Dual EGFR- and TfR-targeted gene transfer for sodium iodide symporter gene therapy of glioblastoma.

Sodium iodide symporter (NIS) gene transfer for active accumulation of iodide in tumor cells is a powerful theranostic strategy facilitating both diagnostic and therapeutic application of radioiodide. In glioblastoma (GBM), the blood-brain barrier (BBB) presents an additional delivery barrier for nucleic acid nanoparticles. In the present study, we designed dual-targeted NIS plasmid DNA complexes containing targeting ligands for the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR), thus providing the potential for active transport across the BBB followed by targeting of tumor cells. In vitro 125I transfection studies confirmed TfR- and EGFR-dependent transfection efficiency and NIS-specific iodide uptake of dual-targeted polyplexes. In vivo gene transfer in mice bearing orthotopic U87 GBM xenografts was assessed at 48 h after intravenous polyplex injection by positron emission tomography (PET) imaging using 18F-labeled tetrafluoroborate (TFB) as tracer. The tumoral 18F-TFB uptake of mice treated with dual-targeted polyplexes (0.56% ± 0.08% ID/mL) was significantly higher compared with mice treated with EGFR-mono-targeted (0.33% ± 0.03% ID/mL) or TfR-mono-targeted (0.27% ± 0.04% ID/mL) polyplexes. In therapy studies, application of 131I induced a superior therapeutic effect of the dual-targeted therapy, demonstrated by a significant delay in tumor growth and prolonged survival.

The sodium iodide symporter (NIS) as theranostic gene: its emerging role in new imaging modalities and non-viral gene therapy.

Cloning of the sodium iodide symporter (NIS) in 1996 has provided an opportunity to use NIS as a powerful theranostic transgene. Novel gene therapy strategies rely on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of therapeutic radionuclides. This review highlights the remarkable progress during the last two decades in the development of the NIS gene therapy concept using selective non-viral gene delivery vehicles including synthetic polyplexes and genetically engineered mesenchymal stem cells. In addition, NIS is a sensitive reporter gene and can be monitored by high resolution PET imaging using the radiotracers sodium [124I]iodide ([124I]NaI) or [18F]tetrafluoroborate ([18F]TFB). We performed a small preclinical PET imaging study comparing sodium [124I]iodide and in-house synthesized [18F]TFB in an orthotopic NIS-expressing glioblastoma model. The results demonstrated an improved image quality using [18F]TFB. Building upon these results, we will be able to expand the NIS gene therapy approach using non-viral gene delivery vehicles to target orthotopic tumor models with low volume disease, such as glioblastoma.Trial registration not applicable.

Selective sodium iodide symporter (NIS) genetherapy of glioblastoma mediatedby EGFR-targeted lipopolyplexes.

Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. 124I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of 131I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using 125I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG24-GE11/NIS or bisDBCO-PEG24-GE11/NIS, and 24-48 h later, 124I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for 131I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG24-GE11/NIS polyplexes 48 h before 124I application was characterized to be the optimal regime in the imaging studies and was therefore used for an 131I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma.

An unusual case of struma ovarii.

SUMMARY: Struma ovarii is a teratoma of the ovaries predominantly composed of thyroid tissue. Hyperthyroidism associated with struma ovarii is rare, occurring in approximately 8% of cases. Due to the rarity of struma ovarii, available data are limited to case reports and small case series.We report on a 61-year-old female patient with known Hashimoto's thyroiditis on levothyroxine replacement therapy for years with transition to clinical and biochemical hyperthyroidism despite antithyroid medication with carbimazole (10 mg/day), new diagnosis of urothelial carcinoma and an adnexal mass suspicious of ovarian cancer. The patient underwent resection of the adnexal mass and histopathology revealed a mature teratoma predominantly composed of thyroid tissue showing high levels of sodium iodide symporter protein expression. Following struma ovarii resection and disappearance of autonomous production of thyroid hormones, the patient developed hypothyroidism with severely decreased thyroid hormone levels fT4 and fT3 (fT4 0.4 ng/dL, reference interval 0.9-1.7 and fT3 < 1.0 pg/mL, reference interval 2.0-4.4). This has previously been masked by continued thyroid-stimulating hormone suppression due to long-term hyperthyroidism pre-surgery indicating secondary hypothyroidism, in addition to primary hypothyroidism based on the known co-existing chronic lymphocytic thyroiditis of the orthotopic thyroid gland. Levothyroxine administration was started immediately restoring euthyroidism.This case illustrates possible diagnostic pitfalls in a patient with two concurrent causes of abnormal thyroid function. LEARNING POINTS: Struma ovarii is an ovarian tumor containing either entirely or predominantly thyroid tissue and accounts for approximately 5% of all ovarian teratomas. In rare cases, both benign and malignant struma ovarii can secrete thyroid hormones, causing clinical and biochemical features of hyperthyroidism. Biochemical features of patients with struma ovarii and hyperthyroidism are similar to those of patients with primary hyperthyroidism. In such cases, thyroid scintigraphy should reveal low or absent radioiodine uptake in the thyroid gland, but the presence of radioiodine uptake in the pelvis in a whole body radioiodine scintigraphy. We give advice on possible diagnostic pitfalls in a case with two simultaneous causes of abnormal thyroid function due to the co-existence of struma ovarii.

Regional Hyperthermia Enhances Mesenchymal Stem Cell Recruitment to Tumor Stroma: Implications for Mesenchymal Stem Cell-Based Tumor Therapy.

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated in vivo in subcutaneous HuH7 mouse xenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6-48 h after or 24-48 h before hyperthermia treatment. Using 123I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of 123I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced by combining CMV-NIS-MSC-mediated 131I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.

Integrin αvß3-dependent thyroid hormone effects on tumour proliferation and vascularisation.

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvß3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvß3, in two murine xenograft tumour models with and without integrin αvß3 expression. Integrin αvß3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvß3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvß3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvß3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Effective control of tumor growth through spatial and temporal control of theranostic sodium iodide symporter (NIS) gene expression using a heat-inducible gene promoter in engineered mesenchymal stem cells.

Purpose: 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 gain local and temporal control of transgene expression, and thereby improve tumor selectivity, we engineered MSCs to express the NIS gene under control of a heat-inducible HSP70B promoter (HSP70B-NIS-MSCs). Experimental Design: NIS induction in heat-treated HSP70B-NIS-MSCs was verified by 125I uptake assay, RT-PCR, Western blot and immunofluorescence staining. HSP70B-NIS-MSCs were then injected i.v. into mice carrying subcutaneous hepatocellular carcinoma HuH7 xenografts, and hyperthermia (1 h at 41°C) was locally applied to the tumor. 0 - 72 h later radioiodine uptake was assessed by 123I-scintigraphy. The most effective uptake regime was then selected for 131I therapy. Results: The HSP70B promoter showed low basal activity in vitro and was significantly induced in response to heat. In vivo, the highest tumoral iodine accumulation was seen 12 h after application of hyperthermia. HSP70B-NIS-MSC-mediated 131I therapy combined with hyperthermia resulted in a significantly reduced tumor growth with prolonged survival as compared to control groups. Conclusions: The heat-inducible HSP70B promoter allows hyperthermia-induced spatial and temporal control of MSC-mediated theranostic NIS gene radiotherapy with efficient tumor-selective and temperature-dependent accumulation of radioiodine in heat-treated tumors.

Integrin αvß3-Mediated Effects of Thyroid Hormones on Mesenchymal Stem Cells in Tumor Angiogenesis.

Background: Several clinical and experimental studies have implicated thyroid hormones in cancer progression. Cancer-relevant effects, including stimulation of tumor growth and new blood vessel formation by angiogenesis, are thought to be mediated by a nonclassical signaling pathway initiated through integrin αvß3 expressed on cancer cells and proliferating endothelium. In an earlier study, we established mesenchymal stem cells (MSCs), important contributors to the fibrovascular network of tumors, as new thyroid hormone-dependent targets. Here, we evaluated the effects of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) versus Tetrac, an integrin-specific inhibitor of thyroid hormone action, on MSCs in tumor angiogenesis. Methods: Modulation of the expression and secretion of angiogenesis-relevant factors by thyroid hormones in primary human MSCs and their effect on endothelial cell tube formation were tested in vitro. We further engineered MSCs to express the sodium iodide symporter (NIS) reporter gene under control of a hypoxia-responsive promoter and the vascular endothelial growth factor (VEGF) promoter to test effects on these pathways in vitro and, for VEGF, in vivo in an orthotopic hepatocellular carcinoma (HCC) xenograft mouse model by positron emission tomography imaging. Results: T3 and T4 increased the expression of pro-angiogenic genes in MSCs and NIS-mediated radioiodide uptake in both NIS reporter MSC lines in the presence of HCC cell-conditioned medium. Supernatant from thyroid hormone-treated MSCs significantly enhanced endothelial cell tube formation. Tetrac and/or inhibitors of signaling pathways downstream of the integrin reversed all these effects. Tumoral radioiodide uptake in vivo demonstrated successful recruitment of MSCs to tumors and VEGF promoter-driven NIS expression. Hyperthyroid mice showed an increased radioiodide uptake compared with euthyroid mice, while tracer uptake was markedly reduced in hypothyroid and Tetrac-treated mice. Conclusions: Our data suggest that thyroid hormones influence angiogenic signaling in MSCs via integrin αvß3 and further substantiate the anti-angiogenic activity of Tetrac in the tumor microenvironment.

Dual-targeted NIS polyplexes-a theranostic strategy toward tumors with heterogeneous receptor expression.

Tumor heterogeneity, within and between tumors, may have severe implications for tumor therapy, especially for targeted gene therapy, where single-targeted approaches often result in limited efficacy and therapy resistance. Polymer-formulated nonviral vectors provide a potent delivery platform for cancer therapy. To improve applicability for future clinical use in a broad range of patients and cancer types, a dual-targeting approach was performed. Synthetic LPEI-PEG2kDa-based polymer backbones were coupled to two tumor-specific peptide ligands GE11 (EGFR-targeting) and cMBP (cMET-targeting). The dual-targeting approach was used to deliver the theranostic sodium iodide symporter (NIS) gene to hepatocellular cancer. NIS as auspicious theranostic gene allows noninvasive imaging of functional NIS gene expression and effective anticancer radioiodide therapy. Enhanced tumor-specific transduction efficiency of dual-targeted polyplexes compared to single-targeted polyplexes was demonstrated in vitro using tumor cell lines with different EGFR and cMET expression and in vivo by 124I-PET-imaging. Therapeutic efficacy of the bispecific concept was mirrored by significantly reduced tumor growth and perfusion, which was associated with prolonged animal survival. In conclusion, the dual-targeting approach highlights the benefits of a bifunctional strategy for a future clinical translation of the bioimaging-based NIS-mediated radiotherapy allowing efficient targeting of heterogeneic tumors with variable receptor expression levels.

TGFB1-driven mesenchymal stem cell-mediated NIS gene transfer.

Based on their excellent tumor-homing capacity, genetically engineered mesenchymal stem cells (MSCs) are under investigation as tumor-selective gene delivery vehicles. Transgenic expression of the sodium iodide symporter (NIS) in genetically engineered MSCs allows noninvasive tracking of MSC homing by imaging of functional NIS expression as well as therapeutic application of 131I. The use of tumor stroma-activated promoters can improve tumor-specific MSC-mediated transgene delivery. The essential role of transforming growth factor B1 (TGFB1) and the SMAD downstream target in the signaling between tumor and the surrounding stroma makes the biology of this pathway a potential option to better control NIS expression within the tumor milieu. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by a synthetic SMAD-responsive promoter (SMAD-NIS-MSCs). Radioiodide uptake assays revealed a 4.9-fold increase in NIS-mediated perchlorate-sensitive iodide uptake in SMAD-NIS-MSCs after TGFB1 stimulation compared to unstimulated cells demonstrating the successful establishment of MSCs, which induce NIS expression in response to activation of TGFB1 signaling using a SMAD-responsive promoter. 123I-scintigraphy revealed significant tumor-specific radioiodide accumulation and thus NIS expression after systemic application of SMAD-NIS-MSCs into mice harboring subcutaneous tumors derived from the human hepatocellular carcinoma (HCC) cell line HuH7, which express TGFB1. 131I therapy in SMAD-NIS-MSCs-treated mice demonstrated a significant delay in tumor growth and prolonged survival. Making use of the tumoral TGFB1 signaling network in the context of MSC-mediated NIS gene delivery is a promising approach to foster tumor stroma-selectivity of NIS transgene expression and tailor NIS-based gene therapy to TGFB1-rich tumor environments.

A Novel Approach for Image-Guided 131I Therapy of Pancreatic Ductal Adenocarcinoma Using Mesenchymal Stem Cell-Mediated NIS Gene Delivery.

The sodium iodide symporter (SLC5A5/NIS) as theranostic gene would allow for non-invasive imaging of functional NIS expression and therapeutic radioiodine application. Genetically engineered mesenchymal stem cells (MSC), based on their tumor-homing abilities, show great promise as tumor-selective NIS gene delivery vehicles for non-thyroidal tumors. As a next step towards clinical application, tumor specificity and efficacy of MSCs were investigated in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). Syngeneic murine MSCs were stably transfected with a NIS-expressing plasmid driven by the CMV-promoter (NIS-MSC). In vivo 123I-scintigraphy and 124I-PET revealed significant perchlorate-sensitive NIS-mediated radioiodide accumulation in PDAC after systemic injection of NIS-MSCs. Active MSC recruitment into the tumor stroma was confirmed using NIS immunohistochemistry (IHC). A therapeutic strategy, consisting of three cycles of systemic MSC-mediated NIS delivery, followed by 131I application, resulted in a significant delay and reduction in tumor growth as compared to controls. Furthermore, IHC analysis of α-SMA and Ki67 revealed differences in the amount and behavior of activated fibroblasts in tumors of mice injected with NIS-MSCs as compared with saline-treated mice. Taken together, MSCs as NIS gene delivery vehicles in this advanced endogenous PDAC mouse model demonstrated high stromal targeting of NIS by selective recruitment of NIS-MSCs after systemic application resulting in an impressive 131I therapeutic effect. IMPLICATIONS: These data expand the prospect of MSC-mediated radioiodine imaging-guided therapy of pancreatic cancer using the sodium iodide symporter as a theranostic gene in a clinical setting.

External Beam Radiation Therapy Enhances Mesenchymal Stem Cell-Mediated Sodium-Iodide Symporter Gene Delivery.

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium-iodide symporter (NIS) to solid tumors. External beam radiation therapy may represent an ideal setting for the application of engineered MSC-based gene therapy, as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7; 1-10 Gy) showed a strong dose-dependent increase in steady-state mRNA levels of CXCL8, CXCL12, FGF2, PDGFB, TGFB1, THBS1, and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration were tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index, mean center of mass, and mean directionality of MSCs toward supernatants was seen from irradiated as compared to non-irradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in quantitative polymerase chain reaction and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2, or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using 123I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. The results demonstrate that external beam radiation therapy enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

EGFR-targeted nonviral NIS gene transfer for bioimaging and therapy of disseminated colon cancer metastases.

Liver metastases present a serious problem in the therapy of advanced colorectal cancer (CRC), as more than 20% of patients have distant metastases at the time of diagnosis with less than 5% being cured. Consequently, new therapeutic approaches are of major need together with high-resolution imaging methods that allow highly specific detection of small metastases. The unique combination of reporter and therapy gene function of the sodium iodide symporter (NIS) may represent a promising theranostic strategy for CRC liver metastases allowing non-invasive imaging of functional NIS expression and therapeutic application of 131I. For targeted NIS gene transfer polymers containing linear polyethylenimine (LPEI), polyethylene glycol (PEG) and the epidermal growth factor receptor (EGFR)-specific ligand GE11 were complexed with human NIS DNA (LPEI-PEG-GE11/NIS). Tumor specificity and transduction efficiency were examined in high EGFR-expressing LS174T metastases by non-invasive imaging using 18F-tetrafluoroborate (18F-TFB) as novel NIS PET tracer. Mice that were injected with LPEI-PEG-GE11/NIS 48 h before 18F-TFB application showed high tumoral levels (4.8±0.6% of injected dose) of NIS-mediated radionuclide uptake in comparison to low levels detected in mice that received untargeted control polyplexes. Three cycles of intravenous injection of EGFR-targeted NIS polyplexes followed by therapeutic application of 55.5 MBq 131I resulted in marked delay in metastases spread, which was associated with improved animal survival. In conclusion, these preclinical data confirm the enormous potential of EGFR-targeted synthetic polymers for systemic NIS gene delivery in an advanced multifocal CRC liver metastases model and open the exciting prospect of NIS-mediated radionuclide therapy in metastatic disease.

Reintroducing the Sodium-Iodide Symporter to Anaplastic Thyroid Carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC), the most aggressive form of thyroid cancer, is unresponsive to radioiodine therapy. The current study aimed to extend the diagnostic and therapeutic application of radioiodine beyond the treatment of differentiated thyroid cancer by targeting the functional sodium-iodide symporter (NIS) to ATC. METHODS: The study employed nanoparticle vectors (polyplexes) based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG) and coupled to the synthetic peptide GE11 as an epidermal growth factor receptor (EGFR)-specific ligand in order to target a NIS-expressing plasmid (LPEI-PEG-GE11/NIS) to EGFR overexpressing human thyroid carcinoma cell lines. Using ATC xenograft mouse models, transfection efficiency by 123I scintigraphy and potential for systemic radioiodine therapy after systemic polyplex application were evaluated. RESULTS: In vitro iodide uptake studies in SW1736 and Hth74 ATC cells, and, for comparison, in more differentiated follicular (FTC-133) and papillary (BCPAP) thyroid carcinoma cells demonstrated high transfection efficiency and EGFR-specificity of LPEI-PEG-GE11/NIS that correlated well with EGFR expression levels. After systemic polyplex injection, in vivo 123I gamma camera imaging revealed significant tumor-specific accumulation of radioiodine in an SW1736 and an Hth74 xenograft mouse model. Radioiodine accumulation was found to be higher in SW1736 tumors, reflecting in vitro results, EGFR expression levels, and results from ex vivo analysis of NIS staining. Administration of 131I in LPEI-PEG-GE11/NIS-treated SW1736 xenograft mice resulted in significantly reduced tumor growth associated with prolonged survival compared to control animals. CONCLUSIONS: The data open the exciting prospect of NIS-mediated radionuclide imaging and therapy of ATC after non-viral reintroduction of the NIS gene. The high tumor specificity after systemic application makes the strategy an attractive alternative for the treatment of highly metastatic ATC.

Systemic tumor-targeted sodium iodide symporter (NIS) gene therapy of hepatocellular carcinoma mediated by B6 peptide polyplexes.

BACKGROUND: Nonviral polymer-based gene transfer represents an adaptable system for tumor-targeted gene therapy because various design strategies of shuttle systems, together with the mechanistic concept of active tumor targeting, lead to improved gene delivery vectors resulting in higher tumor specificity, efficacy and safety. METHODS: Using the sodium iodide symporter (NIS) as a theranostic gene, nonviral gene delivery vehicles based on linear polyethylenimine (LPEI), polyethylene glycol (PEG) and coupled to the synthetic peptide B6 (LPEI-PEG-B6), which specifically binds to tumor cells, were investigated in a hepatocellular carcinoma xenograft model for tumor selectivity and transduction efficiency. RESULTS: In vitro incubation of three different tumor cell lines with LPEI-PEG-B6/NIS resulted in significant increase in iodide uptake activity compared to untargeted and empty vectors. After establishment of subcutaneous HuH7 tumors, NIS-conjugated nanoparticles were injected intravenously followed by analysis of radioiodide biodistribution using 123 I-scintigraphy showing significant perchlorate-sensitive iodide accumulation in tumors of LPEI-PEG-B6/NIS-treated mice (8.0 ± 1.5% ID/g 123 I; biological half-life of 4 h). After four cycles of repetitive polyplex/131 I applications, a significant delay of tumor growth was observed, which was associated with markedly improved survival in the therapy group. CONCLUSIONS: These results clearly demonstrate that systemic in vivo NIS gene transfer using nanoparticle vectors coupled to B6 tumor targeting ligand is capable of inducing tumor-specific radioiodide uptake. This promising gene therapy approach opens the exciting prospect of NIS-mediated radionuclide therapy in metastatic cancer, together with the possibility of combining several targeting ligands to enhance selective therapeutic efficacy in a broad field of cancer types with various receptor expression profiles.

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.

Establishment of an Effective Radioiodide Thyroid Ablation Protocol in Mice.

Due to the high variance in available protocols on iodide-131 ((131)I) ablation in rodents, we set out to establish an effective method to generate a thyroid-ablated mouse model that allows the application of the sodium iodide symporter (NIS) as a reporter gene without interference with thyroidal NIS. We tested a range of (131)I doses with and without prestimulation of thyroidal radioiodide uptake by a low-iodine diet and thyroid-stimulating hormone (TSH) application. Efficacy of induction of hypothyroidism was tested by measurement of serum T4 concentrations, pituitary TSHß and liver deiodinase type 1 (DIO1) mRNA expression, body weight analysis, and (99m)Tc-pertechnetate scintigraphy. While 200 µCi (7.4 MBq) (131)I alone was not sufficient to abolish thyroidal T4 production, 500 µCi (18.5 MBq) (131)I combined with 1 week of a low-iodine diet decreased serum concentrations below the detection limit. However, the high (131)I dose resulted in severe side effects. A combination of 1 week of a low-iodine diet followed by injection of bovine TSH before the application of 150 µCi (5.5 MBq) (131)I decreased serum T4 concentrations below the detection limit and significantly increased pituitary TSHß concentrations. The systemic effects of induced hypothyroidism were shown by growth arrest and a decrease in liver DIO1 expression below the detection limit. (99m)Tc-pertechnetate scintigraphy revealed absence of thyroidal (99m)Tc-pertechnetate uptake in ablated mice. In summary, we report a revised protocol for radioiodide ablation of the thyroid gland in the mouse to generate an in vivo model that allows the study of thyroid hormone action using NIS as a reporter gene.

Thyroid hormones and tetrac: new regulators of tumour stroma formation via integrin αvß3.

To improve our understanding of non-genomic, integrin αvß3-mediated thyroid hormone action in tumour stroma formation, we examined the effects of triiodo-l-thyronine (T3), l-thyroxine (T4) and integrin-specific inhibitor tetrac on differentiation, migration and invasion of mesenchymal stem cells (MSCs) that are an integral part of the tumour's fibrovascular network. Primary human bone marrow-derived MSCs were treated with T3 or T4 in the presence of hepatocellular carcinoma (HCC) cell-conditioned medium (CM), which resulted in stimulation of the expression of genes associated with cancer-associated fibroblast-like differentiation as determined by qPCR and ELISA. In addition, T3 and T4 increased migration of MSCs towards HCC cell-CM and invasion into the centre of three-dimensional HCC cell spheroids. All these effects were tetrac-dependent and therefore integrin αvß3-mediated. In a subcutaneous HCC xenograft model, MSCs showed significantly increased recruitment and invasion into tumours of hyperthyroid mice compared to euthyroid and, in particular, hypothyroid mice, while treatment with tetrac almost completely eliminated MSC recruitment. These studies significantly improve our understanding of the anti-tumour activity of tetrac, as well as the mechanisms that regulate MSC differentiation and recruitment in the context of tumour stroma formation, as an important prerequisite for the utilisation of MSCs as gene delivery vehicles.

Mesenchymal stem cell-mediated, tumor stroma-targeted radioiodine therapy of metastatic colon cancer using the sodium iodide symporter as theranostic gene.

UNLABELLED: The tumor-homing property of mesenchymal stem cells (MSCs) allows targeted delivery of therapeutic genes into the tumor microenvironment. The application of sodium iodide symporter (NIS) as a theranostic gene allows noninvasive imaging of MSC biodistribution and transgene expression before therapeutic radioiodine application. We have previously shown that linking therapeutic transgene expression to induction of the chemokine CCL5/RANTES allows a more focused expression within primary tumors, as the adoptively transferred MSC develop carcinoma-associated fibroblast-like characteristics. Although RANTES/CCL5-NIS targeting has shown efficacy in the treatment of primary tumors, it was not clear if it would also be effective in controlling the growth of metastatic disease. METHODS: To expand the potential range of tumor targets, we investigated the biodistribution and tumor recruitment of MSCs transfected with NIS under control of the RANTES/CCL5 promoter (RANTES-NIS-MSC) in a colon cancer liver metastasis mouse model established by intrasplenic injection of the human colon cancer cell line LS174t. RANTES-NIS-MSCs were injected intravenously, followed by (123)I scintigraphy, (124)I PET imaging, and (131)I therapy. RESULTS: Results show robust MSC recruitment with RANTES/CCL5-promoter activation within the stroma of liver metastases as evidenced by tumor-selective iodide accumulation, immunohistochemistry, and real-time polymerase chain reaction. Therapeutic application of (131)I in RANTES-NIS-MSC-treated mice resulted in a significant delay in tumor growth and improved overall survival. CONCLUSION: This novel gene therapy approach opens the prospect of NIS-mediated radionuclide therapy of metastatic cancer after MSC-mediated gene delivery.

EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene.

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by (123)I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. (124)I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of (131)I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e131; doi:10.1038/mtna.2013.58; published online 5 November 2013.