anaplastic thyroid cancer ( atc ) is rare and represents less than 2% of all thyroid cancer . nevertheless , it is one of the most deadly human cancers which causes more than half of the deaths attributed to thyroid cancer . although intensive multimodalities of treatment shows improved local control , the overall survival remains low . atc does not concentrate iodide due to the loss of expression of sodium iodide symporter ( nis ) gene . 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 . function of iodide transport of nis has been used in radioiodide imaging and therapy . for well - differentiated thyroid cancer , i ablation and whole - body scintillation scan after thyroidectomy is currently the most frequent treatment protocol . 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 . by means of -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 . however , the model in previous study was stable cells established by transient transfection and clonal expansion . 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 . another type of retrovirus , lentivirus , which infects both dividing and nondividing cells has emerged as potent and versatile vectors for gene transfer . 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 . 293 t ( bcrc60019 ) were obtained from the bioresource collection and research center in taiwan . all media and tissue culture supplements were obtained from gibco - brl ( invitrogen , carlsbad , calif , usa ) . all complete media were supplemented with 10% fetal bovine serum ( fbs ) , 2  mm l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin . cells were cultured in a humidified incubator at 37c and 5% co2 : 95% air . the hnis cdna cloned in pcdna3 ( fl*-hnis / pcdna3 ) was kindly provided by dr . 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 . 6  10 293 t 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 293 t 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 4c to pellet cell debris . the virus - containing supernatants ( lv - cmv - hnis and lv - cmv - egfp ) were then passed through a sterile , 0.45  m low protein - binding , millex - hv pvdf filter ( millipore , bedford , mass , usa ) . the flowthrough was collected and overlaid on the top of 4  ml 20% sucrose solution prepared by pbs in the centrifuging tubes . virus concentration was carried out by centrifugation at 26.000  rpm , 4c for 1.5  hrs . after decanting the supernatant , the pellet of viral particles was reconstituted by mixing in the hbss and stored at 80c . the virus titer was assayed by the quicktiter lentivirus quantitation kit ( cell biolabs , inc . cells were seeded and allowed to grow on 22  mm  22  mm coverslips ( marienfeld , lauda - konigshofen , germany ) . after a 24  h incubation , cells were rinsed with pbs for 3 times and then fixed in 4% paraformaldehyde for 15  min at room temperature . after three 5-min washes by pbs , the cells were permeabilized with 0.1% triton x-100 in pbs / cm / bsa ( pbs with 0.1  mm cacl2 , 1  mm mgcl2 , and 0.2% bsa ) for 10  min . after three 5-min washes by pbs , the cells were incubated with a 1  :  100 dilution of mouse anti - hnis polyclonal antibody ( chemicon , temecula , usa ) for 1 hour at room temperature and washed with pbs . a 1  :  200 dilution of fitc - conjugated goat anti - mouse igg ( santa cruz biotechnology , santa cruz , usa ) was then added to cells and incubated for 1 hour at room temperature and followed by pbs wash . 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 . 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 : 94c for 4  min , 94c for 30  s , 56c for 30  s , and 72c 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 , primers that hybridize to a segment of the -actin cdna were included in the same reaction mixture ( -actin forward primer : 5-tcaagatcattgctcctcctgagc-3 ; -actin reverse primer : 5-gggcaccgtaatagagatag-3 ) . 5  10 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  m of nai and 3700 bq of   i at 37c . 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 . for in vivo imaging study , 1  10 aro parental cells and cells transduced with lv - cmv - hnis were subcutaneously injected into the right flank of shoulder of male nod / scid mice . after the tumors reached to 8  mm in diameter , the mice were injected intravenously with i ( 1.85  mbq ) . after one hour , mice were anesthetized with 2% isoflurane ( forane abbott laboratories , queenboroug , kent , england ) and 98% oxygen mixture and secured on the imaging table in the prone position . static images were obtained using the micropet r4 scanner ( siemens preclinical solutions , knoxville , tenn , usa ) for 30  min . fully 3-dimensional list mode data were collected for 30  min using an energy window of 350750  kev and a timing window of 6  ns . for maximum sensitivity , coincidence data were rebinned into 3-dimensional sinograms using the full axial acceptance angle of the scanner ( max . high sampling of the polar angle was used ( span = 3 ) . the resulting 3d sinograms were then rebinned into 2d sinograms using fourier rebinning ( fore ) and reconstructed with 2-dimensional filtered back projection ( fbp ) using a ramp filter with cutoff at nyquist . all images were expressed in the ordinary rainbow color scale with standard visual spectrum installed in the computer of the micropet system . two weeks after cell inoculation , mice bearing tumors were intraperitoneally injected with 55.5  mbq ( 1.5  mci ) i or same volume of normal . tumor volume was measured by caliper and calculated using the equation vt  ( mm ) = l  w  d  0.52 , where vt is the estimated tumor volume , and l , w , and d are the length ( mm ) , width ( mm ) , and depth ( mm ) of the tumor , respectively . 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 . 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 . in figure 3 , transduced cells showed apparent exogenous hnis pcr amplicon with expected size ( lane 5 ) while parental cells had no detected band ( lane 4 ) . rt - pcr was also performed to detect rna expression in parental and transduced cells . after performing reverse transcription and subsequent pcr , expression of exogenous hnis was observed ( lane 2 ) and parental cells showed no detected expression ( lane 1 ) . to evaluate the function of iodide uptake of transduced cells aro parental cell or cells transduced with lv - cmv - hnis or lv - cmv - hnis were incubated in i - containing hbss followed by  counting the radioactivity within cells at different time points . as shown in figure 4 , cells transduced with lv - cmv - hnis accumulated i rapidly in 10 minutes , and the radioactivity reached to the plateau at 20 minutes from beginning . aro parental cells and cells transduced with lv - cmv - egfp showed a minimal i uptake and no higher accumulation as the time increased . at 20 minute , the index of accumulated i in cells transduced with lv - cmv - hnis was ~7 times higher than that of parental cells or cells transduced with lv - cmv - egfp . this result demonstrates the lentiviral - transferred hnis , exhibits its original function , and consequently restores the ability of iodide uptake of anaplastic thyroid cancer cells . in order to know whether aro - transduced cells retain the expression of hnis and the function of iodide uptake , we established the tumors derived from aro parental and transduced cells on nod / scid mice . 1 hour after intravenously injecting i , mice were imaged using micropet scanner . static image of mice bearing tumor derived from aro parental cells ( left ) and transduced cells ( right ) was acquired for 30 minutes and is shown in figure 5 . tumor derived from transduced cells showed more efficient accumulation of i while compared to tumor derived from parental cells . this result indicates transduced cells retain not only the expression of transferred hnis but the ability to accumulate iodide . to know the efficacy of i therapy of hnis - expressing tumor , mice bearing tumors were treated with 55.5 mbq ( 1.5  mci ) of i or saline . result shows that tumor growth was effectively inhibited by treating with 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 . 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 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 . 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 . 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 . reported that i concentrated within thyroid gland continued to increase at 19  h after administration into mice according to a biodistribution experiment . same result was noted by our previous study . by analyzing rois of serial images acquired using a -camera , similar experiment also revealed that within 5 hours from injection , equal level of i accumulation was noted in thyroid gland and in tumors derived from aro stable cells , while only 1/3 of i activity was measured in tumors derived from aro parental cells . 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 . 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 [ 1820 ] . 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 . highly expressed nis alone with sufficient but nonlethal dose of administered 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 . however , several studies demonstrated that with treatment of certain agents , expression of thyroid specific genes could be restored in aro cells . showed that the reverse transcriptase inhibitors recovered the thyrotropin signaling and iodide uptake ability . 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 . 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 . 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 . 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 can not be used to specifically activate downstream gene expression in specific cell type . we previously demonstrated that using a hepatoma chimeric promoter ( eiia enhancer combined with -fetoprotein promoter ) to target hepatocarcinoma cells ( hccs ) resulted in specifically turning on the hnis transgene in hcc , but not in non - hcc . 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 . 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 atc - specific 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 lentivirus - mediated nis radiogene therapy . the transduced cells showed high expression of the transgene and restored the ability of radioiodide accumulation .
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 .