Current perspectives on Vaxinia virus: an immuno-oncolytic vector in cancer therapy.

Viruses are being researched as cutting-edge therapeutic agents in cancer due to their selective oncolytic action against malignancies. Immuno-oncolytic viruses are a potential category of anticancer treatments because they have natural features that allow viruses to efficiently infect, replicate, and destroy cancer cells. Oncolytic viruses may be genetically modified; engineers can use them as a platform to develop additional therapy modalities that overcome the limitations of current treatment approaches. In recent years, researchers have made great strides in the understanding relationship between cancer and the immune system. An increasing corpus of research is functioning on the immunomodulatory functions of oncolytic virus (OVs). Several clinical studies are currently underway to determine the efficacy of these immuno-oncolytic viruses. These studies are exploring the design of these platforms to elicit the desired immune response and to supplement the available immunotherapeutic modalities to render immune-resistant malignancies amenable to treatment. This review will discuss current research and clinical developments on Vaxinia immuno-oncolytic virus.

PET imaging and treatment of pancreatic cancer peritoneal carcinomatosis after subcutaneous intratumoral administration of a novel oncolytic virus, CF33-hNIS-antiPDL1.

Peritoneal carcinomatosis of gastrointestinal malignancies remains fatal. CF33-hNIS-antiPDL1, a chimeric orthopoxvirus expressing the human sodium iodide symporter (hNIS) and anti-human programmed death-ligand 1 antibody, has demonstrated robust preclinical activity against pancreatic adenocarcinoma (PDAC). We investigated the ability of CF33-hNIS-antiPDL1 to infect, help detect, and kill peritoneal tumors following intratumoral (i.t.) injection of subcutaneous (s.c.) tumors in vivo. Human PDAC AsPC-1-ffluc cells were inoculated in both the s.c. space and the peritoneal cavity of athymic mice. After successful tumor engraftment, s.c. tumors were injected with CF33-hNIS-antiPDL1 or PBS. We assessed the ability of CF33-hNIS-antiPDL1 to infect, replicate in, and allow the imaging of tumors at both sites (immunohistochemistry [IHC] and 124I-based positron emission tomography/computed tomography [PET/CT] imaging), tumor burden (bioluminescence imaging), and animal survival. IHC staining for hNIS confirmed expression in s.c. and peritoneal tumors following virus treatment. Compared to the controls, CF33-hNIS-antiPDL1-treated mice showed significantly decreased s.c. and peritoneal tumor burden and improved survival (p < 0.05). Notably, 2 of 8 mice showed complete regression of disease. PET/CT avidity for 124I uptake in s.c. and peritoneal tumors was visible starting at day 7 following the first i.t. dose of CF33-hNIS-antiPDL1. We show that CF33-hNIS-antiPDL1 can help detect and kill both s.c. and peritoneal tumors following s.c. i.t. treatment.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

Reporter gene-engineering of human induced pluripotent stem cells during differentiation renders in vivo traceable hepatocyte-like cells accessible.

Primary hepatocyte transplantation (HTx) is a safe cell therapy for patients with liver disease, but wider application is circumvented by poor cell engraftment due to limitations in hepatocyte quality and transplantation strategies. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSC) are considered a promising alternative but also require optimisation of transplantation and are often transplanted prior to full maturation. Whole-body in vivo imaging would be highly beneficial to assess engraftment non-invasively and monitor the transplanted cells in the short and long-term. Here we report a lentiviral transduction approach designed to engineer hiPSC-derived HLCs during differentiation. This strategy resulted in the successful production of sodium iodide symporter (NIS)-expressing HLCs that were functionally characterised, transplanted into mice, and subsequently imaged using radionuclide tomography.

In vitro study on human umbilical cord mesenchymal stem cells transfected with lentivirus-mediated hNIS-EGFP dual reporter gene and co-labeled with superparamagnetic iron oxide.

The aim of the present study was to establish a stem cell line for multi-mode imaging (in vivo fluorescence imaging, magnetic resonance imaging and 99mTc single-photon emission computed tomography) and to study the biological activity, stemness, proliferative activity and differentiation ability of superparamagnetic iron oxide (SPIO), human sodium/iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) co-labeled human umbilical cord mesenchymal stem cells (hUCMSCs). The EGFP reporter gene was selected to indirectly reflect the expression of target gene hNIS, and hUCMSCs were re-transfected with the successfully constructed recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro. When a stem cell line stably expressing hNIS and EGFP was obtained, the cells were incubated with 30 µg/ml SPIO to obtain hNIS, EGFP and SPIO co-labeled stem cells. The protein expressions of hNIS and EGFP were identified using western blot analysis, and the protein function of hNIS was identified by 125I influx and 125I efflux experiments. hNIS-EGFP-hUCMSCs were labeled with SPIO under the mediation of poly-L-lysine, and SPIO, hNIS and EGFP co-labeled hUCMSCs were established successfully. Staining with Prussian blue confirmed that 98% of cells were successfully labeled with SPIO. Western blotting results demonstrated positive hNIS and EGFP protein expression levels, and 125I influx and 125I efflux experiments confirmed that the protein function of hUCMSCs after expressing hNIS was normal. The uptake of 125I was higher in cell lines hNIS-EGFP-hUCMSCs than in control hUCMSCs (fold change: 16.43±2.30 times; P<0.05). The stemness of hNIS-EGFP-hUCMSCs was found to be slightly decreased but not statistically significant; the overall characteristics of stem cells remained unchanged. The assessments of adipogenic and osteogenic differentiation suggest that hNIS-EGFP-hUCMSCs have no significantly different characteristics compared with primary hUCMSCs.

18F-Fluorosulfate for PET Imaging of the Sodium-Iodide Symporter: Synthesis and Biologic Evaluation In Vitro and In Vivo.

Anion transport by the human sodium-iodide symporter (hNIS) is an established target for molecular imaging and radionuclide therapy. Current radiotracers for PET of hNIS expression are limited to 124I- and 18F-BF4- We sought new 18F-labeled hNIS substrates offering higher specific activity, higher affinity, and simpler radiochemical synthesis than 18F-BF4- METHODS: The ability of a range of anions, some containing fluorine, to block 99mTcO4- uptake in hNIS-expressing cells was measured. SO3F- emerged as a promising candidate. 18F-SO3F- was synthesized by reaction of 18F- with SO3-pyridine complex in MeCN and purified using alumina and quaternary methyl ammonium solid-phase extraction cartridges. Chemical and radiochemical purity and serum stability were determined by radiochromatography. Radiotracer uptake and efflux in hNIS-transduced HCT116-C19 cells and the hNIS-negative parent cell line were evaluated in vitro in the presence and absence of a known competitive inhibitor (NaClO4). PET/CT imaging and ex vivo biodistribution measurement were conducted on BALB/c mice, with and without NaClO4 inhibition. RESULTS: Fluorosulfate was identified as a potent inhibitor of 99mTcO4- uptake via hNIS in vitro (half-maximal inhibitory concentration, 0.55-0.56 µM (in comparison with 0.29-4.5 µM for BF4-, 0.07 µM for TcO4-, and 2.7-4.7 µM for I-). Radiolabeling to produce 18F-SO3F- was simple and afforded high radiochemical purity suitable for biologic evaluation (radiochemical purity > 95%, decay-corrected radiochemical yield = 31.6%, specific activity ≥ 48.5 GBq/µmol). Specific, blockable hNIS-mediated uptake in HCT116-C19 cells was observed in vitro, and PET/CT imaging of normal mice showed uptake in thyroid, salivary glands (percentage injected dose/g at 30 min, 563 ± 140 and 32 ± 9, respectively), and stomach (percentage injected dose/g at 90 min, 68 ± 21). CONCLUSION: Fluorosulfate is a high-affinity hNIS substrate. 18F-SO3F- is easily synthesized in high yield and very high specific activity and is a promising candidate for preclinical and clinical PET imaging of hNIS expression and thyroid-related disease; it is the first example of in vivo PET imaging with a tracer containing an S-18F bond.

Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene.

A whole-body dual-modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment response in different microenvironments.

UNLABELLED: Imaging spontaneous cancer cell metastasis or heterogeneous tumor responses to drug treatment in vivo is difficult to achieve. The goal was to develop a new highly sensitive and reliable preclinical longitudinal in vivo imaging model for this purpose, thereby facilitating discovery and validation of anticancer therapies or molecular imaging agents. METHODS: The strategy is based on breast cancer cells stably expressing the human sodium iodide symporter (NIS) fused to a red fluorescent protein, thereby permitting radionuclide and fluorescence imaging. Using whole-body nano-SPECT/CT with (99m)TcO4(-), we followed primary tumor growth and spontaneous metastasis in the presence or absence of etoposide treatment. NIS imaging was used to classify organs as small as individual lymph nodes (LNs) to be positive or negative for metastasis, and results were confirmed by confocal fluorescence microscopy. Etoposide treatment efficacy was proven by ex vivo anticaspase 3 staining and fluorescence microscopy. RESULTS: In this preclinical model, we found that the NIS imaging strategy outperformed state-of-the-art (18)F-FDG imaging in its ability to detect small tumors (18.5-fold-better tumor-to-blood ratio) and metastases (LN, 3.6-fold) because of improved contrast in organs close to metastatic sites (12- and 8.5-fold-lower standardized uptake value in the heart and kidney, respectively). We applied the model to assess the treatment response to the neoadjuvant etoposide and found a consistent and reliable improvement in spontaneous metastasis detection. Importantly, we also found that tumor cells in different microenvironments responded in a heterogeneous manner to etoposide treatment, which could be determined only by the NIS-based strategy and not by (18)F-FDG imaging. CONCLUSION: We developed a new strategy for preclinical longitudinal in vivo cancer cell tracking with greater sensitivity and reliability than (18)F-FDG PET and applied it to track spontaneous and distant metastasis in the presence or absence of genotoxic stress therapy. Importantly, the model provides sufficient sensitivity and dynamic range to permit the reliable assessment of heterogeneous treatment responses in various microenvironments.

Combined RNA interference of adenine nucleotide translocase-2 and ganciclovir therapy in hepatocellular carcinoma.

PURPOSE: The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model. METHODS: The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate. RESULTS: Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model. CONCLUSIONS: Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma.

Monitoring adenoviral based gene delivery in rat glioma by molecular imaging.

AIM: To determine whether endothelial progenitor cells (EPCs) can be used as delivery vehicle for adenoviral vectors and imaging probes for gene therapy in glioblastoma. METHODS: To use cord blood derived EPCs as delivery vehicle for adenoviral vectors and imaging probes for glioma gene therapy, a rat model of human glioma was made by implanting U251 cells orthotopically. EPCs were transfected with an adenovirus (AD5/carrying hNIS gene) and labeled with iron oxide and inoculated them directly into the tumor 14 d following implantation of U251 cells. Magnetic resonance imaging (MRI) was used to in vivo track the migration of EPCs in the tumor. The expression of gene products was determined by in vivo Tc-99m single photon emission computed tomography (SPECT). The findings were validated with immunohistochemistry (IHC). RESULTS: EPCs were successfully transfected with the adenoviral vectors carrying hNIS which was proved by significantly (P < 0.05) higher uptake of Tc-99m in transfected cells. Viability of EPCs following transfection and iron labeling was not altered. In vivo imaging showed the presence of iron positive cells and the expression of transgene (hNIS) product on MRI and SPECT, respectively, all over the tumors following administration of transfected and iron labeled EPCs in the tumors. IHC confirmed the distribution of EPC around the tumor away from the injection site and also showed transgene expression in the tumor. The results indicated the EPCs' ability to deliver adenoviral vectors into the glioma upon intratumor injection. CONCLUSION: EPCs can be used as vehicle to deliver adenoviral vector to glioma and also act as imaging probe at the same time.

Total Thyroidectomy in the Mouse: the Feasibility Study in the Non-thyroidal Tumor Model Expressing Human Sodium/Iodide Symporter Gene.

PURPOSE: This study sought to probe the feasibility of performing total thyroidectomy in the mouse using a non-thyroidal hNIS expressing tumor model. MATERIALS AND METHODS: Our thyroidectomy protocol included thorough excision of both lobes and the isthmus. For evaluating the completeness of thyroidectomy, we compared the (99m)Tc-pertechnetate scans taken before and after thyroidectomy. The prostate cancer cell line was subcutaneously inoculated 2 weeks after the thyroidectomy. When the tumor reached 5-10 mm in diameter, Ad5/35-E4PSESE1a-hNIS was injected intratumorally, and (131)I scans were performed. The radioiodine uptakes of the neck and the tumor were compared with those of the other regions. RESULTS: Total thyroidectomy was performed in 13 mice. Although 38.5% died during or just after thyroidectomy, the others survived in good health for 2 months. Thyroid tissue was completely eliminated using our protocol; the residual uptake of (99m)Tc-pertechnetate was minimal in the neck area. The neck/background uptake ratio after thyroidectomy was significantly lower than that before thyroidectomy (p < 0.05). Non-thyroidal tumor models were successfully established in all the surviving mice. Radioiodine accumulation in the tumors was visualized on (131)I scans, and the neck uptakes were minimal. CONCLUSION: Using our total thyroidectomy protocol, we successfully established a hNIS-transfected prostate cancer model with a minimal accumulation of radioiodine in the neck. The relatively high mortality after surgery can be a problem, and this might be reduced by minimizing the surgical stress.

Study on hNIS mediated transferring gene99mTcO-4 imaging and mI treatment in xenografted ovariancancer / 中华内分泌代谢杂志

Objective To establish radioiodine therapy in nonthyroid tumor and to investigate 131Ⅰ treatment effect on xenografted ovarian cancer. Methods Based on previous test, xenografted ovarian cancer nude model were established in nude mice. The effects of radioactive isotope 99m TcO-4 imaging and radioiodine 131Ⅰ treatment on xenografted ovarian cancer in vivo were investigated. Results After transferring human sodium/iodide symporter (hNIS) gene, the xenografted ovarian cancer in nude mice was imaged by isotope 99m TcO-4 Moreover,131Ⅰ exerted inhibitory effect on the proliferative activity. Conclusion After the transfection of hNIS gene, 131Ⅰ has inhibitory effect on proliferative activity of xenografted ovarian cancer.

Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells / 핵의학분자영상

PURPOSE: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. MATERIALS AND METHODS: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1+/-4.7%, 54.0+/-6.4%, 85.7+/-8.7%, and 98.4+/-1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. RESULTS: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704+/-6,659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168+/-2,134 picomole/10(6) cells). CONCLUSION: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

Development of Dual Reporter System of Mutant Dopamine 2 Receptor (D2R) and Sodium Iodide Symporter (NIS) Transgenes / 대한핵의학회잡지

PURPOSE: Both human NIS and mutant D2R transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor (D2R) and compared its characteristics. MATERIALS AND METHODS: The recombinant plasmid (pIRES-hNIS/D2R) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter. pIRES-hNIS/D2R was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND (SK-Hep1-hNIS/D2R) cells stably expressing hNIS and D2R was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and D2R genes. The expressions of hNIS and D2R were measured by 125I uptake assays and receptor binding assays. Specific binding of D2R to [3H]spiperone was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. K (d) and B (max) values were estimated. The correlation between hNIS and D2R expression was compared by using each clone. RESULTS: Similar quantities of hNIS and D2R genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. 125I uptake in HEP-ND cells was completely inhibited by KClO4, a NIS inhibitor. Specific binding to HEP-ND cells was saturable and the K (d) and B (max) values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and D2R binding was highly correlated. CONCLUSION: We developed a dual positron and gamma imaging reporter system of hNIS and D2R in a stably transfected cell line. We expect that D2R and hNIS genes can complement mutually as a nuclear reporting system or that D2R can be used as reporter gene when hNIS gene were used as a treatment gene.

Expression of Sodium/iodide Symporter Transgene in Neural Stem Cells / 대한핵의학회잡지

PURPOSE: The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. MATERIALS AND METHODS: F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. RESULTS: The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to 20micro M, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. CONCLUSION: These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene expression of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions.

Clinical Significance of Human Sodium Iodide Symporter mRNA Expressions in Primary and Metastatic Papillary Thyroid Carcinoma / 대한내분비학회지

BACKGROUND: The iodide transport into thyroid cells is an essential step in the biosynthesis of thyroid hormones. The sodium iodide symporter (NIS) which is responsible for iodide transport was cloned recently and identified as a plasma membrane glycoprotein. Recent report suggested the absence of human NIS (hNIS) mRNA expression of papillary carcinoma in thyroid indicates absence of response on radioiodine therapy for distant metastasis. To understand the change of hNIS expression at the stage of metastasis in papillary thyroid carcinomas, we evaluated the expression levels of hNIS mRNA in primary and lymph node metastatic papillary carcinoma tissues. METHODS: Seven pairs of primary and lymph node metastatic tissues were included in this study. The level of hNIS mRNA in lymph node metastatic tissues and primary tissues were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The level of GAPDH mRNA was used as internal control. RESULTS: Two among 6 lymph node metastatic tissues did not show hNIS mRNA even with significant hNIS expressions in papillary carcinoma tissues in thyroid. The levels of hNIS expression of remaining 4 lymph node metastatic tissues were lower than those of corresponding primary tissues. Interestingly, one case showed no hNIS expression in primary tissue, but significant hNIS expression in lymph node metastatic tissue. There was no correlation in hNIS mRNA expression between primary and lymph node metastatic tissues. CONCLUSION: No correlation was found in hNIS mRNA expression between primary and lymph node metastatic tissues, suggesting the measurements of hNIS mRNA level in primary tissues may not predict therapeutic response to radioactive iodine.

Expression of Human Sodium Iodide Symporter mRNA in Papillary Thyroid Carcinoma / 대한내분비학회지

BACKGROUND: The sodium iodide symporter(NIS) is a plasma membrane protein which is respoasibIe for iodide transport into thyroid cell. The cDNA sequence of NIS has recently been cloned from rat and human. Intrinsic ability and its differences in iodide accumulation have been exploited as a useful tool for diagnosis and therapy of thyroid diseases. It is also known that some differentiated thyroid cancers do not take up radioactive iodine at therapeutic dose. METHODS: To understand the expression and regulation of NIS in thyroid tumars, we measured the expressons of human NIS(hNIS), TSH-receptor(R), and thyroglohulin(Tg) mRNAs from papillary thyroid carcinoma(PTC) tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNase protection assay(RPA). RESULT: By RT-PCR analysis, 87% of PTC expressed hNIS mRNA, but the degree of expression were variable. Interestingly, 32% of PTC showed significant level of hNIS expression even though pre-operative technetium thyroid scan of all thyroid tumors were cold but the level was lower than normal control tissues. All of PTC showed the expressions of Tg and TSH-R mRNAs and there was a correlation between hNIS mRNA and TSH-R mRNA(Rsq 0.35, p=0.01). By RPA, the expression of hNIS and TSH-R in normal control tissue were detected with 20microgram and 40microgram of total RNA respectively, but the higher concentrations(> or =60microgram for hNIS and > or =40microgram for TSH-R) were required to detect in PTC, showing that tbe expression of hNIS in FTC was lower than TSH-R expression. CONCLUSION: PTC tends to lose hNIS mRNA expression earlier than TSH-R mRNA and the measurement of hNIS mRNA in PTC may be useful as an indicator of the therapeutic response to radioactive iodine.