2-(n-(7-nitrobenz-2-oxa-1-3-diazol-4-yl)amino)-2-deoxyglucose and Lung-Neoplasms

Malignant pleural effusion (MPE), the presence of malignant cells in pleural fluid, is often the first sign of many cancers and occurs in patients with metastatic malignancies. Accurate detection of tumor cells in pleural fluid is crucial because the presence of MPE denotes an advanced stage of disease and directs a switch in clinical managements. Cytology, as a traditional diagnostic tool, has limited sensitivity especially when tumor cells are not abundant, and may be confounded by reactive mesothelial cells in the pleural fluid. We describe a highly sensitive approach for rapid detection of metabolically active tumor cells in MPE via exploiting the altered glucose metabolism of tumor cells relative to benign cells. Metabolically active tumor cells with high glucose uptake, as evaluated by a fluorescent glucose analog (2-NBDG), are identified by high-throughput fluorescence screening within a chip containing 200,000 addressable microwells and collected for malignancy confirmation via single-cell sequencing. We demonstrate the utility of this approach through analyzing MPE from a cohort of lung cancer patients. Most candidate tumor cells identified are confirmed to harbor the same driver oncogenes as their primary lesions. In some patients, emergence of secondary mutations that mediate acquired resistance to ongoing targeted therapies is also detected before resistance is manifested in the clinical imaging. The detection scheme can be extended to analyze peripheral blood samples. Our approach may serve as a valuable complement to cytology in MPE diagnosis, helping identify the driver oncogenes and resistance-leading mutations for targeted therapies.

Topics: 4-Chloro-7-nitrobenzofurazan; A549 Cells; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Cell Line, Tumor; Deoxyglucose; Diagnosis, Differential; Glucose; High-Throughput Screening Assays; Humans; Leukocytes; Lung Neoplasms; Pleural Effusion; Pleural Effusion, Malignant; Positron-Emission Tomography

To investigate the role of miR-181c in glycolysis of cancer-associated fibroblasts (CAFs) and explore the mechanism.. Human lung CAFs and normal fibroblasts (NFs), isolated from fresh human lung adenocarcinoma tissue specimens by primary culture of tissue explants, were transfected with a miR -181c mimics, a miR-181c inhibitor, a siRNA siRNA-HK2 or the vector HK2-vector via Lipofectamine(TM) 2000. Quantitative real-time PCR was used to analyze the changes in miR-125b expression in the transfected cells; hexokinase-2 (HK2) protein expression in the cells was detected using Western blotting, and the cellular glucose uptake was assessed with 2-NBDG. Lactate production in the cells was examined and expression of HK2 mRNA was detected with dual luciferase reporter gene assay.. No obvious difference was found in the cell morphology between CAFs and NFs. Compared with the NFs, the CAFs showed obviously increased glucose uptake, lactate production and HK2 protein expression with decreased expressions of the miR-181 family (P<0.05). Transfection with the miR-181 inhibito- rsignificantly increased glucose uptake, lactate production and HK2 protein expression in the NFs. In CAFs, transfection with the miR-181 mimics caused significantly lowered glucose uptake, lactate production and HK2 protein expression of. Knockdown of endogenous HK2 by siRNA abolished miR-181 mimics-mediated decrease of glucose uptake and lactate production in CAFs, while transfection with miR-181 mimics suppressed HK2 overexpression-induced enhancement of glucose uptake and lactate production in NFs.. Transfection with miR-181 mimics can suppress glycolysis in CAFs by inhibiting HK2 expression.

Topics: 4-Chloro-7-nitrobenzofurazan; Adenocarcinoma; Adenocarcinoma of Lung; Deoxyglucose; Fibroblasts; Glycolysis; Hexokinase; Humans; Lung Neoplasms; MicroRNAs; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transfection; Tumor Cells, Cultured