The Human Norepinephrine Transporter in Combination with 11C-m-Hydroxyephedrine as a Reporter Gene/Reporter Probe for PET of Gene Therapy
Anne Rixt Buursma, MSc1,*,
Antoine M.J. Beerens, MSc2,*,
Erik F.J. de Vries, PhD1,
Aren van Waarde, PhD1,
Marianne G. Rots, PhD2,
Geke A.P. Hospers, MD, PhD3,
Willem Vaalburg, PhD1 and
Hidde J. Haisma, PhD2
1 Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
2 Department of Therapeutic Gene Modulation, University of Groningen, Groningen, The Netherlands
3 Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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FIGURE 1. (A) Chemical structures of norepinephrine (left) and 11C-mHED (right). (B) Genome of the first-generation adenoviral vector, containing transgenes EGFP (enhanced green fluorescence protein) and hNET, located in region of the E1 deletion. The 2 transgenes are expressed from identical but separate expression cassettes. CMVp = cytomegalovirus promoter; ITR = inverted terminal repeat.
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FIGURE 2. 11C-mHED uptake in COS-7 cells transfected with adenoviral AdTrack-hNET at various MOI. MOI = 0 represents control (cells infected with AdTrack-Luc). Two days after transfection, cells were assayed for 11C-mHED accumulation in presence or absence of 6 µmol/L DMI. Three independent experiments were performed, either in duplicate or triplicate. Average ± SD is shown. *Significantly higher uptake than that of DMI-treated cells (P < 0.05).
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FIGURE 3. 11C-mHED uptake vs. viral titer in COS-7 cells, A2780 cells, and U373 cells. Cells were transduced with AdTrack-hNET at various MOI. MOI = 0 represents control (cells infected with AdTrack-Luc). Two days after infection, cells were assayed for 11C-mHED accumulation. In all cell lines, 11C-mHED uptake increased linearly with viral titer (r2 = 0.997 for COS-7 cells, r2 = 0.949 for A2780 cells, and r2 = 0.994 for U373 cells; P < 0.05). Error bars represent SD.
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FIGURE 4. 11C-mHED uptake vs. amount of hNET protein in U373 cells. hNET protein expression was determined in U373 cells grown and transfected parallel to cells used for 11C-mHED accumulation experiments 2 d after infection. Good correlation between amount of hNET protein and 11C-mHED uptake is evident (r2 = 0.987; P < 0.0001). Error bars represent SD.
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FIGURE 5. 11C-mHED uptake vs. EGFP fluorescence in COS-7 cells (A), A2780 cells (B), and U373 cells (C). Cells were transduced with AdTrack-hNET. Two days after infection, EGFP intensity was measured immediately before the start of 11C-mHED accumulation experiments. In all 3 cell lines, EGFP fluorescence correlated well with 11C-mHED uptake (r2 = 0.965 for COS-7 cells, r2 = 0.989 for A2780 cells, and r2 = 0.979 for U373 cells; P < 0.005). Error bars represent SD.
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FIGURE 6. Anterior 11C-mHED PET image of HSD RH rnu rat bearing 2 U373 tumors that were either transfected with adenoviral vector AdTrack-hNET (top) or control vector (bottom) 2 d before PET study. PET image was obtained 50–60 min after injection of 42 ± 6 MBq of 11C-mHED tracer. Image is summation of all coronal planes, on which animal was visible.
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FIGURE 7. Visualization of EGFP fluorescence in tumor tissue. EGFP expression in tumor tissue was visualized under fluorescence microscope in 4-µm cryosections. (A) Phase-contrast image. (B) EGFP fluorescence. (C) Merged picture.
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Copyright © 2005 by the Society of Nuclear Medicine.
