Analysis Of Breast Cancer Stem Cell Genome Mutation Via Single Cell Sequencing

1.Background Cancer stem cells(CSCs)are a group of cells with the ability of self-renewal and tumorigenic potential.CSCs are the main factor causing intra-tumor heterogeneity.These cells are resistant to commonly applied treatment such as radiotherapy and chemotherapy,and are often an important causing factor of drug resistance and metastasis in tumor patients.Latest Research shows that,the origin of cancer stem cells may be related to epithelial-mesenchymal transformation(EMT),tumor microenvironment hypoxia,chronic inflammation,and metabolic reprogramming.Based on the functional characteristics of cancer stem cells and the tricky problem of clinical drug resistance,as well as the unclear mechanisms of stemness mechanism,it is particularly important to explore the mechanism regulating cancer stemness.Current findings indicate that there are copy number variants(CNVs)in the cancer stem cell genome.For example,it has been proved that there are significantly more CNVs in glioma stem cells than in non-glioma stem cells.Moreover,in glioma stem cells,a considerable number of gene CNVs are associated with changes in their corresponding expression levels.In addition,some cancer stem cell genomic mutations exist,such as MLF2 R158 W,RPL39 A14 V,HN1L P20 L,and HN1 L A106V in breast cancer lung metastasis,and EML4-ALK fusion in lung cancer stem cells.However,most of the reports see the tumor as a whole,finding mutations in one or some genes,and then exploring whether these mutations can induce stemness.In vitro spheroid formation model of tumor cells is used as the enrichment model of cancer stem cells.Along with the development of the single cell sequencing technology,we can separate breast cancer cell as single cells.On the basis of Multiple Displacement Amplification(MDA)method,single cell genome can be amplified and sequenced.This approach enables us to accurately explore the breast cancer stem cells’ genome in depth with high accuracy.Therefore,we choose single cell spheroid formation model to enrichment for breast cancer stem cells,after using MDA amplification techniques for single-cell genome amplification,multiple PCR technology to prepare target library,and targeted sequencing,breast cancer stem cell genome mutation can be characterized.2.Methods(1)(1)Select MDA-MB-231 human breast cancer single cells for in vitro spheroid formation assay,inoculated in a low-adhesion 96-well plate,and cultured with serum-free DMEM/F-12,supplemented with b FGF,EGF,B-27 for 7-10 days;(2)Spheroid stemness phenotype verification.(2)(1)Two non-cancer stem cells and three cancer stem cells were selected and their whole genome was amplified by Multiple Displacement Amplification(MDA);(2)Use Multiplex PCR to prepare target library,then sequence for the known tumor hot spot mutant area;(3)To find the gene loci with high-frequency of mutations in breast cancer stem cells in the target area.(3)(1)Gene functional enrichment analysis of high-frequency mutations in breast cancer stem cells.(4)(1)Single cell transcriptome sequencing;(2)Gene differential expression analysis;(3)Gene pathway enrichment analysis of differentially expressed genes in breast cancer stem cells.(5)(1)Kaplan meier survival analysis website was used to provide post-surgery follow-up information of breast cancer patients,and survival analysis was conducted with genes highly expressed in breast cancer stem cells.3.Results(1)(1)MDA-MB-231 human breast cancer single cells can form spheroid in vitro;(2)In vitro formed spheroids shows high level of SOX-2 and NANOG both in m RNA and protein levels,and shows significant enhanced invasion ability comparing to their non-sphere counterpart.(2)(1)Single breast cancer stem cell genome can be well amplified by MDA method;(2)Single breast cancer stem cell target sequencing data shows deep depth and standard base quality;(3)The mutation frequency of 97 genes such as AGXT2 in breast cancer stem cells was significantly higher than that of non-cancer stem cells.(3)(1)Gene enrichment analysis of high-frequency mutations in breast cancer stem cells shows significant enrichment in GRB2 events in ERBB2 signaling pathway.(4)(1)Single cell transcriptome sequencing yields standard data with high quality;(2)Gene differential expression analysis shows 217 differentially expressed genes;(3)Gene pathway enrichment analysis of differentially expressed genes in breast cancer stem cells shows significant enrichment in “lipid metabolism” and “Peptidyl amino acid modification”.(5)(1)Kaplan meier survival analysis website was used to provide post-surgery follow-up information of breast cancer patients,and survival analysis was conducted with genes highly expressed in breast cancer stem cells.Results shows that genes highly expressed in breast cancer stem cells such as SAR1 A is related to clinical poor prognosis in patients after surgery.4.Conclusions(1)In vitro single cell spheroid formation model can effectively enrich breast cancer stem cells(BCSCs);(2)The mutation frequency of 97 genes such as AGXT2 in breast cancer stem cells was significantly higher than that of non-breast cancer stem cells(NBCSCs);(3)97 mutant genes of breast cancer stem cells were significantly correlated with GRB2 events in ERBB2 signaling pathway;(4)m RNA levels of breast cancer stem cells show significant differences in gene m RNA expression levels;(5)Highly expressed genes in breast cancer stem cells indicates poor clinical prognosis in patients.