Hypoxic Microenvironment Regulates Key Components And Signaling Pathways In The Malignant Phenotype Of Intrahepatic Cholangiocarcinoma

Background and objectiveIntrahepatic cholangiocarcinoma(ICC)is a malignant tumor originating from the epithelial cells of the secondary bile duct and above.In recent years,the morbidity and mortality of ICC have been rising,especially in China,Vietnam and other Asian countries.In general,ICC is highly malignant and difficult to diagnose in early stage.Besides,ICC prefers lymphatic metastasis to hematogenous metastasis.Currently,surgical treatment is the only curable way for cholangiocarcinoma,but the 5-year survival rate after radical surgery is only 15%～40%.With the concept of precision medicine,it is urgent to understand and prevent ICC from the molecular level and tumor microenvironment.In the 2019 edition of WHO Classification of Tumors of the Digestive System,ICC is divided into perihilar and peripheral subtypes based on histological features and cell origins,replacing the traditional classification based on gross pathology,degree of differentiation or mucin phenotypes.Clinical evidence indicated that the patients with perihilar ICC tended to have lymphatic metastasis and poorer prognosis,but there is little understanding of these two types of cholangiocarcinoma at the molecular level,and there are no guidelines or research data for the treatments in the perihilar and peripheral ICC.The difficulties in previous research lie in obtaining clinical samples(the incidence rate is relatively low and some patients have lost the chance of surgery when they are diagnosed),the lack of suitable animal models(no murine cell line),and the understanding of this tumor is based on bulk-seq(little is known about intra-tumor heterogeneity).To address the above difficulties,relying on the clinical sample resources of the Eastern Hepatobiliary Surgery Hospital(Naval Medical University)and the experimental platform of the National Center for Liver Cancer,we applied hydrodynamic tail vein injection to create an orthotopic ICC model in mice.With the help of single-cell RNA sequencing and spatial transcriptomics,we explored the internal heterogeneity and subtype characteristics of ICC,and verified the findings in vivo and in vitro.In summary,this study aimed to explore the characteristics of the intratumoral microenvironment of the perihilar and peripheral ICC,as well as and the mechanism of lymphatic metastasis.We also analyzed the impact of the tumor microenvironment on the overall survival of patients.This study will provide theoretical and experimental basis for the guidance of precise treatment in ICC.Methods1.Collect tumors,para-tumor tissues and lymph nodes of patients with ICC.Apply single-cell RNA sequencing and spatial transcriptome sequencing to sequence the cell suspensions or frozen sections.2.Identify benign and malignant epithelial cells by the Infer CNV algorithm.Apply the monocle pseudo-trajectory algorithm to analyze the cell fates of the epithelial cells.3.Annotate cluster by using canonical cell markers.Analyze the differences between perihilar and peripheral malignant cells by using the GSVA algorithm.Reconstruct the simultaneous gene regulatory network of tumor cells by using the SCENIC algorithm.4.Culture cells in hypoxic chamber and detect the expression of CD47 and HIF-1α in tissue samples by immunohistochemistry and western blot.5.Compare the distribution of specific cell subgroups in tumors and adjacent tissue to identified the ones which are positively correlated with the abundance of tumor cells.Apply the Cell Chat algorithm to study the receptor-ligand relationship.6.Treat tumor cells with galectin-1 recombinant protein,and observe the tumor proliferation,apoptosis and metastasis by flow cytometry,CCK8 experiment,scratch test,migration experiment and q PCR.7.Interfere the expression of LGALS1 in HFF-1 cells by si RNA,observe the proliferation and migration of tumor cells in a co-culture system.Detect the level of cytokines in the culture supernatant by ELISA.8.Apply flow cytometry and multicolor immunofluorescence to observe the distribution of specific cell populations in paraffin embeded samples of ICC.9.Analyze the correlation between specific cell clusters and the prognosis of patients by using the GEO datasets.Analyze the pan-tumor expression of LGALS1 and SIRPA in TISCH2 and TCGA databases.10.Apply erythrophagocytosis and E.coli phagocytosis experiment to study the in vitro phagocytosis function of macrophages under hypoxic conditions.11.Apply Maraviroc,a CCR5 inhibitor,to study the tumorigenesis effect of CCL5/CCR5 axis in macrophages.12.Bear tumor subcutaneously in nude mice to study the effects of LGALS1,CD47 and SIRPα.Detect cell proliferation and apoptosis as well as the quantitative changes of cell components by immunohistochemistry and flow cytometry.13.Apply the XGOF algorithm to identify key cellular components and pathways in the ICC microenvironment,and conduct related analysis around hypoxia pathways and macrophages.14.Apply spatial transcriptome technology to study the colocalization between SIRPα～+ macrophages and lymphatic endothelial cells.15.Apply tube formation experiment to study the effect of macrophages and tumor cells in promoting lymphangiogenesis.Results1.The distribution of cell populations in ICC is highly heterogeneous.The infiltration of T cells,NK cells and dendritic cells in tumor tissues is reduced,while the infiltration of monocytes,macrophages and fibroblasts in tumor tissues is increased.2.We identified the malignant cells with high copy number variation.Trajectory analysis showed that the branch started from normal epithelial cells with the expression of ALB,and divided into two cell fates.3.Perihilar ICC differentially expressed CD47,MMP7,MUC1,TFF1 and CDH1.CD47 high cells are regulated by HIF1 A,a transcription factor.The peripheral subtype upregulated the WNT-CATENIN pathway.The perihilar intrahepatic cholangiocarcinima upregulated the KRAS pathway,as well as the glycolysis and hypoxia pathways.4.Perihilar ICC presented a more hypoxic state,and hypoxia regulated the expression of CD47 through HIF-1α.5.LGALS1～+ fibroblasts and SIRPα～+ macrophages were highly expressed in ICC,and the expression of LGALS1 and SIRPA was correlated with the abundance of tumor cells.6.LGALS1 inhibited tumor cell apoptosis,increased S-phase cells and reduced the ratio of G2/M-phase cells.LGALS1 also promoted tumor invasion and migration by upregulating CCR2,ADAM15 and β-integrin.7.Fibroblasts that highly expressed LGALS1 promoted the migration and invasion of tumor cells by secreting cytokines MMP11,MCP-1 and IL-6.8.SIRPα～+ macrophages were highly expressed in the tumor tissue and PBMCs of the patients with ICC.9.Patients with severe infiltration of LGALS1～+ fibroblasts and SIRPα～+ macrophages tended to have a poorer prognosis.The abundance of LGALS1～+ fibroblasts and SIRPα～+ macrophages correlated with tumor stage significantly.10.Hypoxic environment inhibits the non-specific phagocytosis functions of macrophages to pathogens and cells.11.Tumor cells recruited SIRPα～+ macrophages through CCL5/CCR5 axis,which facilitated the tumor progression.12.Blockade of the expression of LGALS1,CD47 and SIRPα in the xenograft tumor bearing mice could inhibit tumor cell proliferation and promote cell apoptosis.13.Correlation analysis of cell components by XGOF found that effector T cells,activated macrophages and hypoxic cells were highly correlated with ICC.Immune response correlation ontology analysis found that the biological process of macrophage migration was significantly enriched in cholangiocarcinoma.14.Spatial transcriptomics reveals co-localization of SIRPα～+ macrophages and lymphatic endothelial cells.15.SIRPα～+ macrophages could promote tube formation of lymphatic endothelial cells.ConclusionICC is a highly heterogenous malignancy,and to investigate the intratumor heterogeneity of ICC,we analyzed single-cell RNA sequencing data of the primary tumor,adjacent normal tissues and lymph nodes from treatment-na?ve patients.In this study,we found that the hypoxic microenvironment in the perihilar ICC induced the expression of CD47 on the surface of tumor cells.Tumor cells recruited SIRPα～+ macrophages through CCR5/CCL5 axis and mediated an immunosuppression effect.The co-localization between SIRPα～+ macrophages and lymphatic endothelial cells was revealed by spatial transcriptomes.The culture supernatant of tumor cells and macrophages after hypoxic culture could significantly promote lymphangiogenesis,speculating that this may be the reason of why the lymphatic metastasis of perihilar ICC is more frequent than peripheral ICC.In the tumor microenvironment,a group of fibroblast subpopulations marked by LGALS1 have an increased proportion in tumors and up-regulate hypoxia-related pathway genes.The secreted protein galectin-1 encoded by the LGALS1 gene regulates the cell cycle and promotes tumor progression and metastasis,leading to poorer prognosis.Blockade the expression of LGALS1,CD47 and SIRPα on corresponding cells in vitro and in vivo could inhibit tumor proliferation and promote cell apoptosis.Our work will further the understanding of the heterogeneity among ICC tissues and provide novel strategies for the treatment of ICC by targeting the components in the tumor microenvironment.