In vitro and in vivo evaluation of [123I]-VEGF165 as a potential tumor marker

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Abstract

One of the research challenges in oncology is to develop new biochemical methods for noninvasive tumor therapy evaluation to determine whether the chemotherapeutics is effective. Vascular endothelial growth factor (VEGF) was labeled with radioiodine and evaluated in vitro as well as in vivo, using A2058, a melanoma cell line overexpressing VEGFR-1 and -2. Saturation binding analysis with [125I]-VEGF resulted in a Kd of 0.1 nM. Internalization assays indicate the preserved ligand induced internalization and metabolization of the tracer. Biodistribution studies with [123I]-VEGF in wild type and A2058 tumor-bearing athymic mice showed low background activity and a tumor to reference tissue ratio of maximum 6.12. These results suggest that [123I]-VEGF is a potentially suitable tracer for tumor therapy evaluation.

Introduction

Angiogenesis plays an essential role in embryogenesis, normal tissue growth, wound healing and the female reproductive cycle. It also plays a major role in various diseases. Special interest is focused on tumor growth, since tumors cannot grow more than a few millimeters in size without developing a new blood supply.

Among others, vascular endothelial growth factor (VEGF-A) is one of the key molecules for angiogenesis and for the survival of the endothelium. It is a specific endothelial cell mitogen and a strong vascular permeability factor. VEGF-A is a heparin-binding glycoprotein, secreted as a homodimer of 45 kDa by many different cell types. Several variants of VEGF-A have been described, but VEGF165 is the most predominant protein. VEGF-A transcription is highly activated by hypoxia and by oncogenes like H-ras and several transmembrane tyrosine kinases, such as epidermal growth factor receptor and erbB2. Together, these pathways account for a significant up-regulation of VEGF-A in tumors compared to normal tissues and are of prognostic importance [1], [12]. VEGF signaling is mediated mainly by two receptors: VEGFR-1 (Kd=10–20 pM for VEGF165) and VEGFR-2 (Kd=75–125 pM for VEGF165). The affinity for the VEGFR-1 is higher, but the cellular response is mainly dominated by VEGFR-2 [2].

One of the research challenges in oncology is to develop new biochemical methods for tumor therapy evaluation to determine whether the chemotherapeutics is effective [3], [4]. This article presents the evaluation of the [123/125I]-VEGF165 for future use as a radiodiagnostic to evaluate the effect of farnesyl transferase inhibitors in tumor-inoculated athymic mouse models. Although [123I]-VEGF165 and [123I]-VEGF121 have been used before in vitro as well as in patients by Li et al. [5], [6], biodistribution studies in animals, which are of prior importance, are not reported. Li also showed that the labeled VEGF165 isoform showed affinity to more cell lines than the VEGF121 and is therefore preferred in this study.

The synthesis, in vitro evaluation of and biodistribution of [123I]-VEGF165 in wild-type NMRI mice as well as in tumor-bearing athymic mice are reported and discussed. If the parameters of the radiopharmacon, mainly kinetics and tumor uptake ratios, are sufficient, in vivo therapy evaluation is considered for chemotherapeutics that interfere with the VEGFR pathway, such as tyrosine kinase inhibitors or farnesyl transferase inhibitors [1].

Section snippets

Synthesis of [123I]-VEGF and [125I]-VEGF

Synthesis of both radioligands was performed in similar conditions, using the Iodogen technique. Iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycouracil, Pierce, Aalst, Belgium) was coated to polypropylene vials using 70 μg of Iodogen per 200 μl of chloroform per vial (Aldrich, Bornem, Belgium). After evaporation of the solvent under N2-atmosphere at room temperature, the vials were stored at 4°C. Two micrograms of VEGF (Peprotech, London, UK) and the required amount of carrier-free Na123I or Na

Synthesis of [123I]-VEGF165 and [125I]-VEGF165

The overall radiochemical yield using freshly prepared Iodogen vials is 37% for [123I]-VEGF165 and for [125I]-VEGF165. A representative chromatogram of the purification step for the 123I compound is shown in Fig. 1. An elution of purified [123I]-VEGF165 with PBS/BSA on PD-10 gives rise to a total eluted fraction of 80%. Nonspecific adsorption on the PD-10 column cannot be decreased using increasing amounts of BSA. Radiochemical purity of the 123I tracer recovered at end of synthesis was >95%.

Conclusion

Given the preserved receptor–ligand interactions and the high receptor affinity, its low in vivo background activity due to relatively low dehalogenation and good tumor uptake, we conclude that [123I]-VEGF165 is a potent tool to perform VEGF receptor scintigraphy in vivo.

Acknowledgment

The authors thank Alexandra Janssens who performed the labeling and stability studies and assisted in the in vitro and in vivo evaluation.

References (12)

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