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Departments of Internal Medicine II (M.J.W., B.-R.S.S., B.G., C.S.) and Nuclear Medicine (F.-J.G.), Ludwig-Maximilians-University, 81377 Munich, Germany; Department of Nuclear Medicine (I.W., R.S.-S.), Technical University Munich, 80333 Munich, Germany; Department of Carcinogenesis of the Skin (H.-J.S.), German Cancer Research Center, 69120 Heidelberg, Germany; and Division of Endocrinology (J.C.M.), Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Christine Spitzweg, M.D., Klinikum Grosshadern, Medizinische Klinik II, Marchioninistrasse 15, 81377 Muenchen, Germany. E-mail: Christine.Spitzweg{at}med.uni-muenchen.de.
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Abstract |
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Objective: In the current study, we therefore studied the potential of 188rhenium (188Re), as an alternative radionuclide, also transported by NIS, with a shorter half-life and higher energy ß-particles than 131I.
Results: NIS-transfected LNCaP cells (NP-1) concentrated 8% of the total applied activity of 188Re as compared with 16% of 125I, which was sufficient for a therapeutic effect in an in vitro clonogenic assay. 
-Camera imaging of NP-1 cell xenografts in nude mice revealed accumulation of 8–16% injected dose (ID)/g 188Re (biological half-life 12.9 h), which resulted in a 4.7-fold increased tumor absorbed dose (450 mGy/MBq) for 188Re as compared with 131I. After application of 55.5 MBq 131I or 188Re, smaller tumors showed a similar average volume reduction of 86%, whereas in larger tumors volume reduction was significantly increased from 73% after 131I treatment to 85% after application of 188Re.
Conclusion: Although in smaller prostate cancer xenografts both radionuclides seemed to be equally effective after prostate-specific antigen promoter-mediated NIS gene delivery, a superior therapeutic effect has been demonstrated for 188Re in larger tumors.
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Introduction |
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Based upon the effective application of radioiodine that has been used for over 60 yr in the management of follicular cell-derived thyroid cancer due to endogenous expression of the sodium iodide symporter (NIS), cloning of NIS has provided us with a promising suicide gene (3, 4, 5). As one of the oldest targets of molecular imaging and therapy, characterization of NIS as a novel therapeutic gene offers the possibility of NIS gene transfer into nonthyroidal tumors followed by radioiodine application. The dual function of NIS as a diagnostic and therapeutic gene thereby allows easy monitoring of functional NIS expression by scintigraphic imaging before proceeding to the application of a therapeutic 131iodine (131I) dose, an essential prerequisite for the exact planning and controlling of gene therapy applications in the clinical setting.
To ensure tumor specificity of radiation exposure, the application of tumor-specific promoters offers the ability to transcriptionally target NIS gene expression to tumor cells. In earlier studies we have reported the application of the prostate-specific antigen (PSA) and probasin promoters to achieve prostate-specific iodide accumulation in the human prostatic adenocarcinoma cell line LNCaP in vitro and in vivo (6, 7, 8, 9, 10). Furthermore, the amount of accumulated 131I was sufficiently high to selectively kill NIS-transfected LNCaP cells in vitro as well as in vivo in stably transfected LNCaP cell xenografts, with an average tumor volume reduction of more than 95% after application of 3 mCi (111 MBq) 131I (6, 7). A similar therapeutic effect of 131I was observed after adenoviral in vivo NIS gene delivery using the replication-defective tissue-unspecific Ad5-CMV-NIS (11). In preparation of a first phase I clinical study, these data were confirmed in beagle dogs using Ad5-CMV-NIS for local intraprostatic injection. Dosimetry calculations after application of 3 mCi 131I revealed an average absorbed dose to the prostate of 23 ± 42 cGy/mCi 131I, indicating that a dose of 85 mCi 131I would be sufficient to obtain a target dose of 2000 cGy to the prostate (12). These studies clearly showed for the first time that tissue-specific NIS gene expression into nonthyroidal tumors might represent an effective and potentially curative therapy for tumors without endogenous iodide accumulation.
In the thyroid gland thyroid peroxidase-catalyzed oxidation and incorporation of trapped iodide into tyrosyl residues along the thyroglobulin backbone, a process called iodide organification increases the retention time of accumulated radioiodide (5), thereby increasing the therapeutic effect of 131I due to prolongation of its effective half-life. Because extrathyroidal tissues are generally not able to organify iodide after NIS gene transfer, therapeutic efficacy of 131I can be limited by rapid iodide efflux. The application of alternative radioisotopes also transported by NIS with a shorter physical half-life and decay properties superior to 131I may provide a powerful method to enhance therapeutic efficacy of NIS-targeted radionuclide therapy in the presence of a limited effective half-life of 131I. In addition to I–, NIS has been demonstrated to transport other structurally similar anions like ClO3–, SCN–, SeCN–, NO3–, Br–, TcO4–, as well as ReO4– (13, 14). 188Rhenium (188ReO4–) represents a ß-emitting radionuclide with a short physical half-life (16.7 h) that has recently been used in a variety of therapeutic applications in humans, including cancer radioimmunotherapy, palliation of skeletal bone pain, and endovascular brachytherapy after angioplasty (15, 16, 17, 18, 19, 20, 21, 22, 23). Due to its higher energy and shorter physical half-life compared with 131I, administration of 188Re offers the possibility of higher energy deposition in a shorter time period.
In the current study, we therefore examined accumulation and therapeutic efficacy of 188Re in direct comparison to radioiodine in our prostate cancer model after PSA promoter-driven NIS gene transfer in vitro and in vivo (6, 7).
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Materials and Methods |
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Generation of the stably transfected LNCaP cell lines NP-1 (NIS/PSA-pEGFP-1) and P-1 (PSA-pEGFP-1) has been performed as described previously (6, 7).
Cell culture
LNCaP cells were grown in RPMI 1640 medium with 10% fetal bovine serum supplemented with L-glutamine, pen/strep, and 200 µg/ml Geneticin (Invitrogen Inc., Karlsruhe, Germany), and maintained at 37 C and 5% CO2.
Cell viability assay
Cell viability was measured using the commercially available MTS-assay (Promega Corp., Mannheim, Germany) according to the manufacturer’s recommendations as described previously (9).
Radionuclide uptake studies in vitro
Uptake of 188Re and 125I was determined according to the method by Weiss et al. (24), as described previously (7). Radionuclide uptake studies were performed in Hank’s balanced salt solution supplemented with 10 µM NaReO4 or 10 µM NaI, 0.1 µCi (3.7 kBq) Na188Re or Na125I/ml, and 10 mM HEPES (pH 7.3). Results were normalized to cell survival measured by cell viability assay MTS (see previous paragraph) and expressed as cpm/A490 nm.
Radionuclide efflux studies in vitro
Radionuclide efflux was determined according to the method by Weiss et al. (24), as described previously (9).
In vitro clonogenic assay
LNCaP cells stably transfected with the expression vector (NP-1) or the control vector (P-1) were incubated for 7 h with 29.6 MBq (0.8 mCi) 188Re in Hanks’ balanced salt solution supplemented with 10 µM NaReO4 and 10 mM HEPES (pH 7.3) and 37 C. After incubation with 188Re, a clonogenic assay was performed as described previously (6, 25).
Establishment of LNCaP cell xenotransplants
Xenotransplants derived from NP-1 (right flank) and P-1 (left flank) were established in male CD-1 nu/nu mice (Charles River Laboratories, Sulzfeld, Germany) as described previously (6). The experimental protocol was approved by the regional governmental commission for animal protection (Regierung von Oberbayern).
Radionuclide uptake studies in vivo
Eight to 10 wk after sc injection of cells (tumor diameter 
10 mm), mice were switched to a low-iodine diet and received T4 supplementation (5 mg/liter) in their drinking water for 2 wk to maximize radioiodine uptake in the tumor and reduce uptake by the thyroid gland. After ip injection of 111 MBq (3 mCi) 188Re or 18.5 MBq (0.5 mCi) 123I, radionuclide imaging was performed using a -camera (Forte; ADAC Laboratories, Milpitas, CA) equipped with a medium-energy general purpose (MEGP) collimator (188Re) and an ultrahigh-resolution (VXHR, Vantage Extra High Resolution) collimator (123I). Regions of uptake have been quantified and expressed as a fraction of the total amount of applied radionuclide. Radionuclide retention time in the tumor was determined by serial scanning (0.25, 0.5, 1, 3, 5, 10, 16, 24, and 48 h post injectionem). Dosimetric calculations were performed using OLINDA/EXM (26) (these calculations are published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).
Radionuclide therapy studies in vivo
Xenografts of NP-1 and P-1 cells were established in six groups of mice (each group n = 6). Two groups of mice were administered 55.5 MBq (1.5 mCi) 131I or 55.5 MBq (1.5 mCi) 188Re by a single ip injection after 8- to 10-wk tumor growth (tumor volume > 0.2 cm3), and two other groups of mice were administered 55.5 MBq of either 131I or 188Re after 4- to 6-wk tumor growth (tumor volume < 0.2 cm3). Two control groups with larger and smaller tumors were administered saline only. Tumors were measured as described previously (6). All mice were followed for a total of 6 wk. Statistical significance was tested using the Student’s t test (for unpaired samples: when examining between 188Re- and 131I- treated mice; and for paired samples).
Indirect immunofluorescence assay
For immunofluorescence staining, frozen tissue sections were fixed for 5 min in 80% methanol at 4 C and 2 min in acetone at –20 C. After rehydration in PBS, incubation with the following primary antibodies was performed in 12% BSA/PBS at room temperature for 2 h or 4 C overnight: 1) rat monoclonal antibody against mouse CD31 (PharMingen, Heidelberg, Germany; dilution 1:200); 2) rabbit polyclonal antibody against mouse Collagen IV (Novotec, Lyon, France; dilution 1:400); and 3) rabbit polyclonal antibody against human Ki67 (Abcam, Cambridge, UK; dilution 1:1000). Sections were then incubated with appropriate secondary antibodies (Cy-2 or Cy-3 conjugated; Dianova, Hamburg, Germany) along with 5 µg/ml Hoechst bisbenzimide for cell nuclei staining, and embedded in Permafluor (Immunotech, Marseille, France). Stained sections were examined using an Olympus AX-70 microscope (Olympus, Hamburg, Germany) equipped with epifluorescence optics and recorded with a CCD-camera (F-View 12; Soft Imaging Systems, Münster, Germany) applying Analysis Pro 6.0 software (Olympus).
Statistical methods
All experiments were carried out in triplicate. For the clonogenic assay, 12 wells were evaluated for each condition and cell density. Results are presented as means ± SD of triplicates. Statistical significance was tested using the Student’s t test.
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Results |
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Radionuclide uptake was studied in LNCaP cells stably expressing NIS under the control of the PSA promoter (NP-1) and control cell line P-1 after a 48-h incubation with mibolerone, a synthetic androgen (10–9 M) (Fig. 1A
). NP-1 cells concentrated 125I about 47.5-fold and 188Re about 28.6-fold in a perchlorate-sensitive manner compared with control P-1 cells that showed no perchlorate-sensitive radionuclide uptake (P < 0.01). The maximum 188Re uptake was approximately 8% of the total administered dose compared with 16% for 125I (Fig. 1A). Time course experiments showed similar kinetics for radionuclide uptake using 188Re and 125I with half-maximal levels of radionuclide accumulation after 5 min and saturation at 30 min (data not shown).
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Radionuclide efflux was studied in vitro in NP-1 cells after incubation with either 125I or 188Re showing a similar efflux rate (radionuclide release per minute) for both radionuclides (Fig. 1B
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In vitro clonogenic assay
An in vitro clonogenic assay was performed to determine the therapeutic efficiency of 188Re in NIS-transfected LNCaP cells in vitro (Fig. 2). Approximately 98% of NP-1 cells were killed after treatment with 188Re, whereas NIS-negative P-1 cells revealed an unselective killing rate of approximately 60% after exposure to 188Re (P < 0.01). NP-1 cells incubated with 188Re and perchlorate in parallel showed a killing rate similar to P-1 cells (data not shown).
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Radionuclide uptake was determined using a -camera after ip injection of 18.5 MBq (0.5 mCi) 123I or 111 MBq (3mCi) 188Re (each n = 5). In contrast to P-1 tumors, which showed no radionuclide uptake, NP-1 tumors accumulated 123I (Fig. 3A) as well as 188Re (Fig. 3B). As already shown in an earlier study (6), 25–30% injected dose (ID)/g (percentage injected dose per gram tumor) radioiodine was trapped in the NP-1 tumors with an effective half-life of 37 h for 131I. In comparison, 8–16% ID/g 188Re was concentrated in the NP-1 tumors with an effective half-life of 7.3 h.
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Radionuclide therapy studies in vivo
There were 55.5 MBq (1.5 mCi) 131I or 188Re injected in two groups of mice after 8- to 10-wk (tumor volume > 200 mm3; Fig. 4, B and D), or 4- to 6-wk (tumor volume < 200 mm3; Fig. 4, A and C) tumor growth, respectively, whereas the two control groups were administered saline only. While P-1 tumors treated with 131I or 188Re as well as P-1 and NP-1 tumors treated with saline continued their growth throughout the observation period, small NP-1 tumors treated with 131I (mean tumor volume 138 ± 8 mm3) or 188Re (mean tumor volume 121 ± 4 mm3) showed an average tumor volume reduction of 86% (131I: 90 ± 12%, P < 0.01; 188Re: 83 ± 24%, P < 0.01) (Fig. 4, A and C). In smaller tumors 131I seemed to be slightly more efficient than 188Re without reaching statistical significance. In contrast, larger NP-1 tumors treated with 131I (mean tumor volume 472 ± 14 mm3) showed an average tumor volume reduction of only 73 ± 10%, whereas 188Re treated NP-1 tumors (mean tumor volume 466 ± 18 mm3) showed a significantly enhanced therapeutic effect with a tumor volume reduction of approximately 85 ± 6% (P < 0.05) (Fig. 4, B and D). None of the mice showed adverse effects after radionuclide administration.
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Six weeks after treatment, mice were killed, and NP-1 tumors were dissected and processed for immunofluorescence analysis. Immunofluorescence analysis using a Ki67-specific antibody and antibodies against CD31 and collagen type IV (labeling blood vessels) showed striking differences between saline- (Fig. 5, A and D), 131I- (Fig. 5, B and E), and 188Re-treated (Fig. 5, C and F) tumors. While saline-treated tumors showed high blood vessel density as well as high proliferative activity with Ki67-positive cells, particularly surrounding infiltrating blood vessels (Fig. 5, A and D), radionuclide treated tumors exhibited lower intratumoral blood vessel density and a lower proliferation index (Fig. 5, B, C, E, and F) in addition to large areas of acellular necroses (Fig. 5, E and F).
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Discussion |
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An elegant strategy to enhance therapeutic efficacy of NIS-targeted radionuclide therapy in tumors with rapid iodide efflux might be the application of more potent isotopes like ReO4–, which is also transported by NIS, but in contrast to 131I, offers the possibility of higher energy deposition in the tumor in a shorter period of time due to its shorter physical half-life and higher energy (13, 14). Zuckier et al. (33) have demonstrated a similar biodistribution pattern for 125I– and 188ReO4– in mice, with the exception of the thyroid gland, in which only 125I is retained by organification. 188Re is conveniently obtained from a 188W/188Re generator, and represents a powerful ß-emitting radionuclide with a short physical half-life of 16.7 h (vs. 8 d for 131I) and higher energy ß-particles (E = 0.764 vs. 0.195 MeV for 131I) that are effective over a greater range (23–32 vs. 2.6–5 mm for 131I), suggesting superior therapeutic efficacy in medium or large tumors by an enhanced "crossfire effect" (17). In addition, its lower energy and low abundance -photons (155 keV, 15% abundance) are applicable for imaging and even easier to shield than the -photons with higher energy (364 keV) of 131I. These properties of 188ReO4– make it a worthy candidate for investigating its therapeutic efficacy after targeted NIS gene transfer in nonthyroidal cancers.
In our current study, NIS expressing LNCaP cells concentrated 8% of the 188ReO4– provided in the medium in vitro, which was half as much as for 125I, indicating that NIS reveals a higher affinity for iodide than for perrhenate. Radionuclide efflux was quite rapid in vitro with similar kinetics for both radionuclides. These data correlate with the results reported by Kang et al. (34), who showed an 87-fold increase of 188Re-perrhenate uptake in NIS-transfected hepatocellular carcinoma cells in vitro in contrast to a 150-fold increase of 125I uptake. Furthermore, although lower amounts of 188ReO4– were accumulated in NIS-transfected LNCaP cells in our study, the absorbed dose was sufficiently high for a significant selective killing effect of 98% using 188ReO4– in an in vitro clonogenic assay compared with 75% when cells were treated with 131I (6). The superior therapeutic effect of 188ReO4–, despite a lower accumulation rate, results from the higher radiation dose that can be achieved by 188ReO4– compared with 131I, which has also been demonstrated by Kang et al. (34) in NIS-transfected hepatocellular carcinoma cells, in which only 28.9 ± 5.2% of cells survived the treatment with 188ReO4–, whereas 46.3 ± 10.1% survived after treatment with 131I. In this study NIS-negative control cells survived treatment with 188ReO4– to 100%, whereas in our study NIS-negative LNCaP cells showed an unselective killing of approximately 60%, which was not seen with 131I, in which only 20% of the control P-1 cells died (6). The high unselective killing effect of 188ReO4– in LNCaP cells in vitro probably results from the different decay properties of 188Re compared with 131I, i.e. its higher energy and longer path length of emitted ß-particles. In this context it is important to mention that the in vitro monolayer system is quite an artificial system and does not allow to assess fully the therapeutic efficacy of a radionuclide due to the lack of a three-dimensional structure, which was, therefore, further explored in vivo in a LNCaP xenotransplant model. Compared with 123I, in which approximately 25–30% ID/g was accumulated in the NIS-transfected LNCaP xenotransplants with an average biological half-life of 45 h (6), approximately 8–16% ID/g 188ReO4– was accumulated with an average biological half-life of 12.9 h. These data are consistent with the biodistribution study by Kang et al. (34) in a liver cancer xenotransplant model. In our study a tumor absorbed dose of 450 mGy/MBq 188Re was calculated, which was 4.7 times higher than for 131I (6) and correlates well with the study by Dadachova et al. (15), showing a radiation dose 4.5 times higher for 188Re than for 131I in NIS expressing mammary adenocarcinomas in MMTV-NeuT mice. Our comparative 131I and 188Re therapy experiments in vivo in NIS-transfected LNCaP xenografts were performed with a single injection of 55.5 MBq (1.5 mCi) 131I or 188Re and showed a similar effect for both radionuclides in smaller tumors with an average tumor volume reduction of 86%, whereas therapeutic efficacy was significantly higher for 188Re in larger tumors with a tumor volume reduction of 85% compared with only 73% for 131I. This tumor size-dependent therapeutic efficacy with enhanced tumor volume reduction with 188Re in larger tumors is most likely due to the greater path length of the ß-particles emitted by 188Re (23–32 mm) compared with 131I (2.6–5 mm), resulting in an enhanced "crossfire effect" of 188Re. Therefore, the application of 188Re allows stimulation of the bystander effect, which is one of the advantages of NIS gene therapy because not only NIS-transduced cancer cells but also surrounding nontransduced cells are destroyed by the "crossfire effect" of ß-emitting radionuclides, thereby also offering the possibility to compensate for the generally limited in vivo transduction efficiency of currently available vector systems, in particular after systemic application.
In addition, immunofluorescence analysis showed reduced proliferation associated with decreased blood vessel density inside and surrounding the tumor after treatment with 131I or 188Re. Due to the longer path length of 188Re, a higher radiation dose is expected to be delivered to the surrounding stroma, which might cause increased stromal cell damage leading to reduced angiogenesis and secretion of growth-stimulatory factors, thereby explaining, at least in part, the superior therapeutic effect of 188Re in larger tumors.
In support of our data, Shen et al. (35) demonstrated increased survival prolongation in rats harboring NIS-transfected intracerebral gliomas after injection of 188Re compared with 131I. In addition, Dadachova et al. (17) showed a more pronounced growth inhibiting effect in NIS-expressing mammary tumors in a transgenic mouse model after application of 188Re. In this study a protective effect of 188Re to the thyroid was also reported (17), which is primarily caused by the lack of 188Re organification in the thyroid gland, thereby not only reducing radiation damage to the thyroid gland but also increasing tumoral 188Re uptake by the elimination of the thyroid "sink" effect. These data are consistent with the findings in our study, in which the biological half-life of 188Re in the thyroid gland was only approximately 12 h in contrast to more than 48 h for 131I, resulting in an approximate 3.6-fold increased absorbed dose to the thyroid after application of 131I compared with 188Re.
In conclusion, our study is the first in vivo study that directly compared the therapeutic efficacy of 131I and 188Re in LNCaP xenotransplant tumors after PSA promoter-targeted NIS gene delivery. Although the cumulative activity of 188Re was about half the cumulative activity of 131I in vitro as well as in vivo, therapeutic efficacy of 188Re was higher in larger tumors with a similar therapeutic effect in smaller tumors. Considering that in our current study a stably NIS-transfected cell line was used with maximum NIS expression levels, which is not directly applicable for clinical use in humans, the efficacy of 188Re has to be evaluated further in future studies after systemic in vivo NIS gene transfer with usually limited transduction efficiency and a more heterogeneous NIS expression pattern. Provided that these studies will confirm our findings, 188Re may serve as an attractive alternative to 131I, in particular in larger tumors and tumors with short iodide retention time.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 14, 2007
Abbreviations: 131I, Iodine-131; NIS, sodium iodide symporter; PSA, prostate-specific antigen; 188Re, 188rhenium.
Received February 21, 2007.
Accepted August 6, 2007.
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