Production of a replicating retroviral vector expressing Reovirus fast protein for cancer gene therapy.

Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.

Therapeutic Effect of IL-4 Receptor-Targeting Pro-Apoptotic Peptide (AP1-ELP-KLAK) in Glioblastoma Tumor Model.

BACKGROUND: Thermal-responsive self-assembled elastin-like polypeptide (ELP)-based nanoparticles are an emerging platform for controlled delivery of therapeutic peptides, proteins and small molecular drugs. The antitumor effect of bioengineered chimeric polypeptide AP1-ELP-KLAK containing an interleukin-4 receptor (IL-4R) targeting peptide and pro-apoptotic peptide (KLAKLAK) was evaluated in glioblastoma (GBM) in vitro and in vivo. METHODS AND RESULTS: Herein, the therapeutic effect of AP1-ELP-KLAK was tested in advanced, and less curable glioblastoma cells with higher expression of IL-4R. Glioblastoma cell lines stably expressing different reporter systems i.e., caspase-3 sensor (surrogate marker for cellular apoptosis) or effluc/enhanced firefly luciferase (cellular viability) were established to measure cell death non-invasively. Bioluminescence imaging (BLI) of D54/effluc and U97MG/effluc treated with AP1-ELP-KLAK exhibited higher cell death up to 2~3-fold than the control. Treatment with AP1-ELP-KLAK resulted in time-dependent increase of caspase-3 sensor BLI activity in D54/C cells and D54/C tumor-bearing mice. Intravenous injection of AP1-ELP-KLAK dramatically reduced tumor growth by inducing cellular apoptosis in D54/effluc tumor-bearing mice. Further, the immuno-histological examination of the excised tumor tissue confirmed the presence of apoptotic cells as well as caspase-3 activation. CONCLUSION: Collectively, AP1-ELP-KLAK effectively induced cellular apoptosis of glioblastoma cells and non-invasive imaging provides a window for real-time monitoring of anti-tumor effect with the provision of improving therapeutic efficacy in a glioblastoma mice model.

Visualization of the Biological Behavior of Tumor-Associated Macrophages in Living Mice with Colon Cancer Using Multimodal Optical Reporter Gene Imaging.

We sought to visualize the migration of tumor-associated macrophages (TAMs) to tumor lesions and to evaluate the effects of anti-inflammatory drugs on TAM-modulated tumor progression in mice with colon cancer using a multimodal optical reporter gene system. Murine macrophage Raw264.7 cells expressing an enhanced firefly luciferase (Raw/effluc) and murine colon cancer CT26 cells coexpressing Rluc and mCherry (CT26/Rluc-mCherry, CT26/RM) were established. CT26/RM tumor-bearing mice received Raw/effluc via their tail veins, and combination of bioluminescence imaging (BLI) and fluorescence imaging (FLI) was conducted for in vivo imaging of TAMs migration and tumor progression. Dexamethasone (DEX), a potent anti-inflammatory drug, was administered intraperitoneally to tumor-bearing mice following the intravenous transfer of Raw/effluc cells. The migration of TAMs and tumor growth was monitored by serial FLI and BLI. The migration of Raw/effluc cells to tumor lesions was observed at day 1, and BLI signals were still distinct at tumor lesions on day 4. Localization of BLI signals from migrated Raw/effluc cells corresponded to that of FLI signals from CT26/RM tumors. In vivo FLI of tumors demonstrated enhanced tumor growth associated with macrophage migration to tumor lesions. Treatment with DEX inhibited the influx of Raw/effluc cells to tumor lesions and abolished the enhanced tumor growth associated with macrophage migration. These findings suggest that molecular imaging approach for TAM tracking is a valuable tool for evaluating the role of TAMs in the tumor microenvironment as well as for the development of new drugs to control TAM involvement in the modulation of tumor progression.

Inverse Agonist of Estrogen-Related Receptor γ Enhances Sodium Iodide Symporter Function Through Mitogen-Activated Protein Kinase Signaling in Anaplastic Thyroid Cancer Cells.

UNLABELLED: Anaplastic thyroid cancer (ATC), a rare thyroid cancer with poor prognosis, is associated with insufficient function of the sodium iodide symporter (NIS). Estrogen-related receptor γ (ERRγ) is a member of the orphan nuclear receptors with important functions in cell development and homeostasis. However, there are no reports that demonstrate whether ERRγ is related to NIS function. Here, we evaluated the role of ERRγ in the regulation of NIS function in ATC cells using GSK5182, an inverse agonist of ERRγ. METHODS: Two ATC cell lines, BHT-101 and CAL62, were incubated with GSK5182 at various time points and doses. The NIS function in the ATC cells was serially assessed by their uptake of radioiodine. The effects of GSK5182 on ERRγ and the mitogen-activated protein (MAP) kinase pathway, as well as on NIS protein, were evaluated by immunoblot assay. To examine whether the GSK5182-induced NIS functional activity can be affected by inhibition of the MAP kinase pathway, the MAP kinase activity and levels of radioiodine uptake were determined after application of a mitogen-activated protein kinase kinase (MEK) inhibitor to GSK5182-treated cells. Finally, the cytotoxic effect of (131)I was determined by clonogenic assay. RESULTS: Treatment with GSK5182 resulted in dose- and time-dependent increases in iodide uptake in ATC cells, which were accompanied by both the downregulation of ERRγ protein and the activation of extracellular signal-regulated kinase (ERK) 1/2. Both the increased radioiodine uptake and ERK1/2 activation of ATC cells were completely inhibited by the specific MEK inhibitor. GSK5182 treatment enhanced the membrane localization of NIS in both ATC cell lines. Accordingly, preexposure to GSK5182 enhanced the cytotoxic effects of (131)I treatment in ATC cells. CONCLUSION: These findings suggest that the inverse agonist of ERRγ enhances the responsiveness of radioiodine therapy by modulating NIS function in ATC cells via the regulation of ERRγ and the MAP kinase signaling pathway.

Combined Fluorescence and Magnetic Resonance Imaging of Primary Macrophage Migration to Sites of Acute Inflammation Using Near-Infrared Fluorescent Magnetic Nanoparticles.

PURPOSE: This study aimed to track the migration of primary macrophages labeled with near-infrared (NIR) fluorescent magnetic nanoparticles toward chemically induced acute inflammatory lesions in mice and to visualize the effect of anti-inflammatory drugs on macrophage migration using combined fluorescence and magnetic resonance imaging (FLI/MRI). PROCEDURES: Primary macrophages were labeled with NIR fluorescent magnetic nanoparticles, and labeled cells were injected into mice intravenously. One day later, inflammation was induced by subcutaneous injection of 1% carrageenan (CG) solution to footpads of the right hind leg, and phosphate-buffered saline (PBS) as control treatment was subcutaneously injected to footpad of the left hind leg. To evaluate the effect of drug treatment on macrophage migration, a single dose of dexamethasone (DEX) was intraperitoneally administered to the mice immediately after the induction of inflammation and was followed by combined FLI/MRI at predetermined time points. RESULTS: No difference in cellular viability or phagocytic activity was observed between the labeled and parent macrophages. In vivo optical imaging revealed an increase in FLI signals in CG-injected footpads in a time-dependent manner, but not in PBS-treated footpads. DEX treatment inhibited the migration of the labeled macrophages to the CG-injected footpads, with relative decreases in FLI activity. In accordance with FLI, T2*-weighted MR images showed hypo-intense signals in the CG-injected footpads but not in the PBS-injected footpads. The DEX-treated mice did not show a dark signal loss zone on MR images in the CG-treated paw. CONCLUSIONS: We successfully tracked the migration of macrophages to inflammatory lesions using both FLI and MRI with NIR fluorescent magnetic nanoparticles and demonstrated the inhibitory effects of DEX on macrophage migration to inflammation sites.

Noninvasive imaging of apoptosis induced by adenovirus-mediated cancer gene therapy using a caspase-3 biosensor in living subjects.

We attempted to visualize the serial induction of caspase-3-dependent apoptosis mediated by Fas ligand/tumor necrosis factor-related apoptosis-inducing ligand (FasL/TRAIL) adenoviral gene therapy in mice bearing human glioma xenografts using a caspase-3 biosensor and monitored its therapeutic effects. Human D54 glioma cells expressing both the caspase-3 sensor and the Renilla luciferase (Rluc) gene were established (referred to as D54-CR cells). The bioluminescence imaging (BLI) signals of the caspase-3 sensor in the D54-CR cells were increased in a time- and virus dose-dependent manner by Ad-TRAIL or Ad-FasL transduction. Fluorescence-activated cell sorting (FACS) analysis revealed an increase in both cleaved caspase-3 or poly(ADP-ribose) polymerase (PARP) and annexin V- and propidium iodide-positive cells depending on the dosage of administered virus. Ad-FasL treatment resulted in a significant increase in the BLI activity of the caspase-3 sensor in the D54-CR tumors, which were ≈ 8.2, ≈ 12.9, and ≈ 46.6 times higher than those of control at 12 hours, 24 hours, and 96 hours posttreatment, respectively. In contrast, a significant reduction in Rluc activity, as a surrogate marker of cell viability, was detected in the tumors treated with Ad-FasL but not in those treated with Ad-null. Overall, the activation of caspase-3-dependent apoptosis induced by Ad-FasL/Ad-TRAIL gene therapy was successfully monitored by a sensitive imaging platform for caspase-3 activation.

Evaluation of therapeutic effects of natural killer (NK) cell-based immunotherapy in mice using in vivo apoptosis bioimaging with a caspase-3 sensor.

Natural killer (NK) cell-based immunotherapy is a promising strategy for cancer treatment, and caspase-3 is an important effector molecule in NK cell-mediated apoptosis in cancers. Here, we evaluated the antitumor effects of NK cell-based immunotherapy by serial noninvasive imaging of apoptosis using a caspase-3 sensor in mice with human glioma xenografts. Human glioma cells expressing both a caspase-3 sensor as a surrogate marker for caspase-3 activation and Renilla luciferase (Rluc) as a surrogate marker for cell viability were established and referred to as D54-CR cells. Human NK92 cells were used as effector cells. Treatment with NK92 cells resulted in a time- and effector number-dependent increase in bioluminescence imaging (BLI) activity of the caspase-3 sensor in D54-CR cells in vitro. Caspase-3 activation by NK92 treatment was blocked by Z-VAD treatment in D54-CR cells. Transfusion of NK92 cells induced an increase of the BLI signal by caspase-3 activation in a dose- and time-dependent manner in D54-CR tumor-bearing mice but not in PBS-treated mice. Accordingly, sequential BLI with the Rluc reporter gene revealed marked retardation of tumor growth in the NK92-treatment group but not in the PBS-treatment group. These data suggest that noninvasive imaging of apoptosis with a caspase-3 sensor can be used as an effective tool for evaluation of therapeutic efficacy as well as for optimization of NK cell-based immunotherapy.-Lee, H. W., Singh, T. D., Lee, S.-W., Ha, J.-H., Rehemtulla, A., Ahn, B.-C., Jeon, Y.-H., Lee, J. Evaluation of therapeutic effects of natural killer (NK) cell-based immunotherapy in mice using in vivo apoptosis bioimaging with a caspase-3 sensor.