Yukie Morokoshi

Molecular imaging of mesothelioma by detection of manganese-superoxide dismutase activity using manganese-enhanced magnetic resonance imaging

Malignant mesothelioma (MM) is a fatal malignancy with a rapidly increasing incidence in industrialized countries because of the widespread use of asbestos in the past centuries. Early diagnosis of MM is critical for a better prognosis, but this is often difficult because of the lack of disease‐specific diagnostic imaging. Here, we report that manganese‐enhanced magnetic resonance imaging (MEMRI) represents a promising approach for a more selective mesothelioma imaging by monitoring a high‐level expression of manganese‐superoxide dismutase (Mn‐SOD), which is observed in many MM. We found that most human MM cells overexpressed Mn‐SOD protein compared with human mesothelial cells and that NCI‐H226 human MM cells highly expressed Mn‐SOD and augmented Mn accumulation when loaded with manganese chloride (MnCl2). The cells showed marked T1‐signal enhancement on in vitro MRI after incubation with MnCl2 because of the T1 shortening effect of Mn2+. H226 subcutaneous tumor was preferentially enhanced compared with a lung adenocarcinoma cell tumor and another human MM cell tumor in MnCl2‐enhanced T1‐weighted MR image (T1WI), correlating with their respective Mn‐SOD expression levels. Moreover, in a more clinically relevant setting, H226 xenografted pleural tumor was markedly enhanced and readily detected by MEMRI using manganese dipyridoxyl diphosphate (MnDPDP), a clinically used contrast agent, as well as MnCl2. Therefore, we propose that MEMRI can be a potentially powerful method for noninvasive detection of MM, with high spatial resolution and marked signal enhancement, by targeting Mn‐SOD.

Controlled administration of penicillamine reduces radiation exposure in critical organs during 64Cu-ATSM internal radiotherapy: a novel strategy for liver protection.

Purpose 64Cu-diacetyl-bis (N 4-methylthiosemicarbazone) (64Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with 64Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during 64Cu-ATSM IRT. Methods Biodistribution was evaluated in HT-29 tumor-bearing mice injected with 64Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between 64Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after 64Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic 64Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses. Results Penicillamine reduced 64Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after 64Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic 64Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine. Conclusions We developed a novel strategy to reduce radiation exposure in critical organs during 64Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other 64Cu-labeled compounds.

H-ferritin overexpression promotes radiation-induced leukemia/lymphoma in mice

H-ferritin (HF) is a core subunit of the iron storage protein ferritin and is related to the pathogenesis of malignant diseases. HF overexpression is present in human hematologic malignancies, suggesting that HF overexpression may contribute to the development of hematologic cancers. However, in vivo evidence that HF is directly linked to hematologic tumorigenesis has not yet been shown. In this study, we show that transgenic (tg) mice overexpressing the human HF gene (hHF-tg) developed aggressive radiation-induced thymic lymphoma/leukemia (TL) compared with wild-type (WT) mice, providing evidence that HF overexpression promotes leukemia/lymphomagenesis. Fractionated X-irradiation of hHF-tg mice caused a higher incidence and earlier onset of TL compared with WT mice. Immunological and pathological features of TLs were similar in both groups. However, proliferative activity of hHF-tg lymphoma cells was higher than that of WT lymphoma cells, and microarray analyses revealed that some leukemia/lymphoma-related genes were differentially expressed in hHF-tg TLs compared with WT TLs. To investigate whether cell damage induced by irradiation is related to leukemia/lymphomagenesis, we evaluated apoptotic levels in the thymus and bone marrow (BM) of hHF-tg and WT groups after fractionated X-irradiation. Apoptosis was augmented in the hHF-tg BM, but not in the thymus, compared with the WT BM, suggesting a possible linkage between increased BM apoptosis by HF overexpression and accelerated radiation-induced TL development. Our findings indicate that HF overexpression is closely related to the development of leukemia/lymphoma, which could have implications for the prevention of malignant hematologic diseases.

Transcriptomic Signatures of Auger Electron Radioimmunotherapy Using Nuclear Targeting 111In-Trastuzumab for Potential Combination Therapies

(111)In-labeled trastuzumab modified with nuclear localizing signal (NLS) peptides ((111)In-trastuzumab-NLS) efficiently delivers an Auger electron (AE) emitter (111)In into the cell nucleus and is thus a promising radiopharmaceutical in AE radioimmunotherapy (AE-RIT) for targeted killing of HER2-positive cancer. However, further improvement of its therapeutic efficacy is required. In this study, the authors show a transcriptomic approach to identify potential targets for enhancing the cytotoxic effects of (111)In-trastuzumab-NLS. They generated two types of (111)In-trastuzumab-NLS harboring different numbers of NLS peptides, (111)In-trastuzumab-NLS-S and -L. These radioimmunoconjugates (230 and 460 kBq) showed a significant higher cytotoxicity to SKBR3 human breast cancer cells overexpressing HER2 compared to (111)In-trastuzumab. Microarray analysis revealed that NF-kB-related genes (38 genes) were significantly changed in transcription by (111)In trastuzumab-NLS-L (230 kBq) treatment. Quantitative reverse transcription polymerase chain reaction confirmed the microarray data by showing transcriptional alternation of selected NF-κB target genes in cells treated with (111)In-trastuzumab-NLS-L. Interestingly, bortezomib, a drug known as a NF-κB modulator, significantly enhanced the cytotoxicity of (111)In-trastuzumab-NLS-L in SKBR3 cells. Taken together, the transcriptome data suggest the possibility that the modulation of NF-kB signaling activity is a molecular signature of (111)In-trastuzumab-NLS and coadministration of bortezomib may be efficacious in enhancement of AE-RIT with (111)In-trastuzumab-NLS.

Evaluation of ferritin-overexpressing brain in newly developed transgenic mice

Aberrant expression of ferritin, a major iron-binding protein, has shown to be involved in neurodegenerative diseases. In this study, we generated transgenic (Tg) mice of human ferritin heavy chain (FTH) gene and investigated the effects of ferritin overexpression in FTH-Tg brain by (1)H-MRI and (1)H-MRS. The mice displayed no apparent neurological symptoms, and no specific morphological and T(2) alterations were found in the brain by MRI, and not even by histological studies. (1)H-MRS, however, revealed that some metabolic markers were significantly altered in FTH-Tg brains compared to wild-type control brains, such as decreases in myo-inositol and glutamine, and an increase in lactate. Our present studies suggested that despite the absence of neurological, morphological, T(2), and histological signatures, brain metabolisms were significantly affected in FTH-Tg mice. This study also highlights the usefulness of (1)H-MRS in the analysis of transgenic mouse models.

Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging

Radiation‐induced thymic lymphoma (RITL) in mice is induced by fractionated whole‐body X‐irradiation (FX) and has served as a useful model for studying radiation carcinogenesis. In this model, the initial postirradiation cellular events in the thymus and bone marrow (BM) are critically important for tumorigenesis, and BM transplantation (BMT) prevents RITL. However, direct assessment of these events is so far restricted by the lack of noninvasive monitoring techniques. Here, we have developed positron emission tomography (PET) and magnetic resonance imaging (MRI) methods to quantify the events critical for RITL development and the effects of BMT in living animals. Apparent diffusion coefficients (ADCs) were calculated from diffusion‐weighted MRI to evaluate the changes in the BM of mice receiving FX. ADC values dramatically changed in the irradiated BM, corresponding to pathological findings of the irradiated BM, returning to normal levels following BMT sooner than with spontaneous recovery. PET with 4ʹ‐[methyl‐11C]thiothymidine, a novel tracer for cell proliferation, revealed that the irradiated thymus showed significantly higher tracer uptake than the unirradiated thymus 1 week after FX. Interestingly, its increased uptake was completely abolished by BMT, even with very few donor‐derived cells in the thymus. Thereafter, the thymus receiving BMT had significantly increased tracer uptake. These findings suggest that BMT first suppresses FX‐induced aberrant thymocyte proliferation and then accelerates thymic regeneration. This study demonstrates the feasibility of using PET and MRI for noninvasive monitoring of tumorigenic cellular processes in an animal model of radiation‐induced cancer.

Locoregional therapy with α‐emitting trastuzumab against 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 α‐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 double‐strand breaks, and to drastically reduce the tumor burden in another three mice. No bodyweight loss, leukocytopenia, or significant biochemical changes in liver or 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.

Evidence of local concentration of α-particles from 211At-labeled antibodies in liver metastasis tissue

We investigated the local concentration of α-particles from 211At-labeled trastuzumab antibodies against human epidermal growth factor receptor type 2 antigens in liver metastasis tissue of mice. Methods: Mice carrying metastatic cancer in their liver were injected with 211At-agent. After 12 h, the liver was removed and sliced, and 2 tissue samples of liver tissues without lesions and one containing metastatic lesions were mounted on the CR-39 plastic nuclear track detector. Microscope images of the tissues on the CR-39 were acquired. After irradiation for 31 h, the tissues were removed from the CR-39. A microscope image of α-particle tracks on the CR-39 was acquired after chemical etching. The positions of each tissue sample and the emitted α-particle tracks were adjusted to the same coordinates. Results: The positional distribution of α-particle tracks emitted from 211At was consistent within the tissue. The α-particle tracks were mainly allocated in the tumor region of the tissue. The absorbed dose in individual cells segmented by 10-μm intervals was obtained by the spectroscopic analysis of the linear-energy-transfer spectrum. The concentration efficiency—the track density ratio of α-particle tracks in the necrotized tissue, which was the tumor region, to the normal tissue—was found to be 6.0 ± 0.2. In the tumor region, the high–linear-energy-transfer α-particles deposited a large enough dose to cause lethal damage to the cancer cells. Conclusion: The total absorbed dose ranged from 1 to 7 Gy with a peak at around 2 Gy, which would correspond to a 2–3 times higher biologically equivalent dose because of the high relative biological effectiveness of the α-particles emitted from 211At.

α-particle therapy for synovial sarcoma in the mouse using an astatine-211-labeled antibody against frizzled homolog 10

Synovial sarcoma (SS) is a rare yet refractory soft‐tissue sarcoma that predominantly affects young adults. We show in a mouse model that radioimmunotherapy (RIT) with an α‐particle emitting anti‐Frizzled homolog 10 (FZD10) antibody, synthesized using the α‐emitter radionuclide astatine‐211 (211At‐OTSA101), suppresses the growth of SS xenografts more efficiently than the corresponding β‐particle emitting anti‐FZD10 antibody conjugated with the β‐emitter yettrium‐90 (90Y‐OTSA101). In biodistribution analysis, 211At was increased in the SS xenografts but decreased in other tissues up to 1 day after injection as time proceeded, albeit with a relatively higher uptake in the stomach. Single 211At‐OTSA101 doses of 25 and 50 μCi significantly suppressed SS tumor growth in vivo, whereas a 50‐μCi dose of 90Y‐OTSA101 was needed to achieve this. Importantly, 50 μCi of 211At‐OTSA101 suppressed tumor growth immediately after injection, whereas this effect required several days in the case of 90Y‐OTSA101. Both radiolabeled antibodies at the 50‐μCi dosage level significantly prolonged survival. Histopathologically, severe cellular damage accompanied by massive cell death was evident in the SS xenografts at even 1 day after the 211At‐OTSA101 injection, but these effects were relatively milder with 90Y‐OTSA101 at the same timepoint, even though the absorbed doses were comparable (3.3 and 3.0 Gy, respectively). We conclude that α‐particle RIT with 211At‐OTSA101 is a potential new therapeutic option for SS.