Multimodal approach for cancer research using MRI and optical imaging

Multimodal molecular/cellular imaging using magnetic resonance imaging (MRI) and optical imaging has been actively investigated and made a rapid progress in the past decade. These techniques have been shown to contribute to a wide range of cancer research, for example, the development of early diagnosis of cancer and the assessment of therapeutic efficacy. In this symposium, I would like to present our recent progress in multimodal imaging using MRI and optical imaging; noninvasive monitoring of intratumoral transgene expression for gene therapy and targeted detection of mesothelioma based on the biological characteristics of the tumor cells. We developed a method for monitoring in vivo transgene expression mediated by electroporation (EP) in a tumor model. EP has been used in the laboratory for gene delivery to cells in vitro or in vivo, and is recently receiving much attention in clinical cancer gene therapy. Using ferritin MR transgene reporter coupled with an optical gene reporter, we visualized intratumoral transgene expression by MRI and optical imaging. We also developed a method for targeted detection of mesothelioma by manganese-enhanced MRI (MEMRI). Mesothelioma is mainly caused by exposure to asbestos and is one of the intractable cancers because of the difficulty in early diagnosis and resistance to conventional therapy. Manganese-superoxide dismutase, an antioxidant enzyme cofactored with manganese, is overexpressed in mesothelioma and is therefore an attractive target for mesothelioma detection. We applied MEMRI and in vivo fluorescence imaging to detect mesothelioma stably expressing fluorescence protein in a preclinical animal model. Our study suggested that MEMRI represents a promising approach for a more selective mesothelioma imaging.第8回日本分子イメージング学会総会・学術集

Modulating the splicing activitiy of Tetrahymena ribozyme via RNA self-assembly

The internal guiding sequence(IGS)is normally located at the 5\u27 end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalianc ells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides

Detection of mRNA in mammalian cells with a Sprit Ribozyme Reporter

This communication describes a split RNA reporter strategy for sensing RNAs in mammalian cells. A Tetrahymena group I intron ribozyme reporter was split into two halves, and in the presence of the target RNA, the split reporter assembled into an active complex that trans-spliced and produced a reporter enzyme. We show that this split reporter can sense tumor suppressor p53 mutant mRNA in mammalian cells. This system may open up new avenues for detecting and imaging RNA molecules in vivo

Favorable tumor uptake and nuclear transport of Auger electrons by nuclear targeting with 111In-trastuzumab in an intraperitoneal tumor mouse model

Objectives: The 111In-labeled anti-HER2 antibody trastuzumab modified with a nuclear-localizing sequence (NLS) peptide (111In-trastuzumab-NLS) is a radiopharmaceutical candidate for Auger electron radioimmunotherapy (AE-RIT). However, in vivo action of 111In-trastuzumab-NLS is poorly understood in intraperitoneal tumors. We aimed to elucidate the nuclear targeting activity of 111In-trastuzumab-NLS in a mouse model of intraperitoneal tumors. Methods: Trastuzumab, trastuzumab-NLS-S with shorter NLS peptides, and trastuzumab-NLS-L with longer NLS peptides were tested in an intraperitoneal tumor xenograft. The AE-emitting radionuclide 111In was labeled with these antibodies. The cell binding activity, nuclear importation, and cytotoxicity of those radiolabeled antibodies were examined in human cancer cell lines. Analyses of the biodistribution and in vivo nuclear importation of 111In were conducted in a mouse model.Results: The two111In-trastuzumab-NLS variants delivered the radionuclide into the nucleus more efficiently and had a comparable cytotoxicity to 111In-trastuzumab against human gastric cancer cells, although had a lower cell binding affinity. 111In-trastuzumab-NLS-L exhibited both a superior tumor uptake and in vivo nuclear transportation of the radionuclide than 111In-trastuzumab. Conclusions: Nuclear targeting using 111In-trastuzumab-NLS promotes a more efficient tumor cell uptake and subsequent nuclear translocation of the 111In AE-emitting radionuclide in vivo. This radio-immunoconjugate will likely be an effective agent for HER2-targeting by AE-RIT

HER2高発現胃がん肝転移に対する新規標的α線内用療法

Gastric cancer (GC) is a leading cause of cancer related death worldwide. Because of the asymptomatic at early stage, GC patients often already have distant metastasis at the time of diagnosis, and 4-14% of them are liver metastasis of GC (LMGC) with dismal prognosis. Trastuzumab, a humanized anti-HER2 monoclonal antibody, has been clinically used for the treatment of HER2-positive (HER2+) GC, which account for about 20% of GC. Recently, targeted alpha therapy (TAT) is getting higher attention as a novel therapeutic option for metastasis, benefited from the combination of advantages of antibodies, cancer specific targeting, and alpha particles, high cell killing effects and short range. Astatine 211 (At211) is one of the attractive alpha- emitter for clinical use. In this study, we investigated the therapeutic efficacy and toxicity of TAT with At211-trastuzumab using preclinical HER2+ LMGC mice model. Our results provided the proof of concept that TAT with At211-trastuzumab has high potential as a novel therapeutic option against HER2+ LMGC.第78回日本癌学会学術総

Molecular MR Imaging of Cancer Gene Therapy: Ferritin Transgene Reporter Takes the Stage

Molecular imaging using magnetic resonance (MR) imaging has been actively investigated and made rapid progress in the past decade. Applied to cancer gene therapy, the technique\u27s high spatial resolution allows evaluation of gene delivery into target tissues. Because noninvasive monitoring of the duration, location, and magnitude of transgene expression in tumor tissues or cells provides useful information for assessing therapeutic efficacy and optimizing protocols, molecular imaging is expected to become a critical step in the success of cancer gene therapy in the near future. We present a brief overview of the current status of molecular MR imaging, especially in vivo reporter gene imaging using ferritin and other reporters, discuss its application to cancer gene therapy, and present our research of MR imaging detection of electroporation-mediated cancer gene therapy using the ferritin reporter gene

Generation of nuclear localizing antibody for targeted radionuclide therapy

Radionuclide therapy kills tumor cells by the placement of unsealed therapeutic radionuclide in the tumor for the selective delivery of radiation to tumors. Enhanced delivery of therapeutic radionuclide, especially short-range particle emitters, to tumor cell nucleus may induce an increased numbers of lethal DNA damage and cell death, leading to more effective treatment of tumor. Here we report that we generated trastuzumab (Her) modified with nuclear localization signal (NLS) to enhance the delivery of therapeutic radioisotopes to cell nucleus of HER2-overexpressing tumors. A synthetic 13mer peptide including NLS-motif derived from SV40 large T-antigen was attached to Her, a HER2-targeting antibody, by chemical crosslinking. The nuclear localizing Her antibodies (Her-NLS) labeled with auger electron emitters such as iodine-125 were bound to HER2-overexpressing cancer cells and rapidly internalized in the cells. Her-NLS showed enhancement of nuclear localization compared with Her. Nuclear localizing trastuzumab will be useful for HER2-targeting radionuclide therapy using short-range particle emitters such as auger-electron emitters and alpha particle emitters.第72回日本癌学会学術総

Auger electron radioimmunotherapy using 111In-nuclear localizing anti-HER2 antibody: A cell biological study

Objective: Auger electrons have a potential for effective cell killing when the radionuclide is delivered into cell nucleus and located to close to DNA. The use of nuclear localizing signal (NLS) has been proposed to transport Auger-electron emitters to the cell nucleus of tumor cells. To explore the capability of nuclear localized 111In, an Auger-electron emitter, to bring cell ablation to the targeted human cancer cells overexpressing HER2, we evaluated the cytotoxicity of the 111In-labeled anti-HER2 antibody attached with peptides containing NLS derived from simian virus 40, to human breast cancer cells overexpressing HER2.Materials and methods: Trastuzumab, an anti-HER2 antibody, was conjugated with CHX-A’’-DTPA (DTPA) for 111In labeling and derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) to react with 13-mer peptides containing a NLS sequence (CGYGPKKKRKVGG). Two types of NLS-trastuzumab were generated; one carried approximately 4 NLS peptides per antibody (NLS-trastuzumab-S) and another carried 10 (NLS-trastuzumab-L). NLS-trastuzumab-DTPA conjugates and trastuzumab-DTPA were labelled with 111In. Radiochemical labelling of 111In-NLS-trastuzumab and 111In-trastuzumab was evaluated by HPLC. Nuclear accumulation of 111In was measured in NIH-3T3 cells overexpressing human HER2 by subcellular fraction. Cell survival was evaluated by colony-forming assays in NIH-3T3 cells overexpressing human HER2. Cell viability was tested by dye-based cell viability assays in human mammary epithelial cells (HMEpC) and two types of human mammary carcinoma cells, MCF-7 and SK-BR-3 cells with a low and a high expression of HER2, respectively. DNA damages were examined in SK-BR-3 cells by γ-H2AX immunohistochemistry. Results: Nuclear uptake of 111In was increased 1.5-fold and 1.3-fold in cells treated with 111In-NLS-trastuzumab-L and 111In-NLS-trastuzumab-S compared to 111In-trastuzumab, respectively. The clonogenic survival of NIH-3T3 cells overexpressing human HER2 was decreased when treated with 111In-NLS-trastuzumab-L compared to 111In-trastuzumab. Cell viability was significantly reduced with both of 111In-NLS-trastuzumab-L and S (462.5 and 231.25 kBq) compared to 111In-trastuzumab in SK-BR-3 cells with a high expression of HER2. Both of 111In-NLS-trastuzumab had little or no antiproliferative effects on MCF-7 cells and HMEpC that expressed a low level of HER2 protein. Increased number of γ-H2AX foci were found in SK-BR-3 cells reacted with 111In-NLS-trastuzumab-L compared to 111In-trastuzumab.Conclusions: The cytotoxic and antiproliferative effects of 111In-NLS-trastuzumab are higher than 111In-trastuzumab in HER2-overexpressing human mammary carcinoma cells. These data demonstrate that delivery of 111In into cell nucleus by anti-HER2 antibody harboring NLS is an effective strategy for targeted cell killing of the human cancer cells overexpressing HER2.11th Congress of the World Federation of Nuclear Medicine and Biolog