The sodium iodide symporter (NIS): novel applications for radionuclide imaging and treatment

Cloning of the sodium iodide symporter (NIS) 25 years ago has opened an exciting chapter in molecular thyroidology with the characterization of NIS as one of the most powerful theranostic genes and the development of a promising gene therapy strategy based on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of 131I or alternative radionuclides, such as 188Re and 211At. Over the past 2 decades significant progress has been made in the development of the NIS gene therapy concept, from local NIS gene delivery, towards promising new applications in disseminated disease, in particular, through the use of oncolytic viruses, non-viral polyplexes, and genetically engineered MSCs as highly effective, highly selective and flexible gene delivery vehicles. In addition to allowing the robust therapeutic application of radioiodine in non-thyroid cancer settings, these studies have also been able to take advantage of NIS as a sensitive reporter gene that allows temporal and spatial monitoring of vector biodistribution, replication, and elimination - critically important issues for preclinical development and clinical translation.

Taking Advantage of the TGFB1 Biology in Differentiated Thyroid Cancer to Stimulate Sodium Iodide Symporter (NIS)-Mediated Iodide Uptake in Engineered Mesenchymal Stem Cells

The sodium iodide symporter (NIS) mediates the active transport of iodide into thyroid follicular cells, providing the basis for the use of radioiodide for diagnostic imaging and therapy of differentiated thyroid cancer and also non-thyroidal tumors after tumor-selective NIS gene transfer. Based on their excellent tumor-homing capacity, mesenchymal stem cells (MSCs) can be employed as tumor-selective NIS gene delivery vehicles. Transgenic expression of NIS in genetically engineered MSCs allows noninvasive imaging of functional NIS expression as well as therapeutic application of 131I. The use of promoters activated by tumor micromilieu-derived signals to drive NIS expression enhances selectivity and effectiveness, while limiting potential off-target effects. In this study we aimed to exploit the central role of transforming growth factor B1 (TGFB1) in tumor milieu-associated signaling using a TGFB1-inducible synthetic SMAD-responsive promoter to selectively drive NIS-transgene expression in engineered MSCs (SMAD-NIS-MSC) in the context of differentiated thyroid cancer based on the critical role of TGFB1 in the pathogenesis of radioiodine refractory differentiated thyroid cancer. To evaluate the TGFB1 expression in thyroid cancer cell lines, the TGFB1 concentration in conditioned medium (CM) from an array of established human papillary thyroid cancer (PTC) cell lines (BCPAP and K1) was measured by ELISA. BCPAP-CM showed a higher concentration of TGFB1, while a lower concentration was measured in K1-CM. Stimulation of SMAD-NIS-MSCs with PTC-CM showed a significant increase of NIS-mediated radioiodide-125 uptake in these MSCs in vitro. In addition, iodide uptake in SMAD-NIS-MSCs was significantly stimulated by co-culture with thyroid cancer cells. Cell migration assay was performed to validate the effect of PTC-CM in MSC recruitment. MSCs subjected to a gradient between tumor CM and serum free medium showed a directed chemotaxis towards CM with increased forward migration index (FMI) and center of mass (CoM). In a next step, based on the in vitro studies, SMAD-NIS-MSCs will be systemically applied via the tail vein to mice harboring subcutaneous PTC tumors and tumoral iodide uptake will be monitored by 123I-scintigraphy. Taken together, these data indicate the feasibility of commandeering TGF-β/SMAD signaling in the TGFB1-rich tumor environments of radioiodine refractory differentiated thyroid carcinomas to re-establish functional NIS expression using engineered mesenchymal stem cells as therapy vehicles.

SUN-120 Regional Hyperthermia Enhances Selective Mesenchymal Stem Cell Migration Towards the Tumor Stroma

The tumor homing characteristics of mesenchymal stem cells (MSCs) make them attractive vehicles for the tumor-specific delivery of therapeutic agents, such as the sodium iodide symporter (NIS). NIS is a theranostic protein that allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression by radioiodine imaging as well as the therapeutic application of 131I. To enhance the actively recruitment of MSCs to growing tumor stroma and thereby trigger targeted delivery of the NIS gene to the tumor, we examined the combination with regional hyperthermia, as heat induces the secretion of immunomodulatory chemokines, cytokines and growth factors, well-known attractants of MSCs. Human hepatocellular carcinoma cells (HuH7) were heat-treated in a water bath at 41 °C for 1h, followed by incubation at 37 °C for 0-48h. mRNA and protein levels of chemokines involved in MSC migration was analyzed by RT-PCR and ELISA. Chemotaxis of MSCs in relation to a gradient of supernatants was tested in a 3D live cell tracking migration assay. In a subcutaneous HuH7 mouse xenograft tumor model, a single systemic injection of CMV-NIS-MSCs was applied 6h, 24h, 48h after or 24h, 48h before hyperthermia treatment and tumoral 123I accumulation was assessed by 123I-scintigraphy. Ex vivo NIS analysis of tumor sections was performed by RT-PCR and immunohistochemistry. The optimal imaging regime was then used for a 131I therapy study. Chemokine mRNA and protein analysis indicated a substantial increase in expression levels of chemokines and growth factors, involved in MSC tumor homing, after heat exposure. In addition, MSCs showed directed migration towards the supernatant of thermo-stimulated cancer cells. In vivo, with the optimal regime, we observed a significantly increased uptake of 123I in tumors of heat-treated animals (41 °C) when thermostimulated 24h after CMV-NIS-MSC injection compared to control animals (37 °C). Immunohistochemical staining of tumor sections showed strong tumoral NIS-specific immunoreactivity and RT-PCR an increased NIS mRNA expression in heat-treated tumors, thereby confirming tumor-selective, temperature-dependent MSC migration. CMV-NIS-MSC-mediated 131I therapy combined with regional hyperthermia resulted in a reduced tumor growth that was associated with prolonged survival of regional heat-treated animals compared to normothermic mice and to the saline control group. In summary, we have demonstrated a significantly increased, selective MSC migration towards the tumor stroma after regional hyperthermia in the 123I imaging study. The combination of MSC-mediated NIS gene therapy with mild regional hyperthermia resulting in stimulated therapeutic efficacy of NIS-mediated 131I therapy.

MON-512 The Anti-Cancer Agent, Homoharringtonine, Induces the Sodium Iodide Symporter (NIS) Gene Expression in Culture Cells from Papillary Thyroid Cancer, as Well as Non-Thyroid Cancers

BACKGROUND: The therapeutic effect of thyroid cancer by radioiodide treatment is dependent on enhancement of NIS expression by TSH, especially its much greater magnitude in target tissue(s) compared to other healthy tissues. We preliminarily found that a protein synthesis inhibitor cycloheximide (CHX) markedly enhanced NIS mRNA expression, followed by increased iodide uptake, in several cancer cell lines, though CHX is highly toxic for clinical use. Aims: To evaluate the possibility of clinical application of such a pathway to enhance NIS expression, we tried another weak protein synthesis inhibitor, homoharringtonine (HHT), a natural plant alkaloid already utilized as an anti-leukemia agent, in several human cancer cell lines, including thyroid cancer. Methods: BHP 2-7 papillary thyroid cancer cells, MCF7 breast cancer cells, and MKN gastric cancer cells were treated with HHT and/or a p38 inhibitor, and harvested for quantitative RT-PCR of NIS. Results: HHT significantly induced the NIS mRNA expression in all of the cell lines tested, up to 298-fold in BHP cells, 38-fold in MCF7 cells, and 235-fold in MKN cells. Time course experiments indicated a biphasic induction of NIS in BHP cells with two peaks at 48 hours and 96 hours, with the EC50 of 664 ng/mL and 767 ng/mL, respectively. In contrast, NIS induction by HHT was monophasic in MCF7 cells at 24 hours with EC50 of 24.6 ng/mL, as well as MKN cells at 96 hours with EC50 of 255.7 ng/mL. Roles of p38 MAPK in the NIS induction has been reported previously, however, p38 inhibitors, SB239063 (10μM), as well as ML3403 (30μM), did not significantly reduce the NIS expression in HHT-treated BHP cells. Conclusion: These results indicated that HHT has a potential to enhance NIS expression in some NIS-expressing cancer tissues, including papillary thyroid cancer, although its functionality and efficacy are to be validated. The heterogeneity of response in the NIS expression to HHT in three cell lines could be due to differential mechanisms of NIS gene regulation in different tissues.

Cell non-autonomous interactions during non-immune stromal progression in the breast tumor microenvironment

SummaryThe breast tumor microenvironment of primary and metastatic sites is a complex milieu of differing cell populations, consisting of tumor cells and the surrounding stroma. Despite recent progress in delineating the immune component of the stroma, the genomic expression landscape of the non-immune stroma (NIS) population and their role in mediating cancer progression and informing effective therapies are not well understood. Here we obtained 52 cell-sorted NIS and epithelial tissue samples across 37 patients from i) normal breast, ii) normal breast adjacent to primary tumor, iii) primary tumor, and iv) metastatic tumor sites. Deep RNA-seq revealed diverging gene expression profiles as the NIS evolves from normal to metastatic tumor tissue, with intra-patient normal-primary variation comparable to inter-patient variation. Significant expression changes between normal and adjacent normal tissue support the notion of a cancer field effect, but extended out to the NIS. Most differentially expressed protein-coding genes and lncRNAs were found to be associated with pattern formation, embryogenesis, and the epithelial-mesenchymal transition. We validated the protein expression changes of a novel candidate gene, C2orf88, by immunohistochemistry staining of representative tissues. Significant mutual information between epithelial ligand and NIS receptor gene expression, across primary and metastatic tissue, suggests a unidirectional model of molecular signaling between the two tissues. Furthermore, survival analyses of 827 luminal breast tumor samples demonstrated the predictive power of the NIS gene expression to inform clinical outcomes. Together, these results highlight the evolution of NIS gene expression in breast tumors and suggest novel therapeutic strategies targeting the microenvironment.