Detection Of Circulating Tumor Cells Using Single Cell Gel Electrophoresis Based On Radiosensitization Effect

Objective: To establish a technique for quantitative detection of circulating tumor cells by single cell gel electrophoresis using the sensitivity of tumor cells to ionizing radiation.Methods:(1)To establish a method for the detection of circulating tumor cells by single cell gel electrophoresis based on the radiation sensitization effect.Human breast cancer cell line MCF-7,human lymphoma cell line Raji and single nucleated cells extracted from peripheral venous blood of healthy human were treated with different doses of X-rays and different types of radiosensitizers(sodium glyburide,tirazamine,nanogold and mi RNA-148b),and single cell gel electrophoresis was performed to reflect the DNA doublestrand breaks(DSB)of single nucleated cells and tumor cells.(2)Evaluation of the feasibility and reliability of the method.DSBs were detected by flow cytometry using γ-H2 AX fluorescence labeling;tumor cells were determined by cell size,DNA double-strand breaks damage and single cell gel electrophoresis-immunofluorescence(Comet-IF);different amounts of tumor cells were added to healthy human peripheral blood for recovery rate experiments.Results:(1)The treatment with sodium glyburide combined with X-ray caused significant DNA double-strand break damage to human breast cancer cell line MCF-7 and human lymphoma cell line Raji,and low degree of DSBs damage to peripheral blood single nucleated cells,with statistically significant differences in DSBs between tumor cells and single nucleated cells;(2)γ-H2 AX fluorescence-labeled flow cytometry results were consistent with the single cell gel electrophoresis results.MCF-7 and Raji two tumor cells had the highest γ-H2 AX fluorescence intensity under the treatment of sodium glycine bispyrazole combined with X-rays,peripheral blood single nucleated cells had low fluorescence intensity under this treatment,and the highest fluorescence intensity under the treatment of nanogold combined with X-rays;(3)the cell diameter was greater than 10 μm,cell DNA double chain break damage severity and Comet-IF results showing fluorescence were judged as tumor cells;(4)The results of the recovery rate experiment showed that the recovery rates of different tumor determination methods were greater than75%,and the recovery numbers of different methods were well correlated with the accession numbers(Spearman r > 0.9),and there was no statistical difference between the recovery numbers and correlation coefficients of each method.Conclusion: Based on the radiation sensitization effect and single cell gel electrophoresis,the treatment of ionizing radiation combined with radiation sensitizers significantly increased the DSBs damage of tumor cells,and the feasibility and reliability of the method were evaluated by flow cytometry with γ-H2 AX fluorescent labeling and recovery experiments with tumor cells added to peripheral blood,thus we successfully established a rapid,simple,intuitive and quantitative method to detect circulating tumor cells.