Preliminary Study Of Individualized Treatment Of Non-small Cell Lung Cancer Via The Feedback System Control Drug Optimization Technique

PartⅠApplication of Feedback System Control（FSC）technique in the optimization of drug combination for NSCLC cell linesObjective:The Feedback System Control optimization technique will be used to analyze three pathologically major types of NSCLC cells,namely A549,H226 and H460 cells,and to optimize the combinations of drugs.Methods:According to the FSC technique,CCK-8 assay was used to test the survival rate of A549,H226 and H460 cells,which were treated with cisplatin,carboplatin,paclitaxel,docetaxel,pemetrexed and gemcitabine,vinorelbine and erlotinib respectively.We used the Graphpad prism 6.0 vision to calculate the IC₅₀0 for each single drug.The Fractional Factorial Design method was used to study the initial drug combinations at three levels(0,1/5IC₅₀,1/2IC₅₀)of eight drugs.We introduced polynomial fitting model to analyze experiment data and calculate the optimal combinations.Western blot was conducted to evaluate the effect of combination drug on apoptosis related proteins.SPSS 13.0 was used to perform statistical analysis.Results:（1）We optimized the drug combination by testing only 59 combinations for each cell line,accounting for approximately 0.9%（59/6561）of all possible combinations.The ideal regimens identified for A549,H226 and H460 cells,respectively,were Car/Pac/Doc（2.95×10^-44 M Car,1.18×10^-44 M Pac,3.78×10^-55 M Doc）,Cis/Gem/Pac(4.43×10^-44 M Cis,3.13×10^-33 M Gem,1.08×10^-6M Pac)and Cis/Gem/Pem(5.59×10^-44 M Cis,5.45×10^-99 M Gem,7.73×10^-4M Pem).（2）Compared with the initial combination,the cell killing rate was significantly increased（P<0.01）,with the drug dosages decreased.（3）Through Western bolt,we furthermore found that the optimal regimens could significantly promote cancer cell apoptosis by increasing the expression of Bax,cleaved poly（ADP-ribose）polymerase,caspase-9,caspase-3,and caspase-8,and decreasing the expression of Bcl-2 in A549cells,compared with single drug or clinical chemotherapy regimen in common use.Conclusion:We have successfully optimized drug combinations for A549,H226 and H460 cells via FSC technique,which suggests that the this method holdspromise for optimization of anti-cancer combination therapy.Part II Cultivation of human NSCLC stem cellsObjective:To explore and learn the methods and techniques for the isolation,culture,passage,cryopreservation and recovery of NSCLC stem cells from surgery tissue or pleural fluid.We hope to lay the foundation for the future application of FSC technique in the optimization of drug combination and individualized treatment of lung cancer.Methods:Serum free culture method was used to isolate,culture and enrich NSCLC stem cells.Results:（1）18 cases of lung cancer stem cells were successfully cultured by the method of serum free culture.（2）The average time from single suspension cells to lung cancer stem cell sphere was 4.28±2.25 days.Stem cells derived from surgical specimens needed 3.60±1.51 days to sphere while those derived from pleural fluid needed 3.60±1.51 days with statistical difference（P<0.01）.Conclusion:Serum free culture can be used to isolate,culture and enrich lung cancer stem cells.