Revealing Tumor Subclonal Evolution And Heterogeneity In Primary Breast Cancer Based On Single-cell Transcriptomics Sequencing And TCGA Multi-omics Datasets

Breast cancer was characterized for its high degree inter-tumor and intra-tumor heterogeneity,which poses a huge challenge to clinical diagnosis and treatment evaluation.The application of single-cell RNA sequencing(sc RNA-Seq)technology paved the way to study the subclone heterogeneity,the mechanism of drug resistance,the plasticity of the immune microenvironment,and the proposal of new clinical treatment options for breast cancer.However,the current studies of single-cell transcriptome technology in breast cancer mainly focuses on the tumor immune microenvironment and the gene expression heterogeneity,the research on the heterogeneity of subclonal and evolution routine of breast cancer is remain incomplete.This study aims to reveal the subclonal evolutionary routine,mutational and expression pattern heterogeneity of breast cancer using sc RNA-Seq datasets,so as to provide data analysis and theoretical support for the pathogenesis and development of breast cancer.In this study,based on a primary,multi-subtype breast cancer dataset from the GEO database,we constructed a single-cell transcriptome cell map,inferred copy number variation(CNV)base on sc RNA-seq datasets,which is validated by whole exon sequencing dataset.We then defined tumor subclones and construct the evolutional routine of each tumor sample based on the CNV patterns.We build up the Single nucleotide variant(SNV)calling pipeline for sc RNA-seq dataset and revealed the SNV landscape of tumor patients and its subclones.It is found that mitochondrial mutation such as MT-RNR2 is an early and shared mutation event and can be used as a potential marker to distinguish tumor cells from normal cells,and the activation of cytosine deaminase in the AID/APOBEC family of mutation signature may attribute to the occurrence of breast cancer.We also defined the subclone and construct the evolutionary trajectory base on SNV pattern,and found that there is a low concordance of subclonal classification(ARI=0.36)between the two results for the first time.Subsequently,we integrated the gene expression,pathway enrichment and transcriptional regulation patterns of tumor subclones with the subclonal classification results,and found that the expression patterns of subclones were basically corresponding to the results of subclonal classification based on CNV rather than SNV.The results of transcriptional regulation analysis showed that the occurrence and development of breast cancer may be related to the upregulation of transcription factors such as TFAP2 C,RBBP5,RAD21 and SOX4.Consistent clustering and gene enrichment analysis indicated that functional heterogeneous tumor subclusters may be formed during the occurrence and development of breast cancer,which are related to tumor migration or metastasis,proliferation and division,and tumor immune interactive,respectively.In cell-cell interaction analysis,we found that ligand-receptor pairs such as SPP1-CD44 and SPP1-PTGER4 were involved in the interaction of macrophages with other cells,and tumor-immune interactive subcluster has stronger interactions with macrophages and T lymphocytes through ligand-receptor pairs such as ACKR2-CCL4 and ACKR2-CCL5.In addition,TCGA breast cancer genomics and transcriptomics dataset were used to validate the results of the study.TTN,TP5,PIK3 CA,HMCN1 MUC16 gene mutations was consistent with TCGA breast high-frequency top10 gene mutations,most of the key transcription factors such as TFAP2C、RBBP5、SOX4 and RAD21 and differentially expressed genes such as ASCL1,DLK1 and EN2 detected in this study were significantly activated or upregulated in TCGA tumor samples,indicating that the findings of this study are highly consistent with the results of BRCA population samples.In conclusion,based on the public sc RNA-Seq and TCGA multi-omics datasets,this study used various bioinformatics analysis methods to study the tumor evolution,mutational landscape and expression heterogeneity of breast tumor subclone,which is of great significance to understand the occurrence and evolution of breast cancer.The findings of this research will provide novel insights for evaluating the types and abundances of different subclones carried by breast cancer patients by using sc RNA sequencing,it’s promising to research and develop more precise clinical therapies in the future.