Identification Of Rare Metabolically Abnormal Tumor Cells In Body Fluids Of Patients With Lung Caner

In the process of metastasis of malignant tumor,malignant cells usually invade into the body fluid,blood and other circulatory systems.In these samples,identification of malignant cells can directly reflect the tumor metastasis with important clinical significance.Nowadays,the classification of benign and malignant cells mainly depends on cytology,histological examination,cell invasion experiments or xenotransplantation models,and malignant cells could be distinguished from benign cells with the characteristics such as cellular atypia,poorly differentiated phenotype as well as rapid proliferation and highly invasive growth,but the number of malignant cells in body fluids is rare in most patients,so it’s ineffective to detect rare malignant cells with these methods.So we want to develop a more sensitive and simple method for the detection of malignant cells.The metabolic abnormality is one of the basic features of tumor cells,the Warburg effect is one of the most common appearance of metabolic abnormalities in cancer cells: tumor cells uptake large quantities of glucose and rapidly generate energy and biosynthetic intermediates through the aerobic glycolysis pathway.The rapid proliferation of malignant cells requires large amount of energy and metabolites,whereas benign cells with relatively low rates of glucose metabolism.And the fluorodeoxyglucose FDG has been well demonstrated the utility for identification of malignant tumors in clinic trials.with the aid of single-cell technologies,it is possible to measure the metabolism abnormalities at single-cell level for the detection of malignant cells.So according to the characteristics of malignant cells with enhanced anaerobic glycolysis comparing with benign cells,we establish a rapid and sensitive assay for identification of metabolically active tumor cells in malignant pleural effusions(MPE).MPE represents the existence of tumor cells in pleural effusion,which is a pathologic indicator of cancer involvement and metastasis,indicating the metastasis of tumor and the poor prognosis,so detection of tumor cells in pleural effusion with high sensitivity and accuracy has a great clinical importance for planning appropriate management and improving prognosis of MPE patients.However,the sensitivity of cytology examination as a common approach to diagnose malignant pleural effusion is limited,especially for samples with few tumor cells.In this assay candidate tumor cells are revealed by a fluorescent glucose analogue 2-NBDG through enhanced glucose uptake combined with the leukocytes marker CD45.With the help of addressable chip with 200,000 microwells and high-content microscopy imaging system,all the nucleated cells can be rapidly imaged and analyzed with multiple channels,and micromanipulation then applied to retrieve all the suspected tumor cells for single-cell sequencing to confirm the malignancy of each cell.In 10 pleural effusion samples,we successfully identify metabolically active suspected tumor cells and their malignancy are confirmed by singlecell sequencing,what’s more,most of the detected driver gene mutations are consistent with the oncogene mutations present in primary lesions of the patients.Surprisingly,3 MPE samples confirmed by our assay in which conventional cytology fails to detect tumor cells,demonstrating the increased sensitivity of this single-cell glucose uptake detection assay and the potential a complementary to cytology examination.In addition,this method can also be further extended to the detection of circulating tumor cells in peripheral blood samples.More importantly,the results of pleural effusions also prove that the metabolic detection method we established can be used as a novel approach for the identification of malignant cells at single-cell level.