[CANCER RESEARCH 63, 6864 – 6869, October 15, 2003] Subcellular Localization of Radiolabeled Somatostatin Analogues: Implications for Targeted Radiotherapy of Cancer 1 Mu Wang, Amy L. Caruano, Michael R. Lewis, 2 Laura A. Meyer, Robert P. VanderWaal, and Carolyn J. Anderson 3 Mallinckrodt Institute of Radiology [M. W., A. L. C., M. R. L., L. A. M., C. J. A.], and Department of Radiation Oncology [R. P. V.], Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT Copper-64 (T 1/2  12.7 h;   , 17.4%;   , 39%) has been used both in positron emission tomography imaging and in radiotherapy. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. These studies incorporate the use of somatosta- tin receptor-positive AR42J rat pancreatic tumor cells in vitro to understand the cell killing mechanism of 64 Cu by focusing on subcellular distribution of the somatostatin analogues 64 Cu-labeled 1,4,8,11-tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid-octreotide ( 64 Cu-TETA-OC) and 111 In-labeled di- ethylenetriaminepentaacetic acid-octreotide ( 111 In-DTPA-OC). Cell uptake and organelle isolation studies were conducted on 64 Cu-TETA-OC and 111 In-DTPA-OC. Nuclear localization of 64 Cu and 111 In from 64 Cu- TETA-OC and 111 In-DTPA-OC, respectively, increased over time, with 19.5  1.4% and 6.0  1.0% in the cell nucleus at 24 h, respectively. In pulse-chase experiments, in which 64 Cu-TETA-OC was incubated with AR42J cells for 4 h, it was found that the nuclear localization of 64 Cu increased significantly over the next 20 h (from 9.8  1.0% to 26.3  5.4%). In a control pulse-chase experiment, levels of 64 Cu from [ 64 Cu]cupric acetate decreased from 4 to 24 h postadministration (20.6  8.7 to 5.4  1.9), suggesting that the redistribution mechanism, or the kinetics of 64 Cu from 64 Cu-TETA-OC is different from that for 64 Cu from [ 64 Cu]cupric acetate. The amount of 64 Cu from 64 Cu-TETA-OC also increased in the mitochondria over time, with 21.1  3.6% in the mitochondria at 24 h postadministration. These results suggest that localization of substantial quantities of 64 Cu to the cell nucleus and mitochondria may contribute to cell killing with 64 Cu radio- pharmaceuticals. INTRODUCTION Over the last several years, considerable progress has been made in the investigation of radiolabeled somatostatin analogues as radiother- apeutic agents for somatostatin-receptor-positive tumors. Because of the use of radiolabeled somatostatin analogues for targeted radiother- apy of cancer (1, 2), the intracellular fate of the radiolabeled soma- tostatin analogues after binding to cell surface receptors has been a topic of considerable interest (3, 4). For example, in experiments in which 111 In-DTPA-OC, 4 a clinically approved imaging agent for somatostatin-receptor-positive tumors in the United States and Eu- rope, was incubated with cells grown in culture, uptake of 111 In in the cell nuclei was observed (3). This suggests a possible mechanism for the therapeutic efficacy of this Auger electron-emitting radiopharma- ceutical. We are interested in 64 Cu [T 1/2  12.7 h;   , 0.655 MeV (17.4%);   , 0.573 MeV (39%)] because of its decay by   emission for diagnostic imaging by PET, along with decay by   emission for cancer therapy applications. The decay characteristics of 111 In and 64 Cu are summarized in Table 1. 64 Cu-TETA-OC, in which TETA  1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, is a somatostatin analogue that has been shown to have applications for PET imaging and targeted radiotherapy of cancer (5–7). The structures of DTPA-OC and TETA-OC are shown in Fig. 1. In a previous study, we observed enhanced therapeutic efficacy of an internalizing 64 Cu-labeled mAb, 64 Cu-BAT-2IT-1A3, in a tumor- bearing rodent model compared with published studies of 131 I- or 90 Y-labeled mAbs in the same animal model (8). The mechanism for this improved tumor cell killing is unclear. One hypothesis that has been set forth is that, after cellular internalization, 64 Cu may localize to the nuclei of tumor cells because of the dissociation of the radio- metal from macrocyclic chelators in vivo (9). Copper-64 decays by   (and   ) emission, and it would seem that the range of  particles is too large for nuclear uptake to affect tumor cell killing. There is considerable evidence for the binding of Cu(II) to DNA and/or other structures in the nucleus. Copper ions have been suggested to play an important role in the maintenance of nuclear matrix organization and DNA folding (10, 11). Chiu et al. have found that treatment of isolated nuclei with levels as low as 1 M Cu(II) causes nuclear matrix- associated DNA to bind to nuclear matrix proteins and further, Cu(II) causes DNA protein cross-linking, as well as DNA double-strand breaks on subsequent irradiation (12, 13). The fact that copper has been shown to bind to radiation-sensitive areas in the nucleus, such as the nuclear matrix proteins and DNA, suggests that in comparison with other radionuclides, radiopharmaceuticals labeled with copper radioisotopes may have enhanced effectiveness for targeted radio- therapy. To investigate this hypothesis further, we chose to evaluate the subcellular distribution of 64 Cu-TETA-OC compared with 111 In- DTPA-OC, because radiolabeled somatostatin analogues are known to be internalized (14). Other investigators have observed the localiza- tion of 111 In to the nuclei of tumor cells after incubation with 111 In- DTPA-OC (3, 4), and a comparison of 64 Cu-TETA-OC with this agent is warranted. Most importantly, determining the extent of the nuclear localization of 64 Cu from 64 Cu-TETA-OC will be a first step in determining whether the delivery of 64 Cu radiopharmaceuticals to the nuclei of tumor cells has implications for cancer therapy. MATERIALS AND METHODS Materials. High specific activity 64 CuCl 2 was produced from enriched 64 Ni targets on a CS-15 cyclotron at Washington University (St. Louis, MO) as described previously (15). Indium-111 chloride ( 111 InCl 3 ) was obtained from Mallinckrodt, Inc. (St. Louis, MO). Ammonium acetate and ammonium citrate were purchased from Fluka (Buchs, Switzerland). AR42J rat pancreatic tumor cells were obtained from Mallinckrodt, Inc. AR42J cell media [1 DMEM (Cellgro) and Hams F12K], 1 trypsin-EDTA [0.05% trypsin/0.02% EDTA (Cellgro)], and EDTA were obtained from Fisher Scientific (Pittsburgh, PA). The protease inhibitor, Pefabloc-SC, was purchased from Roche Diagnostics Received 4/25/03; revised 7/21/03; accepted 8/5/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grant R01 CA64475 and in part by Depart- ment of Energy Training Grant DE F0101 NE23051 (to A. L. C.). The production of copper radionuclides at Washington University is supported by National Cancer Institute Grant R24 CA86307 (to Dr. Michael J. Welch). 2 Present address: Department of Veterinary Medicine and Surgery, College of Vet- erinary Medicine, University of Missouri-Columbia, Columbia, MO 65211. 3 To whom requests for reprints should be addressed, at Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway Bou- levard, Campus Box 8225, St. Louis, MO 63110. Phone: (314) 362-8427; Fax: (314) 362-9940; E-mail: andersoncj@mir.wustl.edu. 4 The abbreviations used are: DTPA, diethylenetriaminepentaacetic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; OC, octreotide; mAb, mono- clonal antibody; BAT, bromoacetamidobenzyl-TETA; 2IT, 2-iminothiolane; CSK, cy- toskeleton; LDH, lactate dehydrogenase; SSTR2, somatostatin receptor subtype 2; PET, positron emission tomography. 6864