Development And Clinical Application Of The Kit For Anti-cancer Drug Sensitivity Test Based On 3D Cultured Tumor Cells

BackgroundAs we know,chemotherapy is an effective systemic treatment for malignant tumor.However,it has distinctly individual differences in clinical practices,and not only ineffective chemotherapy can't benefit patients,but may also aggravate the patients' physical burden.To find an effective method of anti-cancer drug sensitivity test is the precondition of the accurate chemotherapy for cancer patients.Collagen gel droplet culture drug sensitivity test(CD-DST),which is a new chemotherapy drugs sensitivity detection technology based on three-dimensional cultured primary tumor cells,has been applied in a variety of solid carcinomas,and gradually recognized as an effective chemotherapy drugs sensitivity test method.But,in China,regrettably it is the main reason to restrict the clinical application research that relevant clinical investigations are very few,and localization of industrial kit hasn't be finished.According to this situation,firstly we developed the kit for chemo-sensitivity test based on three-dimensional cultured tumor cells,and investigate the quality control standards for this kit,which will lay the foundation of the kit's industrialization;Secondly,we launched a preliminary evaluation about the clinical performance of self-made kit in four types of high-grade solid cancer,which would accumulate the research data based on the population of Chinese cancer patients.Methods(1)Confirm the main raw material,including of collagen,cell culture medium,stain and their optimal application condition.Regard the NSCLC cell line A549 as the quality control reference,the relevant quality control standards were developed.(2)Collect fresh tissue specimens of BC,NSCLC,CRC and GC,and compare the test performance of self-made kit with control kit.Meanwhile,clinical follow-up was carried out,which will be used to analyze the correlation of in-vitro test results and clinical response.Results(1)We choose collagen type ?,serum free medium and neutral red dye as the raw materials for the main components of this kit.In further tests,we set basic conditions of the kit.The best volume ratio of collagen solution:10×F12 medium:collagen reconstituted buffer for preparation of collagen gel is 8:1:1,and that of the collagen gel solution and cell suspension is not less than 10:1.Collagen gel droplets are cured at 37? incubator,30 minutes.The optimal concentration of cells embedded is 2×105?5×105/mL.The optimal incubation time of serum free medium is 5?7 days.The optimal concentration(1:100)and the optimal dyeing time(2 hour)of neutral red are also determined.We discover that dyeing in 37?,5%CO2 cell incubator can prevent the neutral red crystal.Compared with the present method by manual operation,the improved method by MACS has certain advantages.MACS can improve the isolation efficiency of primary tumor cells in spite of the cytoactive of the two groups is similar.The detection of cell lines A549 in three classes library is also performed,STR test results shows A549 cell line in each class library had stable genetic traits which can be traced back.Contaminated microorganism is not found out from A549 cell line of each level library by mycoplasma detection and sterility detection,respectively.Proliferation rate experiments of A549 cell line shows that cell proliferation ratio is not less than 5-fold,and sensitivity of A549 cell line to cisplatin is highly sensitive.What the proportion of fibroblasts is not more than 70%,is acceptable by our self-made kit.Our investigation also shows that the test performance of self-made kit is similar to control kit,the rate of coarse consistency was 97.53%,Kappa value was 0.936(P<0.01).(2)The overall evaluable rate of four cancer specimens detected by our self-made kits is 92.41%,and the in-vitro sensitivity rate of each cancer to individual drugs is similar to its clinical response rate(r=0.788,p<0.01).Clinical follow-up shows that,in breast cancer,overall survival and progression-free survival between the group with high sensitivity and those with low sensitivity tested by our kits were significant differences(P<0.05),respectively.Meanwhile in colorectal cancer,gastric cancer,and non-small cell lung cancer,significant differences(P<0.05)of progression-free survival while no significant difference(P>0.05)of overall survival between the high sensitivity group and the low one is also observed.Conclusion:We develop the kit for chemotherapeutic drugs sensitivity test,quality control reference and the related standard,based on three-dimensional cultured tumor cells successfully.The detection performance of our kit is similar with imported control kit.The preliminary clinical study shows that there is a significant correlation between the in-vitro-sensitivity rate and the clinical-response rate,and statistically significant to predict the prognosis of clinical chemotherapy.