Noninvasive monitoring of radiation-induced mouse thymic lymphoma by positron emission tomography and magnetic resonance imaging

Here we show in vivo imaging of radiation-induced mouse thymic lymphoma (TL) by positron emission tomography (PET) and magnetic resonance imaging (MRI). Molecular imaging using PET and MRI are widely used not only for clinical cancer diagnostics but also for animal models of cancer. The aim of this study is to establish a method for monitoring mouse thymic lymphoma (TL) development induced by ionizing radiation. For radiation lymphomagenesis, we used fractionated whole body X-ray irradiation protocol and thus irradiated 4 weeks-old C57BL/6 mice at 1.2 Gy weekly for 4 consecutive weeks (total dose: 4.8 Gy). Three to six months after irradiation, affected mice showed body weight loss and difficulty in breathing. To assess the size of thymus of these affected mice, T1-weighted images were acquired by high magnetic field animal 7T-MRI scanner. PET with [18F]fluorodeoxyglucose (FDG) was also applied to mice with an enlarged thymus. FDG (~4MBq) was administrated into an affected mouse intravenously and a 10-min emission scan was started at 50 min after injection using Siemens Inveon small animal PET systems. FDG was highly accumulated in the affected thymus, suggesting that the lesions were metabolically active and malignant. Histopathological studies confirmed that they were T-cell lymphomas. Molecular imaging using PET and MRI allows us to trace the development and progression of mouse TL in living animals. Therefore, we propose that these techniques are powerful tools for detecting radiation-induced thymic lymphoma and would offer a new opportunity to understand the molecular and cellular mechanism(s) of radiation-induced mouse thymic lymphoma development.14th International Congress of Radiation Research(ICRR’2011

Noninvasive detection of early pathophysiological changes of irradiated bone marrow in mouse radiation carcinogenesis by diffusion-weighted imaging

Ionizing radiation is capable of causing various tumors in both mouse and human and thus is a well-known environmental carcinogen. Mouse models have been invaluable to elucidate the mechanism(s) of radiation carcinogenesis. To date, many rodent models have been developed for this purpose. Fractionated whole-body X-ray irradiation (FX) efficiently induces thymic lymphoma (TL) in C57BL/6 mice, which are susceptible to radiation-induced thymic lymphoma (RITL) development, at 3 to 10 months after the last irradiation. This model has long been used as a classical method to study radiation carcinogenesis. In this model, although TL is arising from the thymus, many previous studies have shown that cellular damages of the bone marrow (BM) by FX are critically involved in TL development. BM changes after FX has been examined by histological analyses so far. However, analyzing spatiotemporal dynamics of pathophysiological BM changes induced by FX has been hampered by the lack of a reliable method to monitor them noninvasively. The aim of this study is to explore methods for noninvasive monitoring of the pathophysiological changes of the X-ray irradiated BM in mouse radiation carcinogenesis. Here we report that diffusion-weighted imaging (DWI) noninvasively detects early pathophysiological changes of FX-treated BM in mouse radiation carcinogenesis. Four-weeks-old C57BL/6 mice were X-irradiated with 4 doses of 1.6 Gy (total dose 6.4 Gy) each given at 7-days intervals and the pathophysiological changes of their BM were evaluated by MRI and histopathological analyses 3 days after each irradiation. T2 values were not significantly different between FX-treated and unirradiated normal BM. Interestingly, the apparent diffusion coefficient (ADC) measured by DWIs, which quantitatively represents the diffusivity of protons in tissue, significantly increased in FX-treated BM compared to those in unirradiated normal BM from the first to third irradiation (p < 0.001). These ADC changes were correlated with and well accounted for by the pathological findings, likely reflecting reduced cellularity and altered vascularity in the irradiated BM. These data indicate that DWI is useful in quantitatively evaluating dynamic changes of FX-treated BM in living subjects and may help elucidate the pathogenesis of RITL. In addition, this study further supports the notion that DWI is able to detect altered cellularity and thus may be applied to evaluate BM suppression which often is the limiting factor in clinical fractionated radiotherapy.2012　World Molecular Imaging Congres

Microarray analysis of human breast cancer cells treated with 111In-trastuzumab modified with nuclear localizing signal peptides

111In-labeled trastuzumab modified with nuclear localizing signal (NLS) peptides (111In-trastuzumab-NLS) is a promising therapeutic agent for cancer cells overexpressing HER2 because it can efficiently deliver an Auger electron emitter 111In into tumor cell nucleus and 111In is highly toxic when it decays in the nucleus. However, to further improve its therapeutic efficacy, better understanding of cellular responses to 111In-trastuzumab-NLS is required. The aim of this study is to identify gene expression signatures of cells treated with 111In-trastuzumab-NLS using microarray technology.\nTrastuzumab modified with approximately 10 NLS peptides per antibody was generated. Microarray analyses were performed using RNAs isolated from human breast cancer SKBR3 cells treated for 7 days with the treatments including 231.25 MBq of trastuzumab-NLS. Untreated SKBR3 cells were used as control. The identified genes were imported to the Ingenuity Pathway Analysis (IPA) to characterize gene expression and biofunctional changes induced by 111In-trastuzumab-NLS.\nMicroarray data showed that more than 1,200 probes were up-regulated and down-regulated in treatment of 111In-trastuzumab-NLS in comparison with control and revealed that 338 and 520 probes were specifically up-regulated and down-regulated in treatment of 111In-trastuzumab-NLS, respectively. IPA detected molecular and cellular functions affected by treatment of 111In-trastuzumab-NLS, including cell-to-cell signaling and interaction and cell death/survival.\nOur microarray study identified gene expression signatures of human breast cancer cells treated with 111In-trastuzumab-NLS. These data may be useful for enhancing therapeutic efficacy of 111In-trastuzumab-NLS.2015 Society of Nuclear Medicine and Molecular Imaging Annual Meetin

Locoregional therapy with α-emitting trastuzumab peritoneal metastasis of human epidermal growth factor receptor 2-positive gastric cancer in mice

Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found, however, that locoregionally administered trastuzumab armed with astatine-211 (211At-trastuzumab) is effective against human epidermal growth factor receptor 2 (HER2)-positive PMGC in a xenograft mouse model. We first observed that 211At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in s.c. tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test a-particle radioimmunotherapy with 211At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the i.p. administration of 211At-trastuzumab (1 MBq) was a more efficient means of delivery of 211At into metastatic tumors than i.v.injection; the maximum tumor uptake with i.p. administration was over 60% injected dose per gram of tissue (%ID/g) compared to approximately 18%ID/g with i.v. injection. Surprisingly, a single i.p. injection of 211At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA doublestrand breaks, and to drastically reduce the tumor burden in another three mice.No bodyweight loss, leukocytopenia, or significant biochemical changes in liveror kidney function were observed in the treatment group. Accordingly, locoregionally administered 211At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211At-trastuzumab may offer a new treatment option for HER2-positive PMGC

Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene

H-ferritin (HF) is a core subunit of the iron storage protein ferritin, and plays a central role in the regulation of cellular iron homeostasis. Recent studies revealed that ferritin and HF are involved in a wide variety of iron-independent functions, including regulating biological processes during physiological and pathological conditions, and can be overexpressed in some human diseases. To investigate the in vivo function of HF, we generated transgenic (tg) mice overexpressing the human HF gene (hHF-tg). We established two independent hHF-tg mouse lines. Although both lines of hHF-tg mice were viable, they showed reduced body size compared to wild-type (WT) mice at 4&#8211;12 weeks of age. Serum iron concentration and blood parameters of hHF-tg mice such as hemoglobin and red blood cell counts were comparable to those of WT mice. At 3&#8211;5 weeks of age, hHF-tg mice exhibited temporary loss of coat hair on the trunk, but not on the head or face. Histological analyses revealed that although initial hair development was normal, hHF-tg mice had epidermal hyperplasia with hyperkeratosis, dilated hair follicles, bended hair shafts and keratinous debris during the hairless period. In conclusion, we showed that hHF-tg mice exhibited mild growth retardation and temporary hairless phenotype. Our findings highlight the physiological roles of HF and demonstrate that hHF-tg mice are useful for understanding the in vivo functions of HF

Manganese-enhanced MRI as a molecular imaging of mesothelioma

Purpose: To develop a selective diagnostic imaging of mesothelioma by targeting disease-specific biomarkers.Materials and Methods: All experiments were approved by the Institutional Animal Care and Use Committee. Manganese-superoxide dismutase (Mn-SOD) expression was evaluated in one human mesothelial cells and five human malignant mesothelioma (MM) cells. Mn accumulation in NCI-H226 and MSTO-211H MM cells was examined when loaded with Mn. Mn-enhanced magnetic resonance imaging (MEMRI) of those cell pellets and subcutaneous tumors were conducted using 7 Tesla-MRI. Signal enhancement of H226 xenografted pleural tumors was determined by MEMRI with manganese dipyridoxyl diphosphate (MnDPDP) as well as manganese chloride (MnCl2). Results: We found that 4 of 5 human MM cells over-expressed Mn-SOD protein compared to mesothelial cells, and that H226 MM cells highly expressed Mn-SOD and augmented Mn accumulation when loaded with MnCl2. The cells showed marked T1-signal enhancement on in vitro MRI after incubation with MnCl2. H226 subcutaneous tumors were preferentially enhanced compared to MSTO-211H tumors, which had less Mn-SOD expression, in MnCl2-enhanced T1-weighted MR image (T1WI). H226 pleural tumors were markedly enhanced and readily detected by MEMRI using MnDPDP as well as MnCl2.Conclusion: We propose that MEMRI can be a potentially powerful method for non-invasive detection of MM with high spatial resolution and marked signal enhancement by targeting Mn-SOD.World Molecular Imaging Congress 201

Preclinical evaluation of At-211-labeled trastuzumab, an alpha-particle radioimmunotherapeutic agent, for the treatment of gastric cancer.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths in the world. Cytotoxic chemotherapeutic or molecularly targeted therapy agents for inoperable/progressive GC are currently available, for example, a humanized anti-HER2 antibody trastuzumab has been used for the treatment of HER2-positive GC patients. However, the therapeutic efficacy of pharmacotherapy is limited so far and the prognosis of patients with inoperable/progressive GC is still poor. To improve the clinical outcome of GC patients, novel antitumor strategies are required. Radioimmunotherapy (RIT) combines radiation therapy and immunotherapy, and has been emerged as an effective cancer therapy for non-Hodgkin’s lymphoma. RIT uses target-specific antibody conjugated to radioisotopes emitting particle radiation such as alpha- or beta-particles to achieve the targeted cell killing. Astatine-211 (At-211), an alpha-particle emitter, is of considerable interest for RIT because it deposits a large amount of energy within a few cell diameters and is highly cytotoxic by inducing irreparable DNA injuries. In this study, we investigated the efficacy of alpha-particle RIT using trastuzumab conjugated to At-211 ([At-211]trastuzumab) for the treatment of HER2-positive GC in a mouse xenograft model. Trastuzumab was conjugated with N-succinimidyl 3-trimethylstannyl-benzonate for At-211 labeling. The immunoconjugate (200 μg) was then labeled with At-211 (18.5-74 MBq), and the labeled antibody was purified by spin column. Specific activity of [At-211]trastuzumab was 239.5 ± 139.5 MBq/mg. Radiochemical purity was typically over 96% as determined by methanol precipitation. The stability of [At-211]trastuzumab in serum was 88.7% until 24 hours. The binding of [At-211]trastuzumab to HER2-positive human GC cells NCI-N87 (5 x 106 cells) was 72.8 ± 10.7% of input radioactivity, whereas 1.8 ± 0.5% to HER2-negative human GC cells MKN45 (5 x 106 cells). These results were comparable to the results of other radiolabeled trastuzumab antibodies such as an Indium-111 labeled trastuzumab. In vitro cytotoxicity studies showed that [At-211]trastuzumab (0.37 and 1.85 kBq) had a higher cytotoxicity to NCI-N87 cells compared to free form of At-211 and the equivalent protein amount of unlabeled trastuzumab. To investigate the effects of [At-211]trastuzumab on the tumor growth in vivo and bio-distribution of the radioimmunoconjugate, we established xenografts of NCI-N87 GC cells in nude mice. Bio-distribution studies revealed that the tumor uptake of [At-211]trastuzumab was increased over time, being 12.5 ± 4.0% injection dose/gram of tissue weight at 24 hours after injection of 0.5 MBq of [At-211]trastuzumab. A single administration of 0.5 MBq of [At-211]trastuzumab by intravenous injection significantly suppressed the tumor growth of subcutaneous xenografted tumors and prolonged the survival time compared to the xenograft mice injected with PBS or unlabeled trastuzumab. During the treatment, the counts of white blood cells and body weight in mice injected with 0.5 MBq of [At-211]trastuzumab temporarily decreased around 7 days after injection. However, the decrease was gradually recovered and they reached the comparable levels of control mice thereafter. Taken together, these findings suggest that [At-211]trastuzumab shows antitumor effects against HER2-positive human GC cells in vivo with acceptable toxicity and is a promising radioimmunotherapeutic agent for pharmacotherapy in HER2-positive GC.Tenth AACR-JCA Joint Conference on Breakthroughs in Cancer Research: From Biology to Therapeutic