NIS Expression Research Articles

Abstract 193: CRISPR-mediated Introduction of the Sodium-iodide Symporter to Enable Non-invasive Monitoring of Macaque Induced-pluripotent Stem Cell-derived Cardiomyocytes

Due to the limited regenerative capacity of mature cardiomyocytes, cardiac cell therapies constitute an exciting strategy for myocardial repair. However, there is limited understanding of the spatio-temporal distribution and survival of transplanted cells. Hence, there is demand for technologies enabling long-term non-invasive tracking of transplanted cellular therapeutics. Sodium-iodide symporter (NIS)-based in vivo imaging has many potential advantages, including predicted safety and immunotolerance due to reliance on an endogenous species-specific gene and on widely available imaging technologies. We believe that non-human primates represent ideal models for investigating the biology of allogenic or autologous cellular grafts, because of close physiologic similarity to humans. We report the development of NIS-based in vivo imaging to detect and track rhesus induced pluripotent stem cell (RhiPSC)-derived teratomas as a proof-of-concept model tested in mice, and characterization of NIS-positive RhiPSC (NIS-RhiPSC)-derived cardiomyocytes (CM). NIS-RhiPSCs were generated by CRISPR/Cas9-mediated integration of the rhesus NIS cDNA within the AAVS1 safe harbor locus. NIS was stably expressed and radiotracer uptake by NIS-RhiPSCs was demonstrated in vitro . To evaluate viability of NIS-mediated imaging in RhiPSCs, undifferentiated NIS-RhiPSCs were introduced intramuscularly into immunodeficient mice, and NIS imaging was performed via PET/CT at 2, 4, and 6-weeks post-injection. NIS-positive teratomas were readily detectable as early as 2 weeks post-injection, prior to development of any palpable mass. Using our previously established differentiation protocol, NIS-RhiPS-CMs were derived with high purity, exhibited spontaneous beating in culture, and were similar in all aspects to parental RhiPS-CMs. NIS-RhiPS-CMs maintained stable NIS expression that was comparable to undifferentiated NIS-RhiPSCs, suggesting that in vivo imaging of transplanted NIS-RhiPS-CMs should be feasible. Further functional characterization of NIS-RhiPS-CMs, including in vitro radiotracer uptake, post-transplantation imaging in a mouse myocardial infarction model, and electrophysiologic analysis is ongoing and data will be presented.

Flavonoids, Thyroid Iodide Uptake and Thyroid Cancer-A Review.

Thyroid cancer is the most common malignant tumor of the endocrine system and the incidence has been increasing in recent years. In a great part of the differentiated carcinomas, thyrocytes are capable of uptaking iodide. In these cases, the main therapeutic approach includes thyroidectomy followed by ablative therapy with radioiodine. However, in part of the patients, the capacity to concentrate iodide is lost due to down-regulation of the sodium-iodide symporter (NIS), the protein responsible for transporting iodide into the thyrocytes. Thus, therapy with radioiodide becomes ineffective, limiting therapeutic options and reducing the life expectancy of the patient. Excessive ingestion of some flavonoids has been associated with thyroid dysfunction and goiter. Nevertheless, studies have shown that some flavonoids can be beneficial for thyroid cancer, by reducing cell proliferation and increasing cell death, besides increasing NIS mRNA levels and iodide uptake. Recent data show that the flavonoids apingenin and rutin are capable of increasing NIS function and expression in vivo. Herein we review literature data regarding the effect of flavonoids on thyroid cancer, besides the effect of these compounds on the expression and function of the sodium-iodide symporter. We will also discuss the possibility of using flavonoids as adjuvants for therapy of thyroid cancer.

Tumor suppressor protein p53 exerts negative transcriptional regulation on human sodium iodide symporter gene expression in breast cancer.

Aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) is well documented but the transcription factors (TF) regulating its aberrant expression is poorly known. We identify the presence of three p53 binding sites on the human NIS promoter sequence by conducting genome-wide TF analysis, and further investigate their regulatory role. The differences in transcription and translation were measured by real-time PCR, luciferase reporter assay, site-directed mutagenesis, in vivo optical imaging, and chromatin immunoprecipitation. The relation of NIS and p53 in clinical samples was judged by TCGA data analysis and immunohistochemistry. Overexpression of wild-type p53 as a transgene or pharmacological activation by doxorubicin drug treatment shows significant suppression of NIS transcription in multiple BC cell types which also results in lowered NIS protein content and cellular iodide intake. NIS repression by activated p53 is further confirmed by non-invasive bioluminescence imaging in live cell and orthotropic tumor model. Abrogation of p53-binding sites by directional mutagenesis confirms reversal of transcriptional activity in wild-type p53-positive BC cells. We also observe direct binding of p53 to these sites on the human NIS promoter. Importantly, TCGA data analysis of NIS and p53 co-expression registers an inverse relationship between the two candidates. Our data for the first time highlight the role of p53 as a negative regulator of functional NIS expression in BC, where the latter is a potential targeted radioiodine therapy candidate. Thus, the study provides an important insight into prospective clinical application of this approach that may significantly impact the patient with mutant versus wild-type p53 profile.

Tracking dendritic cell migration into lymph nodes by using a novel PET probe 18F-tetrafluoroborate for sodium/iodide symporter

BackgroundRecently, 18F-tetrafluoroborate (TFB) was used as a substrate for the human sodium/iodide symporter (hNIS) reporter gene. This study evaluated the feasibility of performing molecular-genetic imaging by using the new radiotracer (18F-TFB) for the hNIS gene, to track dendritic cell (DC) migration in live mice. A murine dendritic cell line (DC2.4) co-expressing the hNIS and effluc genes (DC/NF) was established. To confirm the functional cellular expression of both effluc and NIS in the inoculated DC/NF cells by bio-medical imaging, combined bioluminescence imaging (BLI) and 18F-TFB positron emission tomography/computed tomography (PET/CT) imaging was performed after intramuscular injection with parental DCs and DC/NF cells. For DC-tracking, parental DCs or DC/NF cells were injected in the left or right mouse footpad, respectively, and 18F-TFB PET/CT and BLI were performed to monitor these cells in live mice.ResultsIn vivo PET/CT and BLI showed a clear signal in DC/NF injection sites but not in parental DC injection sites. The signal intensity in DC/NF cells was correlated with time. In vivo 18F-TFB PET/CT imaging showed higher radiotracer activity in the draining popliteal lymph nodes (DPLNs) in DC/NF injection sites than those in DC injection sites on day 2. BLI also showed DC/NF cell migration to the DPLNs on day 2 after the injection.ConclusionsMigration of DCs to the lymph nodes was successfully monitored using 18F-TFB PET/CT imaging of the NIS gene and optical imaging of the effluc gene in live mice. These data support the feasibility of using 18F-TFB as a substrate for hNIS reporter gene imaging to track the migration of DCs to the lymph nodes in live animals. The use of 18F-TFB may facilitate enhanced PET imaging of the hNIS reporter gene in small animals and humans in future studies.

SIGNIFICANCE OF IODINE SYMPORTER FOR PROGNOSIS OF THE DISEASE COURSE AND EFFICACY OF NEOADJUVANT CHEMOTHERAPY IN PATIENTS WITH BREAST CANCER OF LUMINAL AND BASAL SUBTYPES

The aim of the research was to study the relation between expression of Na.

Low Iodine in the Follicular Lumen Caused by Cytoplasm Mis-localization of Sodium Iodide Symporter may Induce Nodular Goiter.

Iodine is a key ingredient in the synthesis of thyroid hormones and also a major factor in the regulation of thyroid function. A local reduction of iodine content in follicular lumen leads to overexpression of local thyroid-stimulating hormone receptor (TSHr), which in turn excessively stimulates the regional thyroid tissue, and result in the formation of nodular goiter. In this study, we investigated the relationship between iodine content and sodium iodide symporter (NIS) expression by using the clinical specimens from patients with nodular goiter and explored the pathogenesis triggered by iodine deficiency in nodular goiter. In total, 28 patients were clinically histopathologically confirmed to have nodular goiter and the corresponding adjacent normal thyroid specimens were harvested simultaneously. Western blot and immunohistochemistry were performed to assay NIS expression and localization in thyrocytes of both nodular goiter and adjacent normal thyroid tissues. NIS expression mediated by iodine in follicular lumen was confirmed by follicular model in vitro. Meanwhile, radioscan with iodine-131were conducted on both nodular goiter and adjacent normal thyroid. Our data showed that NIS expression in nodular goiter was significantly higher than that in adjacent normal tissues, which was associated with low iodine in the follicular lumen. Abnormal localization of NIS and lower amount of radioactive iodine-131 were also found in nodular goiter. Our data implied that low iodine in the follicular lumen caused by cytoplasm mis-localization of NIS may induce nodular goiter.

Thyroid-Related Protein Expression in the Human Thymus.

Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS.

Wild-Type P53 Induces Sodium/Iodide Symporter Expression Allowing Radioiodide Therapy in Anaplastic Thyroid Cancer

Aims: Anaplastic thyroid cancer(ATC) is one of the most aggressive solid tumors. Mutations in the p53 gene are common in anaplastic thyroid cancer, but the effects of p53 mutations are yet to be elucidated. Here, we investigated the role of p53 in ATC. Methods: p53 mutation was detect by immunohistochemistry in ATC tissues. Expression of NIS were measured using immunohistochemistry, qRT-PCR, western blot, immunofluorescence in ATC tissues and cell line 8505c. Luciferase reporter assay was performed to examine the effect of wild-type p53 on NIS. Radioiodide uptake assay and flow cytometry analysis were used to detect the role of wild-type p53 on radioiodide uptake.and cell apoptosis in ATC cell line. Results: We showed that the p53 mutation can be detected in ATC tissues. Furthermore, we demonstrated that wild-type p53 transactivated the NIS promoter. In 8505c cells transfected with wild-type p53, treatment with radioiodine resulted in increased radioiodine uptake and increased apoptotic cell death compared with 8505c cells harboring the p53 mutation. Conclusion: In summary, transfection with wild-type p53 can increase the therapeutic effect of radioiodine by regulating the expression of the NIS.

3-Iodothyronamine Decreases Expression of Genes Involved in Iodide Metabolism in Mouse Thyroids and Inhibits Iodide Uptake in PCCL3 Thyrocytes.

3-Iodothyronamine (3-T1AM) is an endogenous decarboxylated thyroid hormone (TH) metabolite. Pharmacological doses of 3-T1AM decrease heart rate, body temperature, and metabolic rate in rodents-effects that are contrary to classic TH excess. Furthermore, a single dose of 3-T1AM was shown to suppress the hypothalamic-pituitary-thyroid (HPT) axis in rats. It was hypothesized that 3-T1AM might play a role in the fine-tuning of TH action and might have a direct regulatory effect on the thyroid gland. This study tested whether repeated 3-T1AM treatment interfered with thyroid function and the HPT axis in mice. Therefore, male C57BL/6 mice were intraperitoneally injected with 5 mg/kg of 3-T1AM or vehicle daily for seven days. Additionally, the effects of 3-T1AM on the differentiated rat thyrocyte cell line PCCL3 were analyzed. Repeated administration of 3-T1AM decreased thyroidal mRNA content of the sodium iodide symporter (Nis), thyroglobulin, and pendrin in mice. No interference with the HPT axis was observed, as determined by unaltered pituitary mRNA levels of triiodothyronine-responsive genes, including thyrotropin subunit β. Furthermore, 3-T1AM treatment did not change transcript levels of hepatic triiodothyronine-responsive genes, such as deiodinase 1. In line with this, serum TH concentrations were not changed after the treatment period of seven days. In concordance with the in vivo findings, 3-T1AM decreased the thyrotropin-dependent expression of Nis and functional iodide uptake in PCCL3 cells in vitro. Additionally, uptake and metabolism of 3-T1AM by PCCL3 cells was observed, as well as 3-T1AM-dependent changes in intracellular Ca2+ concentration that might be involved in mediating the reported effects. In conclusion, 3-T1AM application decreased expression of selected TH synthesis genes by acting directly on the thyroid gland, and it might therefore affect TH synthesis without involvement of the HPT axis.

CORRELATION OF IODINE SYMPORTER EXPRESSION IN HIGHLY AND LOW MALIGNANT CELL LINES OF HUMAN BREAST CANCER DIFFERED IN THEIR SENSITIVITY TO DOXORUBICIN

To investigate the relationship of sodium/iodide symporter (NIS) expression with molecular phenotype of highly and low malignant cell lines of human breast cancer (BC) with different sensitivity to doxorubicin (Dox). The cell lines used in the analysis included T47D, MCF-7, MDA-MB-231, MDA-MB-468, and MCF/Dox. NIS expression was studied by immunocytochemical method. The strongest iodine symporter expression (248 ± 1.9; 272 ± 3.2 and 289 ± 2.8 points, respectively) were found in cells of highly malignant cell lines - MDA-MB-231, MDA-MB-468, and MCF-7/Dox. NIS expression was significantly weaker (< 120 points) in two BC cell lines of low malignancy (MCF-7 and T47D). In addition, the reduced sensitivity to Dox is associated with elevation of NIS expression in both high and low malignant cells. We have demonstrated correlations between NIS levels and certain indices of BC malignancy, namely proliferative activity (r = 0.51), receptor status (estrogen receptor; r = -0.47; and progesteron receptor; r = -0.47) and invasiveness (r = 0.46). Our data evidence that NIS expression level correlates with BC cells indices of malignancy and their sensitivity to Dox. The results obtained suggest the necessity for further studies of NIS expression in BC patients aimed at prognosing disease course and monitoring treatment efficacy with anthracyclines.

Hippo Pathway mediator TAZ acts as a negative regulator of the sodium iodide symporter

Background. TAZ/WWTR1 (transcriptional coactivator with a PDZ-binding domain) is a transcriptional cofactor involved in the Hippo Signalling pathway, which is described to have a key role in the control of cell proliferation, apoptosis, the inhibition of cell-cell contact, stem cell self-renewal and tissue regeneration. TAZ has been reported to regulate these processes through the transactivation of transcription factors in the nucleus, where it has also been shown to interact with Smad2/3-Smad4 complexes favouring nuclear accumulation under TGFβ stimulation. TAZ co-activates Pax8, a master gene of thyroid differentiation, within the thyroglobulin promoter. Furthermore, Pax8 is the main positive regulator of sodium iodide symporter (NIS) expression and it has been reported to interact with Smad3 causing NIS transcriptional repression mediated by TGFβ.Aim. Therefore, the aim of this work was to study the involvement of TAZ in the expression of NIS, since it is an important protein not only for the correct function of the thyroid gland, but also for radioiodide treatment in thyroid cancer.Methods and Results. Strikingly, we observed that TAZ negatively regulates the transcriptional activity of Pax8 within the NIS promoter. Furthermore, we provided evidence that TAZ could play an important role in the downregulation of NIS expression by TGFβ; we detected that TAZ protein is mainly located in the nucleus under treatment with this cytokine and its silencing induces a partial recovery of NIS protein and mRNA levels. Our results also demonstrated an increased expression of TAZ in thyroid carcinoma cell lines, in which NIS levels are typically decreased. Specifically, TAZ nuclear translocation is increased in those thyroid carcinoma cells with mutated BRAFV600E or when this oncogene is conditionally activated. Since we have described that this mutation increases the secretion of TGFβ, this could be connected with the decreased levels of NIS in these cells.Conclusion. This study has shed light on the important role of the Hippo pathway in the regulation of NIS expression in thyroid cells, repressing Pax8 activity and impaired thyroid differentiation. Given that this protein has been identified to be overexpressed in thyroid carcinoma, future research of the role of TAZ in thyroid tumorogenesis will enable development of new strategies to treat thyroid cancer.

Small activating RNA upregulates NIS expression: promising potential for hepatocellular carcinoma endoradiotherapy.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Currently, the clinical strategies available for the treatment of HCC remain insufficient for the poor prognosis. Sodium/iodide symporter (NIS)-based radioiodine therapy is proposed as a promising therapeutic strategy for the treatment of HCC. However, it is difficult for HCC cells to trap iodine for the lower expression of NIS. Small activating RNA (saRNA) is a newly identified small double-stranded RNA (dsRNA) that can induce endogenous gene expression by targeting promoter sequences. Here, we designed an saRNA (saRNA-482) that targeted the NIS promoter sequences. In the cultured HepG2 cells and Hep3B cells, the expressions of NIS were upregulated after transfection of saRNA-482. In addition, the uptake of 125I increased in the cultured HepG2 and Hep3B cells transfected with saRNA-482. Furthermore, the cell viabilities were significantly inhibited in the saRNA-482-transfected HepG2 and Hep3B cells after 131I treatment. Meanwhile, the apoptosis of saRNA-482-transfected HepG2 and Hep3B cells significantly increased after 131I treatment. The results suggest that RNA activation-mediated upregulation of NIS may have an endoradiotherapeutic potential in the treatment of HCC.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium.

Different expression of sodium-iodide importer (NIS) between lactating breast and thyroid tissues may be due to structural difference of thyroid-stimulating hormone receptor (TSHR).

Thyroid-stimulating hormone (TSH) binds TSH receptor (TSHR) on thyroid cell membranes, which will lead activation of cyclic adenosine 3',5'-monophosphate/protein kinase A signaling pathway. Through this pathway, TSHR regulates the expression of sodium-iodide symporter (NIS) to complete iodine intake. In recent studies, it is found that TSHR is widely expressed in a variety of extra-thyroidal tissues. TSHR expressions as well as distribution in normal mammary gland tissues have not been reported. The physiological mechanism of the TSHR in the extra-thyroidal tissues has also been controversial. In this study, immunohistochemistry and immunofluorescence were used to characterize the expression distribution of TSHR protein in lactating breast. DNA sequence of TSHR cDNA from mice lactating breast was determined and then compared with TSHR cDNA from mice thyroidal tissue. A 173 amino acid (AA) fragment deletion was found in the extra-cellular domain of lactating breast TSHR. The expression levels of NIS mRNA were compared between two tissues, and the level of NIS mRNA in lactating breasts was lower than the one in thyroidal tissues. The lower expression of NIS in lactating breast may be due to the 173 AA deletion in the TSHR resulting the lower binding of TSH to the TSHR. For the first time, this finding may explain the reason of the lower NIS expression in lactating breast.

Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery

Adoptively transferred mesenchymal stem cells (MSCs) home to solid tumors. Biologic features within the tumor environment can be used to selectively activate transgenes in engineered MSCs after tumor invasion. One of the characteristic features of solid tumors is hypoxia. We evaluated a hypoxia-based imaging and therapy strategy to target expression of the sodium iodide symporter (NIS) gene to experimental hepatocellular carcinoma (HCC) delivered by MSCs.MSCs engineered to express transgenes driven by a hypoxia-responsive promoter showed robust transgene induction under hypoxia as demonstrated by mCherry expression in tumor cell spheroid models, or radioiodide uptake using NIS. Subcutaneous and orthotopic HCC xenograft mouse models revealed significant levels of perchlorate-sensitive NIS-mediated tumoral radioiodide accumulation by tumor-recruited MSCs using 123I-scintigraphy or 124I-positron emission tomography. Functional NIS expression was further confirmed by ex vivo 123I-biodistribution analysis. Administration of a therapeutic dose of 131I in mice treated with NIS-transfected MSCs resulted in delayed tumor growth and reduced tumor perfusion, as shown by contrast-enhanced sonography, and significantly prolonged survival of mice bearing orthotopic HCC tumors. Interestingly, radioiodide uptake into subcutaneous tumors was not sufficient to induce therapeutic effects. Our results demonstrate the potential of using tumor hypoxia-based approaches to drive radioiodide therapy in non-thyroidal tumors.

Abstract 2879: PIGU modulates radioactive iodine uptake in differentiated thyroid cancers

Abstract Radioactive iodine (RAI) treatment has a significant role in the management of differentiated thyroid carcinoma after surgical treatment. Although there is significant variability between patients, in response to RAI treatment, the mechanism responsible for this phenomenon is unknown. PIGU is an endoplasmic reticulum protein and a component of the GPIT complex, which plays a crucial role in post-translational modification of proteins such as sodium iodide symporter (NIS). Immunohistochemical analysis revealed significantly lower expression of PIGU in papillary carcinomas compared to matched normal thyroid tissue (P&amp;lt;0.001). To study the role of PIGU in RAI uptake of differentiated thyroid cancer, we stably expressed PIGU and mock plasmid in the papillary cancer cell line (K1-PIGU and K1, respectively). Immunofluorescence analysis showed a significant increase in perinuclear expression of PIGU in the K1-PIGU cancer cells compared to controls. Similarly, flow cytometry analysis showed a significant increase in membranous expression of NIS in PIGU transfected cancer cells compared to controls. There were no significant differences in proliferation, invasion or motility between K1-PIGU and K1 cell lines. PIGU expression significantly increased I125 uptake compared to controls both in vitro (1.13 and 1030 ppm, respectively, p&amp;lt; .001) and in vivo (153 and 1.2×105 ppm, respectively, p&amp;lt; .001). Mitogen-activated protein kinase inhibition showed clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine. To address the mechanism underlying PIGU down regulation in RAI resistant tumors, we treated K1 and KI-PIGU cells with U0126 MEK inhibitor. This inhibition resulted in a significant increase in PIGU expression and I125 uptake in vitro. Taken together, our results revealed a pivotal role for PIGU in RAI sensitivity of thyroid cancer cells, by modulation of NIS expression. Citation Format: MORAN AMIT, shorook Na’ara, Tomer Charas, Ziv gil. PIGU modulates radioactive iodine uptake in differentiated thyroid cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2879.

Abstract 2315: Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer

Abstract Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant disease. However, efficacy of solid tumour CAR-T-immunotherapy has proven inconsistent. Limitations include poor T-cell trafficking to tumour deposits, insufficient T-cell longevity in-vivo and the occurrence of both predicted and unanticipated on-target and off-target toxicity. To refine this therapeutic approach, it is desirable to develop systems that allow the monitoring of T-cell location and persistence in-vivo. A retroviral vector named PiN-4 was constructed, which co-expresses: (i) an interleukin (IL)-4-responsive chimeric cytokine receptor (4αβ); (ii) a prostate-specific membrane antigen (PSMA)-targeted CAR (P28z) and (iii) hNIS, which promotes the uptake of technetium-99m pertechnetate (99mTcO4−) in viable cells. IL-4 enriched human 4P28zN+ T-cells were administered intravenously to Nod Scid Gamma (NSG) mice bearing prostate tumour xenografts. Measurement of the tumour xenografts by bioluminescent imaging (BLI) found that only PSMA-expressing tumours responded to 4P28zN+ T-cell treatment. Longitudinal SPECT-CT imaging further confirmed this with the preferential accumulation of 4P28zN+ T-cells in PSMA-expressing tumours compared to PSMA-negative tumours. Use of NIS as a T-cell imaging reporter brings several potential advantages. It promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4−). In addition, the ectopic expression of NIS is well tolerated by host cells and hNIS functions only in viable cells. These data demonstrate proof of concept for the utility of hNIS as an imaging reporter in genetically engineered T-cells. Citation Format: Nia Emami-Shahri, Julie Foster, Jane Sosabowski, John Maher, Sophie Papa. Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2315.

Regression of experimental NIS-expressing breast cancer brain metastases in response to radioiodide/gemcitabine dual therapy.

Treating breast cancer brain metastases (BCBMs) is challenging. Na+/I− symporter (NIS) expression in BCBMs would permit their selective targeting with radioiodide (131I−). We show impressive enhancement of tumor response by combining131I− with gemcitabine (GEM), a cytotoxic radiosensitizer. Nude mice mammary fat-pad (MFP) tumors and BCBMs were generated with braintropic MDA-MB-231Br cells transduced with bicistronically-linked NIS and firefly luciferase cDNAs. Response was monitored in vivo via bioluminescent imaging and NIS tumor expression.131I−/GEM therapy inhibited MFP tumor growth more effectively than either agent alone. BCBMs were treated with: high or low-dose GEM (58 or 14.5 mg/Kg×4); 131I− (1mCi or 2×0.5 mCi 7 days apart); and 131I−/GEM therapy. By post-injection day (PID) 25, 82-86% of controls and 78-83% of 131I−-treated BCBM grew, whereas 17% low-dose and 36% high-dose GEM regressed. The latter tumors were smaller than the controls with comparable NIS expression (~20% of cells). High and low-dose 131I−/GEM combinations caused 89% and 57% tumor regression, respectively. High-dose GEM/131I− delayed tumor growth: tumors increased 5-fold in size by PID45 (controls by PID18). Although fewer than 25% of cells expressed NIS, GEM/131I− caused dramatic tumor regression in NIS-transduced BCBMs. This effect was synergistic, and supports the hypothesis that GEM radiosensitizes cells to 131I−.

4 - Noninvasive Imaging of Cellular and Viral Therapies Using the NIS Reporter Gene

We are developing cellular therapies for a variety of applications and there is an urgent need to have convenient methods for noninvasive monitoring of these transplanted cells in preclinical animal models and in the patients enrolled in the clinical trials. NIS, the thyroidal sodium iodide symporter, is the reporter gene chosen for our virus, gene cell tracking applications. NIS is expressed endogenously in thyroid follicular cells where its role is to concentrate iodine for the synthesis of thyroid hormones. For more than 70 years, thyroidal NIS expression has been provided the basis for 123I gamma camera or SPECT/CT imaging in thyroid disorders. Using NIS imaging, we could monitor viral and cellular delivery to tumors or specific organs in the same animals serially over time out to 200 days. To determine the sensitivity of detection using our preclinical scanncer, MSC from rat, human and canine were collected from adipose tissues or the bone marrow, expanded and transduced with lentiviral vectors encoding the human or species specific NIS genes. NIS function in transduced MSC was first validated in vitro; NIS expressing MSC (MSC_NIS) from multiple species concentrated high levels of I-125 with no side effects. The sensitivity of cell detection was determined by transplanting a known number of MSC_NIS subcutaneously into mice. We can reliably detect 2x105 MSC_NIS in mice using the newly acquired U-SPECT II machine. Canine MSC derived from the bone marrow were surprisingly robust; viable cells were still detected (albeit lower numbers) at day 28 in the athymic mice. In contrast, NIS signals from adipose tissue derived rat or human MSC disappeared by day 7 post transplantation. Using PET imaging and F18-TFB, sensitivity of imaging could be significantly improved to detect lower numbers of cells. We are currently evaluating the use of NIS for tracking natural killer cells and transplanted hemapoietic stem cells and results will be presented.