Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts

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Abstract

c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of 125I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC50 values of peptides were 1.5 μM, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various 125I labeled cMBPs, image contrast and overall quality were unremarkable.

Introduction

Malignant gliomas are the most common types of brain tumors and are the second leading cause of cancer death [1]. With a high proliferation rate, marked neovascularization and extensive local invasion of tumor into the normal brain parenchyma, gliomas are resistant to conventional therapies and rapidly progress to patient death.

c-Met is a receptor tyrosine kinase (RTK) that is known to stimulate the invasive growth of cancer cells and increase their metastatic potential, and is also known to be expressed and mutated in a variety of solid tumors [2], [3]. c-Met is overexpressed in human glioblastomas, and expression levels correlate with glioma malignancy grade and vascularity [4], [5], [6]. The structure of c-Met consists of a disulfide-linked α-β heterodimer with a molecular weight of 190-kDa. The 140-kDa β chain spans the membrane; however, the α chain is only located extracellularly. Both these forms are cleaved from a 170-kDa precursor form [2].

Kim et al. [7] previously reported that monoclonal antibody SFN68 inhibits the hepatocyte growth factor (HGF)–c-Met interaction and blocks the biological function of c-Met mediated by HGF. To define the epitope, they screened out an epitope-mimicking peptide, NH2-Lys1-Ser2-Leu3-Ser4-Arg5-His6-Asp7-His8-Ile9-His10-His11-His12-Ac, from a phage display of a combinatorial peptide library. They found that this peptide binds to c-Met and inhibits the HGF–c-Met interaction.

Although radiolabeled receptor-binding peptides already represent an important class of radiopharmaceuticals, c-Met binding radiopeptides for cancer imaging have not been studied. In the current study, therefore, we evaluated tumor imaging with c-Met-binding peptide (cMBP) in a glioma xenograft model. We also tested whether or not the pharmacokinetics changed when glycine (G) or 8-aminooctanoic acid (AOC) were introduced. Several groups have evaluated the change of pharmacokinetics of small peptides when various linkers are introduced. Parry et al. designed the bombesin analogue containing glycine (G), serine (S), glutamic acid (E) or carbon chain linkers and evaluated them using various tumor models [8], [9]. Based on these studies, it appears that such conjugation could alter tumor uptake and lead to improved image contrast. The aim of this study was to evaluate the utility of radiolabeled cMBP as a molecular imaging probe in mice bearing human glioma cancer xenografts.

Section snippets

Reagents and chemicals

Na125I was obtained from Perkin Elmer Life Science, Inc. (Boston, MA). All chemicals were obtained from Sigma-Aldrich Chemicals (St. Louis, MO), and all solvents were reagent grade and used without further purification. KSLSRHDHIHHH (cMBP), KSLSRHDHIHHH-GGG-SC (cMBP-GGG) and KSLSRHDHIHHH-AOC-C (cMBP-AOC) were synthesized by Peptron using standard Fmoc chemistry (Seoul, Korea). The purity of all samples was 90%.

Preparation of radioiodinated peptides

cMBP, cMBP-GGG and cMBP-AOC were radiolabeled with Na125I using chloramine T.

125I-labeling

The amino acid sequences of the peptides are shown in Fig. 1. The radiochemical purity of the 125I-labeled peptide conjugates were 90% to 95% at 24 h postlabeling. Radiochemical purity following incubation in human serum was over 90% at 1 h and 88% at 4 h.

RT-PCR and Western blot analysis

To confirm the expression of c-Met mRNA in U87MG cells and tumor tissue, total RNA was isolated using the Trizol. As shown in Fig. 2A and B, c-Met mRNA was expressed in both cells and tumor tissue. Western blot analysis (Fig. 2C) showed

Discussion

Rapid progress toward understanding the role of HGF and its receptor in cancer cells and cancer development has been made over the past decade [10], [11], [12], [13]. Cancers that overexpress HGF and/or c-Met are frequently linked to the aggressive nature and show poor clinical outcomes. The signaling pathways downstream of HGF and/or c-Met are becoming clearer, and HGF and/or c-Met may serve as a potential prognostic indicator for aggressiveness and angiogenesis when considered together with

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This work was supported by the Nuclear R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Science and Technology (M20702000003-08N0200-00310).

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