In Vivo Epithelial Metabolic Imaging Using a Topical Fluorescent Glucose Analog.

The demanding metabolic needs of cancer cells are met by aerobic glycolysis. While whole-body PET imaging methods exist for evaluating this metabolic response, these are not ideal for local, more detailed regions such as mucosal surfaces. Fluorescence imaging of glucose analogs with similarities to radiolabeled deoxyglucose used in PET, namely, fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), offers such an alternative, particularly as this glucose analog may be delivered by local topical delivery. In this chapter, methods for in vivo epithelial imaging in a preclinical hamster model for oral cancer and oral epithelial dysplasia are described. Outlined are methods for preparation and in vivo delivery of 2-NBDG by topical application to the oral mucosa followed by fluorescence imaging to compare fluorescence responses between neoplasia and control mucosa or to monitor changes in fluorescence signal with time in both groups.

Reduced UDP-glucose Levels Are Associated with P-glycoprotein Over-expression in L1210 Cells and Limit Glucosylceramide Synthase Activity.

BACKGROUND/AIM: P-glycoprotein (Pgp) expression in neoplastic cells is known to reduce cell sensitivity to several cytotoxic Pgp substrates. A member of the ABC transporter family, Pgp, represents the most frequently described membrane efflux pump and its expression in neoplastic cells is responsible for multi-drug resistance. Several lines of evidence indicate that the expression and increased function of both Pgp and glucosylceramide synthase (GCS, an enzyme responsible for ceramide pathway de-activation in the regulation of apoptosis progression) enhance the resistance of Pgp-positive cells. Previously, we described a reduction in the uridine diphosphate (UDP)-glucose contents of mouse leukemia cells (R) expressing Pgp due to vincristine selection compared to parental L1210 cells (S). The reduced availability of UDP-glucose as a glucose donor in R cell glycosylation reactions could limit GCS-catalyzed ceramide glycosylation. Consequently, the over-expression of Pgp in Pgp-positive L1210 cells may be associated with reduced ceramide glycosylation. MATERIALS AND METHODS: To test this idea, we measured the expression and activities of Pgp and GCS, UDP-glucose levels, cellular uptake of C12-NBD-ceramide (a fluorescent analogue of ceramide) and ceramide-induced cell death in S and R cells. T-cells, another Pgp-positive variant of L1210 cells that express Pgp due to their transfection with a gene encoding human Pgp were also used in this study. RESULTS: We detected significantly reduced levels of C12-NBD-ceramide glycosylation and reduced UDP-glucose contents in Pgp-positive R and T-cells compared to S cells. C12-NBD-ceramide uptake assays revealed nearly identical dynamics of uptake time-dependency curves. The Pgp-positive L1210 variants (R and T) are more sensitive than Pgp-negative S cells to ceramide-induced cell damage, as measured by an fluorescein isothiocyanate-labeled annexin V and propidium iodide apoptosis necrosis kit. Short chain C2-ceramide was more effective at inducing cell damage than ceramide analogues with longer chains. CONCLUSION: These evidence indicates that the down-regulation of UDP-glucose contents in Pgp-positive L1210 cells is responsible for their collateral sensitivity to ceramide-induced apoptosis.

Delivery-corrected imaging of fluorescently-labeled glucose reveals distinct metabolic phenotypes in murine breast cancer.

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.

Delivery rate affects uptake of a fluorescent glucose analog in murine metastatic breast cancer.

We demonstrate an optical strategy using intravital microscopy of dorsal skin flap window chamber models to image glucose uptake and vascular oxygenation in vivo. Glucose uptake was imaged using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). SO2 was imaged using the differential absorption properties of oxygenated [HbO2] and deoxygenated hemoglobin [dHb]. This study was carried out on two sibling murine mammary adenocarcinoma lines, 4T1 and 4T07. 2-NBDG uptake in the 4T1 tumors was lowest when rates of delivery and clearance were lowest, indicating perfusion-limited uptake in poorly oxygenated tumor regions. For increasing rates of delivery that were still lower than the glucose consumption rate (as measured in vitro), both 2-NBDG uptake and the clearance rate from the tumor increased. When the rate of delivery of 2-NBDG exceeded the glucose consumption rate, 2-NBDG uptake decreased with any further increase in rate of delivery, but the clearance rate continued to increase. This inflection point was not observed in the 4T07 tumors due to an absence of low delivery rates close to the glucose consumption rate. In the 4T07 tumors, 2-NBDG uptake increased with increasing rates of delivery at low rates of clearance. Our results demonstrate that 2-NBDG uptake in tumors is influenced by the rates of delivery and clearance of the tracer. The rates of delivery and clearance are, in turn, dependent on vascular oxygenation of the tumors. Knowledge of the kinetics of tracer uptake as well as vascular oxygenation is essential to make an informed assessment of glucose demand of a tumor.

Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose.

The clinical value of assessing tumor glucose metabolism via F-18 fluorodeoxyglucose (FDG) PET imaging in oncology is well established; however, the poor spatial resolution of PET is a significant limitation especially for early stage lesions. An alternative technology is optical molecular imaging, which allows for subcellular spatial resolution and can be effectively used with topical contrast agents for imaging epithelial derived cancers. The goal of this study was to evaluate the potential of optical molecular imaging of glucose metabolism to aid in early detection of oral neoplasia. Fluorescently labeled deoxyglucose (2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose)) was applied topically to tissue phantoms, fresh oral biopsies (n = 32) and resected tumors specimens (n = 2). High-resolution imaging results show that 2-NBDG can be rapidly delivered to oral epithelium using topical application. In normal epithelium, the uptake of 2-NBDG is limited to basal epithelial cells. In contrast, high-grade dysplasia and cancers show uptake of 2-NBDG in neoplastic cells throughout the lesion. Following 2-NBDG labeling, the mean fluorescence intensity of neoplastic tissue averages 3.7 times higher than that of matched nonneoplastic oral biopsies in samples from 20 patients. Widefield fluorescence images of 8-paired oral specimens were obtained pre and postlabeling with 2-NBDG. Prior to labeling, neoplastic samples showed significantly lower autofluorescence than nonneoplastic samples. The fluorescence of neoplastic samples increased dramatically after labeling; the differential increase in fluorescence was on average 30 times higher in neoplastic samples than in normal samples. Topical application of 2-NBDG can therefore provide image contrast in both widefield and high-resolution fluorescence imaging modalities, highlighting its potential in early detection of oral neoplasia.

Adiponectin gene therapy of streptozotocin-induced diabetic mice using hydrodynamic injection.

BACKGROUND: Adiponectin (Adipo), an adipocyte hormone involved in the regulation of glucose and lipid metabolism, has already been identified as a potential therapeutic target for the treatment of diabetes. However, successful delivery of Adipo to the receptors is difficult due to their peptide characteristics. Receptors for Adipo are abundantly expressed in the liver and skeletal muscle. METHODS: Uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in hepatoblastoma HepG2 cells expressing Adipo was examined. Adipo-expressing plasmid DNA (10-50 microg) in saline solution (0.1 ml/g body weight) was rapidly injected into the tail vein of 4-week-old diabetic mice after 4-6 weeks of treatment with streptozotocin (STZ). Uptake of glucose in diabetic mice also was measured using a planar positron imaging system featuring 18-fluorodeoxyglucose. RESULTS: HepG2 cells expressing Adipo exhibited significantly increased 2-NBDG uptake compared with cells transfected with control plasmid even in the absence of insulin. STZ-induced diabetic mice showed decreased serum Adipo levels compared with non-diabetic mice. A single hydrodynamic injection of 10-50 microg Adipo-expressing plasmid DNA into diabetic mice led to approximately 10-15-fold elevation in serum Adipo levels, and resulted in decreased serum levels of glucose and triglyceride. As well as exhibiting higher levels of Adipo expression, diabetic mice also had higher hepatic glucose uptake than similar mice injected with control plasmid. CONCLUSIONS: We report that STZ-induced diabetic mice exhibited decreased Adipo levels and hyperglycemia which may be alleviated by hydrodynamic injection of the Adipo gene. This type of gene delivery system to the liver offers a different approach in developing novel treatments for type 1 and 2 diabetes.

Regional differences in glucose transport in the mouse hippocampus.

In order to observe glucose transport into the brain, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG), a non-metabolizable and fluorescent glucose analogue, was injected intravenously into mice. After ascertaining that this glucose analogue is non-metabolizable in the brain, the NBDG contents in the blood and brain were measured quantitatively by spectrofluorimetry at 0, 0.5, 2, 5, 10 and 30 min after intravenous injection. The NBDG content in the blood decreased markedly with time, whereas in the brain it rapidly decreased, then gradually increased after 2 min. Glucose transport into the hippocampus was observed with a confocal laser scanning microscope. At 0.5 min, NBGD was seen to be highly concentrated on the vascular wall. Using the confocal mode, it was found that the fluorescence was unevenly distributed on the microvessel wall, suggesting local differences of glucose transport in the vascular wall. At 5 min, the fluoresent intensity of the vascular wall was markedly decreased, whereas relatively intense fluorescence was observed in the cerebral parenchyma of the stratum lacunosum-moleculare and stratum pyramidale of Ca3. At 10 min, a weak fluoresence was diffusely distributed in the hippocampus. As to the localization of NBDG in the brain, capillary endothelium (luminal and abluminal membrane), basement membrane, and the feet of the astrocytes are discussed.