Investigating Tumor Spatial Heterogeneity And Stemness Of Hepatocellular Carcinoma By Single-cell RNA Sequencing

Objective:Liver cancer is one of the most common malignant tumors in China,and it’s the sixth in the malignant tumor incidence and the third in tumor mortality globally.Among them,hepatocellular carcinoma(HCC)is the main type of primary liver cancer.In recent years,people have made great progress in the research of liver cancer,but the overall survival of liver cancer patients has not improved significantly.The 5-year survival rate of patients with advanced liver cancer is less than 10%.Tumor heterogeneity is the main reason for tumor resistance,metastasis,and recurrence.Cancer stem cells(CSC)generated with the progression of cancer have the ability to proliferate and differentiate indefinitely,produce different subclonal progeny,and promote tumor heterogeneity.In addition to the accumulation of self-mutation,the generation of CSC is also inseparable from the role of the tumor immune microenvironment(TIME).In this study,single-cell transcriptomic sequencing technology(sc RNA-seq)was used to explore the spatial heterogeneity of liver cancer,and to find that DNA Topoisomerase II Alpha(TOP2A)maintains the stemness of HCC.This study is mainly divided into three parts:Investigating the spatial heterogeneity of HCC by sc RNA-seq;mechanism of TOP2A promoting HCC progression through Hippo signaling pathway;network pharmacology screening of effective drugs against TOP2A.Methods:(1)After sequencing HCC samples,we performed data quality control.Individual cell types are identified from their RNA-seq data through the marker genes they express.We selected epithelial cells from the main cell clusters for subsequent analysis.Tumor cells were identified from epithelial cells by cellular heterogeneity and copy number variation.Functional clustering of all tumor cells was performed by consensus non-negative matrix factorization(c NMF).Intratumor heterogeneity analysis and differentiation potential entropy were used to detect the stemness of tumor cell clusters.Subsequently,evolutionary changes in different tumor cell clusters were detected by pseudo-time trajectory analysis.The CSC-specific expression genes were validated by the TCGA cohort.(2)T cells,B cells,and myeloid cells were selected in TIME for sc RNA-seq analysis.After dimensionality reduction,the distribution of T cells and B cells in different spatial locations was compared.Then,developmental analysis and functional enrichment analysis were performed for T and B cell clusters.Myeloid cells are divided into subclusters based on the marker genes.Among them,macrophages undergo more detailed division and functional analysis.Cell communication assays are used to detect interactions between tumor cells and other cells.Co-culture experiments verified the stemness-promoting effect of macrophages on tumor cells.(3)TOP2A mutation ratio was detected by c Bio Portal.Weighted correlation network analysis(WGCNA)predicts TOP2A-related biological functions.The TOP2A m RNA and protein expression levels in HCC tissues and cell lines were detected by q RT-PCR and Western blot.CCK-8,clone formation,wound healing,Transwell,and flow cytometry was performed to detect the role of TOP2A in HCC cell proliferation,invasion,migration,cell cycle,and apoptosis.Western blot was used to detect the expressions of cell proliferation-related proteins,cycle-related proteins,and epithelial-mesenchymal transition-related proteins.A subcutaneous xenograft model was established to verify the effect of TOP2A on tumorigenesis in vivo.(4)We used bioinformatics to predict TOP2A downstream signaling pathways and used pathway inhibitors to demonstrate whether TOP2A promotes the tumor-malignant phenotype of HCC through key pathways.Then,we used STRING to find the mediators that TOP2A acts on the signaling pathway.Nucleocytoplasmic isolation and Western blot were used for specific mechanistic research.Co-immunoprecipitation(co-IP)was used to verify the specific mechanism of action.Results:(1)We captured 82,030 cells by sc RNA-seq from 15 samples,categorized into5 major types,including epithelial cells,T cells,B cells,myeloid cells,and endothelial cells.Subsequently,we identified tumor cell clusters from epithelial cells characterized by heterogeneity and copy number variation.Among them,we found that tumor cells have spatial distribution characteristics.Some tumor cell clusters(Cluster 12,15,17,20)showed high stemness and high differentiation potential and were considered as CSC.c NMF confirmed CSC clusters function and discovered immune-related tumor cell clusters.In addition,tumor-initiating cells(TIC)with immunomodulatory effects and TOP2A~+CENPF~+CSC in the late stage of development were found in tumor cell development analysis,both of which were distributed at the tumor margin.(2)The immune cells in TIME mainly include T cells,B cells,and myeloid cells.T cells are divided into 11 clusters;among them,the infiltrating T cells in the tumor are mainly proliferative exhausted T cells(Tex)in the late stage of development.B cells are divided into 7 clusters;Plasma＿2,4,and 6 existed in the tumor,and their functions are enriched in oxidative phosphorylation,which is patient-specific.The dimensionality reduction of myeloid cells is 11 clusters,which are divided into 6categories,among which macrophages and Kupffer cells are spatially specific;macrophages are divided into two subtypes,M1 and M2,according to the expression of Marker genes and functional enrichment.Cell communication analysis found that CD168~+M2-like macrophages had strong interactions with tumor cells and CSC subsets;TIC could recruit CD168~+M2-like macrophages through chemokines.Co-culture experiments demonstrated that M2 macrophages could promote the up-regulation of tumor stemness genes in HCC cells.(3)TOP2A has a high mutation rate in liver cancer and is closely related to cell cycle,DNA replication,and other functions.We found that TOP2A was upregulated in HCC tissues and cell lines.TOP2A knockdown inhibited the proliferation,invasion,and migration of HCC cells,and changed the HCC cell cycle and promoted cell apoptosis.Knockdown of TOP2A inhibited the growth of subcutaneous tumors in nude mice.TOP2A overexpression promoted HCC cell proliferation,invasion,migration,and inhibited cell apoptosis.In vivo experiments demonstrated that up-regulation of TOP2A promoted the growth of subcutaneous tumors in nude mice.Altering TOP2A expression affected the expression of proliferation-related proteins,cyclins,and epithelial-mesenchymal transition-related proteins in HCC cells.(4)Functional enrichment found that TOP2A exerted biological effects through the Hippo pathway.We found that Verteporfin(VP)inhibited the proliferation,invasion and migration of HCC cells,and reversed the malignant phenotype of HCC tumor cells caused by TOP2A overexpression.Knockdown or overexpression of TOP2A alters the expression level of YAP1 in the Hippo signaling pathway.Nucleocytoplasmic separation and co-IP experiments showed that the overexpressed TOP2A in the nucleus adsorbsβ-catenin,resulting in the release of YAP1 into the nucleus and promoting the transcription of genes downstream of the Hippo pathway.Furthermore,TEAD4knockdown reduced TOP2A expression.TOP2A and hippo form a loop.Conclusion:(1)There is spatial heterogeneity of tumor cells in HCC solid tumors;tumor cell clusters included early-stage immunomodulatory TIC and late-stage LCSC that promote tumor heterogeneity.(2)In the tumor immune microenvironment,T cells,B cells,and myeloid cells have spatial distribution characteristics;most of the infiltrating immune cells in the tumor are highly proliferative immune-exhausted cells;among them,CD168~+macrophages present the M2 phenotype,which promotes tumor cell stemness.(3)TOP2A is abnormally highly expressed in tumor tissues and HCC cell lines,and plays a malignant role in promoting tumor cell proliferation,invasion,migration,cell cycle,and apoptosis.(4)TOP2A affects the Hippo signaling pathway throughβ-catein and promotes HCC progression.