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

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

When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO2) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, "RD", reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG60/RD) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG60/RD was increased in 4T1 tumors relative to 4T07 tumors yet average SO2 was comparable, suggesting a shift toward a "Warburgian" (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG60/RD increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo.

Topics: 4-Chloro-7-nitrobenzofurazan; Adenocarcinoma; Animals; Blood Glucose; Breast Neoplasms; Cell Line, Tumor; Deoxyglucose; Female; Fluorescent Dyes; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Optical Imaging; Oxygen Consumption

The availability of confocal endomicroscopy motivates the development of optical contrast agents that can delineate the morphologic and metabolic features of gastrointestinal neoplasia. This study evaluates 2-NBDG, a fluorescent deoxyglucose, the uptake of which is associated with increased metabolic activity, in the identification of Barrett's-associated neoplasia. Surveillance biopsies from patients with varying pathologic grades of Barrett's esophagus were incubated ex vivo at 37°C with 2-NBDG and imaged with a fluorescence confocal microscope. Images were categorized as neoplastic (high grade dysplasia, esophageal adenocarcinoma) or metaplastic (intestinal metaplasia, low grade dysplasia) based on the degree of glandular 2-NBDG uptake. Classification accuracy was assessed using histopathology as the gold standard. Forty-four biopsies were obtained from twenty-six patients; 206 sites were imaged. The glandular mean fluorescence intensity of neoplastic sites was significantly higher than that of metaplastic sites (p<0.001). Chronic inflammation was associated with increased 2-NBDG uptake in the lamina propria but not in glandular epithelium. Sites could be classified as neoplastic or not with 96% sensitivity and 90% specificity based on glandular mean fluorescence intensity. Classification accuracy was not affected by the presence of inflammation. By delineating the metabolic and morphologic features of neoplasia, 2-NBDG shows promise as a topical contrast agent for confocal imaging. Further in vivo testing is needed to determine its performance in identifying neoplasia during confocal endomicroscopic imaging.

Topics: 4-Chloro-7-nitrobenzofurazan; Adenocarcinoma; Administration, Topical; Algorithms; Area Under Curve; Barrett Esophagus; Biopsy; Contrast Media; Deoxyglucose; Drug Evaluation, Preclinical; Esophageal Neoplasms; Esophagoscopy; Esophagus; Fluorescent Dyes; Goblet Cells; Humans; Microscopy, Confocal; ROC Curve