Research Of The Heterogeneity Of Tumor Microenvironment And The Mechanisms Related To Metastasis Of Intrahepatic Cholangiocarcinoma

Background and Purpose:Intrahepatic cholangiocarcinoma(ICC)ranks as the second most common malignant tumor of the liver,accounting for approximately 10%-20%of primary liver cancers.Over the past two decades,its incidence has been steadily rising globally,posing a significant threat to human health.The etiology of ICC is complex,involving factors such as intrahepatic bile duct stones,viral hepatitis,and liver fluke infection,as well as metabolic disorders such as obesity,diabetes,and non-alcoholic fatty liver disease.As research progresses,it has been found that ICC can originate from bile duct epithelial cells,peribiliary glands,hepatocytes,and multipotent stem cells.While curative surgical resection offers potential for cure in ICC patients,its aggressive and often occult nature,propensity for lymphatic metastasis(with up to 45%-62%of ICC patients presenting with lymph node metastasis at initial diagnosis),and the limited eligibility for surgical resection(only about 20%-30%of patients being eligible at initial diagnosis)remain significant challenges.Even after radical surgery,the risk of recurrence and metastasis remains high,with a 5-year recurrence rate of up to 60%-70%,leading to an overall 5-year survival rate of only 20%-40%.For advanced ICC patients,the median overall survival with first-line chemotherapy regimens such as gemcitabine plus cisplatin is only about 11.7 months.Although targeted therapies such as pemigatinib targeting fibroblast growth factor receptor 2(FGFR2)gene fusions and ivosidenib targeting isocitrate dehydrogenase 1(IDH1)gene mutations can improve the prognosis to some extent,their benefits are limited to less than 40%of ICC patients due to low mutation rates.Similarly,immune checkpoint inhibitors have shown promising objective response rates in clinical trials in cohorts of cholangiocarcinoma patients with mismatch repair deficiency,high microsatellite instability,and high tumor mutational burden,yet less than 5%of cholangiocarcinoma patients have these features.The significant heterogeneity of ICC,resulting from diverse etiological factors,origins,and genomic alterations,leads to considerable variations in treatment response among different patients,underscoring the urgent need for more effective therapies through dissecting the tumor microenvironment heterogeneity and identifying novel therapeutic targets to improve patient outcomes.ICC is highly invasive,with a high incidence of lymph node metastasis.Lymph nodes draining from tumors are the most common sites of extrahepatic metastasis in ICC.Lymph nodes,rich in immune cells such as T cells,B cells,and macrophages,play a crucial role in anti-tumor immunity but paradoxically often serve as the first sites of metastatic spread for most malignant tumors.Moreover,tumor cells that have metastasized to lymph nodes tend to gain enhanced survival abilities and increased invasiveness after"education"within the lymph node microenvironment.Previous studies on lymph node metastasis have mainly focused on lymphangiogenesis and tumor cell migration and invasion,while overlooking how the lymph node microenvironment rich in immune cells permits tumor cell survival under adverse conditions and how tumor cells reshape the lymph node microenvironment to evade immune recognition and killing.The concept of tumor immune editing suggests that the immune system can both restrict and promote tumor progression,evolving dynamically through a process of tumor-immune system interaction from immune elimination,immune equilibrium,to immune escape.Studies have shown that lymph nodes without metastasis respond to PD-L1 antibody treatment by inducing the activation and proliferation of precursor-depleted CD8+T cells and intermediate-depleted CD8+T cells.However,there is no such response in lymph nodes with metastasis.During the process of ICC lymph node metastasis,do tumor cells reshape the lymph node microenvironment,induce immune tolerance,and promote immune escape,leading to the occurrence of metastasis?Therefore,applying single-cell transcriptome sequencing technology to analyze the cellular composition and gene expression differences between non-metastatic lymph nodes and metastatic lymph nodes in ICC patients will help us explore potential immune escape mechanisms during ICC lymph node metastasis and identify potential therapeutic targets to improve patient prognosis.In this study,we aim to comprehensively analyze the tumor microenvironment heterogeneity of ICC through single-cell transcriptome sequencing of ICC patients’primary tumors,adjacent liver tissues,and lymph node tissues(metastatic and non-metastatic).Methods:We collected tumor tissues,adjacent liver tissues,and lymph node tissues(metastatic and non-metastatic)from ICC patients for single-cell transcriptome sequencing.Additionally,bulk transcriptome sequencing was performed on another 87 pairs of ICC patient samples(tumor and adjacent liver tissues),and spatial transcriptome sequencing was conducted on the junction between tumor and adjacent non-tumor tissues in 4 ICC patients.The processed data underwent quality control,batch correction,dimensionality reduction clustering,cell type annotation,and tumor cell identification using the infer CNV algorithm.Cell subtypes were identified based on marker genes from previous literature and databases,and their distribution in different types of samples and different types of patients was analyzed.Functional analysis of different subtypes was conducted using GSVA and GO enrichment.Cell trajectory analysis was performed using Monocle and RNA velocity.Cell-cell interaction analysis was conducted using Cell Phone DB.Transcription factor activity analysis was performed using the SCENIC algorithm.Transcriptional feature clustering was conducted using non-negative matrix factorization algorithms.Validation was performed through experiments such as lentiviral transfection,q PCR,western blotting,proliferation,migration,invasion assays,as well as xenograft tumor models(subcutaneous implantation,splenic injection for liver metastasis,and orthotopic liver implantation).Results:1.There is significant heterogeneity in the tumor microenvironment of ICC lymph node metastasis,and the distribution of various cell subgroups in different types of samples and different types of patients shows high heterogeneity.2.Macrophages can be divided into 5 subgroups,including AIF1~+Macro,CD3D~+Macro,IL1B~+Macro,LYVE1~+Macro,and SPP1~+Macro;among them,SPP1~+Macro is significantly more infiltrated in the metastatic lymph nodes than in the non-metastatic lymph nodes,and it has a significant ability to promote angiogenesis.3.Neutrophils can be divided into 3 subgroups,including S100A9~+Neu,MIF~+Neu,and HLA-DR~+Neu,among which MIF~+Neu is the senescent subgroup of neutrophils.4.Dendritic cells can be divided into 3 subgroups,including DC1,DC2,and DC,among which DC3 highly expresses CD274,and the transcription factors HIVEP1 and IRF1 may play an important role in regulating the expression of CD274 in dendritic cells.5.CD4~+T cells can be divided into 4 subgroups,including Treg＿FOXP3,CD4＿IL2,CD4＿LEF1,and CD4＿BTLA,among which Treg＿FOXP3 cell subgroup is increased in infiltrated in the metastatic lymph nodes.6.CD8~+T cells can be divided into 6 subgroups,including CD8＿IL7R,CD8＿KLRG1＿TNF,CD8＿KLRG1＿IFNG,CD8＿GZMK,CD8＿LAG3,and CD8＿HAVCR2.According to the evolutionary trajectory,CD8＿IL7R and CD8＿KLRG1＿TNF are relatively immature stages of CD8+T cells,and CD8＿LAG3 and CD8＿HAVCR2 are in the exhausted stage.And the metabolism of CD8~+T cells is significantly different in different types of samples,with CD8~+T cells in the metastatic lymph nodes showing highly active amino acid metabolism.7.The transcriptional characteristics of ICC tumor cells can be clustered into 5 different feature modules,namely proliferation feature,immune regulation feature,metastasis feature,metabolism feature,and epithelial feature.8.ICC tumor cells can be classified into 5 subtypes based on transcription factor activity,namely angiogenesis subtype,metabolism subtype,inflammation subtype,immune suppression subtype,and stem cell-like subtype.Integration of bulk RNA-seq data and clinical prognosis data reveals that Scissor+cells associated with poor prognosis mainly distribute in the inflammation subtype and stem cell-like subtype.ASPH is highly expressed in Scissor+cells,and high ASPH expression correlates with poor prognosis in ICC patients.Subsequent experimental results further demonstrate that knockdown of ASPH significantly inhibits the proliferation,migration,and invasion abilities of ICC cells in vitro.Additionally,knockdown of ASPH markedly suppresses the growth and metastasis of ICC cells in vivo in immunocompetent mice.9.Tumor cells in metastatic lymph nodes tend to exhibit a mesenchymal phenotype,with enhanced epithelial-mesenchymal transition(EMT).These tumor cells in metastatic lymph nodes show high expression of MHC Ⅱ molecules but almost no expression of co-stimulatory molecules involved in T cell activation,indicating a lack of the second signal required for T cell activation.Furthermore,tumor cells in metastatic lymph nodes exhibit high expression of MAL2 molecules.In vitro experiments demonstrate that MAL2expression has no significant impact on ICC growth in T cell-deficient nude mice but significantly inhibits ICC growth in immunocompetent mice.Conclusion:Through comprehensive analysis of single-cell transcriptome sequencing results from ICC patient tumor tissues,adjacent liver tissues,and lymph node tissues(metastatic and non-metastatic),we have uncovered significant heterogeneity in the ICC tumor microenvironment.CD8+T cells exhibit highly active amino acid metabolism in metastatic lymph nodes,while Treg＿FOXP3 T cell subtypes and SPP1+Macrophage subtypes infiltrate to form an immune-suppressive tumor microenvironment in metastatic lymph nodes.Tumor cells in metastatic lymph nodes tend to adopt a mesenchymal phenotype with enhanced EMT.These tumor cells in metastatic lymph nodes express high levels of MHC Ⅱ molecules as"bait"molecules to induce CD4+T cells into a state of immune incompetence.Additionally,high expression of MAL2 molecules promotes ICC growth in immunocompetent mice,suggesting potential immune escape mechanisms during ICC lymph node metastasis.Overall,this study provides a deeper understanding of the changes in the microenvironment during ICC lymph node metastasis and potential immune escape mechanisms,offering broader insights and theoretical basis for developing more effective therapeutic targets.