| Background&Aims:Primary liver cancer(PLC)is highly heterogeneous.This heterogeneity is demonstrated by the formation of cell clusters with different molecular and functional characteristics in tumor cells during tumorigenesis and evolution.At present,it is believed that this heterogeneity from tumor and tumor microenvironment(TME)is a key factor in tumor lethality,immune and targeted therapy failure,and drug resistance.Therefore,accurate assessment of the heterogeneity of PLC is extremely important for developing effective therapies and prolonging patient prognosis.Tumor heterogeneity runs through the whole process of tumor evolution in both temporal and spatial heterogeneity dimensions.In PLC,temporal heterogeneity refers to the differences in tumor cells at different time stages,while spatial heterogeneity includes three characteristics,namely tumor heterogeneity between different patients,heterogeneity between different tumor nodules in the same patient,and heterogeneity in different regions within the same tumor nodules in one patient.Therefore,analyzing the tumor evolution process of PLC requires a comprehensive assessment of PLC heterogeneity from different dimensions of time and space.The widespread use of single cell RNA-seq(sc RNA-seq)technology,which has been developed in recent years,has revealed the heterogeneous characteristics of PLC at the single cell level,including tumor cell typing,immune cell infiltration,tumor-associated stromal cells,and other aspects.However,spatial information of tumor cells is lost during the preparation of tissues into single-cell suspensions,which makes the study of tumor spatial heterogeneity difficult.The recently developed spatial transcriptomics(ST)technology could overcome this shortcoming of sc RNA-seq.By positioning histological cryosections on arrayed reverse transcription primers with unique positional barcodes,ST provides high-quality genome-wide transcriptome data with intact two-dimensional positional information and achieves a resolution of 55?m in diameter per capture spot.The aim of this project is to study the spatial heterogeneity characteristics of PLC by using high-resolution ST technology,to find the cellular and molecular characteristics of tumor lesions and their TME in different spatial locations,and to provide new therapeutic strategies for the treatment of PLC.Methods:1.Tumor tissues were collected from 7 patients with PLC,including 4 cases of hepatocellular carcinoma(HCC),1 case of cholangiocarcinoma(ICC),1 case of combined hepatocellular cholangiocarcinoma(c HC)and 1 case of HCC with portal vein carcinoma thrombus;subsequently,a total of 21 samples of these tumor tissues at different spatial locations were obtained for ST sequencing and analysis to fully characterize the spatial heterogeneity of PLC;2.To characterize the heterogeneity of TME in the leading-edge area,we evaluated the difference in immune cell distribution and parenchymal cell function in normal liver tissue and tumor tissue on both sides of the tumor border line;3.To characterize the intratumoral heterogeneity of PLC tumors,we evaluated the dominant gene expression,cell function,prognosis,copy number variations(CNV)and ligand-receptor interactions of the clusters in tumor;4.By analyzing the expression of different marker genes of cancer stem cells(CSC)in each sample,we screened and veried the CSCs associated with tumor invasion and migration;5.We screened for a gene set that could be used to identify tertiary lymphoid structures(TLS)based on H&E staining and ST data;subsequently,we validated the feasibility for predicting tumor prognosis in a public database.Results:1.The intratumor clusters in PLC have two spatial distribution patterns:regional distribution and staggered distribution.Different spatial patterns are associated with different clonal evolutionary processes;2.The tumor fibrous capsule correlates with the heterogeneity of the immune microenvironment in the leading-edge area of PLC.Specifically,when tumor capsule present in the leading-edge area,the scores of immune cells in normal liver tissues outside the capsule were more than that in tumor tissues;when tumor capsule absent,the degree of infiltration of immune cells in the leading-edge area was enhanced accompanied by an increase in exhausted T cells,and the distributions of immune cells on both sides of the tumor capsule were more disordered;3.Different clusters within the tumor have different dominant gene expression,cell function,prognosis,and cloning sources,accompanied by extensive ligand-receptor interactions in their contact area;4.The enrichment of CD47~+and PROM1~+CSCs is positively correlated with tumor invasion and migration of HCC;5.We have developed a new gene set TLS-50 for the identification of tertiary lymphatic structure,and verified that the high-scoring TLS-50 is associated with a better prognosis of HCC in the TCGA database.Conclusion:In this study,we found two spatial distribution patterns of tumor clusters in PLC:regional and staggered distribution.In the leading-edge area,the presence of fibrous capsule was correlated with immune cell infiltration and cluster distribution patterns.Within the tumor,different tumor clusters have distinct genetic characteristics,but there is significant cell-cell communication in their contact area.The enrichment of CD47~+and PROM1~+CSC is related to the invasion and migration of HCC tumors.We developed a new TLS identified gene set(TLS-50),and HCC prognosis can be assessed by evaluating intra-tumor TLS-50scores. |
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