Research ArticleBasic Science Investigation

In Vivo Small-Animal PET/CT of EphB4 Receptors Using 64Cu-Labeled Peptide

Chiyi Xiong, Miao Huang, Rui Zhang, Shaoli Song, Wei Lu, Leo Flores, Juri Gelovani and Chun Li
Journal of Nuclear Medicine February 2011, 52 (2) 241-248; DOI: https://doi.org/10.2967/jnumed.110.081943

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Structure of 64Cu-DOTA-TNYL-RAW and natCu-DOTA-TNYL-RAW peptides.

  • FIGURE 2.
    FIGURE 2.

    SPR sensorgrams of natCu-DOTA-TNYL-RAW and scrambled peptide on sensor chips coated with EphB4. Peptides were injected as ten 2-fold concentration series from 1.6 to 800 nM and were analyzed in duplicate binding cycles. Datasets (shown in black) are overlaid with curves fit to 1:1 mass transfer interaction model (red lines). Vertical axes in response units represent binding of each peptide to immobilized EphB4.

  • FIGURE 3.
    FIGURE 3.

    (A) Fluorescence photomicrographs of PC-3M cells treated with FITC-TNYL-RAW (10 μM, green) or scrambled peptide FITC-sc-TNYL-RAW (10 μM, green) for 20 min at room temperature. Cells were also stained with phycoerythrin-conjugated anti-EphB4 antibody (red) for expression of EphB4 receptors. For blocking experiment, FITC-TNYL-RAW (10 μM) was coincubated with TNYL-RAW (1 mM). Cell nuclei were counterstained with DAPI (blue). Bar = 20 μm. (B) Uptake of 64Cu-DOTA-TNYL-RAW in EphB4-expressing PC-3M cells. Cell-to-medium uptake ratio is expressed as (counts/min/μg of protein in pellet)/(counts/min/μg of medium). 64Cu-DOTA-TNYL-RAW exhibited increased uptake over time in PC-3M cells. This uptake was blocked by parent TNYL-RAW peptide. PE = phycoerythrin.

  • FIGURE 4.
    FIGURE 4.

    Representative small-animal PET/CT images of mice bearing CT26, PC-3M, and A549 tumors after intravenous administration of 64Cu-DOTA-TNYL-RAW (n = 4). Arrows refer to tumors.

  • FIGURE 5.
    FIGURE 5.

    Representative small-animal PET/CT images showing blocking of 64Cu-DOTA-TNYL-RAW uptake in CT26 tumors at 4 h and in PC-3M tumors at 24 h after radiotracer injection. For blocking experiment, 64Cu-DOTA-TNYL-RAW was coinjected with cold TNYL-RAW (50 μg/mouse). Arrows refer to tumors.

  • FIGURE 6.
    FIGURE 6.

    (A) Biodistribution of 64Cu-DOTA-TNYL-RAW in mice bearing PC-3M and A549 tumors at 24 h after injection. Data are presented as %ID/g and are expressed as mean ± SD (n = 4). (B) Tumor-to-muscle-uptake ratio. In blocking group, mice were injected with 64Cu-DOTA-TNYL-RAW (7.4 MBq [200 μCi]) and cold TNYL-RAW peptide (20 μg/mouse). Data are presented as mean ± SD (n = 6). Data were obtained at 4 h after injection in mice with CT26 tumors and at 24 h after injection in mice with PC-3M tumors.

Tables

  • TABLE 1

    Association and Dissociation Rates and KD of Peptides Interacting with Immobilized EphB4 Receptors Obtained from SPR Analysis

    Analytekon (M−1s−1)koff (s−1)KD (M)χ2
    TNYL-RAW4.26 × 1051.3 × 10−33.06−90.155
    DOTA-TNYL-RAW1.10 × 1052.56 × 10−32.33−80.281
    natCu-DOTA-TNYL-RAW6.89 × 1061.31 × 10−21.98−91.86

    • koff = dissociation rate constant; kon = association rate constant.

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