A transporter gene (sodium iodide symporter) for dual purposes in gene therapy: imaging and therapy

Radioiodide uptake (RAIU) in thyroid follicular epithelial cells, mediated by a plasma membrane transporter, sodium iodide symporter (NIS), provides a first step mechanism for thyroid cancer detection by radioiodide injection and effective radioiodide treatment for patients with invasive, recurrent, and/or metastatic thyroid cancers after total thyroidectomy. NIS gene transfer to tumor cells may significantly and specifically enhance internal radioactive accumulation of tumors following radioiodide administration, and result in better tumor control. NIS gene transfers have been successfully performed in a variety of tumor animal models by either plasmid-mediated transfection or virus (adenovirus or retrovirus)-mediated gene delivery. These animal models include nude mice xenografted with human melanoma, glioma, breast cancer or prostate cancer, rats with subcutaneous thyroid tumor implantation, as well as the rat intracranial glioma model. In these animal models, non-invasive imaging of in vivo tumors by gamma camera scintigraphy after radioiodide or technetium injection has been performed successfully, suggesting that the NIS can serve as an imaging reporter gene for gene therapy trials. In addition, the tumor killing effects of 131I after NIS gene transfer have been demonstrated in in vitro clonogenic assays and in vivo radioiodide therapy studies, suggesting that NIS gene can also serve as a therapeutic agent when combined with radioiodide injection. Better NIS-mediated tumor treatment by radioiodide requires a more efficient and specific system of gene delivery with better retention of radioiodide in tumor. Results thus far are, however, promising, and suggest that NIS gene transfer followed by radioiodide treatment will allow non-invasive in vivo imaging to assess the outcome of gene therapy and provide a therapeutic strategy for a variety of human cancers.     

X-ray-visible microcapsules containing mesenchymal stem cells improve hind limb perfusion in a rabbit model of peripheral arterial disease

The therapeutic goal in peripheral arterial disease (PAD) patients is to restore blood flow to ischemic tissue. Stem cell transplantation offers a new avenue to enhance arteriogenesis and angiogenesis. Two major problems with cell therapies are poor cell survival and the lack of visualization of cell delivery and distribution. To address these therapeutic barriers, allogeneic bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in alginate impregnated with a radiopaque contrast agent (MSC-Xcaps). In vitro MSC-Xcap viability by a fluorometric assay was high (96.9% ± 2.7% at 30 days postencapsulation) and as few as 10 Xcaps were visible on clinical x-ray fluoroscopic systems. Using an endovascular PAD model, rabbits (n = 21) were randomized to receive MSC-Xcaps (n = 6), empty Xcaps (n = 5), unencapsulated MSCs (n = 5), or sham intramuscular injections (n = 5) in the ischemic thigh 24 hours postocclusion. Immediately after MSC transplantation and 14 days later, digital radiographs acquired on a clinical angiographic system demonstrated persistent visualization of the Xcap injection sites with retained contrast-to-noise. Using a modified TIMI frame count, quantitative angiography demonstrated a 65% improvement in hind limb perfusion or arteriogenesis in MSC-Xcap-treated animals versus empty Xcaps. Post-mortem immunohistopathology of vessel density by anti-CD31 staining demonstrated an 87% enhancement in angiogenesis in Xcap-MSC-treated animals versus empty Xcaps. MSC-Xcaps represent the first x-ray-visible cellular therapeutic with enhanced efficacy for PAD treatment.     

Sodium/iodide symporter: physiopathological aspects and therapeutic perspectives

The existence of the natriuric/iodide symporter (NIS) represents a new view to understand the thyroid metabolism of iodide. Due to its cellular localisation on basolateral membrane, this transporter exerts an essential role in the biological functions of the thyroid, especially the capacity to accumulate iodide into the thyrocytes. Clinical perspectives of NIS activity modulation would ameliorate the diagnosis and the treatment of thyroid diseases by using radioisotopes transported by the NIS (131 iodide, 99m technetium, 188 rhenium). The study of the regulation pathways modulating the expression and the activity of the symporter NIS, would allow to understand pathogeny of benign or malignant diseases of the thyroid gland. The relative facility of the therapy management by 131 iodide and its good efficiency associated to the recent advance of NIS function also give an interesting perspective to the gene therapy treatment of the nonthyroid cancers despite existent methodological problems.     

荷淋巴瘤裸鼠放射免疫治疗99mTc-Annexin V凋亡显像的研究

探讨99mTc-Annexin Ⅴ凋亡显像在评价淋巴瘤131I-Rituximab放射免疫治疗疗效中的价值及其最佳显像时间。荷淋巴瘤裸鼠经肿瘤间质、尾静脉注射131I-Rituximab治疗后不同时间点进行99mTc-Annexin Ⅴ凋亡显像,并分离解剖肿瘤组织进行流式细胞术和免疫组织化学检查,根据其与凋亡显像的吻合程度确定显像最佳时间。结果显示：131I-Rituximab经肿瘤间质注射后24h、36h、48h、96h99mTc-Annexin Ⅴ凋亡显像肿瘤/非肿瘤组织摄取比（T/NT）分别为6.0±1.08、8.4±1.68、8.1±1.89、7.0±0.95,高于相同时间点静脉注射组（3.4±0.27、5.1±0.82、6.7±0.58、5.8±0.91）,且均与相同时间点细胞凋亡率、bax/bcl-2变化基本一致。结论：99mTc-Annexin Ⅴ凋亡显像作为无创性手段,能有效监测淋巴瘤131I-Rituximab放射免疫治疗后细胞凋亡情况,且放射免疫治疗后36h~48h是凋亡显像较佳时间。     

NIS基因转染对肝细胞癌NIS蛋白表达及摄碘功能的影响

目的:探讨肝细胞癌细胞核素靶向治疗方法。方法:提取NIS基因片段并构建重组质粒pcDNA3/NIS,将重组质粒导入肝细胞癌HepG2细胞。转染后24 h利用Western-Blot法检测HepG2细胞NIS蛋白表达,采用125I结合试验评估转染后细胞的摄碘率,采用DAPI染色法评估HepG2细胞摄125I后的凋亡情况。实验组与对照组之间NIS蛋白表达、摄碘率以及细胞凋亡率比较采用t检验。结果:蛋白电泳表明经NIS基因转染后,实验组HepG2细胞已表达NIS蛋白,表达强度显著高于对照组（t=2.693, P<0.05）。实验组HepG2细胞摄碘率B/T%平均为（18.4±5.8）%,显著高于对照组（t=36.842, P<0.05）。结合125I后24 h,实验组凋亡细胞数多于对照组,平均凋亡率为（19.2±5.3）%,显著高于对照组（t=3.086, P<0.05）。结论:转染外源性NIS基因可上调肝细胞癌HepG2细胞NIS蛋白表达,使其具备摄碘功能,加快细胞凋亡,为放射性碘靶向治疗提供实验依据。     

Quantification of metastatic tumor growth in bleomycin-injured lungs

The lung is a common target organ in experimental models of tumor metastasis in which quantification usually involves counting labeled tumor cells shortly after injection, or enumeration of grossly visible pleural tumors. In this study, these approaches were used in addition to autoradiographic and morphometric methods to analyse the effect of bleomycin-mediated injury on the development, distribution and quantification of pulmonary metastases. One day after intravenous injection of 2 × 105 fibrosarcoma cells, the lungs of C57 bl/6 mice, pretreated with bleomycin (120mg/kg i.v., 5 days before) contained about nine times as many [131 I] iododeoxyuridine-labeled cells as the lungs of control animals given saline injections. At this time, autoradiographic counts of [³H] thymidine-labeled tumor cells in lung sections showed a similar increase in tumor cell localization after bleomycin, with labeled cells distributed equally between parenchymal and pleural areas. However, subsequent tumor growth was demonstrated microscopically to be predominantly in pleural and peribronchial areas, especially at sites of lung injury induced by bleomycin. Counts of grossly visible pleural tumors failed to demonstrate a difference between bleomycin groups and controls at 7 days whereas counts of nodules in lung sections, and quantification of lung area occupied by tumor both showed significantly greater tumor involvement in bleomycin-treated animals. As tumors became confluent, morphometric measurements demonstrated tumor growth in the lung more accurately than did nodule counts. We conclude that bleomycin-induced injury greatly enhances metastatic tumor growth and that morphometric methods are more sensitive than lung colony counts in their ability to quantify pulmonary metastases. Morphometry and autoradiography have also demonstrated that while there is a uniform distribution of arrested tumor cells in the lung initially, there is preferential development of metastatic tumors at sites of pulmonary damage, in particular at the pleura.     

Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy

The tumor tropism of mesenchymal stem cells (MSCs) makes them an excellent delivery vehicle used in anticancer therapy. However, the exact mechanisms of MSCs involved in tumor microenvironment are still not well defined. Molecular imaging technologies with the versatility in monitoring the therapeutic effects, as well as basic molecular and cellular processes in real time, offer tangible options to better guide MSCs mediated cancer therapy. In this study, an in situ breast cancer model was developed with MDA-MB-231 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP). In mice breast cancer model, we injected human umbilical cord-derived MSCs (hUC-MSCs) armed with a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk), renilla luciferase (Rluc) and red fluorescent protein (RFP) into tumor on day 13, 18, 23 after MDA-MB-231 cells injection. Bioluminescence imaging of Fluc and Rluc provided the real time monitor of tumor cells and hUC-MSCs simultaneously. We found that tumors were significantly inhibited by hUC-MSCs administration, and this effect was enhanced by ganciclovir (GCV) application. To further demonstrate the effect of hUC-MSCs on tumor cells in vivo, we employed the near infrared (NIR) imaging and the results showed that hUC-MSCs could inhibit tumor angiogenesis and increased apoptosis to a certain degree. In conclusion, hUC-MSCs can inhibit breast cancer progression by inducing tumor cell death and suppressing angiogenesis. Moreover, molecular imaging is an invaluable tool in tracking cell delivery and tumor response to hUC-MSCs therapies as well as cellular and molecular processes in tumor.     

Bone marrow stromal cells induce changes in pain behavior after sciatic nerve constriction

Peripheral nerve injury, i.e. a single ligature nerve constriction (SLNC), triggers neuropathic pain. Bone marrow stromal cells (MSCs) have been observed to migrate to the injured tissues and mediate functional recovery following brain, spinal cord and peripheral nerve lesions. We have recently shown MSC selective migration to the ipsilateral lumbar (L3-6) dorsal root ganglia (DRGs) after a sciatic nerve SLNC. In this study, we have analyzed the thermal and mechanical sensitivities of animals subjected to a SLNC of the sciatic nerve and an ipsilateral intraganglionic MSC injection, using the von Frey and Choi tests. Control animals were subjected to the nerve lesion either alone or followed by the administration of phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs). All the animals were tested both before surgery and after 1, 3, 7, 14, 21, 28 and 56 days. Animals subjected to the sciatic nerve constriction developed ipsilateral mechanical and thermal allodynia already 3 days after the lesion. The allodynic responses were maintained even after 56 days. MSC administration prevented the generation of mechanical allodynia and reduced the number of allodynic responses to cold stimuli. On the contrary, the injection of either PBS or BNMCs could not counteract allodynia. These results suggest that MSCs may modulate pain generation after sciatic nerve constriction. The underlying mechanisms by which MSCs exert their actions on pain behavior need to be clarified.     

Antitumoral activity and osteogenic potential of mesenchymal stem cells expressing the urokinase-type plasminogen antagonist amino-terminal fragment in a murine model of osteolytic tumor

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.     

Intervertebral Disc Cell Therapy for Regeneration: Mesenchymal Stem Cell Implantation in Rat Intervertebral Discs

This study explores the use of mesenchymal stem cells (MSCs) for intervertebral disc regeneration. We used an in vivo model to investigate the feasibility of exogenous cell delivery, retention, and survival in the pressurized disc space. MSC injection into rat coccygeal discs was performed using 15% hyaluronan gel as a carrier. Injections of gel with or without MSCs were performed. Immediately after injection, fluorescently labeled stem cells were visible on sections of cell-injected discs. Seven and 14 days after injection, stem cells were still present within the disc, but their numbers were significantly decreased. At 28 days, a return to the initial number of injected cells was observed, and viability was 100%. A trend of increased disc height compared to blank gel suggests an increase in matrix synthesis. The results indicate that MSCs can maintain viability and proliferate within the rat intervertebral disc.     

骨髓间充质干细胞移植对溃疡性结肠炎大鼠结肠的修复作用

目的 探讨大鼠骨髓间充质干细胞(MSCs)移植后对溃疡性结肠炎(UC)大鼠结肠的修复作用.方法 分离培养、荧光标记大鼠MSCs.SD大鼠45只随机分为3组,A、B组用免疫复合法诱导UC模型,C组为正常对照组.A组移植MSCs,B组移植等量PBS.移植后3d、7d、14d取结肠组织制作石蜡切片,观察病理变化;另取A组结肠...     

Radionuclide 131I labeled reduced graphene oxide for nuclear imaging guided combined radio- and photothermal therapy of cancer

Nano-graphene and its derivatives have attracted great attention in biomedicine, including their applications in cancer theranostics. In this work, we develop 131I labeled, polyethylene glycol (PEG) coated reduced nano-graphene oxide (RGO), obtaining 131I-RGO-PEG for nuclear imaging guided combined radiotherapy and photothermal therapy of cancer. Compared with free 131I, 131IRGO- PEG exhibits enhanced cellular uptake and thus improved radio-therapeutic efficacy against cancer cells. As revealed by gamma imaging, efficient tumor accumulation of 131I-RGO-PEG is observed after its intravenous injection. While RGO exhibits strong near-infrared (NIR) absorbance and could induce effective photothermal heating of tumor under NIR light irradiation, 131I is able to emit high-energy X-ray to induce cancer killing as the result of radio ionization effect. By utilizing the combined photothermal therapy and radiotherapy, both of which are delivered by a single agent 131IRGO- PEG, effective elimination of tumors is achieved in our animal tumor model experiments. Toxicology studies further indicate that 131I-RGO-PEG induces no appreciable toxicity to mice at the treatment dose. Our work demonstrates the great promise of combing nuclear medicine and photothermal therapy as a novel therapeutic strategy to realize synergistic efficacy in cancer treatment.     

Therapeutic potential of bone marrow-derived mesenchymal stem cells producing pigment epithelium-derived factor in lung carcinoma

Specific and efficient gene delivery to target cells and the subsequent expression of the RNA and protein is crucial to the success of gene-based therapy for cancer. Mesenchymal stem cells (MSCs) represent novel and efficient tools for delivery of therapeutic agents to tumor cells. In this study, we evaluated the potential of bone marrow-derived mesenchymal stem cells, genetically modified to express pigment epithelium-derived factor (PEDF) for the treatment of Lewis lung carcinoma (LLC). MSCs derived from murine bone marrow were efficiently engineered to express human PEDF by adenoviral transduction, and the expression and bioactivity of the transgenic protein from engineered MSCs were confirmed in vitro. Animal experiments showed that the systemic administration of MSCs treated with PEDF dramatically reduced the growth of LLC tumors and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-PEDF-treated animals indicated an increase in apoptosis and a decrease in microvessel density. ELISA showed that the group of MSCs treated with PEDF had relatively higher expression levels of PEDF in tumor tissue and lower levels in serum compared with the free Ad-PEDF group. These results suggest that MSCs have potential use as effective delivery of vehicles for therapeutic genes in the treatment of LLC.     

Mesenchymal stroma cells trigger early attraction of M1 macrophages and endothelial cells into fibrin hydrogels,stimulating long bone healing without long-term engraftment

Implantation of mesenchymal stroma cells (MSCs) is an attractive approach to stimulate closure of large bone defects but an optimal carrier has yet to be defined. MSCs may display trophic and/or immunomodulatory features or stimulate bone healing by their osteogenic activity. The aim of this study was to unravel whether fibrin hydrogel supports early actions of implanted MSCs, such as host cell recruitment, immunomodulation and tissue regeneration, in long bone defects. Female rats received cell-free fibrin or male MSCs embedded in a fibrin carrier into plate-stabilized femoral bone defects. Removed callus was analyzed for host cell invasion (day 6), local cytokine expression (days 3 and 6) and persistence of male MSCs (days 3, 6, 14 and 28). Fibrin–MSC composites triggered fast attraction of host cells into the hydrogel while cell-free fibrin implants were not invaded. A migration front dominated by M1 macrophages and endothelial progenitor cells formed while M2 macrophages remained sparse. Only MSC-seeded fibrin hydrogel stimulated early tissue maturation and primitive vessel formation at day 6 in line with significantly higher VEGF mRNA levels recorded at day 3. Local TNF-α, IL-1β and IL-10 expression indicated a balanced immune cell activity independent of MSC implantation. Implanted MSCs persisted until day 14 but not day 28. Our results demonstrate that fibrin hydrogel is an attractive carrier for MSC implantation into long bone defects, supporting host cell attraction and pro-angiogenic activity. By this angiogenesis, implant integration and tissue maturation was stimulated in long bone healing independent of long-term engraftment of implanted MSCs.     

14S,21R-dihydroxy-docosahexaenoic acid treatment enhances mesenchymal stem cell amelioration of renal ischemia/reperfusion injury

Bone marrow mesenchymal stem cells (MSCs) have shown potential to improve treatment of renal failure. The prohealing functions of MSCs have been found to be enhanced by treatment with the lipid mediator, 14S,21R-dihydroxy-docosa4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21R-diHDHA). In this article, using a murine model of renal ischemia/reperfusion (I/R) injury, we found that treatment with 14S,21R-diHDHA enhanced MSC amelioration of renal I/R injury. Treated MSCs more efficiently inhibited I/R-induced elevation of serum creatinine levels, reduced renal tubular cell death, and inhibited infiltration of neutrophils, macrophages, and dendritic cells in kidneys. Conditioned medium from treated MSCs reduced the generation of tumor necrosis factor-α and reactive oxygen species by macrophages under I/R conditions. Infusion of treated MSCs more efficiently reduced I/R-damage to renal histological structures compared with untreated MSCs (injury score: 7.9±0.4 vs. 10.5±0.5). Treated MSCs were resistant to apoptosis in vivo when transplanted under capsules of I/R-injured kidneys (active caspase-3+ MSCs: 4.2%±2.8% vs. 11.7%±2.4% of control) and in vitro when cultured under I/R conditions. Treatment with 14S,21R-diHDHA promoted viability of MSCs through a mechanism involving activation of the phosphoinositide 3-kinase -Akt signaling pathway. Additionally, treatment of MSCs with 14S,21R-diHDHA promoted secretion of renotrophic hepatocyte growth factor and insulin growth factor-1. Similar results were obtained when 14S,21RdiHDHA was used to inhibit apoptosis of human MSCs (hMSCs) and to increase the generation of renotrophic cytokines from hMSCs. These findings provide a lead for new strategies in the treatment of acute kidney injury with MSCs.     

体外模拟肿瘤微环境中致大鼠MSCs瘤化因子分析

 目的 探讨模拟肿瘤微环境中骨髓间充质干细胞（MSCs）肿瘤转化的发生与HGF、IL-6、BFGF关系。方法 实验组为C6胶质瘤与MSCs间接共培养模拟肿瘤微环境下MSCs的生长,对照组为星型胶质细胞与MSCs间接共培养模拟正常微环境下MSCs的生长,空白对照组MSCs单独培养;QPCR,免疫荧光检测各组的P53和MDM2的基因和蛋白的表达;ELISA检测各组HGF、IL-6、BFGF的表达;免疫荧光检测对应升高的细胞因子作用于MSCs后的STAT3蛋白的表达。结果 实验组的MSCs形态肿瘤化;MSCs的MDM2 mRNA是对照组的1.56±0.21(P<0.05),MDM2蛋白表达率比对照组升高90±7%(P<0.01);MSCs的P53的mRNA表达是对照组的0.55±0.10, 而P53突变蛋白表达率比对照组升高87±5%(P<0.01);HGF、IL-6水平比对照组中达水平升高(P<0.05);过量IL-6处理组的MSCs的STAT3激活蛋白表达率比正常量IL-6处理组明显升高(P<0.01)。结论 IL-6参与MSCs在肿瘤微环境下的肿瘤转化的发生。     

Radioimmunoimaging of human small cell lung carcinoma with I-131 tumor specific monoclonal antibody

In this study, an IgM monoclonal antibody (MAb600D11) directed against human small cell lung cancer (NCI-H69) was radiolabeled with iodine-131, and the biodistribution and image quality of the radiolabeled antibody was evaluated. Radiolabeling was achieved in a solid-phase system consisting of 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Labeling efficiencies and protein purification were accomplished using gel exclusion chromatography while radioimmunoreactivity was determined using a solid-phase radioimmunoassay procedure. The biodistribution of I-131-labeled MAbs was determined in Sprague-Dawley rats up to 7 days after injection. Highest organ concentrations were observed in liver (3.91 +/- 0.47 (SD) and 0.17 +/- 0.04 (SD) mean percent injected dose at 1-7 days after injections) and in thyroid (5.33 +/- 0.71 (SD) and 5.32 +/- 2.01 (SD) mean percent injected dose at 1-7 days after injection). Nude mice, bearing either a small cell lung tumor (NCI-H69) or a nonspecific tumor (adenocarcinoma), were injected with 400-800 microCi of I-131 labeled monoclonal antibody. Optimum tumor visualization was observed 2-4 days after injection with tumor concentrations as high as 10.4% of the initial injected dose. The results demonstrated that radioimmunoimaging of human small cell lung carcinoma was feasible with the tumor-specific IgM I-131-labeled MAb.