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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.

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.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 39-deoxy-39-L-[ 18 F]fluorothymidine ([ 18 F]FLT). This study investigates whether [ 18 F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [ 18 F] FLT-PET in estrogen receptor positive (ER + )-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [ 18 F]FLT transport was demonstrated in all cell lines. In vitro [ 18 F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 6 9 vs. 147 6 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [ 18 F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 6 0.05 vs. 0.89 6 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [ 18 F]FLT retention in ER + tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [ 18 F]

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