GC patients is usually fewer than 6 months.Peritoneal metastasis is found in 10–20% of GC patients at the time of GC diagnosis,and develops in 40-60% GC patients even after curative surgical resection.Peritoneal metastasis has the characteristics of high incidence and poor prognosis in GC.However,the underlying molecular mechanism responsible for the development of peritoneal metastasis for GC is still not fully understood,which is an important factor restricting the development of effective treatment and improvemnt of prognosis of GC.Therefore,exploring the molecular pathology mechanism of peritoneal metastasis and finding effective biomarkers and therapeutic targets are of great value for clinical practices for GC.GC peritoneal metastasis is a multi-stage and complex cellular process involving the participation of various cells and molecular signals.Recently,the "seed and soil" hypothesis is a widely accepted as a fundamental theory explaining peritoneal metastasis in GC.The peritoneal microenvironment acts as a "soil" role in the process of peritoneal metastasis in GC and is a highly complex tumor microenvironment(TME)system,which is composed of various cells,extracellular matrix and signaling molecules and provides a suitable environment for tumor cell growth and plays a vital role in tumor occurrence and development.As "seed",free GC cells in peritoneal cavity is the initiating factor of peritoneal metastasis in GC,but GC cells need to face harsh conditions such as hypoxia and immune surveillance after entering the peritoneal cavity.The GC cells need to resist anoikis and break through the inherent defense barrier of the peritoneum for subsequent peritoneal metastasis.Moreover,the interaction between GC cells and the peritoneal microenvironment is an important factor in the occurrence of peritoneal metastasis in GC.Single-cell RNA sequencing(sc RNA-seq)technology can perform transcriptome sequencing and analyze the genetic information at the single-cell level.Based on this advantage,sc RNA-seq technology can reveal the tumor heterogeneity of various tumors and draw single-cell maps for these diseases.Therefore,sc RNA-seq technology will provide a strong technical support to explore peritoneal microenvironment,and a comprehensive dissection of the dynamic heterogeneity of peritoneal microenvironment will help to better understand the underlying molecular mechanism of peritoneal metastasis in GC,and will provide new insights into the future therapy strategies.In this study,we collected a total of 35 clinical samples of peritoneal lavage fluid and malignant ascites from different disease stages,including benign hysteromyoma,early GC and advanced GC and peritoneal metastasis,to simulate the process of peritoneal metastasis in GC.Then,we applied high-throughput sc RNA-seq technology to reveal the landscape of peritoneal microenvironment during peritoneal metastasis in GC,aiding exploration of the molecular mechanism and find of key molecular biomarkers and therapeutic targets for peritoneal metastasis in GC,which will provide a scientific basis for the precise diagnosis and treatment of peritoneal metastasis in GC.Methods: In this study,single-cell suspension preparation and cell concentration/viability were performed for each peritoneal lavage fluid and ascites samples,and sc RNA-seq technology was applied to perform single-cell RNA sequencing.Cell Ranger Software obtained from the 10 x Genomics official website for demultiplexing,barcode processing,read alignment to GRCh38 human reference genome,single-cell 3’ gene counting,and generation of feature(gene)-barcode expression matrix.Quality of cells was assessed based total UMI counts per cell,total detected genes per cell and proportion of mitochondrial genes per cell,and low-quality cells were filtered.Besides,we used the “Doublet Finder” analysis to remove potential doublets.Cells were clustered using an unsupervised cell clustering method,and uniform manifold approximation and projection(UMAP)was applied for visualization of cell clustering.Cell annotations were performed for immune cells and tumor cells based on differential genes,well-known marker genes and copy number variations(CNV)analyses.Then,we explored the heterogeneity and dynamic changes of immune cells and tumor cells during peritoneal metastasis in GC on the baisis of the cell functional score analysis,gene set enrichment analysis(GSEA)enrichment analysis and SCENIC transcription factor analysis.Moreover,“Monocle” pseudotime trajectory analysis was conducted to infer potential cell lineage trajectory and to explore the underlying molecular regulatory mechanisms between diverse cell phenotypes in peritoneal microenvironment.In addition,Cell Phone DB cell-cell interaction analysis and gene set variation analysis(GSVA)metabolic pathway activity analysis were used to explore the intercellular interactions between GC cells and immune cells,and the therapy-induced evolution of GC cells.Finally,we cultured patient-derived ascites organoids,and then performed in vitro validation experiments and drug intervention experiments based on the organoid models and TCGA database to explore the blocking effect of related inhibitors on peritoneal metastasis in GC.Results: We characterized single-cell RNA transcriptomes of 191,987 high-quality immune cells/GC cells from 35 patients with/without GC peritoneal metastasis from benign hysteromyoma patients and different stages of GC,and comprehensively dissected the landscape and their dynamic changes of the peritoneal microenvironment during peritoneal metastasis.There were multiple immune cell types in the peritoneal microenvironment,such as dendritic cells,macrophages,CD4 T cells CD8 T cells and other cell types,showing cellular diversity,complexity and heterogeneity.We identified a unique proliferative CD14+/CD1C+ dendritic cells(DC)characterized by high MCM4,MCM6,and PCNA expression,with lower antigen-presenting capacity and higher proangiogenic capacity(characterized by SPP1 and STAB1 expression).Besids,the increased cell proportion,reduced antigen-presenting capacity and increased proangiogenic capacity were exhibited during peritoneal metastasis.M2 phenotype tumorassociated macrophages(TAMs)were significantly enriched and their pro-angiogenic capacity was also significantly enhanced during peritoneal metastasis.T cells had dynamic changes and were differentially remodeled durinh GC peritoneal metastasis progression.The proportions of Treg and na(?)ve CD4 T cells increased,and the proportion of CD8 T cytotoxic cells and their cytotoxic capacity reduced.We observed proliferative cycling T cells,characterized by high MKI67,MCM2 and PCNA expression,in the peritoneal microenvironment.These cells exhibited low expression of cytotoxic(GNLY,IFNG,and PRF1)and high expression of inhibitory markers(TIGIT,TNFRSF9,CD47,ENTPD1,and CTLA4),which may represent an exhausted and dysfunctional stage and were enriched during peritoneal metastasis.Moreover,there were cell development and differentiation trajectories among dendritic cells and among T cells.Metabolic reprogramming and functional remodeling occurred in the process of development and differentiation along with immune dysfunction.Therefore,myeloid cells and lymphocytes contribute to the formation of peritoneal immunosuppressive microenvironment favorable for GC peritoneal metastasis via cellular state and function remodeling.In addition,the GC cells in the peritoneal cavity were highly heterogeneous,and we identified a subtype of GC cells with high plasticity that evolved to highproliferative GC cells through an autophagy-dependent plasticity program paligenosis against the cell pressure caused by the harsh peritoneal microenvironment.The autophagy-related gene MARCKS specifically biomarked the high-plasticity GC cells which were associated with poor prognosis,and autophagy inhibitors could significantly induce patient-derived ascites organoids apoptosis and inhibit the growth.GC cells underwent therapy-induced evolution,manifested by cell function remodeling and metabolic reprogramming after anti-tumor therapy.The crosstalk between immune cells and GC cells could maintain stemness and plasticity of GC cells.Conclusion: Our study comprehensively dissects the single-cell landscape and their dynamic changes of peritoneal microenvironment during peritoneal metastasis in GC,exhibiting high cellular complexity and heterogeneity.We identify multiple immune cell subtypes in pro-angiogenic and immunosuppressive states associated with peritoneal metastasis,remodeling of peritoneal microenvironment into a pro-metastatic microenvironment.GC cells acquire proliferative and metastatic ability through cell remodeling and metabolic reprogramming,and autophagy inhibitors can effectively block the growth of GC cells via apoptosis.Our findings will help to reveal the underlying molecular mechanism and regulatory network of peritoneal metastasis in GC,and provide new insights into future therapeutic strategy in precision medicine.