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BackgroundCyclooxygenase-2 (COX-2) is the inducible isoform of the cyclooxygenase enzyme family. COX-2 is involved in tumor development and progression, and frequent overexpression of COX-2 in a variety of human cancers has made COX-2 an important drug target for cancer treatment. Non-invasive imaging of COX-2 expression in cancer would be useful for assessing COX-2-mediated effects on chemoprevention and radiosensitization using COX-2 inhibitors as an emerging class of anti-cancer drugs, especially for colorectal cancer. Herein, we describe the radiopharmacological analysis of [18F]Pyricoxib, a novel radiolabeled COX-2 inhibitor, for specific PET imaging of COX-2 in colorectal cancer.MethodsUptake of [18F]Pyricoxib was assessed in human colorectal cancer cell lines HCA-7 (COX-2 positive) and HCT-116 (COX-2 negative). Standard COX-2 inhibitors were used to test for specificity of [18F]Pyricoxib for COX-2 binding in vitro and in vivo. PET imaging, biodistribution, and radiometabolite analyses were included into radiopharmacological evaluation of [18F]Pyricoxib.ResultsRadiotracer uptake in COX-2 positive HCA-7 cells was significantly higher than in COX-2 negative HCT-116 cells (P < 0.05). COX-2 inhibitors, celecoxib, rofecoxib, and SC58125, blocked uptake of [18F]Pyricoxib in HCA-7 cells in a concentration-dependent manner. The radiotracer was slowly metabolized in mice, with approximately 60 % of intact compound after 2 h post-injection. Selective COX-2-mediated tumor uptake of [18F]Pyricoxib in HCA-7 xenografts was confirmed in vivo. Celecoxib (100 mg/kg) selectively blocked tumor uptake by 16 % (PET image analysis; P < 0.05) and by 51 % (biodistribution studies; P < 0.01).ConclusionsThe novel PET radiotracer [18F]Pyricoxib displays a promising radiopharmacological profile to study COX-2 expression in cancer in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0192-9) contains supplementary material, which is available to authorized users.

Elevated growth in breast cancer (BC) activates hypoxia-inducible factor (HIF1α) and downstream, facilitative glucose transporter 1 (GLUT1), which can be visualized with 2-deoxy-2-[F]fluoro-d-glucose ([F]FDG). GLUT5 (fructose) and GLUT2 (glucose/fructose) might provide alternative targets for BC imaging as to why effects of hypoxia on GLUT1/2/5 levels and function were examined in human BC models. GLUT1/2/5 and HIF1α mRNA was analyzed in BC patient biopsies. In MCF10A, MCF7, and MDA-MB231 cells, [F]FDG, 6-deoxy-6-[F]fluoro-d-fructose (6-[F]FDF) and [F]-fluoroazomycin arabinoside were used in radiotracer experiments, whereas GLUT1/2/5 mRNA was analyzed with real-time PCR and protein levels determined via Western blot/immunohistochemistry. Positron emission tomography imaging was performed in MCF7 and MDA-MB231 tumor-bearing mice. Glucose/fructose/cytochalasin B reduced cellular 6-[F]FDF uptake by 50%, indicating functional involvement of GLUT2. With GLUT5 staining lower than GLUT1, 6-[F]FDF revealed lower uptake than [F]FDG [standardized uptake value (SUV) 0.77 ± 0.06 vs. SUV 1.08 ± 0.07] in MDA-MB231 tumors and was blocked by 20% with cytochalasin B after 10 min. Whereas correspondence between 6-[F]FDF uptake and GLUT5 protein was low, high GLUT2 levels were detected in all cell lines and tumor models. Besides GLUT1, GLUT5 seems to be regulated under hypoxia on the molecular and functional level. Additionally, results strongly support a functional involvement of GLUT2 in fructose metabolism, possibly by compensating for the weaker expression and function of GLUT5 in BC.-Hamann, I., Krys, D., Glubrecht, D., Bouvet, V., Marshall, A., Vos, L., Mackey, J. R., Wuest, M., Wuest, F. Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

IntroductionLysyl oxidase (LOX; ExPASy ENZYME entry: EC 1.4.3.13) and members of the LOX-like family, LOXL1–LOXL4, are copper-dependent enzymes that can modify proteins of the extracellular matrix. Expression of LOX is elevated in many human cancers, including breast cancer. LOX expression correlates with the level of tissue hypoxia, and it is known to play a critical role in breast cancer metastasis. The goal of the present study was to target LOX with (1) molecular probe fluorescent labeling to visualize LOX in vitro and (2) a radiolabeled peptide to target LOX in vivo in three different preclinical models of breast cancer.MethodsGene expression of all five members of the LOX family was analyzed at the transcript level via microarray analysis using tissue biopsy samples from 176 patients with breast cancer. An oligopeptide sequence (GGGDPKGGGGG) was selected as a substrate-based, LOX-targeting structure. The peptide was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy experiments with the murine breast cancer cell line EMT-6. In vivo molecular imaging experiments were performed using a C-terminal amidated peptide, GGGDPKGGGGG, labeled with a short-lived positron emitter, fluorine-18 (18F), for positron emission tomography (PET) in three different breast cancer models: EMT6, MCF-7 and MDA-MB-231. The PET experiments were carried out in the presence or absence of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX.ResultsImmunostaining experiments using a LOX-specific antibody on EMT-6 cells cultured under hypoxic conditions confirmed the elevation of LOX expression in these cells. An FITC-labeled oligopeptide, FITC-Ava-GGGDPKGGGGG-NH2, was found to be localized in different cellular compartments under these conditions. After injection of [18F]fluorobenzoate-GGGDPKGGGGG-NH2, radioactivity uptake was visible in all three breast cancer models in vivo. Tumor uptake was reduced by predosing the animals with 2 mg of BAPN 4 h or 24 h before injection of the radiotracer.ConclusionsThe present data support further investigation into the development of LOX-binding radiolabeled peptides as molecular probes for molecular imaging of LOX expression in cancer.

Use of [F]FDG-positron emission tomography (PET) in clinical breast cancer (BC) imaging is limited mainly by insufficient expression levels of facilitative glucose transporter (GLUT)1 in up to 50% of all patients. Fructose-specific facilitative hexose transporter GLUT5 represents an alternative biomarker for PET imaging of hexose metabolism in BC. The goal of the present study was to compare the uptake characteristics of selected hexose-based PET radiotracers in murine BC model EMT6. Uptake of 1-deoxy-1-[F]fluoro-d-fructose (1-[F]FDF), 6-deoxy-6-[F]fluoro-d-fructose (6-[F]FDF), 1-deoxy-1-[F]fluoro-2,5-anhydro-mannitol (1-[F]FDAM), 2-deoxy-2-[F]fluoro-d-glucose (2-[F]FDG), and 6-deoxy-6-[F]fluoro-d-glucose (6-[F]FDG) was studied in EMT6 cells, tumors, and muscle and correlated to GLUT1 and GLUT5 expression levels. Fructose-derivative 6-[F]FDF revealed greater tumor uptake than did structural analog 1-[F]FDF, whereas 1-[F]FDAM with locked anomeric configuration showed similar low tumor uptake to that of 1-[F]FDF. Glucose-derivative 6-[F]FDG reached maximum tumor uptake at 20 minutes, with no further accumulation over time. Uptake of 2-[F]FDG was greatest and continuously increasing owing to metabolic trapping through phosphorylation by hexokinase II. In EMT6 tumors, GLUT5 mRNA expression was 20,000-fold lower compared with GLUT1. Whereas the latter was much greater in tumor than in muscle tissue (GLUT1 50:1), the opposite was found for GLUT5 mRNA expression (GLUT5 1:6). GLUT5 protein levels were higher in tumor versus muscle tissue as determined by Western blot and immunohistochemistry. Our data suggest that tumor uptake of fructose metabolism-targeting radiotracers 1-[F]FDF, 6-[F]FDF, and 1-[F]FDAM does not correlate with GLUT5 mRNA levels but is linked to GLUT5 protein levels. In conclusion, our results highlight the importance of detailed biochemical studies on GLUT protein expression levels in combination with PET imaging studies for functional characterization of GLUTs in BC.