Heterogeneity Of Breast Cancer Revealed By Single Cell Sequencing

Backgrounds: Breast cancer has become the most common cancer and radiotherapy plays an important role in its treatment.Different tumor tissues or tumor cells in the same tumor tissue responses to ionizing radiation(IR)differently.Our understanding on transcriptional regulation of tumor cells responding to IR has mostly come from bulk sequencing.However,bulk sequencing mainly aims at a large number of cells,which can only reflect the average characteristics of the main cell populations.In addition,tumor has obvious heterogeneity,which is not only reflected in the transcriptome level,but also in the differences of copy number variation and abnormal DNA methylation among tumor cells.Tumor heterogeneity will make the heterogeneous responses to radiotherapy more complex.Single cell sequencing is a powerful tool to help understand the heterogeneity.Therefore,in this study,we first explored the heterogeneous cellular responses to IR and effect of the ATM gene on the heterogeneous responses using single cell transcriptome sequencing in a relatively homogeneous cell line.Then,we investigated the heterogeneity at multiple levels in breast cancer tissues with single cell multi-omics sequencing.And this will lay a foundation for the study of heterogeneous responses to IR in breast cancer patients.Methods: Single cell transcriptome sequencing was performed in the breast cancer cell line MDA-MB-231 both with and without IR treatment.Single cells in each group were isolated with mouth pipetting.The modified Smart-Seq2 method was used for single cell library construction and single cell transcriptome sequencing.Then,the differentially expressed genes(DEGs)were found.The GO analysis and STRING network analysis were performed according to the DEGs.t-SNE analysis and GSEA analysis were used to analyze the heterogeneous responses to IR.To further understand how ATM,a major hub protein required for an optimal DNA damage response,affected the heterogeneous IR responses.We also knocked down the ATM gene for single cell transcriptome sequencing before and after IR and analyzed the response changes of ATM knockdown to IR at the single cell level.Finally,three significantly upregulated genes(MCM3,MCM4 and SLBP)after IR treatment were selected for functional verification.Then,in the heterogeneity study of breast cancer,the primary tumor and positive lymph nodes of breast cancer patients were analyzed at multiple levels(genome,transcriptome,DNA methylation)with sc Trio-seq2 method.Single cells were isolated with mouth pipetting.DNA and RNA from single cells were separated using magnetic beads.The DNA methylation library and RNA transcriptome library were constructed and sequenced for each single cell.Copy number variations,DNA methylation and gene expression were analyzed for each cell simultaneously.The heterogeneity characteristics at multiple levels of each single cell were further analyzed.Results: GO analysis of the DEGs between Control group and IR group showed that the upregulated genes were highly enriched in GO terms associated with G1/S phase and ribosome.While the downregulated genes were highly enriched in GO terms associated with antibiotic metabolism.And the STRING network analysis further confirmed the results of GO analysis.Single cell t-SNE analysis showed four clusters of breast cancer cells responding to IR in distinctive ways: Cluster 1 had the least change and was identified as a subpopulation of cells less-responsive or non-responsive to IR;Cluster 2 responded to IR by upregulating ribosome associated genes,while Cluster 4 upregulated both ribosome and G1/S phase associated genes;Cluster 3appeared only in irradiated cells,which implied that Cluster 3 might be a new subpopulation of cells appearing after IR treatment.In the absence of the ATM kinase,cells displayed much less transcriptional changes after IR.The number of DEGs and significantly DEGs decreased significantly.And Cluster 4 in wild-type cells,which changed the greatest number of genes in response to IR,was not present in the ATM knock-down cells.Functional analysis of upregulated genes induced by IR showed that the cells in sh MCM3,sh MCM4 and sh SLBP groups were more sensitive to IR than control group.The single cell copy number variation(CNV)analysis in breast cancer tissues only identified few CNV regions.The single cells of primary tumor and lymph node were mainly classified as one subclone,suggesting that CNVs were clonal at the genomic level.CNV pattern analysis showed the amplification of Chr 1q and deletion of Chr 9p and Chr 16 q.DNA methylation analysis showed different DNA methylation patterns of primary tumor and lymph node.The DNA methylation correlation and global methylation level of lymph node were significantly higher than those of primary tumor.Transcriptome analysis showed that the gene expression profiles of primary tumor and lymph node were significantly distinct,and they were divided into two different clusters,which was consistent with the results of DNA methylation.GO enrichment analysis showed that the upregulated genes in lymph node were mainly enriched in the GO terms of clinical basic body plasma membrane docking,regulation of neuron migration,TORC1 signaling,autophagy,etc.The downregulated genes in lymph node were mainly enriched in GO terms such as: extracellular matrix organization;cell morphogenesis involved in differentiation;actin filtration based process;muscle structure development,etc.Conclusions: Our single cell sequencing analysis has revealed a heterogeneous cellular response to DNA damage induced by IR and identified potential biomarkers of radiation sensitivity in breast cancer cells.The results also revealed the genetic,transcriptomic,and epigenetic heterogeneity and clonal evolution in human breast cancer patents.This will lay a foundation for studying the heterogeneous responses to treatment in breast cancer patients.