Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of cotransfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Cotransfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.
Malignant glioma; sodium iodide symporter; hTERT promoter; tumor imaging
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.
human sodium iodide symporter (hNIS); radioiodine therapy; codon optimization; reporter; gene therapy.
Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers may be treated with radio-iodine following transfection with the human sodium/iodide symporter (hNIS) gene. The glial fibrillary acidic protein (GFAP) promoter is an effective tumor-specific promoter for gene expression and thus may be useful in targeted gene therapy of malignant glioma. The present study used GFAP promoter-modulated expression of the hNIS gene in an experimental model of radioiodine-based treatment for malignant glioma. Cells were transfected using a recombination adeno-virus and evaluated in cells by studying the transfected transgene expression through western blot analysis, 125I uptake and efflux, clonogenicity following 131I treatment and radioiodine therapy using a U87 xenograft nude mouse model. Following transfection with the hNIS gene, the cells showed 95–70-fold higher 125I uptake compared with the control cells transfected with Ad-cytomegalovirus (CMV)-enhanced green fluorescent protein (EGFP). The western blotting revealed bands of ∼70, 49 and 43 kDa, consistent with the hNIS, GFAP and β-actin proteins. The clonogenic assay indicated that, following exposure to 500 μCi of 131I-iodide for 12 h, >90% of cells transfected with the hNIS gene were killed. Ad-GFAP-hNIS-transfected and 2 mCi 131I-injected U87 xenograft nude mice survived the longest of the three groups. The hNIS-expressing tumor tissue accumulated 99mTcO4 rapidly within 30 min of it being intraperitoneally injected. The experiments demonstrated that effective 131I therapy was achieved in the malignant glioma cell lines following the induction of tumor-specific iodide uptake activity by GFAP promoter-directed hNIS gene expression in vitro and in vivo. 131I therapy retarded Ad-GFAP-hNIS transfected-tumor growth following injection with 131I in U87 xenograft-bearing nude mice.
malignant glioma; sodium iodide symporter; glial fibrillary acidic protein promoter; radioiodine therapy
Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of the thyroid-specific expression of the human sodium iodide symporter (hNIS). Here, we explore the efficacy of a novel form of gene therapy using prostate-specific membrane antigen (PSMA) promoter-mediated hNIS gene transfer followed by radioiodine administration for the treatment of castration-resistant prostate cancer (CRPC). The androgen-dependent C33 LNCaP cell line and the androgen-independent C81 LNCaP cell line were transfected by adenovirus. PSMA promoter-hNIS (Ad.PSMApro-hNIS) or adenovirus.cytomegalovirus–hNIS containing the cytomegalovirus promoter (Ad.CMV-hNIS) or a control virus. The iodide uptake was measured in vitro. The in vivo iodide uptake by C81 cell xenografts in nude mice injected with an adenovirus carrying the hNIS gene linked to PSMA and the corresponding tumor volume fluctuation were assessed. Iodide accumulation was shown in different LNCaP cell lines after Ad.PSMApro-hNIS and Ad.CMV-hNIS infection, but not in different LNCaP cell lines after adenovirus.cytomegalovirus (Ad.CMV) infection. At each time point, higher iodide uptake was shown in the C81 cells infected with Ad.PSMApro-hNIS than in the C33 cells (P < 0.05). An in vivo animal model showed a significant difference in 131I radioiodine uptake in the tumors infected with Ad.PSMApro-hNIS, Ad.CMV-hNIS and control virus (P < 0.05) and a maximum reduction of tumor volume in mice infected with Ad.PSMApro-hNIS. These results show prostate-specific expression of the hNIS gene delivered by the PSMA promoter and effective radioiodine therapy of CRPC by the PSMA promoter-driven hNIS transfection.
genetic therapy; prostate-specific membrane antigen (PSMA); prostatic neoplasms; sodium-iodide symporter
To investigate the role of 188Re in human sodium iodide symporter (hNIS) theranostic gene-mediated human glioma imaging and therapy in model mice.
The human glioma cell line U87 was transfected with recombinant lentivirus encoding the hNIS gene under the control of cytomegalovirus promoter (U87-hNIS). The uptake and efflux of 188Re were determined after incubating the cells with 188Re. 188Re uptake experiments in the presence of various concentrations of sodium perchlorate were carried out. In vitro cell killing tests with 188Re were performed. U87-hNIS mediated 188Re distribution, imaging and therapy in nude mice were also tested.
U87-hNIS cell line was successfully established. The uptake of 188Re in U87-hNIS cells increased up to 26-fold compared to control cells, but was released rapidly with a half-life of approximately 4 minutes. Sodium perchlorate reduced hNIS-mediated 188Re uptake to levels of control cell lines. U87-hNIS cells were selectively killed following exposure to 188Re, with a survival of 21.4%, while control cells had a survival of 92.1%. Unlike in vitro studies, U87-hNIS tumor showed a markedly increased 188Re retention even 48 hours after 188Re injection. In the therapy study, there was a significant difference in tumor size between U87-hNIS mice (317±67 mm3) and control mice (861±153 mm3) treated with 188Re for 4 weeks (P<0.01).
The results indicate that inserting the hNIS gene into U87 cells is sufficient to induce specific 188Re uptake, which has a cell killing effect both in vitro and in vivo. Moreover, our study, based on the function of hNIS as a theranostic gene allowing noninvasive imaging of hNIS expression by 188Re scintigraphy, provides detailed characterization of in vivo vector biodistribution and level, localization, essential prerequisites for precise planning and monitoring of clinical gene therapy that aims to individualize gene therapy concept.
Using an adenoviral system as a delivery mediator of therapeutic gene, we investigated the therapeutic effects of the use of combined MDR1 shRNA and human NIS (hNIS) radioiodine gene therapy in a mouse colon xenograft model. In vitro uptake of Tc-99m sestamibi was increased approximately two-fold in cells infected with an adenovirus vector that expressed MDR1 shRNA (Ad-shMDR1) and I-125 uptake was 25-fold higher in cells infected with an adenovirus vector that expressed human NIS (Ad-hNIS) as compared with control cells. As compared with doxorubicin or I-131 treatment alone, the combination of doxorubicin and I-131 resulted in enhanced cytotoxicity for both Ad-shMDR1- and Ad-hNIS-infected cells, but not for control cells. In vivo uptake of Tc-99m sestamibi and Tc-99m pertechnetate was twofold and 10-fold higher for Ad-shMDR1 and Ad-hNIS-infected tumors as compared with tumors infected with a control adenovirus construct that expressed β-galactrosidase (Ad-LacZ), respectively. In mice treated with either doxorubicin or I-131 alone, there was a slight delay in tumor growth as compared to mice treated with Ad-LacZ. However, combination therapy with doxorubicin and I-131 induced further significant inhibition of tumor growth as compared with mice treated with Ad-LacZ. We have shown successful therapeutic efficacy of combined MDR shRNA and hNIS radioiodine gene therapy using an adenoviral vector system in a mouse colon cancer model. Adenovirus-mediated cancer gene therapy using MDR1 shRNA and hNIS would be a useful tool for the treatment of cancer cells expressing multi-drug resistant genes.
MDR1; shRNA; hNIS; Radioiodine gene therapy; imaging
Oncolytic adenoviruses can be engineered for better tumor selectivity, gene delivery and be armed for imaging and concentrating radionuclides into tumors for synergistic oncolysis. We constructed Ad5/3-hTERT-hNIS where replication is controlled by hTERT-promoter. Ad5/3-hTERT-hNIS expresses hNIS for imaging of transgene expression and for treatment of infected tumors by radioiodine. Ad5/3-hTERT-hNIS efficiently killed prostate cancer cells and induced iodine uptake in vitro and in vivo after intratumoral virus administration. Survival of mice treated with intravenous Ad5/3-hTERT-hNIS significantly prolonged survival over mock or radioiodine only but the combination of virus with radioiodine was not more effective than virus alone. Temporal and spatial changes in hNIS-expression during therapy were detected with SPECT, demonstrating feasibility of evaluation of the combination therapy with hNIS-expressing adenoviruses and radioiodide.
The heat shock protein 90 inhibitor, tanespimycin, is an anticancer agent known to increase iodine accumulation in normal and cancerous thyroid cells. Iodine accumulation is regulated by membrane proteins such as sodium iodide symporter (NIS) and pendrin (PDS), and thus we attempted to characterize the effects of tanespimycin on those genes.
Cells were incubated with tanespimycin in order to evaluate 125I accumulation and efflux ability. Radioiodine uptake and efflux were measured by a gamma counter and normalized by protein amount. RT-PCR were performed to measure the level of gene expression.
After tanespimycin treatment, 125I uptake was increased by ∼2.5-fold in FRTL-5, hNIS-ARO, and hNIS-MDA-MB-231 cells, but no changes were detected in the hNIS-HeLa cells. Tanespimycin significantly reduced the radioiodine efflux rate only in the FRTL-5 cells. In the FRTL-5 and hNIS-ARO cells, PDS mRNA levels were markedly reduced; the only other observed alteration in the levels of NIS mRNA after tanespimycin treatment was an observed increase in the hNIS-ARO cells.
These results indicate that cellular responses against tanespimycin treatment differed between the normal rat thyroid cells and human cancer cells, and the reduction in the 125I efflux rate by tanespimycin in the normal rat thyroid cells might be attributable to reduced PDS gene expression.
Thyroid cancer; Radioiodine therapy; Tanespimycin (17-AAG); Sodium-iodide symporter; Pendrin
Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor–expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier 131I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer.
Oneal and colleagues report on two novel conditionally replicating adenoviral vectors that combine 131I therapy with conventional virotherapy, a treatment termed radiovirotherapy. They show that these vectors can specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor-expressing prostate cancer cell lines. In vivo efficacy studies show that treatment of tumor-bearing xenograft mice with these vectors results in a significant oncolytic effect.
To develop a model for endogenous thyroid autoantigen presentation, we transfected EBV-transformed B lymphoblastoid cell lines (EBV-LCL), established from patients with autoimmune thyroid disease and normal controls, with cDNA for the human thyroid autoantigen thyroid peroxidase (hTPO). hTPO-antigen presentation to patient peripheral blood T cells was demonstrated after stimulation in vitro for 7 d with irradiated hTPO-transfected or untransfected autologous EBV-LCL. Anti-hTPO-reactive T cells were subsequently cloned in the presence of irradiated, autologous hTPO-transfected EBV-LCL and IL-2.10 T cell-cloned lines exhibited specific hTPO-induced proliferation (stimulation indices of 2.1-7.9) towards autologous hTPO-transfected EBV-LCL, and were subjected to human T cell receptor (hTCR) V gene analysis, using the PCR for the detection of V alpha and V beta hTcR gene families. The results indicated a preferential use of hTCR V alpha 1 and/or V alpha 3 in 9 of the 10 lines. In contrast, hTCR V beta gene family use was more variable. These data demonstrate a model for the endogenous presentation of human thyroid peroxidase in the absence of other thyroid specific antigens. The high frequency of antigen-specific T cells obtained from PBMC using this technique will facilitate further studies at both the functional and hTCR V gene level.
This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter.
The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP-producing cells and in AFP-nonproducing cells was investigated using 125I uptake assay and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of 131I on AdAFPhNIS-infected HCC cells was studied using an in vitro clonogenic assay. In addition, tumor-bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained.
The expression of hNIS was efficiently demonstrated by 125I uptake assay in AFP-producing cells, but not in AFP-nonproducing cells. AFP-producing HCC-targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP-producing cells caused more sensitivity to 131I than that in AFP-nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging.
An AFP-producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP-producing HCC-specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system.
Hepatocellular carcinoma (HCC); Alpha-fetoprotein (AFP) promoter; Adenovirus; Tumor-targeted gene expression; Systemic delivery; Radioiodide therapy
Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC) can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). In this study, we used superparamagentic iron oxide (SPIO)-labeled APCs to carry the human sodium iodide symporter (hNIS) gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m) scan.
Three million human breast cancer (MDA-MB-231) cells were subcutaneously implanted in the right flank of nude mice. APCs, isolated from fresh human cord blood, were genetically transformed to carry the hNIS gene using adenoviral vectors and magnetically labeled with ferumoxides-protamine sulfate (FePro) complexes. Magnetically labeled genetically transformed cells were administered intravenously in tumor bearing mice when tumors reached 0.5 cm in the largest dimension. MRI and single photon emission computed tomography (SPECT) images were acquired 3 and 7 days after cell injection, with a 7 Tesla animal MRI system and a custom built micro-SPECT using Tc-99m, respectively. Expression of hNIS in accumulated cells was determined by staining with anti-hNIS antibody. APCs were efficiently labeled with ferumoxide-protamine sulfate (FePro) complexes and transduced with hNIS gene. Our study showed not only the accumulation of intravenously administered genetically transformed, magnetically labeled APCs in the implanted breast cancer, but also the expression of hNIS gene at the tumor site. Tc-99m activity ratio (tumor/non-tumor) was significantly different between animals that received non-transduced and transduced cells (P < 0.001).
This study indicates that genetically transformed, magnetically labeled APCs can be used both as delivery vehicles and cellular probes for detecting in vivo migration and homing of cells. Furthermore, they can potentially be used as a gene carrier system for the treatment of tumor or other diseases.
Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid-specific expression of the sodium iodide symporter (NIS) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non–small cell lung cancer (NSCLC) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase (TPO) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO-mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS-based radioiodide concentrator gene therapy for locally advanced lung cancer.
Gene therapy; NIS/TPO; lung cancer
Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.
hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.
Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.
The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted 131I radio-ablative therapy is aimed.
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.
Cord blood endothelial progenitor cells; Adenovirus; Human sodium iodide symporter; Single photon emission computed tomography; Magnetic resonance imaging
Viral vectors used for cancer gene therapy are usually delivered by direct intratumoral administration. We studied the role of hyperthermia (HT) in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread).
Materials and methods
Replication-deficient adenoviruses containing either the human sodium-iodide symporter (Ad5-CMV-hNIS) or green fluorescent protein (Ad5-CMV-eGFP) as reporter genes were used. For in vitro studies, human lung cancer A549 cells were transfected with the virus and assayed for hNIS expression by radioactive pertechnetate uptake or green fluorescence activity using a gamma-counter or fluoroscopy respectively in the presence and absence of HT. For in vivo studies, A549 tumors were established intramuscularly in CD1 athymic mice. The adenoviral constructs (1010 viral particles/tumor) were injected intratumorally when the tumors reached 10–11 mm in diameter. Different timing sequences of HT were examined and viral spread was assessed using technetium-autoradiography or GFP-fluorescence microscopy.
In the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT.
Improvements in intratumoral adenoviral spread in response to hyperthermia were not consistently observed in a mouse tumor model using two quantitative endpoints of gene expression.
Hyperthermia; viral dissemination
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 99mTc-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 131I scans were performed. The radioiodine uptakes of the neck and the tumor were compared with those of the other regions.
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 99mTc-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 131I scans, and the neck uptakes were minimal.
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.
Mouse; Total thyroidectomy; Surgical technique; Human sodium/iodide symporter
The aim of this study was to design a method of radionuclide for imaging and therapy of nasopharyngeal carcinoma (NPC) using the transferred human sodium/iodide symporter (hNIS) gene.
A stable NPC cell line expressing hNIS was established (CNE-2-hNIS). After 131I treatment, we detected proliferation and apoptosis of NPC cells, both in vitro and vivo. In vivo, the radioactivity of different organs of nude mice was counted and 99mTc imaging using SPECT was performed. The apparent diffusion coefficient (ADC) value changes of tumor xenografts were observed by diffusion-weighted magnetic resonance imaging (DW-MRI) within 6–24 days of 131I treatment. The correlation of ADC changes with apoptosis and proliferation was investigated. Post-treatment expression levels of P53, Bax, Bcl-2, Caspase-3, and Survivin proteins were detected by western blotting.
131I uptake was higher in CNE-2-hNIS than in CNE-2 cells. The proliferation and apoptosis rate decreased and increased respectively both in vitro and vivo in the experimental group after 131I treatment. The experimental group tumors accumulated 99mTc in vivo, leading to a good visualization by SPECT. DW-MRI showed that ADC values increased in the experimental group 6 days after treatment, while ADC values were positively and negatively correlated with the apoptotic and Ki-67 proliferation indices, respectively. After treatment, CNE-2-hNIS cells up-regulated the expression of P53 and Survivin proteins and activated Caspase-3, and down-regulated the expression of Bcl-2 proteins.
The radionuclide imaging and therapy technique for NPC hNIS-transfected cell lines can provide a new therapy strategy for monitoring and treatment of NPC.
Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153.
GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer.
GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET.
Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.
Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.
Methods and Results
Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.
EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.
The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.
First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, 124I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using 99mTcO4− radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell's differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI.
The expression of both imaging reporter genes was functional and specific. An elution of 99mTcO4− from the cells was observed, with 31% retention after 3 h. After labeling cells with 124I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using 124I small-animal PET, CLI and BLI.
This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.
It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model.
A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay.
For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy.
Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.
Human sodium iodide symporter (hNIS); Radioiodine gene therapy; Adenine nucleotide translocase-2 (ANT2); Short hairpin RNA (shRNA); Radiation-induced immune response; Cytotoxic T cells (CTLs)
We have constructed a prostate tumor specific conditionally replicating adenovirus (CRAd), named Ad5PB_RSV-NIS that expresses the human sodium iodine symporter gene (hNIS). LNCaP tumors were established in nude mice and infected with this CRAd to study tumor viral spread, NIS expression, and efficacy. Using quantitative polymerase chain reaction (QPCR) we found a linear correlation between the viral dose and viral genome copy numbers recovered after tumor infection. Confocal microscopy showed a linear correlation between adenovirus density and NIS expression. Radioiodine uptake vs. virus dose-response curves revealed that the dose response curve was not linear and displayed a lower threshold of detection at 107 vp and an upper plateau of uptake at 1011 vp. The outcome of radiovirotherapy was highly dependent upon viral dose. At 1010 vp no significant differences were observed between virotherapy alone or radiovirotherapy. However, when radioiodine therapy was combined with virotherapy at a dose of 1011 vp, significant improvement in survival was observed, indicating a relationship between viral dose-response uptake and the efficacy of radiovirotherapy. The reasons behind the differences in radioiodine therapy efficacy can be ascribed to more efficient viral tumor spread and a decrease in the rate of radioisotope efflux. Our results have important implications regarding the desirables and undesirable characteristics of vectors for clinical translation of virus-mediated NIS transfer therapy
prostate cancer; probasin; adenovirus; sodium iodide symporter; virotherapy; gene therapy
The authors evaluated whether the cytotoxicity of CD8+CTLs generated by combined hMUC1 vaccination and hNIS radioiodine gene therapy was enhanced in the presence of CpG in an established tumor model.
CMNF cells (CT26 cells expressing hMUC1, hNIS and Firefly luciferase) were transplanted into BALB/c mice. Four and 10 days later, tumor-bearing mice were immunized intramuscularly with pcDNA3.1 or pcDNA-hMUC1 or pcDNA-hMUC1+CpG, and subsequently administered PBS or 131I [five groups (seven mice/group): referred to as the pcDNA3.1+PBS, phMUC1+PBS, pcDNA3.1+131I, phMUC1+131I, and phMUC1+131I+CpG groups]. The number of CD8+IFNr+ T cells of splenocytes as well as the number of CD8+IFNr+ T cells of splenocytes re-stimulated with CD11c+ cells was determined using FACS analysis. The activities of cytotoxic T cells (CTLs) from splenocytes were investigated.
Marked tumor growth inhibition was observed in the phMUC1+131I and phMUC1+131I+CpG groups, but not in the other three single therapy groups. Particularly the number of CD8+IFN-γ+ T cells of splenocytes was more increased in the phMUC1+131I+CpG group than in the phMUC1+131I group. The number of CD8+IFN-γ+ T cells of splenocytes stimulated with CD11c+ cells was the most enhanced in the phMUC1+131I+CpG group among the five groups. Concurrently, the activities of hMUC1-associated CTLs obtained from splenocytes in the phMUC1+131I+CpG group were significantly greater than in the other four groups (pcDNA+PBS, phMUC1+PBS, pcDNA+131I, phMUC1+131I, and phMUC1+131I+CpG, 16 ± 2%, 20 ± 1%, 30 ± 2%, 60 ± 2%, and 87 ± 2%, respectively, P < 0.01).
Our data suggest that adjuvant CpG ODNs can increase the killing activities of CTLs generated by combined hMUC1 DNA vaccination and hNIS radioiodine gene therapy.
CpG oligodeoxynucleotides; hMUC1 vaccination; hNIS radioiodine gene therapy
We studied the concordance of transgene expression in the transplanted heart using bicistronic adenoviral vector coding for a transgene of interest (human carcinoembryonic antigen: hCEA - beta human chorionic gonadotropin: βhCG) and for a marker imaging transgene (human sodium iodide symporter: hNIS).
Inbred Lewis rats were used for syngeneic heterotopic cardiac transplantation. Donor rat hearts were perfused ex vivo for 30 minutes prior to transplantation with University of Wisconsin (UW) solution (n=3), with 109 pfu/ml of adenovirus expressing hNIS (Ad-NIS; n=6), hNIS-hCEA (Ad-NIS-CEA; n=6) and hNIS-βhCG (Ad-NIS-CG; n=6). On post-operative day (POD) 5, 10, 15 all animals underwent micro-SPECT/CT imaging of the donor hearts after tail vein injection of 1000 μCi 123I and blood sample collection for hCEA and βhCG quantification.
Significantly higher image intensity was noted in the hearts perfused with Ad-NIS (1.1±0.2; 0.9±0.07), Ad-NIS-CEA (1.2±0.3; 0.9±0.1) and Ad-NIS-CG (1.1±0.1; 0.9±0.1) compared to UW group (0.44±0.03; 0.47±0.06) on POD 5 and 10 (p<0.05). Serum levels of hCEA and βhCG increased in animals showing high cardiac 123I uptake, but not in those with lower uptake. Above this threshold, image intensities correlated well with serum levels of hCEA and βhCG (R2=0.99 and R2=0.96 respectively).
These data demonstrate that hNIS is an excellent reporter gene for the transplanted heart. The expression level of hNIS can be accurately and non-invasively monitored by serial radioisotopic single photon emission computed tomography (SPECT) imaging. High concordance has been demonstrated between imaging and soluble marker peptides at the maximum transgene expression on POD 5.
Gene therapy; heart transplantation; sodium iodide symporter (NIS); single photon emission computed tomography (SPECT); molecular imaging