Study On Stability Of Sensitivity Parameters Of Cells To Radiotherapy By Clone Formation Assay

Background:In the field of radiation oncology,in order to individualize radiotherapy for patients,such as higher dose radiotherapy for patients with radiation tolerance,we need an objective and stable index to describe the radiotherapy sensitivity of cells,so as to achieve individualized radiotherapy dose formulation for cancer diagnosis.Therefore,there is an urgent need to develop a genetic spectrum to predict cancer radiosensitivity.However,one of the research obstacles to meeting this need is the lack of big data related to radio sensitivity of cancer cells that can be used for analysis in combination with genomic data.Clonal formationtestingis the gold standard for in vitro radiosensitivity evaluation.Although we found that a large number of studies have reported results and parameters of clonal formation tests that reflect radiosensitivity of cancer cells,differences in experimental conditions for clonal formation have prevented the process of comparing and integrating these radiosensitivity parameters between studies.Therefore,it is necessary to screen out which experimental conditions are likely to affect the results of clone formation and which experimental parameters are relatively stable to reflectthe sensitivity of cell lines to radiotherapy among different independent experimental results.Objectives:The stability of sensitivity parameters of cells to radiotherapy was determined by clone formation assay.Methods:Humanlung cancer A549 and human esophageal cancer KYSE30,KYSE150 and TE-1 cells cultured in vitro were taken as the research objects.Radiosensitivity parameters were obtained by clone formation method,and comprehensive evaluation was conducted.The tumor cells with good condition and fast growth rate were selected for the clone formation experiment.By setting the difference of inoculation density,inoculation time,inoculation and radiotherapy sequence,clone growth time and other conditions,the survival fraction curve of tumor cells was fitted by clicking the multi-target model and linear quadratic model.Sfl,SF2,SF3,SF4,SF5,DO,D10,D50,α,βand other radiotherapy sensitivity parameters were calculated by two models,and the differences betweengroups were statistically analyzed.KYSE30 cells were treated with PARP inhibitors,and the radiotherapy sensitivity parameters of the control group and the experimental group were determined to determine whether PARP inhibitors could change the radiotherapy sensitivity of esophageal cancer cells,and the stability of the parameters was determined as the control results.Results:In this study,we conducted a clone formation experiment on KYSE30 cells and found that the inoculation density,the sequence of inoculation and radiotherapy,and the inoculation time had no obvious effect on the results of clone formation.The CV of DO,D10,SF2 and D50 were all lower than30%in various experimental Settings,showing good stability.Conclusions:We found that in the experiment of clone formation,the inoculation density,the growth time of clone,the sequence of inoculation and radiotherapy,and the inoculation time had no obvious effect onthe parameters of clone formation.The values of D0,D10,SF2 and D50 obtained by using the clone formation experiment are highly consistent and relatively stable among different experimental conditions.These data and conclusions can be used to support inter-study comparison and comprehensive analysis of the stability of published clonal analysis data which will contribute to the advancement of radiation oncology research in the era of big data.