Article Figures & Data
Figures
- FIGURE 1.
Concept of hypoxia imaging using 123I-IPOS. PTD enables 123I-IPOS to be delivered to normoxic and hypoxic tissue. In normoxic tissue, POS is degraded in manner similar to HIF-1α degradation, and 123I-IBB is cleared. In contrast, in HIF-1-active tissues, POS escapes degradation and radioactivity is retained within cell. Thus, POS enables specific imaging of HIF-1-active hypoxic region.
- FIGURE 2.
Analysis of fusion protein expression with sodium dodecylsulfate-polyacrylamide gel electrophoresis (lane M, molecular weight marker; lane 1, Escherichia coli lysate before IPTG induction; lane 2, E. coli lysate after IPTG induction; lane 3, protein solution after purification). New band was expressed in lane 2 at about 60 kDa, corresponding to GST-fused POS. After purification, only 1 band was detected in lane 3 at 34 kDa. IPTG = isopropyl-β-d-thiogalactopyranoside.
- FIGURE 3.
Binding of 125I-IBB to POS. (A) Size-exclusion HPLC chromatogram after incubation of 125I-IBB with POS. Symbols represent radioactivity of each eluate collected every 1 min. (B) Concentration-dependent inhibition of 125I-IBB binding to POS by D-biotin. Symbols and bars represent mean and SD.
- FIGURE 4.
(A) HIF-1-dependent luciferase activity in HeLa, MM1, and FM3A cells. Data were normalized by protein concentration of cells. Results are shown as ratio of activity after hypoxia to activity after normoxia. Columns and bars represent mean and SD (n = 3–6 **P < 0.01 vs. normoxia). (B) Intracellular radioactivity in HeLa, MM1, and FM3A cells after 24-h incubation under either normoxic or hypoxic conditions. Radioactivity was normalized by protein concentration of cells. Columns and bars represent mean and SD (n = 3–6 **P < 0.01 vs. normoxia). (C) Degradation of intracellularly accumulated IPOS after reoxygenation. HeLa cells were incubated with 125I-IPOS for 24 h under hypoxic conditions. Then, medium was replaced with fresh medium, and cells were subjected to further incubation under normoxic or hypoxic conditions. Results are shown as percentage of radioactivity at start of second incubation. Symbols and bars represent mean and SD (n = 3–5; *P < 0.05 vs. normoxia).
- FIGURE 5.
Typical planar images of FM3A-implamted mice at 24 or 48 h after injection of 123I-IPOS. Tumors were clearly visualized in both images (arrow). Arrowheads indicate liver.
- FIGURE 6.
Correlation between accumulation of 125I-IPOS and HIF-1 activity within same tumor. Ordinate represents accumulated radioactivity (%ID), and abscissa represents HIF-1-dependent luciferase activity. Correlation coefficient (R) was 0.71, indicating highly significant correlation (P < 0.05).
- FIGURE 7.
Comparison between intratumoral distribution of 125I-IPOS and pimonidazole-positive hypoxic region. Typical autoradiogram (A) and pimonidazole immunohistochemical staining (B) in identical section are shown. Merged image (C) is also presented. Black arrowheads indicate area of 125I-IPOS accumulation in A and pimonidazole-positive area in B. Black arrows show areas in which both signals are positive, and red arrowheads indicate pimonidazole-positive areas without accumulation of 125I-IPOS. No signal was observed in pimonidazole-untreated tumor (D).
Tables
Time after injection (h) Organ 1 6 24 48 Blood 15.55 ± 1.06 3.14 ± 0.54 0.18 ± 0.12 0.01 ± 0.01 Liver 32.97 ± 1.03 13.80 ± 0.87 0.97 ± 0.44 0.30 ± 0.06 Spleen 17.29 ± 2.35 6.07 ± 0.73 1.21 ± 0.53 0.19 ± 0.05 Kidney 5.51 ± 0.36 2.79 ± 0.47 0.74 ± 0.60 0.09 ± 0.02 Stomach 2.25 ± 0.57 0.62 ± 0.32 0.49 ± 0.31 0.04 ± 0.02 Neck 2.39 ± 0.06 0.80 ± 0.08 0.09 ± 0.04 0.00 ± 0.01 Intestine 7.96 ± 0.09 9.82 ± 1.61 0.91 ± 0.13 0.24 ± 0.08 Tumor 1.47 ± 1.02 1.49 ± 0.38 0.28 ± 0.08 0.07 ± 0.05 Muscle 0.94 ± 0.65 0.28 ± 0.05 0.07 ± 0.06 0.00 ± 0.01 Tumor/blood 0.10 ± 0.07 0.49 ± 0.18 1.79 ± 0.59 6.72 ± 6.61 Organ uptake values are expressed as %ID/g of tissue except for tumor/blood. Values are mean ± SD; n = 3–5.
- TABLE 2
Effect of POS Concentration on Biodistribution of 125I-IPOS in FM3A-Implanted Mice at 24 Hours After Injection
Protein concentration (μg) Organ 0.05 0.5 5 30 Blood 0.12 ± 0.04 0.16 ± 0.10 0.16 ± 0.01 0.27 ± 0.07 Liver 1.92 ± 0.24 1.27 ± 0.53 5.53 ± 1.05 13.61 ± 1.90 Spleen 1.07 ± 0.30 1.15 ± 0.41 2.33 ± 0.25 7.87 ± 2.20 Kidney 0.35 ± 0.20 0.59 ± 0.51 1.03 ± 0.10 4.32 ± 1.87 Stomach 0.28 ± 0.14 0.55 ± 0.42 0.52 ± 0.20 0.86 ± 0.27 Neck 0.05 ± 0.04 0.09 ± 0.04 0.11 ± 0.02 0.23 ± 0.08 Intestine 4.57 ± 0.82 1.95 ± 2.03 2.17 ± 0.24 2.18 ± 0.28 Tumor 0.23 ± 0.08 0.25 ± 0.07 0.49 ± 0.09 1.37 ± 0.33 Muscle 0.05 ± 0.03 0.05 ± 0.05 0.07 ± 0.01 0.10 ± 0.03 Tumor/blood 2.04 ± 0.61 1.61 ± 0.10 3.00 ± 0.68 5.14 ± 0.34 Organ uptake values are expressed as %ID/g of tissue except for tumor/blood. Values are mean ± SD; n = 5.
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