Identification Of Rare Tumor Cell Using A Novel Method Based On Differences In Transcriptional Activity

Background&ObjectiveRare tumor cells in body fluids can provide rich information about tumors,which is of great significance for tumor diagnosis,treatment guidance and recurrence monitoring.At present,the commonly used methods for identifying rare tumor cells are mainly immunocytochemical methods and morphological methods.These methods have low sensitivity,complicated operation,high cost and poor stability,which greatly limit the practical application of rare tumor cells in clinicTherefore,there is an urgent need to develop a more ideal,sensitive and universal new method for the identification of rare tumor cells.Studies have found that there are abnormal changes in energy metabolism in tumor cells,and research have also shown that there may be a potential correlation between tumor abnormal metabolism and its ability of invasion and metastasis.The study of metabolically active tumor cells is of great significance.In addition,the nucleotide metabolism in tumor tissues was significantly more active than that in normal tissues.Therefore,abnormal nucleotide metabolism is expected to provide a new idea for the detection of tumor cells.In recent years,some researchers have introduced "click chemistry" to develop a new method for analyzing RNA synthesis-a click chemical reaction based on 5-ethynyluridine(EU)to label newborn RNA.EU is a kind of uracil nucleoside analogue,which can be incorporated into the newly synthesized RNA under the action of RNA polymerase,and then EU reacts with fluorescent dye with azide group to detect RNA.This method has been widely used to detect the transcriptional activity of cells.Based on the above research background,in view of the difference in cell transcriptional activity,based on the click chemical reaction of EU labeled newborn RNA,a novel rapid,sensitive and universal identification technique for tumor cell labeling was proposed,and the diagnostic value of this method was demonstrated in clinical serous effusion.Methods1.The effect of EU on the viability of MCF7 tumor cells was analyzed by CellCounting-Kit-8.Through different combinations of concentration and incubation time,flow cytometry was used to compare the fluorescence intensity of leukocytes and tumor cells co-incubated with EU under different conditions to optimize the experimental conditions.2.The labeling efficiency and imaging performance of EU on various tumor cells and leukocytes were analyzed by confocal microscope imaging and flow cytometry.Colocalized imaging was analyzed by immunofluorescence staining and Hoechst staining.The labeling efficiency and imaging performance of EU labeling and immunofluorescence staining were compared by confocal microscope imaging and ImageJ software analysis.3.Taking MCF7 cells and MG63 cells as examples,different numbers of tumor cells were mixed into leukocytes to carry out the recovery experiment.4.24 cases of benign and malignant serous effusion samples were collected,and the pleural and ascites samples were stained,imaged and analyzed based on the click chemistry reaction of EU-labeled nascent RNA,and the diagnostic value of this new method of labeling tumor cells in benign and malignant serous effusion was studied.Results1.EU shows good biocompatibility.With the analysis by flow cytometry,it was found that the average fluorescence intensity of tumor cells was 38 times higher than that of leukocytes when cells were incubated with 1 mM EU alone for 1 h.Therefore,this condition was chosen as the the most suitable experimental condition.2.By analyzing the fluorescence images of each celline,it was found that EU had high labeling efficiency and excellent imaging performance on tumor cells.Flow cytometry quantitative analysis showed that after tumor cells were mixed with leukocytes and co-incubated with EU,the average fluorescence intensity of tumor cells was 3.3-5.5 times higher than that of white blood cells.Co-localization imaging analysis showed that EU+/CK+cells were tumor cells.Through cell imaging and ImageJ software analysis,compared with immunofluorescence method,EU can label various kinds of metabolically active tumor cells with different phenotypes,showing bright red fluorescence and superior imaging performance.3.The recovery rate of MCF7 cells was 94.2±2.9%and MG63 cells was 92.8±3.6%.Both of them had high recovery rate and identification efficiency.4.24 cases of benign and malignant serous effusion were stained and analyzed by EU.In benign serous effusion,the number of EU+ cells ranged from 0-4/mL,while in malignant hydrothorax and ascites,the number of EU+cells increased significantly,up to 6-1435/mL,this difference was statistically significant(p<0.0001).Confocal imaging showed that EU could specifically label and distinguish hypermetabolic tumor cells.The new method based on EU-labeled tumor cells has a good diagnostic value in the identification of benign and malignant serous effusions.ConclusionBased on the different transcriptional activity between tumor cells and normal cells,a novel method for rapid,sensitive and universal identification of tumor cells by using the click chemical reaction of EU labeling newborn RNA was successfully developed.This method can be used to label and identify metabolically active tumor cells with different phenotypes,and has been successfully applied to the differential diagnosis of benign and malignant serous effusions,providing a new strategy for early diagnosis and metabolic monitoring of tumors.