125I-F56 Peptide as Radioanalysis Agent Targeting VEGFR1 in Mice Xenografted with Human Gastric Tumor

Abstract

¹²⁵I-Radiolabeled F56 peptide was designed as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind with VEGFR1 receptor. It was synthesized in high radiochemical yield and specific activity. The in vitro stability of ¹²⁵I-F56 was tested, and the bioactivity of ¹²⁵I-F56 was confirmed by both cell uptake and binding affinity measurement in VEGFR1 positive BGC-823 cells. The time-radioactivity relationship and biodistribution of ¹²⁵I-F56 tracer were conducted using nude mice bearing human gastric carcinoma BGC-823, by noninvasive micro-SPECT/CT imaging. The tracer's tumor uptake was further confirmed by autoradiography and HE stain of ¹²⁵I-F56 in tumor tissues ex vivo. Those results demonstrated that ¹²⁵I-F56 holds great potential as a diagnostic agent in both molecular imaging and radioanalysis probe for gastric cancer.

Keywords: 125I−F56; gastric cancer; radioanalysis; vascular endothelial growth factor receptor 1.

Scheme 1. Schematic Illustration of ¹²⁵I-Radiolabeling of F56 Peptide by Iodogen Vessel

The blue dotted circle indicates the reactive amino acids where radioactive ¹²⁵I may be attached.

Figure 1

HPLC chromatograms of ¹²⁵I–F56 performed with an RP-18 column. HPLC analysis revealed that the radiochemical purity of ¹²⁵I–F56 was 94%. (A) UV detection showed that the chromatographic peak of unreacted F56 peptide appeared at 7.8 min. (B) Radiation detection showed that the radiation peak of ¹²⁵I–F56 appeared at 8.5 min.

Figure 2

In vitro BGC-823 cell uptake. The cell uptake data were expressed as the percentage of the radioactivity measured with respect to the total radioactive compound used. **P < 0.01 from student’s t test.

Figure 3

Binding affinity assay of ¹²⁵I–F56 to BGC-823 cells. (A) BGC-823 cells were exposed to increasing concentrations of ¹²⁵I–F56 peptide for 4 h at 37 °C, showing saturation binding. The binding constant was 66 nM, according to a one-site binding model. (B) BGC-823 cells were incubated with increasing concentrations of F56 peptide for 2 h at 37 °C, and the F56 peptide concentration was determined by the antibody against F56 peptide, but conjugated HRP label, measured at 490 nm absorbance.

Figure 4

Biodistribution of ¹²⁵I–F56 in mice xenografted with BGC-823 gastric cancer cells.

Figure 5

Micro-SPECT images of ¹²⁵I–F56 in human gastric cancer cell BGC-823 bearing nude mice at 4 h postinjection and 5 h postcoinjection with 0.1 mg of F56 peptide as block agent. Left, coronal imaging; middle, sagittal imaging; right, coronal imaging of blocked group. The arrow indicates the location of the tumor.

Figure 6

MIP Micro-SPECT/CT imaging of ¹²⁵I–F56 (left) and SPECT images of ¹²⁵I–F56 coadministration with 0.1 mg of F56 peptide as block agent (right) in human gastric cancer cell BGC-823 bearing nude mice. P means prone position. The red arrow indicates the location of the tumor. The white dotted square (and oval) shows the position and the extension of the tumor, which were further examined by autoradiography imaging in dissected tumor tissue.