Identification And Characterization Of Tumor Cells By Liquid Biopsy In Lung Adenocarcinoma Patients

Objective:Analyses of circulating tumor cells(Circulating tumor cells;CTC)has paved new diagnostic avenues and are,to date,the cornerstones of liquid biopsy,but unfortunately,the CTC enrichment process may lead to the loss and damage of CTC.Due to the lack of specific markers for tumor cells identification,great challenges has been brought to the process of CTC detection.Metabolism abnormality is one of the universal characteristics of tumors.We hypothesize that cells with enhanced glucose uptake and without expression of leukocyte markers in pleural effusion(PE)or peripheral blood samples are highly likely to be malignant cells that can be confirmed via single-cell sequencing.A highthroughput metabolic-based assay is developed for rapid detection of rare metabolically active tumor cells.We can quickly find suspected tumor cells with high metabolic activity via this method.As a functional detection,cell activity was not damaged by this method.Finally,all cells assayed are addressable,and the candidate tumor cells will therefore be retrieved individually for single-cell sequencing to confirm their malignancy and reveal their targetable driver oncogenes.Methods and Results:Part one: We demonstrate a high-throughput single-cell metabolic method,called microchip-based metabolic cytomtery(MMC),to assay metabolic states of nucleated cells in the PE samples for detection and phenotyping of metabolically active tumor cells via exploiting altered metabolism in malignant tumor cells.MMC uses fluorescent metabolic markers 2-NBDG/CD45/EthD-1 to detect and classify metabolically active tumor cells into distinct subsets.MMC assays cells on the chip at a high throughput way,leading to ~500,000 single-cell metabolic measurements in each PE sample.Metabolically active tumor cells are individually retrieved by a micromanipultor for genomic and transcriptomic sequencing.The results showed that the candidate tumor cells with high glucose uptake were detected in 10 pleural effusion samples.The collected cells for malignancy confirmation were confirmed via single-cell sequencing.Two candidate CTCs were identified based on the same criteria,and confirmed to be malignant cells bearing the same mutational status found in PE and primary lesions.Part 2: We performed MMC in a 26 PE samples from lung cancer patients that exhibited varied metabolic signatures(2-NBDG and Resazurin).Single-cell sequencing confirmed the malignancy of CD45 negative,metabolically active cells via the oncogenic driver mutation and copy number variation(CNV)profiles.The transcriptomic sequencing analyzes different metabolic phenotypes identified in the PE samples.Metabolic mapping of nucleated cells in PE allows to not only detect PE with enhanced sensitivity but also provide information for prognosis and prediction of treatment response.Conclusions:We developed a simple and informative technique for diagnosing malignant ccells in PE and peripheral blood.The MMC platform is a high-throughput single-cell metabolic assay and uses metabolic markers of aerobic glycolysis(2-NBDG)and Krebs cycle(Resazurin)for metabolically phenotyping of tumor cells in PE and thereby assessing the metabolic signatures of PE samples.As a complementary method to cytology,MMC has the capability to increase detection sensitivity of malignant PE and provide valuable information for prognosis and prediction of treatment response.