Evaluation of Lentiviral-Mediated Expression of Sodium Iodide Symporter in Anaplastic Thyroid Cancer and the Efficacy of In Vivo Imaging and Therapy

Anaplastic thyroid carcinoma (ATC) is one of the most deadly cancers. With intensive multimodalities of treatment, the survival remains low. ATC is not sensitive to 131I therapy due to loss of sodium iodide symporter (NIS) gene expression. We have previously generated a stable human NIS-expressing ATC cell line, ARO, and the ability of iodide accumulation was restored. To make NIS-mediated gene therapy more applicable, this study aimed to establish a lentiviral system for transferring hNIS gene to cells and to evaluate the efficacy of in vitro and in vivo radioiodide accumulation for imaging and therapy. Lentivirus containing hNIS cDNA were produced to transduce ARO cells which do not concentrate iodide. Gene expression, cell function, radioiodide imaging and treatment were evaluated in vitro and in vivo. Results showed that the transduced cells were restored to express hNIS and accumulated higher amount of radioiodide than parental cells. Therapeutic dose of 131I effectively inhibited the tumor growth derived from transduced cells as compared to saline-treated mice. Our results suggest that the lentiviral system efficiently transferred and expressed hNIS gene in ATC cells. The transduced cells showed a promising result of tumor imaging and therapy.


Introduction
Anaplastic thyroid cancer (ATC) is rare and represents less than 2% of all thyroid cancer [1]. Nevertheless, it is one of the most deadly human cancers which causes more than half of the deaths attributed to thyroid cancer [2]. Currently, treatment for ATC is mainly surgery combined with chemotherapy and radiotherapy. Although intensive multimodalities of treatment shows improved local control, the overall survival remains low [3]. Other strategy for treating ATC including new drugs is urgently underway.
ATC does not concentrate iodide due to the loss of expression of sodium iodide symporter (NIS) gene. NIS is a membrane protein with 13 putative transmembrane domain [4]. This symporter is driven by low internal sodium concentration and symports 2 sodium ions for every iodide ion into cells. For thyroid cells, NIS mediated iodide transport is the first step of triiodothyronine (T3) and thyroxine (T4) synthesis [5]. Function of iodide transport of NIS has been used in radioiodide imaging and therapy. For well-differentiated thyroid cancer, 131 I ablation and whole-body scintillation scan after thyroidectomy is currently the most frequent treatment protocol [6]. After NIS gene had been cloned, several investigators used it as an imaging and therapeutic tool in gene therapy. By introducing NIS gene into variety of cancer cells which do not concentrate iodide, it showed a promising therapeutic effect after administrating therapeutic isotopes [7]. By means of γ-camera or positron emission tomography with radioiodide, the accumulation of tracer which reflects the expression of transferred NIS gene can be monitored. We have shown the effectiveness of NIS-mediated radioiodide therapy on tumors derived from ATC cell line, ARO [8]. However, the model in previous study was stable cells established by transient transfection and clonal expansion.
Virus-mediated gene delivery has been used for decades in biological research. Retrovirus has been the mainstay of current gene therapy approach due to its high efficiency of infection and high expression of transferred gene. Long-term expression of delivered gene attributed to genome integration is also a useful advantage. However, the use of retrovirus requires the cells being in an actively dividing status [9]. Another type of retrovirus, lentivirus, which infects both dividing and nondividing cells has emerged as potent and versatile vectors for gene transfer [10]. In this study, we aimed to establish a lentiviral system to deliver NIS gene into ARO cells and evaluate the efficacy of radioiodide imaging and therapy.

Cell
Culture. 293T (BCRC60019) were obtained from the Bioresource Collection and Research Center in Taiwan. The ARO thyroid cancer cell line was kindly provided by Dr. Chin-Wen Chi (National Yang-Ming University, Taiwan). All media and tissue culture supplements were obtained from Gibco-BRL (Invitrogen, Carlsbad, Calif, USA). 293T cells were grown in minimum essential medium (MEM). ARO cells were maintained in RPMI-1640 medium. All complete media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/ mL streptomycin. Cells were cultured in a humidified incubator at 37 • C and 5% CO 2 : 95% air.

Vector Construction and Virus
Production. The hNIS cDNA cloned in pcDNA3 (FL * -hNIS/pcDNA3) was kindly provided by Dr. Sissy Jhiang (Ohio State University). The CMV promoter linked with hNIS in pcDNA3 vector was enzymatically removed and then ligated into LentiLox 3.7 vector (pLL3.7) with proper restriction enzyme to create pLL3.7-hNIS ( Figure 1). Lentiviral particles were produced following the protocol published by Tiscornia et al. with some modification [11]. 6 × 10 6 293T packaging cells per 100 mm tissue culture plate were seeded and cultured in DMEM supplemented with 10% FBS for 24 hours. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif, USA) following the manual instruction. Plasmid DNA and reagent were mixed and incubated for 20 minutes at room temperature to form the DNA-Lipofectamine complexes. The mixture was then added to 293T cell culture and incubated overnight. On the next day, the media were removed and replaced with fresh culture media containing 1% (w/v) BSA. At 72 hour after transfection, the viral particle-containing supernatant was harvested in sterile, capped conical tubes, and centrifuged at 300 g for 15 minutes at 4 • C to pellet cell debris. The virus-containing supernatants (LV-CMV-hNIS and LV-CMV-EGFP) were then passed through a sterile, 0.45 μm low protein-binding, Millex-HV PVDF filter (Millipore, Bedford, Mass, USA was then added to cells and incubated for 1 hour at room temperature and followed by PBS wash. Nuclei were stained using Bisbenzimide H33342 for 30 minutes at room temperature. After three final PBS washes (5 min each), the cells were mounted with mounting solution. Fluorescent images of cells were observed using a Leica TCS SP5 confocal microscope.

Polymerase Chain Reaction (PCR) for Detecting the Integrated Transgene and Gene Expression.
Transduced ARO cells and parental cells were expanded in culture, and total genomic DNA was isolated using the DNeasy kit (Qiagen, Valencia, Calif, USA) following the manufacturer's instruction. Integrated hNIS DNA was amplified by PCR with designed forward primer, 5 -TCAGCACAGCATCCACCAG-3 and reverse primer, 5 -GGGCACCGTAATAGAGATAG-3 . The PCR protocol was set as follow: 94 • C for 4 min, 94 • C for 30 s, 56 • C for 30 s, and 72 • C for 30 s, with repeating reaction cycle for 30 times. For the detection of RNA expression, cells were lysed, and total RNA was isolated using RNeasy mini kit (Qiagen, Valencia, Calif, USA). cDNA synthesis was carried out using MMLV reverse transcriptase (Epicentre Biotechnologies, Madison, Wlis, USA) according to the manufacturer's instruction. In the subsequent PCR reaction, the primers previously described were used to amplify the hNIS gene. As internal controls, primer that hybridize to a segment of the β-actin cDNA were included in the same reaction mixture (β-actin forward primer: 5 -TCAAGATCATTGCTCCTCCTGAGC-3 ; β-actin reverse primer: 5 -GGGCACCGTAATAGAGATAG-3 ). The annealing temperature for β-actin was 54 • C.

In Vitro 125 I Uptake.
In vitro cellular 125 I uptake was measured according to Weiss et al. [12] with little modification. 5 × 10 5 cells per well were seeded in 12-well plates the day before experiment. After rinsed with Hanks' balanced salt solution (HBSS), cells were incubated with 0.5 mL HBSS containing 10 μM of NaI and 3700 Bq of 125 I at 37 • C. At 5, 10, 20, 30, and 60 minute, cells were washed quickly with 1 mL of iodide-free icecold HBSS. Cells were then detached by trypsin, and all cells were collected and resuspended in 1 mL HBSS in counting tubes. The radioactivity was measured using a Cobra II autogamma counter (Packard instrument, Conn, USA) following the manual procedure.

Immunocytochemistry Study.
To evaluate the hNIS expression in transduced ARO cells, we performed immunofluorescent staining. Using mouse anti-hNIS primary antibody coupled with FITC-conjugated goat anti mouse IgG, fluorescence emitted from FITC fluorophore was shown localized on the cellular surface of transduced cells, compared with the location of cell nuclei stained by H33342 (Figure 2(b)). ARO parental cells treated with the same staining procedure showed no FITC-related fluorescent signal, indicating no or low expression of hNIS in these cells (Figure 2(a)). This result indicates the efficient transfer and subsequent expression of hNIS by lentiviral transduction.

PCR for Detecting the Integrated Viral Vector and Gene
Expression. To verify the lentiviral integration of exogenous hNIS construct in transduced ARO cells, we have previously performed PCR with designed primers to detect the integrated construct in genomic DNA isolated from ARO parental cells and transduced cells [13]. In Figure 3

In Vitro Radioiodide Uptake.
To evaluate the function of iodide uptake of transduced cells, we have previously performed the sequential in vitro 125 I uptake experiment [13].

In Vivo 131 I Therapy.
To know the efficacy of 131 I therapy of hNIS-expressing tumor, mice bearing tumors were treated with 55.5 MBq (1.5 mCi) of 131 I or saline. Result shows that tumor growth was effectively inhibited by treating with 131 I, as compared to tumors treated with saline ( Figure 6). This data as well as result of in vivo imaging indicate tumor derived from transduced cells that retain the expression and function of hNIS even in the in vivo environment.

Discussion
Using lentiviral vector, we have successfully transduced ATC cells with NIS gene which was markedly expressed and well functioning in the transduced cells. Tumors established by transduced cells retained high expression of hNIS which enable tumors to be well treated by 131 I and monitored with radioiodide imaging. Our previous data showed that stable cells offered a good model to test the feasibility of NIS-mediated gene therapy. However, gene delivery by viral transduction is more applicable in vivo. Lentivitral gene transfer was intensively used due to its advantages, including stable integration of transgene, capability of transducing dividing and nondividing cells and long-term transgene expression. Kim et al. showed effective long-term monitoring and radionuclide therapy of colon cancer cells in living organism by using a lentiviral vector system carrying hNIS controlled by UbC promoter [14]. Dingli et al. also used lentivectors to target and express hNIS in myeloma cells for 131 I therapy.
Results showed that tumor xenografts treated with 131 I were completely eradicated without recurrence [15]. Reports above indicate the potential of lentiviral vector combined with NIS gene therapy on cancer. Our data is the first demonstration of lentiviral NIS-mediated gene therapy on anaplastic thyroid cancer. Iodide is retained within thyroid gland for a period of time due to multiple processes of thyroid hormone synthesis. Zuckier et al. reported that 131 I concentrated within thyroid gland continued to increase at 19 h after administration into mice according to a biodistribution experiment [16]. Same result was noted by our previous study [8]. By analyzing ROIs of serial images acquired using a γ-camera, radioactivity in thyroid gland reached the peak around 20 hours after 131 I injection. Similar experiment also revealed that within 5 hours from injection, equal level of 131 I accumulation was noted in thyroid gland and in tumors derived from ARO stable cells, while only 1/3 of 131 I activity was measured in tumors derived from ARO parental cells. Radioactivity within tumors decreased after 5 hours in contrast to increase in thyroid gland. Results showed that without further steps of T3 and T4 synthesis, iodide pumped into cell by NIS is rapidly effluxed. This drawback greatly reduces the feasibility of NIS in gene therapy.
To solve the problem of short retention of radioiodide, Huang et al. coexpressed NIS and TPO in nonsmall cell lung cancer which increased retention time of radioiodide within cells as compared to cells expressing NIS alone [17]. Furuya et al. delivered TTF-1 (thyroid transcription factor 1) to NIStransfected cells. TTF-1 is the thyroid specific transcription factor and able to turn on the expression of downstream thyroid specific genes such as Tg, TPO, and NIS [18][19][20]. However, delivery of more than one gene into cells greatly increases the difficulty. Despite the short retention of radioiodine within transduced cells, the growth of tumors still showed significant inhibition in this study and our previous data [8]. Highly expressed NIS alone with sufficient but nonlethal dose of administered 131 I may compensate the problem of short retention of radioiodide.
Recently, a comprehensive DNA sequence profiling analysis has pointed that ARO cells are highly identical with HT-29, a colon cancer cell line [21]. However, several studies demonstrated that with treatment of certain agents, expression of thyroid specific genes could be restored in ARO cells. Landriscina et al. showed that the reverse transcriptase inhibitors recovered the thyrotropin signaling and iodide uptake ability [22]. Upon treatment of histone deacetylase (HDAC) inhibitors such as depsipeptide and trichostatin A (TSA), thyroid-specific gene expression and accumulation of radioiodide through the iodination of generic cellular proteins were detected in ARO cells [23]. ARO cells were also shown to express thyroid stimulatory hormone receptor (TSHR), and the population which expressed TSHR increased following treatment of TSH in a dose-dependent manner [24]. Despite the published results which demonstrated that ARO cells retain the function and specific gene expression related to thyroid cells, more evidences are needed to clarify the identity of ARO cells.
Lentiviral gene transfer is considered as a promising approach to cancer gene therapy. Cancer-specific targeting is one of key point in the in vivo gene therapy. In this study, CMV promoter which has been reported as one of the strongest promoters in vitro was used in our transduction system. However, in the in vivo system, silence of gene governed by CMV was noted within a few weeks in several organs [25,26]. In addition, CMV is a universal promoter which cannot be used to specifically activate downstream gene expression in specific cell type. Therefore, a promoter which specifically target ATC is urgently needed. We previously demonstrated that using a hepatoma chimeric promoter (EIIA enhancer combined with α-fetoprotein promoter) to target hepatocarcinoma cells (HCCs) resulted in specifically turning on the hNIS transgene in HCC, but not in non-HCC [13]. Thyroglobulin promoter was reported to target thyroid cancer. With transfer and specific expression of TK gene driven by the Tg promoter in thyroid cancer cells, result showed marked killing ability in the presence of GCV, little in vivo toxicity, and should be useful in the future for treating thyroid Tg-producing cancers [27]. ATC is known as a poorly differentiated thyroid carcinoma with reduced or silenced expression of several thyroid-specific genes, including NIS and Tg. Therefore, to design an ATCspecific lentiviral targeting system, promoter of Tg gene can not work for this purpose. Our future work will focus on searching for a suitable promoter for ATC in our lentivirusmediated NIS radiogene therapy.

Conclusion
We have established a lentiviral system for delivering hNIS to ARO cell. The transduced cells showed high expression of the transgene and restored the ability of radioiodide accumulation. Tumors derived from the transduced cells showed effective in vivo imaging and therapy. This study offers a promising, applicable way to treat ATC.