Sensitive Drug Screening For Triple-negative Breast Cancer Based On Conditionally Reprogrammed Cell Culture

Objective:Breast cancer is the most common malignancy in women.Triple-negative breast cancer(TNBC)refers to breast cancer which has negative ER,PR and HER-2 expression.TNBC is characterized by rapid development,high invasiveness,and high possibility of recurrence and metastasis.At present,the adjuvant treatment for TNBC is far from satisfying,and there are few effective treatment targets,so individual and effective treatment targets are urgently needed.Conditional reprogramming culture(CRC)model is a tumor cell model which can achieve immortalization of tumor cells and normal epithelial cells without transferring oncogenic genes.The CRC could be easily cultured from the tumor tissue at a low cost.CRC has the potential to be used in the personalized tumor sensitivity prediction.In this study,we constructed CRCs of TNBC from surgical specimens,and used the CRCs to carry out high-throughput drug sensitivity screening test,aiming to explore the use of CRC in the personalized treatment of TNBC.Methods:We prospectively collected information of patients diagnosed with breast cancer and planned to undergo surgical treatment at the Department of Breast Surgery,Peking Union Medical College Hospital from March 1,2019 to March 1,2020.During the operation,specimens of tumor tissue and matched normal breast tissue were collected and used to culture CRCs.After surgery,phenotype of breast cancer is validated by pathology and CRCs of TNBCs were used for further analysis.The DNA mutation profile and transcriptome expression profile of CRCs of TNBC and matched tumor tissues were analyzed by whole exon sequencing(WES)and transcriptome sequencing(RNA-Seq).The sensitivity of the CRCs to 110 drugs was detected by a high-throughput drug sensitivity test,and compared with the WES and RNA-Seq results.We obtained high-throughput mRNA chip and RNA-Seq data of TNBC in the GEO database and TCGA database,and searched for prognostic biomarkers of TNBC through differential gene screening,gene set enrichment analysis and survival analysis.Gene set variation analysis(GSVA)and survival analysis of GSVA scores were performed to find biological pathways which are significantly related to the prognosis of TNBC patients.Results:(1)Six CRCs of TNBC and six CRCs of matched normal breast tissue were constructed.CRCs of TNBC and the original tumor tissue have similar DNA mutation profiles,and the transcriptome gene expression of the two group also showed a strong correlation(R=0.855).(2)PF-04691502,a PI3K-Akt and mTOR dual-target inhibitor,is the most effective drug according to the RRA comprehensive drug sensitivity score,and among the other top ten sensitive drugs,there are 2 mTOR inhibitors,2 Akt targeted drugs,1 Syk(spleen tyrosine kinase)inhibitor,1 topoisomerase inhibitor,1 EGFR inhibitor,1 plant-derived small molecule drug(Polyphyllin VI).DSS scores of sirolimus,temsirolimus,ridaforolimus,and torinl had a high correlation(R=0.82?0.97).The DSS scores of afatinib,gefitinib,and lapatinib have a high correlation(R=0.61?0.93).(3)The DSS score of ipatasertib was significantly correlated with the GSVA score of the mTOR pathway(R=0.587,P=0.045).The DSS score of torin 1 was significantly correlated with the GSVA score of the mTOR pathway(R=0.62,P=0.030).The DSS score of gefitinib was significantly correlated with the GSVA score of the EGF pathway(R=0.60,P=0.039).(4)The GEO and TCGA data showed that the activation of Aktl-mTOR signaling pathway,HIF signaling pathway,VEGF signaling pathway and adipogenesis-related pathways is associated with poor overall survival of patients with TNBC.Patients with high GSVA scores in the T cell antigen receptor signaling pathways or the antigen processing and presentation pathways have a good overall survival.Conclusion:(1)The CRCs of TNBC in this study showed high consistency with the original tumor tissue.The CRC is suitable as an individualized tumor model for the study of TNBC mechanism and drug screening test.(2)The results of the CRC drug sensitivity screening are stable,which can be used to predict the drug sensitivity of TNBC.The CRCs in this study is sensitive to drugs targeting PI3K-Akt,mTOR,EGFR,Syk or TOPII.(3)The GSVA scores of PI3K-Akt and mTOR pathways of CRCs are correlated with the results of CRC drug screening.The GSVA scores of PI3K-Akt and mTOR pathways in TNBC tissue are correlated with the overall survival of TNBC patients,thus the GSVA scores could be used in the prediction of drug sensitivity of TNBC and the evaluation of overall survival of TNBC patients.