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Short communication Treatment of cultured glioma cells with the EGFR-TKI gefitinib (“Iressa”, ZD1839) increases the uptake of astatinated EGF despite the absence of gefitinib-mediated growth inhibition Åsa Liljegren Sundberg, Ylva Almqvist, Vladimir Tolmachev, Jörgen Carlsson Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Received: 8 January 2003 / Accepted: 16 January 2003 / Published online: 7 March 2003 © Springer-Verlag 2003

Abstract. The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib (“Iressa”, ZD1839), a reversible growth inhibitor of EGFRexpressing tumour cells, has been shown to enhance the antitumour effect of ionising radiation, and also to increase the uptake of radioiodinated EGF. Thus, combination of gefitinib treatment and radionuclide targeting is an interesting option for therapy of brain tumours that are difficult to treat with conventional methods. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF (211At-EGF) to cultured glioma U343 cells, which express high levels of EGFR. The growth of U343 cells in the presence of gefitinib was investigated, and it was found that gefitinib does not significantly inhibit the growth of these cells. Nevertheless, the uptake of 211At-EGF in U343 cells was markedly increased (up to 3.5 times) in cells pre-treated with gefitinib (1 µM). This indicates that a combination of gefitinib treatment and radionuclide targeting to EGFR might be a useful therapeutic modality, even for patients who do not respond to treatment with gefitinib alone. Keywords: Gefitinib – EGFR – Glioma Eur J Nucl Med Mol Imaging (2003) 30:727–729 DOI 10.1007/s00259-003-1129-x

Introduction Glioblastoma multiforme tumours rarely produce metastases outside the central nervous system. However, these Jörgen Carlsson (✉) Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden e-mail: [email protected] Fax: +46-18-4713432

brain tumours are very malignant and even the most aggressive treatment protocols fail to prolong the median survival beyond 2 years [1]. Tumour recurrences most often arise from metastatic tumour cells in the brain, which are impossible to detect and eradicate by conventional methods. The use of radionuclide targeting is a possible strategy in glioblastoma treatment since it provides an opportunity to deliver cytotoxic radionuclides to malignant cells with minimal damage to normal brain tissue. Locoregional intracranial administration of targeting vectors makes it possible to overcome problems associated with penetration of the blood-brain barrier. Clinical studies utilising radiolabelled antibodies against tenascin, a glioma-associated extracellular matrix component, have been initiated [2, 3, 4]. There is, however, some concern that the diffusion of the relatively bulky immunoglobulin molecules in brain tissue is inefficient, which might be a limitation for radioimmunotherapy. Targeting of low-grade gliomas with the yttrium-90 labelled somatostatin analogue DOTATOC has demonstrated that short peptides are sufficiently diffusible to target distant brain tumour foci [5]. Unfortunately, expression of somatostatin receptors is low in high-grade gliomas, and the use of DOTATOC would, in this case, probably be inefficient. Another possible target structure on glioblastoma cells is the epidermal growth factor receptor (EGFR), which is expressed at high levels by a high percentage of glioblastomas [6, 7, 8, 9]. A natural ligand to the EGFR, the EGF, is a peptide with a molecular weight of about 6 kDa, which might allow a good diffusion through brain tissues. When coupled to a cytotoxic nuclide, e.g. the alpha-emitter astatine-211 (T1/2=7.2 h), EGF might be an efficient radiopharmaceutical against glioblastoma. Preclinical studies on the use of labelled EGF have been initiated in our laboratory. One strategy in the treatment of EGFR-expressing tumours is to inhibit the tyrosine kinase activity of EGFR, which blocks mitogenic signalling. It has been demonstrated that the EGFR-TKI (tyrosine kinase inhibitor) ge-

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fitinib [“Iressa” (a trademark of the AstraZeneca group of companies), ZD1839] inhibits tumour growth both in vitro and in vivo, and that the use of gefitinib enhances the antitumour effect of ionising radiation [10]. Moreover, it has been observed that treatment of human carcinoma A431 cells with gefitinib increased the uptake of radioiodinated EGF [11]. One could therefore expect it to be beneficial to combine radionuclide targeting therapy of brain tumours with simultaneous gefitinib treatment. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF to cultured glioma U343 cells.

Materials and methods Cell growth during gefitinib exposure. A431 (human squamous carcinoma cells, ATCC CRL1555, approximately 106 EGFR per cell) and U343 (human glioma cells, U343 MGaCl2:6, approximately 105 EGFR per cell) were seeded in Petri dishes (diameter 3 cm) at a density of about 10,000 and 45,000 cells per dish, respectively. After 48 h of incubation, gefitinib (kindly provided by AstraZeneca), 0 or 1 µM in cell culture medium, was added to the cells. The cells were then counted daily for 5 days. Fresh gefitinibcontaining medium was added to the cell dishes daily to ensure continuous exposure to gefitinib. Gefitinib exposure for the A431 cells was interrupted in some of the dishes after 48 h (fresh culture medium was added) to determine whether the cells could recover their growth capacity after growth inhibition with gefitinib. Labelling of EGF with astatine. Astatine was produced by alphaparticle irradiation of bismuth and separated from the target material by dry distillation. Labelling of human recombinant EGF (Chemicon International, Temecula, Calif.) was performed using conjugation with N-succinimidyl p-[211At]astatobenzoate according to Wilbur et al. [12], with some modifications. Time pattern after continuous binding with 211At-EGF during gefitinib exposure. U343 cells were seeded in 12-well plates (approximately 31,000 cells/well) and incubated at 37°C for 4 days. The medium in the cell dishes was then changed to fresh culture medium containing 0 or 1 µM gefitinib and the incubation was continued for 24 h. After washing the cells once, approximately 17 ng 211At-EGF in culture medium, containing 0 or 1 µM gefitinib, was added to the dishes. In addition to this, 100 times excess of unlabelled EGF was added to some of the dishes in order to determine the amount of unspecific binding. The dishes were incubated with 211At-EGF for 1–21 h at 37°C. They were then washed six times with serum-free medium, trypsinised with 0.5 ml trypsinEDTA at 37°C for 15 min and resuspended with 1 ml of culture medium. Part of the cell suspension (0.5 ml) was used for counting the cells in an electronic cell counter, and the radioactivity in the remaining 1 ml was measured in an automated gamma counter.

Results and discussion Growth inhibition experiments demonstrated that gefitinib does not significantly inhibit growth of cultured U343 glioma cells at a concentration of 1 µM in culture medi-

Fig. 1a, b. Influence of gefitinib on the growth of U343 (a) and A431 cells (b). Cells were grown in the presence (open squares) or absence (control, closed diamonds) of 1 µM gefitinib. Each data point is an average value from three dishes ± maximum error

um (Fig. 1a). This differs from the behaviour of another EGFR-expressing cell line, A431, where reversible growth inhibition was observed (Fig. 1b). Experiments on cellular binding of astatinated EGF to U343 cells were nevertheless performed. Binding of 211At-EGF to U343 cells was compared in cells pre-treated with 1 µM gefitinib and non-treated cells. Binding of EGF to cells in both groups was receptor-specific since it could be blocked by an excess amount of non-labelled EGF (Fig. 2). Interestingly, the uptake of 211At-EGF was significantly higher in gefitinib pre-treated cells than in non-treated cells (Fig. 3) throughout the study (21 h, approximately three half-lives of 211At). This increased uptake was especially pronounced during the first two half-lives of astatine, when the main part of the 211At-derived dose was delivered to the targeted cells. Elevated uptake of radioiodinated EGF has been observed earlier in gefitinib-treated cells [11]. However, these observations have been made for A431 cells, which show growth inhibition following gefitinib exposure. Generally, the increased uptake might be of value for targeted radionuclide therapy since one could


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References

Fig. 2. Specificity of 211At-EGF binding in U343 cells. Cells were pre-treated with 0 or 1 µM gefitinib for 24 h, then incubated with astatinated EGF in the presence or absence of gefitinib for a further 7 h. EGFR on cells in control dishes were blocked with an excess of non-labelled EGF to determine the unspecific binding. The values on the y-axis give the percentage of added radioactivity per 105 cells. Each data point is an average value from three dishes ± maximum error

Fig. 3. Binding of 211At-EGF to cultured glioma U343 cells pretreated with 0 µM (closed diamonds) or 1 µM (open squares) gefitinib. The values on the y-axis give the percentage of added radioactivity per 105 cells. Each data point is an average value from three dishes ± maximum error. The values are corrected for radioactive decay

expect additive and supra-additive effects of the combined treatment. Our results demonstrate that treatment with the EGFR-TKI gefitinib (“Iressa”, ZD1839) increases the uptake of radionuclides delivered by EGF even if the tumour cells are not growth inhibited. A three to four times increase in the uptake of a cytotoxic nuclide appreciably increases the chance of a successful targeted radionuclide therapy. This creates an opportunity for improving the outcome of patients with tumours that do not respond to other therapeutic methods. Acknowledgements. Gefitinib was kindly provided by AstraZeneca, Macclesfield, UK. Financial support was provided by the Swedish Cancer Society.

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