microPET of tumor integrin alphavbeta3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4)

Abstract

In vivo imaging of alpha(v)beta(3) expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin alpha(v)beta(3)-binding affinity due to the polyvalency effect. Here we report an example of (18)F-labeled tetrameric RGD peptide for PET of alpha(v)beta(3) expression in both xenograft and spontaneous tumor models.

Methods: The tetrameric RGD peptide E{E[c(RGDyK)](2)}(2) was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate alpha-amino group. NH(2)-mini-PEG-E{E[c(RGDyK)](2)}(2) (PRGD4) was labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer (18)F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies.

Results: The decay-corrected radiochemical yield for (18)F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from (18)F-F(-). The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. (18)F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of (18)F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK).

Conclusion: The tetrameric RGD peptide tracer (18)F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin alpha(v)beta(3) expression in cancer and other angiogenesis related diseases.

FIGURE 1

(Upper) Radiosynthesis scheme of ¹⁸F-FPRGD4. (Lower) Chemical structure of ¹⁸F-FPRGD4.

FIGURE 2

(A) Decay-corrected whole-body coronal microPET images of athymic female nude mice bearing U87MG tumor at 5, 15, 30, 60, 120, and 180 min after injection of ¹⁸F-FPRGD4 (3.7 MBq [100 μCi]). (B) Decay-corrected whole-body coronal microPET images of c-neu oncomice at 30, 60, and 150min (5-min static image) after intravenous injection of ¹⁸F-FPRGD4. (C) Decay-corrected whole-body coronal microPET images of orthotopic MDA-MB-435 tumor-bearing mouse at 30, 60, and 150 min after intravenous injection of ¹⁸F-FPRGD4. (D) Decay-corrected whole-body coronal microPET images of DU-145 tumor-bearing mouse (5-min static image) after intravenous injection of ¹⁸F-FPRGD4. (E) Coronal microPET images of a U87MG tumor-bearing mouse at 30 and 60 min after coinjection of ¹⁸F-FPRGD4 and a blocking dose of c(RGDyK). Arrows indicate tumors in all cases.

FIGURE 3

Time–activity curves of major organs after intravenous injection of ¹⁸F-FPRGD4. Data were derived from a multiple time-point microPET study. ROIs are shown as the %ID/g ± SD (n = 3).

FIGURE 4

Comparison between uptake of ¹⁸F-FPRGD2 and ¹⁸F-FPRGD4 in U87MG tumor, kidneys, liver, and muscle over time. Data were derived from multiple time-point microPET study. ROIs are shown as %ID/g ± SD (n = 3).

FIGURE 5

Immunofluorescent staining of β₃ and CD31 for tumor, liver, kidney, and lung. For β₃ staining, frozen tissue slices (5-μm thick) were stained with a hamster antimouse β₃ primary antibody and a Cy3-conjugated goat antihamster secondary antibody. For CD31 staining, frozen tissue slices were stained with a rat antimouse CD31 primary antibody and a FITC-conjugated goat antirat secondary antibody (×200). Arrowheads indicate overlay area in all cases.