papillary thyroid carcinomas ( ptc ) and follicular thyroid carcinomas ( ftc ) , often referred to together as well - differentiated thyroid cancer ( wdtc ) , account for more than 90  % of all thyroid cancers . generally , ptc and ftc are two of the most curable cancers : they have a favourable prognosis with 10-year overall survival rates of 93  % and 85  % , respectively . anaplastic thyroid cancer ( atc ) , or so - called undifferentiated thyroid cancer ( utc ) , is a rare , aggressive and lethal malignancy with rapid progression . patients with atc have a median survival time of no more than 68  months [ 4 , 5 ] . in terms of morphological appearance and biological behaviour , poorly differentiated thyroid cancer ( pdtc ) represents intermediate entities in the progression from wdtc to atc , and has an intermediate prognosis . conventional management of patients with thyroid cancer involves surgical removal of the tumour followed by radioiodine ablative therapy . the efficacy of radioiodine ablative therapy depends upon the uptake of i by local residual or metastatic lesions . several distinct molecules cooperate in the process of thyroid hormone synthesis leading to iodide accumulation and retention in the cells . these molecules include sodium iodide symporter ( nis ) that pumps iodide into the cells and thyroperoxidase ( tpo ) that oxidizes iodide to iodine and iodinates thyroglobulin ( tg ) . expression of these genes is lower in pdtcs than in wdtcs and can not be detected in atcs [ 7 , 8 ] , and is thought to be the major cause of the failure of i therapy . the cancer stem cell ( csc ) model is based on the hypothesis that there is a subset of cancer cells possessing the ability to self - renew and to generate differentiated progeny . these cells , which resemble normal stem cells and are hierarchically more primitive , express stem cell - related marker genes and are highly resistant to standard chemotherapeutic agents as well as radiation [ 9 , 10 ] . cd133 , also known as prominin-1 , is a five - transmembrane domain glycoprotein specifically expressed on the surface of haematopoietic stem and progenitor cells . previous studies have shown that cd133 is a marker of cscs in different types of cancer including brain tumours , colon cancer [ 13 , 14 ] and melanoma . in terms of the relationship between cd133 and thyroid cancer , zito et al . found that two human atc cell lines , aro and kat-4 , contain subpopulations of cd133 cells which exhibit stem cell - like features such as rapid proliferation , the ability of self renewal and to form colonies , and increased resistance to chemotherapy - induced apoptosis in vitro . friedman et al . reported that cd133 atc cells are solely responsible for tumour growth in immunodeficient mice in response to thyroid - stimulating hormone ( tsh ) . these results strongly suggest that cd133 cells in thyroid cancers represent a more undifferentiated population and possess several stem cell phenotypic characteristics . however , it is not known whether these cd133 cells in thyroid cancers , even in wdtc , are the major source of cells that survive i treatment and thus potentially contribute to recurrence or metastasis of thyroid cancer . to date , as far as we know no information is available on whether the presence of cd133 cells correlates with failure of i treatment of thyroid cancer . the aim of this study was to investigate the existence of cd133 cells in thyroid cancer and to explore their role in the radiation treatment of thyroid cancer . we hypothesized that the cd133 populations in thyroid cancer cells are responsible for the resistance to radiation treatment of thyroid cancers . the thyroid cancer cell lines used in this study included cells lines of the follicular type ( wro and cgth ) , the papillary type ( cg3 ) and the anaplastic type ( aro ) . louis , mo ) supplemented with 10  % fetal bovine serum ( fbs ; invitrogen , grand island , ny ) . tissue samples obtained from patients diagnosed with ptc after they had provided informed consent were used for primary culture of ptc cells . the collected tissues were minced and washed with phosphate - buffered saline ( pbs ; sigma - aldrich , st . then the minced tissues were digested with collagenase ii at a final concentration of 2  mg / ml at 37  c for 30  min . after centrifugation , the cells were cultured in f-12k medium ( invitrogen , grand island , ny ) supplemented with 10  % fbs , hydrocortisone ( 5    10  m ) , tsh ( 5    10  iu / ml ) , insulin ( 10  g / ml ) and glycyl - l - histidyl - l - lysine acetate ( 10  ng / ml ) ( sigma - aldrich , st . louis , mo ) . the cd133 cell population was isolated by fluorescence - activated cell sorting ( facs ) or magnetic selection . for facs isolation , the cells were washed with pbs containing 0.1  % fbs and 2  mm edta , and then incubated with antibody against cd133/1 conjugated with phycoerythrin ( pe ; miltenyi biotec , bergisch gladbach , germany ) on ice in the dark for 20  min . after the cells had been washed with pbs , the cd133 and cd133 populations were isolated by facs using an aria cell sorter ( bd biosciences ) . for magnetic selection , the cells were labelled with 0.5  ml micromagnetic beads conjugated with cd133/1 antibodies and isolation was carried out using a macs column and isolator ( miltenyi biotec ) . for annexin v and cd133 double staining , cells were trypsinized and washed with 1 binding buffer and then incubated with fitc annexin v ( bd pharmingen ) for 15  min at room temperature in the dark . after annexin v staining , cd133-pe staining was performed on the same cells following the procedure described above . , the cells were irradiated using a cs source ( model 143 - 68 blood product irradiator ; jl shepherd and associates , san fernando , ca ) to give different doses . after 714  days of incubation , colonies were fixed with 3.7  % formaldehyde and stained with crystal violet . the surviving fraction at each dose was calculated following the equation : number of colonies/(number of cells seeded  plating efficiency ) , where plating efficiency is ( number of formed colonies / number of cells seeded ) . the surviving fraction at each dose point was determined in triplicate and the results are shown as means  sd . total rna was extracted from cells or tumour tissue using trizol reagent ( invitrogen , grand island , ny ) and was then reverse - transcribed into first strand cdna . all reactions were prepared by mixing 50  ng first strand cdna with taqman fast universal pcr master mix ( applied biosystems , foster city , ca ) and corresponding primers . real - time quantitative polymerase chain reaction ( pcr ) was carried out using a steponeplus real - time system ( applied biosystems , mannheim , germany ) with the following programme : activation at 95  c for 20  s followed by 50  cycles of 95  c for 1  s and 60  c for 20  s. all the intron - spanning primers were designed at the universal probelibrary assay design center ( roche applied science ; http://www.roche-applied-science.com/sis/rtpcr/upl/ezhome.html ) . the primer sequences and probe numbers from universal probelibrary were as follows : nis ( forward tctctcagtcaacgcctctg ; reverse gcgtccattcctgagctg ; probe no . 10 ) , tsh receptor ( tshr ) ( forward cctgacagttattgacaaagatgc ; reverse ctggtttgagacacgtccag ; probe no . 25 ) , thyroid - specific transcription factor 1 ( ttf1 ) ( forward ctcggactacggcaccat ; reverse gcgtcctctcaccaggtc ; probe no . 39 ) , paired box gene 8 ( pax8 ) ( forward tgcctcacaactccatcaga ; reverse aggtctgccattcacaaagg ; probe no . aro cells that stably express human nis gene ( aro - hnis ) had been established in our previous work . a total of 5    10 aro - hnis cells were implanted subcutaneously into the right shoulder of 6-week - old severe combined immunodeficiency ( scid ) mice . two weeks later , when the diameter of the tumours was about 5  mm , the mice were i treated . specifically , five mice were injected intraperitoneally with 37  mbq ( 1  mci ) of i and another five mice were injected with the same volume of normal saline . tumour volume was measured and recorded on days 0 , 4 , 7 and 11 . on day 11 , tumours were removed after the mice had been killed by injection of an overdose of pentobarbital . gene expression was analysed as described above . for dosimetric calculation , two scid mice with developed aro parental and hnis - expressing tumours ( about 500  mm ) on the left and right shoulders , respectively , were subjected to scintigraphic imaging after administration of 1.85  mbq ( 50  ci ) of i . serial images were acquired for 20  min at 1  h , 3  h , 5  h , 7  h , 14  h , 28  h and 48  h after injection using a gamma camera ( ecam ; siemens , hoffman estates , il ) . accumulated event counts in a selected tumour area on each image were converted to radioactivity values ( megabecquerels ) by calibrating the gamma camera with defined activities . time  activity curves of both aro parental and hnis - expressing tumours were fitted to an exponential function and the accumulation of radioactivity was integrated according to the fitted function over a time interval from 0.5 to 60  h. integrated radioactivity in hnis - expressing and parental tumours were 1.69 and 0.36  mbqh , respectively . dosimetric calculation was based on the assumption that the tumours were spherical with a density of 1  g / ml and homogeneous radionuclide distribution . the absorbed dose of the tumours was calculated following the mird ( medical internal radiation dose ) schema using olinda 1.0 software . in vivo animal spect / ct imaging was performed on a triple - head czt gamma camera equipped with a built - in ct component ( triumph - spect ; gamma medica , northridge , ca ) and a high - resolution multiple pinhole collimator ( focal length 75.0  mm , aperture diameter 1.0  mm ) . this system applied circular scanning protocols for both spect and ct acquisition , with a translation stage invariable axial imaging range . before imaging , each mouse was injected with 1.85  mbq ( 50  ci ) of i via a tail vein . at 60  min after injection , the mice were fixed in a prone position on the imaging table under gaseous anaesthesia ( 2  % isoflurane and 98  % oxygen ) and imaged with x - ray ct with a current of 0.5  ma and voltage of 80  kvp . for spect imaging , 32 projections ( 28  s per projection , radius of rotation 4  cm , field of view 5.28  cm ) the energy window was set at 159  kev peak with a 20  % band . the spect image dataset was then reconstructed using the ordered - subset expectation maximization algorithm with standard - mode parameters as provided by the manufacturer . the images were viewed and presented using amide software ( free software provided by source forge ) . the thyroid cancer cell lines used in this study included cells lines of the follicular type ( wro and cgth ) , the papillary type ( cg3 ) and the anaplastic type ( aro ) . louis , mo ) supplemented with 10  % fetal bovine serum ( fbs ; invitrogen , grand island , ny ) . tissue samples obtained from patients diagnosed with ptc after they had provided informed consent were used for primary culture of ptc cells . the collected tissues were minced and washed with phosphate - buffered saline ( pbs ; sigma - aldrich , st . then the minced tissues were digested with collagenase ii at a final concentration of 2  mg / ml at 37  c for 30  min . after centrifugation , the cells were cultured in f-12k medium ( invitrogen , grand island , ny ) supplemented with 10  % fbs , hydrocortisone ( 5    10  m ) , tsh ( 5    10  iu / ml ) , insulin ( 10  g / ml ) and glycyl - l - histidyl - l - lysine acetate ( 10  ng / ml ) ( sigma - aldrich , st . louis , mo ) . the cd133 cell population was isolated by fluorescence - activated cell sorting ( facs ) or magnetic selection . for facs isolation , the cells were washed with pbs containing 0.1  % fbs and 2  mm edta , and then incubated with antibody against cd133/1 conjugated with phycoerythrin ( pe ; miltenyi biotec , bergisch gladbach , germany ) on ice in the dark for 20  min . after the cells had been washed with pbs , the cd133 and cd133 populations were isolated by facs using an aria cell sorter ( bd biosciences ) . for magnetic selection , the cells were labelled with 0.5  ml micromagnetic beads conjugated with cd133/1 antibodies and isolation was carried out using a macs column and isolator ( miltenyi biotec ) . for annexin v and cd133 double staining , cells were trypsinized and washed with 1 binding buffer and then incubated with fitc annexin v ( bd pharmingen ) for 15  min at room temperature in the dark . after annexin v staining , cd133-pe staining was performed on the same cells following the procedure described above . cultured cells were trypsinized , counted and plated . after 24  h of incubation , the cells were irradiated using a cs source ( model 143 - 68 blood product irradiator ; jl shepherd and associates , san fernando , ca ) to give different doses . after 714  days of incubation , colonies were fixed with 3.7  % formaldehyde and stained with crystal violet . the surviving fraction at each dose was calculated following the equation : number of colonies/(number of cells seeded  plating efficiency ) , where plating efficiency is ( number of formed colonies / number of cells seeded ) . the surviving fraction at each dose point was determined in triplicate and the results are shown as means  sd . total rna was extracted from cells or tumour tissue using trizol reagent ( invitrogen , grand island , ny ) and was then reverse - transcribed into first strand cdna . all reactions were prepared by mixing 50  ng first strand cdna with taqman fast universal pcr master mix ( applied biosystems , foster city , ca ) and corresponding primers . real - time quantitative polymerase chain reaction ( pcr ) was carried out using a steponeplus real - time system ( applied biosystems , mannheim , germany ) with the following programme : activation at 95  c for 20  s followed by 50  cycles of 95  c for 1  s and 60  c for 20  s. all the intron - spanning primers were designed at the universal probelibrary assay design center ( roche applied science ; http://www.roche-applied-science.com/sis/rtpcr/upl/ezhome.html ) . the primer sequences and probe numbers from universal probelibrary were as follows : nis ( forward tctctcagtcaacgcctctg ; reverse gcgtccattcctgagctg ; probe no . 10 ) , tsh receptor ( tshr ) ( forward cctgacagttattgacaaagatgc ; reverse ctggtttgagacacgtccag ; probe no . 25 ) , thyroid - specific transcription factor 1 ( ttf1 ) ( forward ctcggactacggcaccat ; reverse gcgtcctctcaccaggtc ; probe no . 39 ) , paired box gene 8 ( pax8 ) ( forward tgcctcacaactccatcaga ; reverse aggtctgccattcacaaagg ; probe no . aro cells that stably express human nis gene ( aro - hnis ) had been established in our previous work . a total of 5    10 aro - hnis cells were implanted subcutaneously into the right shoulder of 6-week - old severe combined immunodeficiency ( scid ) mice . two weeks later , when the diameter of the tumours was about 5  mm , the mice were i treated . specifically , five mice were injected intraperitoneally with 37  mbq ( 1  mci ) of i and another five mice were injected with the same volume of normal saline . tumour volume was measured and recorded on days 0 , 4 , 7 and 11 . on day 11 , tumours were removed after the mice had been killed by injection of an overdose of pentobarbital . gene expression was analysed as described above . for dosimetric calculation , two scid mice with developed aro parental and hnis - expressing tumours ( about 500  mm ) on the left and right shoulders , respectively , were subjected to scintigraphic imaging after administration of 1.85  mbq ( 50  ci ) of i . serial images were acquired for 20  min at 1  h , 3  h , 5  h , 7  h , 14  h , 28  h and 48  h after injection using a gamma camera ( ecam ; siemens , hoffman estates , il ) . accumulated event counts in a selected tumour area on each image were converted to radioactivity values ( megabecquerels ) by calibrating the gamma camera with defined activities . activity curves of both aro parental and hnis - expressing tumours were fitted to an exponential function and the accumulation of radioactivity was integrated according to the fitted function over a time interval from 0.5 to 60  h. integrated radioactivity in hnis - expressing and parental tumours were 1.69 and 0.36  mbqh , respectively . dosimetric calculation was based on the assumption that the tumours were spherical with a density of 1  g / ml and homogeneous radionuclide distribution . the absorbed dose of the tumours was calculated following the mird ( medical internal radiation dose ) schema using olinda 1.0 software . in vivo animal spect / ct imaging was performed on a triple - head czt gamma camera equipped with a built - in ct component ( triumph - spect ; gamma medica , northridge , ca ) and a high - resolution multiple pinhole collimator ( focal length 75.0  mm , aperture diameter 1.0  mm ) . this system applied circular scanning protocols for both spect and ct acquisition , with a translation stage invariable axial imaging range . before imaging , each mouse was injected with 1.85  mbq ( 50  ci ) of i via a tail vein . at 60  min after injection , the mice were fixed in a prone position on the imaging table under gaseous anaesthesia ( 2  % isoflurane and 98  % oxygen ) and imaged with x - ray ct with a current of 0.5  ma and voltage of 80  kvp . for spect imaging , 32 projections ( 28  s per projection , radius of rotation 4  cm , field of view 5.28  cm ) the energy window was set at 159  kev peak with a 20  % band . the spect image dataset was then reconstructed using the ordered - subset expectation maximization algorithm with standard - mode parameters as provided by the manufacturer . the images were viewed and presented using amide software ( free software provided by source forge ) . it has been reported that aro cells contain 57  % cd133 cells , and these have been demonstrated to act as cscs in thyroid cancer . we wondered if the percentage of cd133 population varies across thyroid cancer cell lines with various levels of differentiation . four thyroid cancer cell lines were analysed in this study , namely wro , cgth ( ftc ) , cg3 ( ptc ) and aro ( atc ) . using flow cytometry , the anaplastic type aro line comprised about 60  % cd133 cells , while the other three lines representing wdtc had less than 5  % of cd133 cells ( fig . we assessed the sensitivity to irradiation of the various cell lines with different cd133 populations . a clonogenic radiation survival assay was carried out by irradiating cells with different doses and counting the number of colonies formed after irradiation . the radiation survival curves of the four cell lines showed that aro cells exhibited a markedly higher radioresistance than the other three wdtc lines ( fig . 1b).fig . 1cd133 population and radiosensitivity in thyroid cancer cell lines . a four thyroid cancer cell lines , aro , wro , cg3 and cgth were analysed to explore their cd133 population using flow cytometry . cd133 populations were determined according to the binding of the isotype - matched control in each cell line . b radiosensitivity was assessed by clonogenic survival assays after the cells had been -irradiated at different doses . the surviving fractions are shown as means  sd ( n  =  3 ) cd133 population and radiosensitivity in thyroid cancer cell lines . a four thyroid cancer cell lines , aro , wro , cg3 and cgth were analysed to explore their cd133 population using flow cytometry . cd133 populations were determined according to the binding of the isotype - matched control in each cell line . b radiosensitivity was assessed by clonogenic survival assays after the cells had been -irradiated at different doses . the surviving fractions are shown as means  sd ( n  =  3 ) as cd133-expressing cells are the putative cscs in these thyroid cancer cell lines , we speculated that the cd133 population contributes to the radioresistance of bulk cancer cells . as shown in fig .  2b , cd133 cells were significantly more radioresistant than cd133 cells , indicating that these two populations of aro cells possessed different levels of radiosensitivity . cd133 cells also expressed a higher level of oct4 gene , which is known to be responsible for the maintenance of the pluripotency and the proliferation of embryonic stem ( es ) cells . the expression of lin28 , another marker of es cells , was also higher in cd133 cells than in cd133 cells . furthermore , cd133 cells showed higher expression of glut1 , which indicates dedifferentiation and an unfavourable prognosis of thyroid cancer [ 20 , 21 ] . thus , cd133 cells represent a relatively primitive and more radioresistant population than cd133 cells within the same bulk cancer cell population , suggesting that radiotherapy would result in uneven cell killing.fig . 2radiosensitivity and gene expression of cd133 and cd133 populations of the aro thyroid cancer cell line . the proportion of cd133 cells was determined by flow cytometry before and after cell sorting . b the surviving fractions of isolated cd133 and cd133 populations were assessed by clonogenic assays after irradiation . the surviving fractions are shown as means  sd ( n  =  3 ) c the sorted cd133 and cd133 cells were analysed for cd133 , oct4 , lin28 and glut1 mrna expression using real - time quantitative pcr . expression of each gene was normalized to that of gapdh mrna and is presented as 2 . the relative mrna expression levels are shown as means  sd ( n  =  2 ) radiosensitivity and gene expression of cd133 and cd133 populations of the aro thyroid cancer cell line . the proportion of cd133 cells was determined by flow cytometry before and after cell sorting . b the surviving fractions of isolated cd133 and cd133 populations were assessed by clonogenic assays after irradiation . the surviving fractions are shown as means  sd ( n  =  3 ) c the sorted cd133 and cd133 cells were analysed for cd133 , oct4 , lin28 and glut1 mrna expression using real - time quantitative pcr . expression of each gene was normalized to that of gapdh mrna and is presented as 2 . the relative mrna expression levels are shown as means  sd ( n  =  2 ) to investigate whether radiation treatment of cancer cells affects the cd133 population , aro , cg3 and wro cells were irradiated and then analysed by flow cytometry to assess their cd133 expression . at 48  h after -irradiation at 10  gy , the cd133 population had increased by 14  % ( 61  % to 75  % ) , 4  % ( 5  % to 9.2  % ) and 7.5  % ( 1.5  % to 9  % ) in aro , wro and cg3 cells , respectively ( fig . we further analysed the annexin v binding of these cells to elucidate whether cd133 or cd133 cells are more susceptible to apoptosis after radiation treatment . at 48  h after irradiation with 0 , 10 or 20  gy -radiation , aro cells were double - stained with cd133-pe and annexin v - fitc and analysed by flow cytometry . among the cd133 cells , the cd133/annexin v subpopulation remained relatively unchanged after -irradiation : 6.6  % , 5  % and 6.7  % after irradiation with 0 , 10 and 20  gy , respectively . among the cd133 cells , the cd133/annexin v subpopulation showed quantitative increases after -irradiation : 8  % , 10  % and 18  % increases after irradiation with 0 , 10 and 20  gy , respectively ( fig . the cd133 cells were more resistant to apoptosis than the cd133 cells after radiation treatment which may the enrichment of the cd133 population.fig . a aro , wro and cg3 cells were analysed for cd133 populations by flow cytometry before and 48  h after -irradiation . b apoptosis in the cd133 and cd133 populations was determined by analysis of annexin v binding . similar results were observed in two independent experiments effect of radiation on the cd133 populations . a aro , wro and cg3 cells were analysed for cd133 populations by flow cytometry before and 48  h after -irradiation . b apoptosis in the cd133 and cd133 populations was determined by analysis of annexin v binding . similar results were observed in two independent experiments radiation treatment also preferentially induced more apoptosis in cd133 cells than in cd133 cells in vitro . in order to elucidate whether clinical i treatment of thyroid cancer has a similar effect , we used aro - hnis , an aro cell line that stably expresses the human hnis gene   in an in vivo study . aro - hnis cells were injected subcutaneously into the shoulder of 6-week - old mice . in vivo radioiodide uptake by the aro - hnis tumours was assessed using a micropet / spect / ct imager after the mice had been injected with 1.85  mbq ( 50  ci ) i. the spect / ct images clearly showed accumulation of i in tumour ( fig . 4a ) , indicating that the re - expression of hnis in aro cells efficiently restores the ability to take up i. based on this result , mice were treated with an intraperitoneal injection of 37  mbq ( 1  mci ) i , while a control group were treated with the same volume of normal saline . according to the dosimetric calculation , hnis - expressing aro tumours treated with i received a dose of approximately 0.195  gy / mbq , which was five times higher than that received by aro parental tumours ( 0.0421  gy / mbq ) . tumour growth was recorded and on day 11 after i administration , the volumes of the tumours in the experimental group were significantly reduced compared to those in the saline - treated group ( fig . this efficient inhibition of tumour growth confirms the killing effect of i accumulated in the tumour cells . the expression levels of cd133 , glut1 , the es cell - related regulator oct4 , nanog and lin28 were elevated in i - treated tumours compared to the levels in saline - treated tumours ( fig . the results of this in vivo study strongly suggests that the more primitive of the cd133 cells have a greater ability to survive radiation treatment.fig . a aro cells stably expressing hnis ( aro - hnis , established in our previous study ) were injected subcutaneously into the right shoulder of scid mice . radioiodine uptake by the aro - hnis tumours was verified by intravenous injection of 1.85  mbq ( 50  ci ) i followed by in vivo imaging using a micropet / spect / ct . b the mice bearing the aro - hnis tumours received an intraperitoneal injection of 37  mbq ( 1  mci ) i. mice injected with normal saline formed the control group . tumour volume was measured on various days . on day 11 , the mice were killed and the tumours were excised for subsequent gene expression analysis . c after tumours had been minced and digested , total rna was isolated , reverse - transcribed and the expression levels of cd133 , glut1 , oct4 , nanog and lin28 mrna were determined by real - time quantitative pcr using designed primers . expression of each gene was normalized to that of gapdh mrna and is presented as 2 . the relative mrna expression levels are shown as means  sd ( n  =  2 ) gene expression profile after in vivo i treatment of an aro - hnis tumour . a aro cells stably expressing hnis ( aro - hnis , established in our previous study ) were injected subcutaneously into the right shoulder of scid mice . radioiodine uptake by the aro - hnis tumours was verified by intravenous injection of 1.85  mbq ( 50  ci ) i followed by in vivo imaging using a micropet / spect / ct . b the mice bearing the aro - hnis tumours received an intraperitoneal injection of 37  mbq ( 1  mci ) i. mice injected with normal saline formed the control group . , the mice were killed and the tumours were excised for subsequent gene expression analysis . c after tumours had been minced and digested , total rna was isolated , reverse - transcribed and the expression levels of cd133 , glut1 , oct4 , nanog and lin28 mrna were determined by real - time quantitative pcr using designed primers . expression of each gene was normalized to that of gapdh mrna and is presented as 2 . the relative mrna expression levels are shown as means  sd ( n  =  2 ) cells with heterogeneous phenotypes in a bulk tumour are commonly seen in various types of cancer . we have demonstrated that a cd133 population exists in different proportions in different thyroid cancer cell lines and this cd133 population shows higher radioresistance than the cd133 population . we analysed paraffin - embedded tissues from utc and ptc by immunohistochemical staining using anti - human cd133 antibody . cd133 expression was detectable in these types of thyroid cancer , and utcs showed more positive staining than ptcs ( fig .  5 ) . however , cd133 expression was also detectable in ptcs , even though this type of thyroid cancer is relatively well differentiated . furthermore , cells from primary cultures of fresh resected ptcs expressed the thyroid - specific genes nis and tpo after culture for a number of passages ( fig . cd133 and cd133 cells were separated from the ptc primary cultures and the gene expression profile was analysed . cd133 gene expression levels in the separated cd133 and cd133 populations from five primary lines are shown in fig . cd133 expression was 4- to about 16-fold higher in the cd133 population than in the cd133 population . the cd133 population also showed higher expression levels of various genes related to es cells , including oct4 ( cd133 about 8- to 24-fold higher ) , nanog ( about 3- to 71-fold higher ) , lin28 ( about 1- to 1,000-fold higher ) and sox2 ( about 10- to 68-fold higher ) ( fig . thyroid - specific gene expression was lower in the cd133 population than in the cd133 population , including nis ( cd133 about 2- to 5-fold higher ) , tg ( about 1- to 5-fold higher ) , tpo ( about 1- to 4-fold higher ) , tshr ( about 0.8- to 2.5-fold higher ) , pax8 ( about 0.8- to 3-fold higher ) and ttf1 ( about 1- to 3-fold higher ) ( fig . these results clearly indicate the existence of a cd133 population in primary ptcs which are more undifferentiated.fig . cd133 expression was detected in paraffin - embedded sections of two utcs ( poor ) and two ptcs ( papillary)fig . a clinical ptcs removed from patients were minced , digested and then subjected to primary culture . after total rna isolation , the thyroid origin of the cells was confirmed by examining tg and tpo expression using rt - pcr and ethidium bromide - stained agarose gel electrophoresis . aro cells are known to lack thyroid - specific gene expression and served as the negative control . b cd133 and cd133 populations isolated from primarily cultured cells by facs were subjected to total rna isolation and first - strand cdna transcription . expression levels of cd133 mrna by the isolated populations were analysed by real - time quantitative pcr and normalized to that of gapdh mrna . c expression levels of oct4 , nanog , lin28 and sox2 were assessed in the cd133 and cd133 cell populations by real - time quantitative pcr . d expression levels of nis , tg , tpo , tshr , ttf1 and pax8 were assessed in cd133 and cd133 cell populations by real - time quantitative pcr . data shown are the ratios of gene expression levels in the cd133 population to those in the cd133 population immunohistochemical staining for cd133 in clinical . cd133 expression was detected in paraffin - embedded sections of two utcs ( poor ) and two ptcs ( papillary ) gene expression profiles of cd133 and cd133 cells from primarily cultured ptcs . a clinical ptcs removed from patients were minced , digested and then subjected to primary culture . after total rna isolation , the thyroid origin of the cells was confirmed by examining tg and tpo expression using rt - pcr and ethidium bromide - stained agarose gel electrophoresis . aro cells are known to lack thyroid - specific gene expression and served as the negative control . b cd133 and cd133 populations isolated from primarily cultured cells by facs were subjected to total rna isolation and first - strand cdna transcription . expression levels of cd133 mrna by the isolated populations were analysed by real - time quantitative pcr and normalized to that of gapdh mrna . c expression levels of oct4 , nanog , lin28 and sox2 were assessed in the cd133 and cd133 cell populations by real - time quantitative pcr . d expression levels of nis , tg , tpo , tshr , ttf1 and pax8 were assessed in cd133 and cd133 cell populations by real - time quantitative pcr . data shown are the ratios of gene expression levels in the cd133 population to those in the cd133 population i radiotherapy is an important part of thyroid cancer management and its efficacy is closely relevant to future recurrence and metastasis . although cd133 cells have been proposed as thyroid cscs , their role in radioiodide therapy of thyroid cancer has not been explored . the present study unequivocally demonstrated that cd133 thyroid cancer cells are able to survive radiotherapy and these surviving cells may potentiate future recurrence . cd133 thyroid cancer cells had a higher radioresistance than cd133 cells , and the population of cd133 thyroid cancer cells was enriched after radiation treatment . also , these cells showed higher expression of es cell - related genes ( oct4 , nanog and lin28 ) and lower expression of thyroid - specific genes ( nis , tg , tpo and tshr ) than cd133 cells . this indicates that these cd133 cells resemble stem cells and may possess higher tumour - initiating ability . it has been reported that parallel expression of various thyroid - specific genes ( nis , tg , tpo , tshr , ttf1 and pax8 ) is inversely correlated with differentiation and malignant phenotypes [ 22 , 23 ] . this gene expression pattern has been found in clinical and pathological samples and indicates a stepwise progression of thyroid cancer from wdtc to utc or atc . an area of ptc in atcs has been shown to undergo dedifferentiation , and this process has been reported to be correlated with gaining an additional p53 mutation in braf - mutated ptcs [ 25 , 26 ] . however , how the tumour cells dedifferentiate and how these genes mutate during thyroid cancer progression remains unknown . several studies have demonstrated the existence of thyroid cscs marked by cd133 expression [ 16 , 17 ] , by aldehyde dehydrogenase expression   or as a side population in flow cytometry . together with our results , these cells showing gene expression related to an undifferentiated status are resistant to radiation and chemotherapy drugs and exhibit stem cell - like behaviour . however , these putative thyroid cscs have only been demonstrated to exist as a more primitive population within heterogeneous thyroid cancer . whether these cscs participate in the dedifferentiation during thyroid tumour progression , we demonstrated that the expression levels of several genes in the aro - hnis tumours were altered following i treatment . aro - hnis tumour growth was efficiently inhibited by i treatment , following which the expression levels of es cell regulators / markers as well as glut1 were upregulated . we attribute this phenomenon to the selection effect of i radiation within the heterogeneous cancer cells which resulted in the greater survival of a radioresistant population , namely cd133 cells in this study . cd133 populations , both in thyroid cancer cell lines and in primary cultured ptc cells ( data not shown ) , were enriched after irradiation . this enrichment was due to the higher radioresistance of a group of cells primarily comprising cd133-expressing cells . in addition to the selective killing effect , i treatment has also been reported to induce dedifferentiation of thyroid cancer cells . after pretreatment with i , ftc-133 , a dct cell line showed decreased ability to accumulate radioiodide , which was correlated with the downregulation of nis , tg , tpo and tshr expression . taken together , despite the effectiveness of i therapy in thyroid cancer , its adverse effects , selective killing and induction of dedifferentiation should be generally considered in the management of thyroid cancer . dedifferentiation of thyroid cancer is characterized by a reduction or loss of thyroid - specific gene expression . as a result , the tumours become insensitive to radioiodide therapy due to a loss of radioiodide uptake and accumulation in the cells . in the present study , after in vitro -irradiation , isolated cd133 cells showed a higher survival rate than isolated cd133 cells . also , among the bulk cells , the cd133 population was enriched after -irradiation due to a lower rate of apoptosis . these results indicate that cd133 cells have a higher innate radioresistance in addition to their diminished ability to take up and accumulate iodide . in our in vivo animal aro - hnis tumour model , i treatment efficiently inhibited tumour growth and led to a significant elevation in cd133 expression compared to saline treatment . this result clearly demonstrates the enrichment of cscs after i radiation and also implies the possible role of these cells in future recurrence and metastasis . cd133-expressing cells were found in thyroid cancer cell lines , thyroid cancer tissues and primarily cultured ptcs , suggesting that cd133 expression contributes to the heterogeneity of thyroid cancer . purified aro cd133 cells recapitulated the parental cd133 percentage more efficiently than purified cd133 cells ( data not shown ) . also , it has also been reported that cd133 aro cells undergo asymmetric division and give rise to both cd133 and cd133 progeny . these findings indicate that in thyroid cancers , the cd133 and cd133 populations are in dynamic equilibrium during cancer progression . we characterize the cd133 population as a more undifferentiated and radioresistant population , and suggest that the proportion of cd133 cells within the thyroid cancer population may determine the degree of malignancy of the cancer . whether cd133 expression can serve as a prognostic factor should be intensively investigated . several clinical trials have used retinoic acid , bexarotene , valproic acid , suberoylanilide hydroxamic acid or isotretinoin to reinduce the susceptibility of thyroid cancer to i therapy , the concept of  redifferentiation  therapy [ 31 , 32 ] . these agents preferentially enhance the expression of thyroid - specific genes including nis , and lead to the restoration of radioiodide uptake . it remains to be elucidated whether these agents have a differentiation effect on the cd133 population or cscs in thyroid cancer . nevertheless , cd133-expressing thyroid cancer cells may be a potential target for thyroid cancer treatment . in summary , this study demonstrated the role of cd133-expressing cells in the therapeutic resistance of thyroid cancers . this specific population detected in thyroid cancers exhibited undifferentiated , radioresistance and survived i therapy . these findings provide a new perspective in the therapy of thyroid cancer and point to targeting cd133-expressing cells in the development of a therapeutic strategy . in summary , this study demonstrated the role of cd133-expressing cells in the therapeutic resistance of thyroid cancers . this specific population detected in thyroid cancers exhibited undifferentiated , radioresistance and survived i therapy . these findings provide a new perspective in the therapy of thyroid cancer and point to targeting cd133-expressing cells in the development of a therapeutic strategy . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .
purpose131i therapy is regularly used following surgery as a part of thyroid cancer management . despite an overall relatively good prognosis , 
 recurrent or metastatic thyroid cancer is not rare . 
 cd133-expressing cells have been shown to mark thyroid cancer stem cells that possess the characteristics of stem cells and have the ability to initiate tumours . 
 however , no studies have addressed the influence of cd133-expressing cells on radioiodide therapy of the thyroid cancer . 
 the aim of this study was to investigate whether cd133 + cells contribute to the radioresistance of thyroid cancer and thus potentiate future recurrence and metastasis.methodsthyroid cancer cell lines were analysed for cd133 expression , radiosensitivity and gene expression.resultsthe anaplastic thyroid cancer cell line aro showed a higher percentage of cd133 + cells and higher radioresistance . 
 after -irradiation of the cells , the cd133 + population was enriched due to the higher apoptotic rate of cd133 cells . in vivo 131i treatment of aro tumour resulted in an elevated expression of cd133 , oct4 , nanog , lin28 and glut1 genes . 
 after isolation , cd133 + cells exhibited higher radioresistance and higher expression of oct4 , nanog , sox2 , lin28 and glut1 in the cell line or primarily cultured papillary thyroid cancer cells , and lower expression of various thyroid - specific genes , namely nis , tg , tpo , tshr , ttf1 and pax8.conclusionthis study demonstrates the existence of cd133-expressing thyroid cancer cells which show a higher radioresistance and are in an undifferentiated status . 
 these cells possess a greater potential to survive radiotherapy and may contribute to the recurrence of thyroid cancer . a future therapeutic approach for radioresistant thyroid cancer may focus on the selective eradication of cd133 + cells .