Methotrexate, Paclitaxel, and Doxorubicin Radiosensitize HER2-Amplified Human Breast Cancer Cells to the Auger Electron–Emitting Radiotherapeutic Agent 111In-NLS-Trastuzumab Danny L. Costantini*1,2, Daniela F. Villani*3, Katherine A. Vallis4, and Raymond M. Reilly1,5,6 1Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada; 2Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, Ontario, Canada; 3Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada; 4Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom; 5Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and 6Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada Our goal in this study was to elucidate the mechanisms by NLS-trastuzumab, compared to treatment with 111In-NLS- which methotrexate radiosensitizes HER2-positive human breast trastuzumab alone, significantly increased residual gH2AX foci cancer cells to the Auger electron emitter 111In-trastuzumab in 231-H2N and TrR1 cells but not in MDA-MB-231 or TrR2 cells modified with nuclear-localization sequence peptides (111In- or in any cell line treated concurrently with paclitaxel and 111In- NLS-trastuzumab) and to compare these mechanisms with the NLS-trastuzumab. Cells exposed to low-dose methotrexate accu- potential sensitizing effects of paclitaxel and doxorubicin when mulated in the G1/S phase of the cell cycle, whereas low-dose combined with this radiopharmaceutical. Methods: Experiments paclitaxel or doxorubicin caused cells to arrest in the G2/M phase. were performed in MDA-MB-231 human breast cancer cells, their Conclusion: Low-dose methotrexate, paclitaxel, or doxorubicin HER2-transfected subclones (231-H2N), and 2 trastuzumab- potently sensitized HER2-overexpressing human breast cancer resistant variants (trastuzumab-resistant-1 and -2 [TrR1 and TrR2]). cells, with and without acquired trastuzumab-resistance, to the Effects of coexposure of these cells to 111In-NLS-trastuzumab Auger electron emissions from 111In-NLS-trastuzumab through and low-dose, radiosensitizing methotrexate, paclitaxel, or cell cycle distribution changes and in part through the inhibitory ef- doxorubicin were assessed by clonogenic cell-survival assay. fects of these agents on DNA damage repair. Quantification of residual DNA damage was measured by the Key Words: Auger electron; radiosensitizer; trastuzumab; breast gH2AX-immunofluorescence assay, and cell cycle distribution cancer; radioimmunotherapy was measured by fluorescence-activated cell sorting analysis. J Nucl Med 2010; 51:477–483 The radiation-enhancement ratio was calculated as the ratio DOI: 10.2967/jnumed.109.069716 of the surviving fraction (SF) of cells treated with 111In-NLS- trastuzumab alone to that of cells treated concurrently with 111In-NLS-trastuzumab and methotrexate, paclitaxel, or doxoru- bicin. Results: A reduction in the SF in HER2-positive 231-H2N (55.7% 6 1.3%) and TrR1 (62.6% 6 6.5%) cells was demon- strated after exposure to 111In-NLS-trastuzumab (;0.2 MBq/mg, 100 nmol/L) but not in MDA-MB-231 or TrR2 cells expressing Despite the success of radioimmunotherapy for ad- low levels of HER2 (SF . 90%, P . 0.05). Coadministration of vanced lymphohematopoietic malignancies, the treatment methotrexate, paclitaxel, or doxorubicin enhanced the cyto- of patients with solid tumors such as breast cancer has been toxicity of 111In-NLS-trastuzumab toward 231-H2N and TrR1 limited because of the small amounts of antibody that can cells but not toward MDA-MB-231 or TrR2 cells. The radiation- be targeted to tumors. Also of concern in treatment are the ir- enhancement ratios for methotrexate, paclitaxel, and doxorubicin radiation and killing of normal cells (e.g., bone marrow stem for 231-H2N or TrR1 cells were 2.0–2.2, 1.6–1.8, and 2.7–2.8, cells) by the moderate-energy and long-range (2–10 mm) respectively. Methotrexate or doxorubicin combined with 111In- b-particles emitted by 131Iand90Y, which have been com- monly conjugated to radiotherapeutic agents (1,2). Therefore, Received Aug. 23, 2009; revision accepted Nov. 24, 2009. For correspondence or reprints contact: Raymond M. Reilly, Leslie Dan considerable attention has been given to the combination of Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, radioimmunotherapy with drugs that are known radiosen- Ontario M5S 3M2, Canada. E-mail: [email protected] sitizers and that can amplify the lethal effects of ionizing *Contributed equally to this work. radiation on cancer cells while having minimal or no toxicity COPYRIGHT ª 2010 by the Society of Nuclear Medicine, Inc. on tissues at the low concentrations used (3). 111IN-NLS-TRASTUZUMAB RADIOSENSITIZATION • Costantini et al. 477 This strategy may be especially relevant for patients breast cancer patients in combination with trastuzumab and whose tumors display amplification of the HER2 receptor are also known radiosensitizers (4). Therefore, we also tyrosine kinase. HER2 overexpression occurs in 25%230% sought to determine whether paclitaxel and doxorubicin of breast cancers (4), has been correlated with resistance to would radiosensitize HER2-postive breast cancer cells to hormonal therapy (5–7) and chemotherapy (8), and is 111In-NLS-trastuzumab. The radiosensitizing effects of these directly associated with poor long-term survival (4). Tras- agents, when combined with 111In-NLS-trastuzumab, were tuzumab (Herceptin; Hoffmann-La Roche) is a humanized evaluated and compared with those caused by methotrexate. anti-HER2 monoclonal antibody approved for immunother- apy of HER2-amplified breast cancer (4). However, only MATERIALS AND METHODS 50% of patients with tumors exhibiting moderate to high Cell Culture (21 to 31) immunohistochemistry scores or having greater MDA-MB-231 human breast cancer cells were obtained from than 3 copies of the HER2 gene by fluorescence in situ the American Type Culture Collection, and the 231-H2N and hybridization are predicted to respond to trastuzumab when trastuzumab-resistant 1 and 2 (TrR1 and TrR2) cell lines were given in combination with chemotherapy (9,10). Moreover, kindly provided by Dr. Robert S. Kerbel (Sunnybrook Health the short duration of response to trastuzumab (9–12 mo) Sciences Centre). The 231-H2N cell line was derived from MDA- and the rapid development of drug resistance limit the MB-231 cells that were transfected to stably overexpress c-erbB-2 effectiveness of trastuzumab treatment in this patient (HER2), whereas TrR1 and TrR2 cells were isolated from 231- population (11,12). In an attempt to enhance its antineo- H2N tumors in athymic mice with acquired trastuzumab resistance plastic activity, our group has been studying Auger electron (19). 231-H2N and TrR1 cells express high levels of HER2 (5.0– 5 radioimmunotherapy of HER2-amplified breast cancer 6.0 · 10 receptors per cell), which is approximately 10-fold greater than MDA-MB-231 and TrR2 cells, which express 0.4–0.6 · using 111In-trastuzumab modified with 13 mer peptides 105 HER2 receptors per cell (16). All cell lines were cultured in (CGYGPKKKRKVGG) harboring the nuclear-localization Dulbecco’s minimal essential medium (Ontario Cancer Institute) sequence (NLS) of SV-40 large T antigen, which promotes supplemented with 10% fetal bovine serum (Sigma-Aldrich) con- its nuclear importation after HER2-mediated internalization taining 100 U of penicillin per milliliter and 100 mgofstreptomycin into breast cancer cells (13). Auger electrons are highly per milliliter at 37°C in an atmosphere of 5% CO2. damaging to DNA when they decay near the cell nucleus, making them exquisitely selective and highly potent for Trastuzumab and Radiosensitizers killing targeted single cancer cells (14). Indeed, we recently Trastuzumab was reconstituted according to the package insert. reported that 111In-NLS-trastuzumab, compared with non- Methotrexate and doxorubicin were prepared by dissolving 1 mg radiolabeled trastuzumab (Herceptin), could slow the of the drug (Sigma-Aldrich) in 1 mL of alkalinized (pH 11.0) or growth of HER2-positive breast cancer xenografts in mice, neutral (pH 7.0) distilled, deionized water, respectively. The with minimal harm to normal tissues, and prolong survival methotrexate stock solution was adjusted to pH 7.0 before use. Paclitaxel (Sigma-Aldrich) was prepared by dissolving 1 mg of (15). In in vitro studies, we further found that 111In-NLS- the drug in 100 mL of dimethyl sulfoxide. Stock solutions were trastuzumab was able to kill breast cancer cells that are diluted in culture medium to the desired final concentration. The resistant to trastuzumab and that the potency of the radio- final concentration of dimethyl sulfoxide in the culture medium pharmaceutical could be enhanced by coexposing these cells was 0.1% (v/v) or less. to low, noncytotoxic concentrations of methotrexate (16). 111 Understanding the mechanisms resulting in tumor cell In-Trastuzumab Modified with NLS-Peptides radiosensitization caused by the interaction of chemother- Trastuzumab (Herceptin) was modified with synthetic 13 mer NLS-peptides (CGYGPKKKRKVGG) and labeled with 111InCl apeutic drugs and Auger electron radiotherapeutics would 3 (MDS-Nordion) using diethylenetriaminepentaacetic acid (DTPA) be useful in the development of rational clinical protocols (Sigma-Aldrich) to a specific activity of 201 6 6 MBq/mg (13). that combine these agents. The first objective of this study, Briefly, trastuzumab (500 mg,