Study On The Mechanism Of Compound Sophora Flavescens Injection In The Treatment Of Triple-negative Breast Cancer Based On Single-cell Transcriptom

BackgroundBreast cancer is the most common female malignancy and the second leading cause of cancer-related death.Triple-negative breast cancer(TNBC)is the most malignant and aggressive subtype of breast cancer,which is pathologically featured by the lack of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER2)expression.TNBC as a highly heterogeneous breast cancer subtype has a high risk of early relapse and visceral metastasis,a shorter disease-free survival,and a dismal prognosis.Due to the lack of well-recognized therapeutic targets and enough treatment options,conventional chemotherapy remains the standard treatment for TNBC.Immune infiltration within the tumor microenvironment(TME)plays a crucial role in tumor development and could impact the clinical outcomes of cancer patients.Comprehensive dissecting of tumor-infiltrating immune cells would illuminate the mechanism of immune evasion in cancer,thus providing powerful opportunities to develop new therapeutic strategies.Single-cell technologies are now emerging as powerful tools in the field of cancer research.These technologies characterize the molecular state of each cell within a tumor,enabling novel exploration of tumor heterogeneity,the changes of cellular composition in the TME,and the cell state transitions that influence therapeutic response—particularly in the context of tumor immunotherapy.Compound kushen injection(CKI)is a widely used anticancer Chinese patent medicine in China,which is extracted from the roots of two medicinal plants Kushen(Radix Sophorae Flavescentis)and Baituling(Rhizoma Heterosmilacis)through standardized Good Manufacturing Practice(GMP).CKI alone or it combined with chemotherapy or radiotherapy has been widely used in the treatment of breast cancer and improved therapeutic and prognostic benefits.However,the immunoregulatory effects of CKI on the TME of TNBC are unclear.ObjectiveThis study aimed to comprehensively explore the mechanism of CKI for the treatment of TNBC by applying single-cell RNA-sequencing(scRNA-seq),bulk RNA sequencing(RNAseq),proteome,integrative bioinformatics and in vitro and in vivo experiments.Methods1 Integrative bioinformatics analysisThe RNA-seq data of CKI-perturbed TNBC cells were acquired from public databases.The differential expression genes(DEGs)were calculated,and the gene set variation analysis(GSVA)algorithm was used to explore significantly changed pathways regulated by CKI.The single sample gene set enrichment analysis(ssGSEA)algorithm was used to quantify immune cell abundance in TNBC patients,and these patients were classified into distinct immune infiltration subgroups by unsupervised clustering.Then,prognosis-related genes were screened from DEGs among these subgroups and were further overlapped with the DEGs regulated by CKI.Finally,a predictive immunotherapy signature of CKI on TNBC was constructed based on the LASSO regression algorithm to predict mortality risks of TNBC patients,and the signature was also validated in another TNBC cohort.2 In vitro and in vivo experimentsUltra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS)was conducted to identify the components of CKI.The MDA-MB-231 cell line was used to investigate the anti-cancer effect of CKI.The CCK-8,clone formation and wound-healing assays were applied to detect the effect of CKI on the proliferation,clone formation and migration of MDA-MB-231 cells.A mouse breast cancer model in BALB/c mice with 4T1 cells was established to evaluate the antitumor efficacy and toxicity of CKI combined with cisplatin(DDP)or paclitaxel(PTX).The effect of CKI combined with chemotherapy on tumor cell apoptosis was investigated by HE staining and TUNEL staining,and the in vivo safety of cotreatment was evaluated by HE staining.Flow cytometry was used to detect the effect of cotreatment on the lymphocyte populations in the spleens and tumors of 4T1 tumor bearing mice.3 Bulk RNA-seq and proteomic analysesThe CKI-treated MDA-MB-231 cells were used for bulk RNA-seq and proteomics experiments.The differential expression,gene set enrichment,survival and tumor immune infiltration analyses were used to systematically study the effect of CKI on the genes and proteins of MDA-MB-231 cells.RNA-seq was performed on the tumor tissues of 4T1 tumor bearing mice to observe the modulation of CKI plus chemotherapy on the TME of the mice.4 Single cell RNA sequencing analysisThe scRNA-seq data of TNBC patients receiving immunotherapy were acquired from public databases.The key cell subsets closely associated with treatment response were identified after performing multi-sample integration,dimension reduction,clustering,cell type identification and cell-cell communication analyses.Based on the genes related to CD8+T cells and treatment response,a prognostic signature was built based on the LASSO regression algorithm to predict relapse risks of TNBC patients.The droplet-based scRNA-seq technology was utilized to characterize the influence of CKI combined with PTX on the TME of breast tumor-bearing mice.The tumor tissues of the mice were prepared as a single cell suspension under standard tissue culture conditions,and scRNAseq was conducted using the ChromiumTM Single Cell 3’ Solution from 10x Genomics.The scRNA-seq data were analyzed by performing quality control,multi-sample integration,dimension reduction,clustering,differential expression analysis,cell type identification,repartition of T lymphocyte subsets,GSVA and GSEA.Results1 Study on the immunotherapeutic biomarkers for TNBC based on scRNA-seqTwo scRNA-seq datasets comprising TNBC patients receiving immunotherapy were collected from public databases.After performing multi-sample integration,dimension reduction,clustering and cell type identification,a total of 90747 cells with 12 cell types(including tumor,stromal and immune cells)in the BioKey cohort and 1 18339 CD45+immune cells with nine immune cell types in the GSE169246 cohort were identified.The proportion of CD8+ T cells in the patients with T cell expansion was significantly higher than that in the patients without T cell expansion during anti-PD-1 treatment.The proportion of CD8+T cells was significantly increased in the responders treated with anti-PD-L1+chemotherapy or chemotherapy compared to these patients before treatment.The results suggested that CD8+ T may play an important role in the response to cancer treatment.The strength of cell-cell communication in the patients with T cell expansion was significantly increased during antiPD-1 treatment,and the strength of cell-cell communication of the patients who had a partial response was significantly increased after anti-PD-L1+chemotherapy or chemotherapy.The CD8+ T cells were extracted for differential expression analysis among multiple groups,and 102 key genes related to therapeutic response were eventually obtained.Lastly,a signature comprising 10 genes associated with the response to immunotherapy in TNBC,which could predict the relapse risks of TNBC patients with a good performance.2 High throughput transcriptome data analysis reveals immunotherapy biomarkers of CKI for TNBCA total of 3692 DEGs were detected after analyzing the RNA-seq data of CKI-perturbed TNBC cells,and CKI significantly regulated the biological pathways correlated with cell cycle,metabolism and immune function.The relative intratumoral immune infiltration levels of 28 immune cells in TNBC patients were estimated by the ssGSEA algorithm.The patients were classified into three distinct immune cell infiltration patterns with the consensus clustering method,and then 1593 DEGs were detected among the subgroups,in which 304 genes were correlated with prognosis.The prognosis-related genes were further overlapped with the DEGs regulated by CKI,and eventually two genes with HR>1 that can be down-regulated by CKI and 26 genes with HR<1 that can be up-regulated by CKI were selected as key immune-and prognosis-related genes regulated by CKI.Lastly,a five-gene prognostic signature comprising two risky genes that can be down-regulated by CKI and three protective genes that can be upregulated by CKI was developed,and it showed a good performance in both training and test sets.3 The anti-tumor effect and mechanism of CKI on TNBC cell lines in vitroA total of 25 compounds in CKI were identified by UPLC-MS analysis.CKI showed obvious anticancer activity against MDA-MB-231 cells in vitro.CKI significantly inhibited the proliferation,clone formation and migration of MDA-MB-231 cells.This study found 2896 significantly differential genes and 1322 significantly differential proteins that can be perturbed by CKI.Analysis of Hallmark gene signatures highlighted that the proliferation-related pathways were significantly down-regulated after CKI treatment.Meanwhile,CKI significantly inhibited multiple biological processes associated with cell cycle regulation.The immune function-related gene sets were significantly enriched in the CKI-treated group.The result suggested the potential immunomodulatory activity of CKI.Moreover,this study found 19 genes and 10 proteins with HR>1 that can be down-regulated by CKI,and 18 genes and 28 proteins with HR<1 that can be up-regulated by CKI.These genes and proteins are not only associated with the overall survival(OS)of TNBC patients but also the relapse-free survival(RFS),implying that CKI has the potential to improve patient outcomes by regulating them.Interestingly,most of the genes and proteins up-regulated by CKI were positively correlated with the relative abundance of tumor-infiltrating immune cells,suggesting that CKI may modulate anti-tumor immunity.4 The antitumor effect of CKI in vivo and the impact of CKI on the TME of 4T1 tumor bearing mice based on scRNA-seqCKI significantly enhanced the anticancer activity of chemotherapy in vivo with no obvious side effects.Flow cytometry results revealed an elevated percentage of CD3+,CD4+,CD8+ T lymphocytes and NK cells in the spleens and tumors of the mice with combination therapy,suggesting the immunomodulatory effects of CKI when it was combined with chemotherapy.The bulk RNA-seq results of the mouse tumor tissues illuminated that the gene sets correlated with T,cytotoxic CD8+T and NK cells were significantly activated in the combination therapy group versus the monotherapy group(DDP+CKI vs.DDP;PTX+CKI vs.PTX),so CKI may enhance anti-tumor immunity by modulating the immune cell populations in TME.According to the scRNA-seq results of the mouse tumor tissues,the mice treated with PTX+CKI showed a higher percentage of total T and CD8+T cells compared with the mice merely treated with PTX.The mice in the PTX+CKI and CKI groups showed a higher average expression of canonical T cell,CD8+ T cell and cytotoxic CD8+ T cell markers(Cd3d,Cd3e,Cd3g,Cd8a,Cd8b1,Gzma,Gzmb,Gzmk,Prfl,Fasl,Ifng,Nkg7)than the mice without CKI intervention.GSEA and GSVA analyses demonstrated that the pathways and biological processes associated with the activation of immune response,lymphocytes,T cells,cytotoxic T cells and NK cells were significantly enriched in the PTX+CKI group versus the PTX group.The scRNA-seq results further verified that CKI could improve the TME of TNBC.ConclusionsGuided by the concept of integrated big data,this study applied in vitro and in vivo experiments,scRNA-seq,RNA-seq,proteome,machine learning algorithms and integrative bioinformatics methods to explore the mechanism of CKI for the treatment of TNBC.This study provides novel insights for the study on the mechanism of Chinese medicine.The study shows that CKI combined with chemotherapy triggers effective antitumor immunity by activating immune cells in a murine breast cancer model,and the combination of CKI and chemotherapy might provide a higher efficiency and lower toxicity strategy than a single chemotherapy drug for TNBC.This is the first study that employs the scRNA-seq technology to reveal the efficacy of CKI plus chemotherapy on the TME of TNBC.Our findings may be useful in further deeply exploring the effect of Chinese medicine on TME,and our analytic methods would provide novel insights for further dissecting the mechanism of Chinese medicine from the single cell level.