In Vitro Cytotoxicity of 211At-Astatide and 131I-Iodide to Glioma Tumor Cells Expressing the Sodium/Iodide Symporter
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BASIC SCIENCE INVESTIGATIONS In Vitro Cytotoxicity of 211At-Astatide and 131I-Iodide to Glioma Tumor Cells Expressing the Sodium/Iodide Symporter Sean Carlin, PhD; Gamal Akabani, PhD; and Michael R. Zalutsky, PhD Department of Radiology, Duke University Medical Center, Durham, North Carolina The sodium/iodide symporter (NIS) has been identified as an It is now well established that the accumulation of iodide attractive target for cancer therapy. The efficacy of 131I-iodide by the thyroid gland is mediated by the sodium/iodide for NIS-expressing tumor therapy may be limited by a combi- symporter (NIS), an integral membrane protein expressed nation of poor cellular retention and unfavorable physical char- acteristics (long physical half-life and low linear-energy-transfer on the basolateral surface of the thyrocyte. Iodide is trans- [LET] radiative emissions). On the other hand, 211At-astatide is ported across the cell membrane against a concentration also transported by NIS and offers several therapeutic advan- gradient in a sodium-dependent manner, in a process elec- tages over 131I-iodide due to its physical characteristics (short trochemically coupled to the energy-dependent Naϩ/Kϩ- ␣ half-life, high LET -particle emissions). The objective of this adenosinetriphosphatase (1). The cloning of the NIS gene study was to directly compare the radiotoxicity of both radio- has allowed investigations into the effect of plasmid- or nuclides using a NIS-transfected cultured cell model. Methods: Cytotoxicity was determined by colony-forming assays. Also, a viral-mediated NIS expression on the iodide-concentrating first-order pharmacokinetic model was used to simulate the capacity in a variety of thyroid and nonthyroid cell types closed compartmental system between the medium and cells. (2). These studies have indicated that NIS transgene expres- Experimental data were then fitted to this model and used to sion, under the control of a variety of promoters, confers estimate the transfer coefficients between medium and cells, iodide uptake capacity in a wide range of cell lines, allowing kc , and between cells and medium, km. Using the pharmacoki- m c the possibility of radioiodide therapy for many tumor types. netic model, the cumulated activity concentrations in the me- dium and cells were calculated. Monte Carlo transport methods However, several of these studies also report that the efflux were then used to assess absorbed doses from 131I and 211At. of iodide from NIS-transfected cells, including anaplastic Results: 211At-Astatide was significantly more cytotoxic than thyroid cells, is extremely rapid (3–6). This observation is 131I-iodide in this closed compartmental system. For 211At-as- of fundamental importance for any endoradiotherapeutic tatide, absorbed doses per unit administered activity were 54- strategy, as radiation-absorbed dose (and thus treatment to 65-fold higher than for 131I-iodide. Both NIS-expressing and control cells showed increased sensitivity to 211At over 131I, with efficacy) is a function of the cellular residence time and physical half-life (t ) of the radionuclide (7), with optimal significantly lower D0 (absorbed dose required to reduce the 1/2 Ϫ1 survival fraction to e ) and SF2 (2-Gy survival fraction) values, conditions reached when the radiologic t1/2 matches the ␣ highlighting the higher intrinsic cytotoxicity of -particles. How- biologic t1/2 (i.e., cellular retention). Dosimetric calculations ever, NIS-independent (nonspecific) binding of 211At-astatide and subsequent in vivo studies, using a murine xenograft 131 was higher than that of I-iodide, therefore, yielding a lower model, have indicated that successful treatment of low- absorbed dose ratio between NIS-transfected and -nontrans- 131 fected cells. Conclusion: Treatment of NIS-expressing cells retention NIS-expressing tumor deposits with I-iodide ϭ with 211At-astatide resulted in higher absorbed doses and in- (t1/2 8.04 d) may not possible without the administration creased cytotoxicity per unit administered activity than that of prohibitively high activities of radioiodide (8–10). In observed with 131I-iodide. These results suggest that 211At-as- addition, the effectiveness of 131I in curing micrometastatic tatide may be a promising treatment strategy for the therapy of disease decreases with tumor size, due to the relatively long NIS-expressing tumors. path length of the -particle (11), resulting in an increased J Nucl Med 2003; 44:1827–1838 probability of disease recurrence. Astatine (astatide), the heaviest of the halogens, exhibits a pronounced accumulation in the thyroid gland in halide form and in the stomach (12). We have previously demon- Received Apr. 11, 2003; revision accepted Jul. 22, 2003. For correspondence or reprints contact: Michael R. Zalutsky, PhD, Depart- strated NIS-mediated accumulation of astatide, with uptake ment of Radiology, Box 3808, Duke University Medical Center, Durham, NC 27710. and efflux characteristics almost identical to those of iodide 211 E-mail: [email protected] (4). Fortunately, At has a physical t1/2 of 7.214 h, which 211AT-ASTATIDE:DOSIMETRY AND RADIOTOXICITY • Carlin et al. 1827 closely matches the biologic t1/2 reported for iodide in a G-418 sulfate (0.5 mg/mL) (Gibco) in addition to the conditions variety of NIS-expressing xenograft models (9,13,14), un- described above. 131 211 like the physical t1/2 of I. In addition, the decay of At ␣ External Beam Irradiations results in the emission of -particles with a mean linear Cells were trypsinised, counted, and resuspended in sterile tubes energy transfer (LET) of 97 keV/ m. This value, represent- containing 5 mL fresh medium to a final concentration of 5 ϫ 105 ing the ionizing energy deposited by the particulate emis- cells per milliliter. Irradiations (0–9 Gy) were performed using a sion per unit distance, is approximately 500 times that of the 6-MV external photon beam accelerator at a dose rate of 0.5 therapeutic -emitting radionuclides 90Y and 131I. Upon Gy-minϪ1 with the appropriate bolus to ensure full buildup of the interaction with DNA, this high density of ionization events radiation beam. Cells were plated for colony-forming assay in result in a high incidence of generally irreparable double- 6-well plates (growth area, 9.6 cm2) and grown at 37°Cina5% strand DNA breaks, which accounts for the extreme radio- CO2 atmosphere for 7–10 d. Colonies were then stained and toxicity of this radionuclide (15). Also, the short range of counted, with colonies comprising 50 or more cells scored as 211At ␣-particles in tissue (maximum range, 70 m) makes viable. such emissions more effective for the sterilization of micro- Radionuclides metastatic tumor clusters than -particles. Another advan- The radionuclide 211At was produced at the Duke University tage of ␣-particle therapy is that cell survival is independent Medical Center CS-30 cyclotron via the 209Bi(␣, 2n)211At reaction of dose rate, which is of particular significance in relation to using the MIT-1 internal target system as previously described the rapid uptake and efflux kinetics of radiohalides in NIS- (12). 211At was distilled from the molten bismuth target and expressing cells lacking an organification mechanism. trapped in a cooled condenser. It was then isolated from the This article describes in vitro studies directed at evaluat- condenser by washing with approximately 1 mL phosphate-buff- ing the hypothesized cytotoxic advantages of 211At-astatide ered saline (PBS), pH 7.4. After isolation, Na2SO3 (Mallinckrodt) ϫ Ϫ4 over 131I-iodide for the treatment of NIS-expressing cells was added to a final concentration of 2 10 mol/L, to minimize the formation of higher oxidation state species. Sodium 131I-iodide lacking a halide organification mechanism. Experiments (no carrier added) was purchased from Dupont–New England were performed using the human glioma cell line UVW, Nuclear. transfected with human NIS complementary DNA (cDNA) (called NIS6 hereafter), using the parental cell line (UVW) Pharmacokinetics as a negative control. The sensitivity of each cell line to Experiments were carried out to assess the uptake and efflux external beam irradiation was established by clonogenic pharmacokinetics of radiohalides. Cells were plated in 24-well ϫ 4 assay, allowing comparison of intrinsic radiosensitivity in plates at approximately 5 10 cells per well and allowed to both cell lines and also of the cellular sensitivity to both low attach overnight. For radionuclide uptake curves, cells were incu- bated for 1–30 min in wells with 0.5 mL PBS containing either and high LET particle-emitting radionuclides. Sensitivity of 211 131 211 131 At-astatide (100 kBq/mL) or I-iodide (50 kBq/mL). At- both cell lines to varying activity concentrations of I- astatide uptake and 131I-iodide uptake were terminated by the 211 iodide and At-astatide was also established by colony- removal of the radiohalide-containing incubation buffer, followed forming assay. A pharmacokinetic model was derived to by 3 rapid washes in ice-cold PBS. Cells were then solubilized by describe the closed compartmental system between the me- the addition of 0.5 mL lysis buffer (0.1 mol/L NaOH, 1% sodium dium and cells. Experimentally derived kinetic data were dodecyl sulfate, 2% Na2CO3) and assessed for radioactivity using then fitted to this model and used to estimate transfer an LKB 1282 ␥-counter (LKB), in dual-channel mode. For radio- coefficients between medium and cells. Using the pharma- halide efflux studies, cells were plated as described above in PBS cokinetic model, the cumulated activity concentrations in containing 211At-astatide (100 kBq/mL) and 131I-iodide (50 kBq/ the medium and cells were calculated. Monte Carlo trans- mL) for 30 min. Radioactive incubation buffer was then removed, ϫ Ϫ3 port methods were then used to assess absorbed doses from and cells were incubated in PBS containing 1 10 mol/L thiocyanate ion (SCNϪ) to inhibit the reuptake of radiohalide for 131I and 211At, which were correlated with the radionuclide the various time intervals to ensure the same conditions used in the sensitivity data, allowing the determination and comparison colony-forming assay.