Spatial Transcriptomics Sequencing Reveals Tumor Microenvironment Heterogeneity In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common histological type of primary liver cancers.Its morbidity and mortality are among the forefront in the world,and the medical burden remains heavy.Postoperative recurrence and metastasis of HCC are the core cause of high mortality at present,and HCC heterogeneity is the key to the discrepency in the prognosis and drug sensitivity of patients with advanced liver cancers,which is also an important direction of accurate treatment and prognostic analysis of HCC.In recent years,the emerging spatial transcriptomics technology is an important breakthrough technology after single-cell transcriptomics due to its unique advantage of capturing spatial information of gene expression,and has been widely applied to study spatial gene expression patterns in the heterogeneity and microenvironment of various solid tumors.In this study,10×Genomics Visium spatial transcriptomics sequencing was performed on HCC tissue samples taken from three different sites of one HCC patient,and each tissue features with clear boundaries between distinct carcinoma,para-carcinoma and tumor boundary regions.We first classified the 12,545 captured regions(spots)described by sequencing into 20 subsets,and investigated the distribution of these subsets in three tissue specimens.UMAP dimension reduction analysis,n Count number,cellular marker gene distribution,differentially expressed gene analysis and other bioinformatics techniques were used to explain the overall landscapes of spatial expression patterns and the intratumoral heterogeneity within the three tissues.Subsets with malignant phenotypes were identified by cell cycle phase and epithelial-mesenchymal transformation analysis.The heterogeneity of HCC is closely related to the tumor microenvironment(TME),for example,tumor-associated macrophages(TAMs),T cells and B cells and other immune cells play an important role in the progression of HCC,however,the spatial relationship between HCC heterogeneity and tumor microenvironment is still unclear.In this study,we first explored the relationship between intratumoral heterogeneity(ITH)and spatial gene expression through the pseudotime analysis of subsets and gene expression.The tumor core region is defined as the set of subgroups with high n Count abundance and at the terminal stage of pseudotime differentiation.Through differential expression gene analysis(DEGA)in tumor core region,we believe that the immunosuppressive microenvironment formed in this region is caused by the significantly high expression of chemokine CCL15.Further mechanism studies in vitro show that CCL15 promotes the formation of immunosuppressive microenvironment via recruitment and polarization of M2-type macrophages,thus affecting the prognosis of HCC patients.Clinical tissue analysis verifies that CCL15 and M2-type macrophage marker CD163 are significantly correlated with the prognosis of HCC patients.The higher the expression level of CCL15 or CD163,the worse the prognosis of HCC patients.HCC patients with high expression of combined CCL15and CD163 have the worst overall survival(OS)and recurrence-free survival(RFS).Received operator characteristic curve(ROC)analysis indicates that the combined application of CCL15 and CD163 have higher clinical predictive value for the prognosis of HCC patients.The interaction between inflammatory factors,immune cells and HCC cells in the TME as well as their functions and role in the progression of HCC are still not fully understood.In this study,we also evaluated the infiltration degree and spatial distribution of various immune cells including T cells,B cells,NK cells and myeloid cells in 20 subsets.Four subsets with high abundance of immune cells are defined as immune infiltration enrichment(IIE),among which T cells and B cells are the most dominent.In contrast to the tumor core region,the differentially upregulated genes are characterized by immunoglobulin family genes and two chemokines,CCL19 and CCL21.Further analysis shows that there is a strong correlation between CCL19 and CCL21 in terms of original sources,biological functions and spatial expression patterns.High expression of combined CCL19 and CCL21 can predict better prognosis of HCC patients.We then constructed adeno-associated viruses(AAV)with overexpressing CCL19 and CCL21 and explored them in subcutaneous tumor xenograft and drug-induced liver cancer animal models.Flow cytometry,western blotting,quantitative real-time PCR and tissue immunofluorescence experiments further demonstrate that CCL19 and CCL21 could inhibit HCC growth by affecting the infiltration degree of CD3~+T cells and CD20~+B cells,and hence predict a good prognosis.Conclusively,these results further indicate that the spatial expression patterns of some key molecules,such as CCL15,CCL19,and CCL21,in specific regions of HCC affect the infiltration of different immune cells and their respective functions,jointly promote the formation of intracomatous heterogeneity in the microenvironment,and hence affect the prognosis of HCC patients.In recent years,many studies have shown that m6A modification plays an important role in the treatment and progression of HCC.However,the functions of relatively modified enzymes or proteins,especially the YTHDF family as well as their expression and mechanism of action in HCC,are still controversial.Consequently,we first analyzed the spatial expression pattern of 21 m6A modified enzymes or proteins in three tissue specimens,and found that the expression of YTHDF family in HCC is closely related to each other in this study.Further mechanism studies,such as gene-expressing cells,luciferase reporter assay,RNA immunoprecipitation and RNA stability experiment have shown that YTHDF1 regulates the expression of YTHDF2 by affecting the stability of YTHDF2 m RNA,but its regulation effect on YTHDF3 is not obvious.We next confirm that both YTHDF1 and YTHDF2 are upregulated in 30 pairs of HCC tissues,and the expressions of them are indeed strongly correlated either at the protein level or the transcriptional level.Immunohistochemical staining analysis was further performed on clinical tissue arrays.We found that high expression of YTHDF1 and YTHDF2respectively in carcinoma tissues are associated with poor prognosis.Combinatively high expression of them is associated with aggressive clinicopathological features and predicts the worst prognosis.Combined use also shows better value in predicting the prognosis of HCC patients.In conclusion,this study performs ST sequencing on the three diversely-located tissue pieces from one HCC case and excavates spatial gene expression pattern based on the bioinformatics analysis.We also determine the tumor core region and immune infiltration enrichment region which are associated with immune microenvironment and explore the innate relations between gene expression patterns and tumour heterogeneity within the region.Key molecules such as CCL15,CCL19 and CCL21 have been discovered,and their biological functions and clinical significances have been preliminarily verified through multi-layer experiments of molecular,cell,zoopery,and clinical tissue samples.In addition,we also discuss the basic function of the controversial YTHDF family and their roles in HCC.Based on spatial transcriptomics technology,this study preliminarily reveals intratumoral heterogeneity of HCC and its relationship with immune microenvironment,emphasizes the clinical significance of gene expression patterns and spatial heterogeneity.This study provides some insights into the internal relations of the YTHDF family and their functional roles in HCC,and provides theoretical basis for developing new prognostic markers and therapeutic targets for HCC.