基本情報

所属

• 遺伝子病制御研究所　病因研究部門

職名

• 特任講師

学位

• 博士(医学)（2017年03月 東京大学）

J-Global ID

• 201801010953174492

研究キーワード

• 合成生物学   組織透明化   

研究分野

• ライフサイエンス / 薬理学
• ライフサイエンス / 実験病理学
• ライフサイエンス / システムゲノム科学

職歴

• 2021年05月 - 現在 北海道大学 遺伝子病制御研究所 特任講師
• 2020年04月 - 2021年04月 東京大学 医学系研究科分子病理学分野 特任研究員
• 2017年04月 - 2020年03月 東京大学医学系研究科 分子病理学分野 日本学術振興会 特別研究員
• 2014年04月 - 2017年03月 東京大学医学系研究科 システムズ薬理学教室 日本学術振興会 特別研究員
• 2013年04月 - 2014年03月 東京大学医学系研究科 システムズ薬理学教室 博士課程
• 2007年04月 - 2013年03月 名古屋大学 医学部

研究活動情報

論文

• DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation

  Seiichiro Naito, Hiroki Tanaka, Jing-Jing Jiang, Masato Tarumi, Ari Hashimoto, Yuki Tanaka, Kaoru Murakami, Shimpei I. Kubota, Shintaro Hojyo, Shigeru Hashimoto, Masaaki Murakami

  Biochemical and Biophysical Research Communications 703 149666 - 149666 2024年04月 [査読有り]
  
• Click3D: A whole-organ 3D imaging method utilizing tissue clearing-compatible click reaction

  Iori Tamura, Daichi M. Sakamoto, Bo Yi, Yutaro Saito, Naoki Yamada, Jumpei Morimoto, Yoichi Takakusagi, Masafumi Kuroda, Shimpei I. Kubota, Hiroshi Harada, Kazuki Tainaka, Shinsuke Sando

  2024年02月12日 

  Click chemistry stands as an invaluable chemical technology, offering a multitude of applications through highly selective and efficient bioorthogonal reactions under biological environments. In the realm of bioimaging, pre-targeting strategies have often been employed, utilizing click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated state-of-the-art tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution 3D fluorescence imaging of the target molecules. Nevertheless, these techniques have faced a challenge in terms of limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thorough staining of whole tissues and whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed click staining depth assay. The Click3D protocol, optimized by incorporating these conditions, exhibits a substantially greater staining depth compared to conventional methods. Through the implementation of Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. Remarkably, we have also accomplished whole-brain imaging of hypoxia by employing the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier. The Click3D method, leveraging the versatility of click chemistry, is a foundational technology and is expected to have diverse applications employing various clickable probes.
• Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing

  Daichi M. Sakamoto, Iori Tamura, Bo Yi, Sho Hasegawa, Yutaro Saito, Naoki Yamada, Yoichi Takakusagi, Shimpei I. Kubota, Minoru Kobayashi, Hiroshi Harada, Kenjiro Hanaoka, Masayasu Taki, Masaomi Nangaku, Kazuki Tainaka, Shinsuke Sando

  ACS Nano 18 6 5167 - 5179 2024年02月01日 [査読有り]
  
• High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore

  Kaoru Murakami, Shimpei I. Kubota, Kumiko Tanaka, Hiroki Tanaka, Keiichiroh Akabane, Rigel Suzuki, Yuta Shinohara, Hiroyasu Takei, Shigeru Hashimoto, Yuki Tanaka, Shintaro Hojyo, Osamu Sakamoto, Norihiko Naono, Takayui Takaai, Kazuki Sato, Yuichi Kojima, Toshiyuki Harada, Takeshi Hattori, Satoshi Fuke, Isao Yokota, Satoshi Konno, Takashi Washio, Takasuke Fukuhara, Takanori Teshima, Masateru Taniguchi, Masaaki Murakami

  Lab on a Chip 23 22 4909 - 4918 2023年11月 [査読有り]
   

  Our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants including the next pandemic.
• Computer model of IL-6-dependent rheumatoid arthritis in F759 mice

  Reiji Yamamoto, Satoshi Yamada, Toru Atsumi, Kaoru Murakami, Ari Hashimoto, Seiichiro Naito, Yuki Tanaka, Izuru Ohki, Yuta Shinohara, Norimasa Iwasaki, Akihiko Yoshimura, Jing-Jing Jiang, Daisuke Kamimura, Shintaro Hojyo, Shimpei I Kubota, Shigeru Hashimoto, Masaaki Murakami

  International Immunology 2023年05月25日 

  Abstract The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.
• GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation

  Shiina Matsuyama, Reiji Yamamoto, Kaoru Murakami, Nobuhiko Takahashi, Rieko Nishi, Asuka Ishii, Junko Nio-Kobayashi, Nobuya Abe, Kumiko Tanaka, Jing-Jing Jiang, Tadafumi Kawamoto, Toshihiko Iwanaga, Yuta Shinohara, Takeshi Yamasaki, Izuru Ohki, Shintaro Hojyo, Rie Hasebe, Shimpei I. Kubota, Noriyuki Hirata, Daisuke Kamimura, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami

  The Journal of Immunology 211 1 34 - 42 2023年05月22日 

  Abstract We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common β chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.
• Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

  Hajime Senjo, Shinpei Harada, Shimpei I Kubota, Yuki Tanaka, Takahiro Tateno, Zixuan Zhang, Satomi Okada, Xuanzhong Chen, Ryo Kikuchi, Naoki Miyashita, Masahiro Onozawa, Hideki Goto, Tomoyuki Endo, Yuta Hasegawa, Hiroyuki Ohigashi, Takahide Ara, Yoshinori Hasegawa, Masaaki Murakami, Takanori Teshima, Daigo Hashimoto

  Blood 2023年05月22日 

  Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal-Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory-Tex), expressing both inhibitory receptors and effector molecules, into terminal-Tex, and inhibited tolerance induction. Adoptive transfer of transitory-Tex, but not terminal-Tex, into secondary recipients developed chronic GVHD. Transitory-Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory-Tex, not terminal-Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.
• In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method.

  Fayrouz Naim, Rie Hasebe, Shintaro Hojyo, Yukatsu Shichibu, Asuka Ishii, Yuki Tanaka, Kazuki Tainaka, Shimpei I Kubota, Katsuaki Konishi, Masaaki Murakami

  Bio-protocol 13 7 e4644  2023年04月05日 

  Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.
• Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients

  Takeshi Yamasaki, Noriyuki Nagata, Toru Atsumi, Rie Hasebe, Yuki Tanaka, Izuru Ohki, Shimpei Kubota, Yuta Shinohara, Yong Bin Teoh, Nozomu Yokoyama, Noboru Sasaki, Kensuke Nakamura, Hiroshi Ohta, Takehiko Katsurada, Yoshihiro Matsuno, Shintaro Hojyo, Shigeru Hashimoto, Mitsuyoshi Takiguchi, Masaaki Murakami

  International Immunology 35 7 313 - 326 2023年03月18日 

  Abstract Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn’s disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.
• Combination therapy with bevacizumab and a CCR2 inhibitor for human ovarian cancer: An in vivo validation study

  Tianyue Zhai, Takashi Mitamura, Lei Wang, Shimpei I. Kubota, Masaaki Murakami, Shinya Tanaka, Hidemichi Watari

  Cancer Medicine 12 8 9697 - 9708 2023年02月22日
• Dupuytren’s contracture-associated SNPs increase SFRP4 expression in non-immune cells including fibroblasts to enhance inflammation development

  Hiroaki Kida, Jing-Jing Jiang, Yuichiro Matsui, Ikuko Takahashi, Rie Hasebe, Daisuke Kawamura, Takeshi Endo, Hiroki Shibayama, Makoto Kondo, Yasuhiko Nishio, Kinya Nishida, Yoshihiro Matsuno, Tsukasa Oikawa, Shimpei I Kubota, Shintaro Hojyo, Norimasa Iwasaki, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami

  International Immunology 35 7 303 - 312 2023年01月31日 [査読有り]
   

  Abstract Dupuytren’s contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and βTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.
• An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis.

  Kei Takahashi, Ko Abe, Shimpei I Kubota, Noriaki Fukatsu, Yasuyuki Morishita, Yasuhiro Yoshimatsu, Satoshi Hirakawa, Yoshiaki Kubota, Tetsuro Watabe, Shogo Ehata, Hiroki R Ueda, Teppei Shimamura, Kohei Miyazono

  Nature communications 13 1 5239 - 5239 2022年09月12日 

  The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.
• A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

  Adam K Glaser, Kevin W Bishop, Lindsey A Barner, Etsuo A Susaki, Shimpei I Kubota, Gan Gao, Robert B Serafin, Pooja Balaram, Emily Turschak, Philip R Nicovich, Hoyin Lai, Luciano A G Lucas, Yating Yi, Eva K Nichols, Hongyi Huang, Nicholas P Reder, Jasmine J Wilson, Ramya Sivakumar, Elya Shamskhou, Caleb R Stoltzfus, Xing Wei, Andrew K Hempton, Marko Pende, Prayag Murawala, Hans-Ulrich Dodt, Takato Imaizumi, Jay Shendure, Brian J Beliveau, Michael Y Gerner, Li Xin, Hu Zhao, Lawrence D True, R Clay Reid, Jayaram Chandrashekar, Hiroki R Ueda, Karel Svoboda, Jonathan T C Liu

  Nature methods 19 613 - 619 2022年05月11日 [査読有り][通常論文]
   

  Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
• Visualization of the cancer cell cycle by tissue-clearing technology using the Fucci reporter system.

  Kei Takahashi, Ryo Tanabe, Shogo Ehata, Shimpei I Kubota, Yasuyuki Morishita, Hiroki R Ueda, Kohei Miyazono

  Cancer science 112 9 3796 - 3809 2021年06月19日 

  Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.
• Heterogenous expression of endoglin marks advanced renal cancer with distinct tumor microenvironment fitness.

  Yusaku Momoi, Jun Nishida, Kosuke Miyakuni, Masafumi Kuroda, Shimpei I Kubota, Kohei Miyazono, Shogo Ehata

  Cancer science 112 8 3136 - 3149 2021年06月06日 

  Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor β and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.
• Whole-organ analysis of TGF-β-mediated remodelling of the tumour microenvironment by tissue clearing

  Shimpei I. Kubota, Kei Takahashi, Tomoyuki Mano, Katsuhiko Matsumoto, Takahiro Katsumata, Shoi Shi, Kazuki Tainaka, Hiroki R. Ueda, Shogo Ehata, Kohei Miyazono

  Communications Biology 4 1 294 - 294 2021年03月 [査読有り]
   

  Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-β (TGF-β) in cancer metastasis. TGF-β-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-β-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-β-stimulated cancer cells, suggesting that TGF-β might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.
• Protocol for Imaging and Analysis of Mouse Tumor Models with CUBIC Tissue Clearing

  Kei Takahashi, Shimpei I. Kubota, Shogo Ehata, Hiroki R. Ueda, Kohei Miyazono

  STAR Protocols 1 3 100191 - 100191 2020年12月 [査読有り]
  
• Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution

  Shimpei I. Kubota, Kei Takahashi, Jun Nishida, Yasuyuki Morishita, Shogo Ehata, Kazuki Tainaka, Kohei Miyazono, Hiroki R. Ueda

  CELL REPORTS 20 1 236 - 250 2017年07月 [査読有り][通常論文]
   

  Stochastic and proliferative events initiated from a single cell can disrupt homeostatic balance and lead to fatal disease processes such as cancer metastasis. To overcome metastasis, it is necessary to detect and quantify sparsely distributed metastatic cells throughout the body at early stages. Here, we demonstrate that clear, unobstructed brain/ body imaging cocktails and computational analysis (CUBIC)-based cancer (CUBIC-cancer) analysis with a refractive index (RI)=optimized protocol enables comprehensive cancer cell profiling of the whole body and organs. We applied CUBIC-cancer analysis to 13 mouse models using nine cancer cell lines and spatiotemporal quantification of metastatic cancer progression at single-cell resolution. CUBIC-cancer analysis suggests that the epithelial-mesenchymal transition promotes not only extravasation but also cell survival at metastatic sites. CUBIC-cancer analysis is also applicable to pharmacotherapeutic profiling of anti-tumor drugs. CUBIC-cancer analysis is compatible with in vivo bioluminescence imaging and 2D histology. We suggest that a scalable analytical pipeline with these three modalities may contribute to addressing currently incurable metastatic diseases.
• Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling

  Kazuki Tainaka, Akihiro Kuno, Shimpei I. Kubota, Tatzya Murakami, Hiroki R. Ueda

  ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 32 32 713 - 741 2016年 [査読有り][通常論文]
   

  Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.
• Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

  Kazuki Tainaka, Shimpei I. Kubota, Takeru Q. Suyama, Etsuo A. Susaki, Dimitri Perrin, Maki Ukai-Tadenuma, Hideki Ukai, Hiroki R. Ueda

  CELL 159 4 911 - 924 2014年11月 [査読有り][通常論文]
   

  The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.
• Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse

  Keisuke Kuroda, Shinnosuke Yamada, Motoki Tanaka, Michiro Iizuka, Hisashi Yano, Daisuke Mori, Daisuke Tsuboi, Tomoki Nishioka, Takashi Namba, Yukihiko Iizuka, Shimpei Kubota, Taku Nagai, Daisuke Ibi, Rui Wang, Atsushi Enomoto, Mayu Isotani-Sakakibara, Naoya Asai, Kazushi Kimura, Hiroshi Kiyonari, Takaya Abe, Akira Mizoguchi, Masahiro Sokabe, Masahide Takahashi, Kiyofumi Yamada, Kozo Kaibuchi

  HUMAN MOLECULAR GENETICS 20 23 4666 - 4683 2011年12月 [査読有り][通常論文]
   

  Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N-and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of similar to 100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

講演・口頭発表等

• 組織透明化関連で自動化できたらいいなと思っていること

  久保田晋平

  ラボラトリーオートメーション月例勉強会 2023年06月
• 光シート顕微鏡を用いた三次元病理学解析

  久保田晋平, 村上正晃

  光シート顕微鏡ワークショップ 2022年11月
• 組織透明化技術を基盤とした三次元病理学解析  [招待講演]

  久保田晋平

  異分野融合研究セミナー iSeminar 2022年11月
• 組織透明化による一細胞解像度での病理学解析  [通常講演]

  久保田晋平

  第81回日本癌学会 2022年09月
• ゲートウェイ反射による組織特異的な炎症性疾患の誘導機構

  久保田晋平, 長谷部理絵, 村上薫, 田中勇希, 村上正晃

  第102回 北海道医学大会 病理分科会 2022年09月
• 組織透明化を用いたがん微小環境の全細胞解析  [招待講演]

  久保田晋平

  第21回遺伝子・デリバリー研究会 2022年08月
• 組織透明化技術を用いた三次元病理学解析  [招待講演]

  久保田 晋平

  第111回日本病理学会総会 シンポジウム・ワークショップパネル（指名）
• 三次元病理解析に資する組織透明化手法の開発  [招待講演]

  Shimpei I. Kubota, Kei Takahashi, Kazuki Tainaka, Shogo Ehata, Hiroki R. Ueda, Kohei Miyazono

  第110回日本病理学会総会 シンポジウム・ワークショップパネル（指名）

その他活動・業績

• 三次元病理解析に資する組織透明化手法の開発

  久保田晋平, 高橋恵生, 田井中一貴, 江幡正悟, 上田泰己, 宮園浩平 日本病理学会会誌 110 (1) 2021年
• シングルセル解析でなにがわかるか

  竹山 春子, 細川 正人 組織透明化を用いた全身全細胞解析の最前線 2020年07月 [査読無し][通常論文]
  
• 組織透明化による一細胞解像度での癌転移解析

  久保田晋平, 高橋恵生, 西田純, 江幡正悟, 宮園浩平 日本癌学会学術総会抄録集(Web) 79th 2020年
• 組織透明化によるがん転移解析基盤の確立

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 田井中 一貴, 上田 泰己, 宮園 浩平 日本薬理学会年会要旨集 93 (0) 1 -YIA-16 2020年 [査読無し][通常論文]
   

  It has been recognized that interactions between cancer cells and stroma are important for survival of cancer cells and metastasis at distant organs. In addition, the complexity within tumor microenvironment contributes to resistance of conventional therapy. Therefore, despite decades of cancer researches, it is still difficult to elucidate the complexity of tumor microenvironment at whole organ or whole body level. To understand tumor microenvironment and overcome cancer, it is necessary to detect and quantify sparsely distributed metastatic cells throughout the body or organ at single-cell resolution. 

  Here, we demonstrate that CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis)-based cancer (CUBIC-Cancer) analysis with refractive-indices (RI) optimized protocol enables comprehensive cancer cell profiling in whole body and organs. We applied CUBIC-Cancer analysis to a dozen mouse models using several cancer cells and spatio-temporal quantification of metastatic cancer progression at single-cell resolution. As a result, metastatic foci can be observed and quantified through whole organ or whole body at single-cell resolution. In addition, three-dimensional (3D) monitoring revealed that the patterns of metastasis were dependent upon cancer cell or metastatic organs. Comparing two cancer cell lines, we quantified the difference of metastatic processes between angiogenesis and vessel cooption. Whole-organ profiling with single-cell resolution also enables to quantify the early steps of lung metastasis formation and rejection.CUBIC-Cancer analysis suggests that the epithelial-mesenchymal transition promotes not only extravasation but also cell survival at metastatic sites. CUBIC-Cancer analysis is applicable to pharmacotherapeutic profiling of anti-tumor drugs. CUBIC-Cancer analysis is compatible with in vivo bioluminescence imaging and 2D histology.We suggest that a scalable analytical pipeline with these three modalities may contribute to addressing currently incurable metastatic diseases.

• TGF-βにより細胞周期が停止した口腔扁平上皮がん細胞の運動能ならびに転移能は亢進する(TGF-β-induced cell cycle arrest is associated with increased migration and metastasis of oral squamous carcinoma cells)

  高橋 和樹, 井上 カタジナアンナ, 戒田 篤志, 高橋 恵生, 久保田 晋平, 須河内 昭成, 内橋 俊大, 田中 晋, 古郷 幹彦, 三浦 雅彦, 宮園 浩平, 渡部 徹郎 日本癌学会総会記事 78回 J -3010 2019年09月 [査読無し][通常論文]
  
• 組織透明化を用いたがん微小環境の解析(Visualization of tumor microenvironment using tissue-clearing technology)

  高橋 恵生, 久保田 晋平, 江幡 正悟, 宮園 浩平 日本癌学会総会記事 78回 E -3064 2019年09月 [査読無し][通常論文]
  
• 新しいがん治療に向けたがん転移機構の解明 がん微小環境によるがん転移機構の同所性移植モデルを用いた解析(Mechanisms of cancer metastasis for new cancer treatments Analyses of cancer metastasis regulated by tumor microenvironments using orthotopic transplantation models)

  宮園 浩平, 高橋 恵生, 久保田 晋平, 宮國 昂介, 西田 純, 江幡 正悟 日本癌学会総会記事 78回 CS4 -5 2019年09月 [査読無し][通常論文]
  
• 組織透明化を用いたがん転移の臓器丸ごとプロファイリング(Whole-organ quantitative analysis of cancer metastasis by tissue clearing)

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 宮園 浩平 日本癌学会総会記事 77回 1950 -1950 2018年09月 [査読無し][通常論文]
  
• 見る脂質のページ〈透明化による全細胞解析〉組織透明化による全身全細胞解析

  久保田晋平, 高橋恵生, 上田泰己, 宮園浩平 The Lipid 2018年07月 [査読無し][招待有り]
  
• 組織透明化技術CUBICを用いる肺がん細胞の転移解析

  高橋恵生, 久保田晋平, 上田泰己, 宮園浩平 Lung Perspectives 26 72 -76 2018年 [査読無し][招待有り]
  
• CUBIC-癌分析法を用いた癌転移の全身/全組織的特性解析(Whole-body/organ profiling of cancer metastasis with CUBIC-Cancer analysis)

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 田井中 一貴, 上田 泰己, 宮園 浩平 生命科学系学会合同年次大会 2017年度 [2P -1450] 2017年12月 [査読無し][通常論文]
  
• CUBIC-癌分析法を用いた癌転移の全身/全組織的特性解析(Whole-body/organ profiling of cancer metastasis with CUBIC-Cancer analysis)

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 田井中 一貴, 上田 泰己, 宮園 浩平 生命科学系学会合同年次大会 2017年度 [2P -1450] 2017年12月 [査読無し][通常論文]
  
• CUBICによる抗がん剤治療効果の臓器丸ごとプロファイリング

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 宮園 浩平 日本癌学会総会記事 76回 J -1041 2017年09月 [査読無し][通常論文]
  
• CUBICによる臓器透明化を用いたがん微小転移の観察

  高橋 恵生, 久保田 晋平, 西田 純, 江幡 正悟, 宮園 浩平 日本癌学会総会記事 76回 J -3097 2017年09月 [査読無し][通常論文]
  
• 癌微小転移の臓器まるごと病理学的解析

  久保田 晋平, 高橋 恵生, 西田 純, 江幡 正悟, 宮園 浩平 日本癌学会総会記事 75回 J -3054 2016年10月 [査読無し][通常論文]
  
• タンパク質可視化・制御の新技術 細胞局所から個体まで CUBIC 化学混合液を用いた単一細胞レベルでの全器官と全組織のイメージング法(CUBIC: whole-organ, whole-body imaging with single-cell resolution using chemical cocktails)

  田井中 一貴, 洲崎 悦生, 久保田 晋平, 上田 泰己 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2S4 -5] 2015年12月 [査読無し][通常論文]
  
• 一細胞解像度個体全身三次元イメージング

  久保田晋平, 田井中一貴, 上田泰己 細胞工学 34 278 -282 2015年03月 [査読無し][招待有り]
  
• 1細胞解像度個体全身三次元イメージング (特集 次世代生物学の扉を開く 1細胞解析法)

  久保田 晋平, 田井中 一貴, 上田 泰己 細胞工学 34 (3) 278 -282 2015年 [査読無し][通常論文]
  
• 生体組織透明化・脱色化試薬による一細胞解像度個体丸ごとイメージング技術の開発 2

  久保田晋平, 田井中一貴, 上田泰己 日本薬理学会関東部会プログラム・要旨集 131st 67 2014年 [査読無し][通常論文]
  
• 生体組織透明化・脱色化試薬による一細胞解像度個体丸ごとイメージング技術の開発 1

  田井中一貴, 久保田晋平, 上田泰己 日本薬理学会関東部会プログラム・要旨集 131st 66 2014年 [査読無し][通常論文]
  
• 統合失調症発症脆弱因子DISC1の神経細胞における局在と機能解析

  久保田晋平, 森大輔, 黒田啓介, 坪井大輔, 貝淵弘三 生化学 ROMBUNNO.2P-0958 2010年 [査読無し][通常論文]
  

受賞

• 2023年04月 文部科学省 令和５年度 科学技術分野の文部科学大臣表彰 若手科学者賞
   全身全細胞解析に資する組織透明化手法の研究
• 2021年10月 一般財団法人 エヌエフ基金 2021年度 研究開発奨励賞
   組織透明化手法の開発による神経-免疫連関機構の計測
• 2019年03月 花王芸術・科学財団 平成31年度花王科学奨励賞
   

  受賞者: 久保田 晋平
• 2018年05月 第6回 後藤喜代子・ポールブルダリ科学賞 特別賞
   

  受賞者: 久保田 晋平
• 2018年03月 細胞ダイバース第一回若手ワークショップ 最優秀発表賞
   

  受賞者: 久保田 晋平
• 2018年02月 第34回 井上研究奨励賞
   

  受賞者: 久保田 晋平
• 2017年06月 第31回 独創性を拓く 先端技術大賞受賞 (フジテレビジョン賞)
   

  受賞者: 久保田 晋平
• 2017年01月 第7回 日本学術振興会育志賞
   

  受賞者: 久保田 晋平
• 2016年10月 平成28年度 Tadamitsu Kishimoto International Travel Award
   

  受賞者: 久保田 晋平
• 2016年09月 QBiC Symposium travel fellowship
   

  受賞者: 久保田 晋平
• 2015年03月 日本薬理学会 第88回日本薬理学会年会優秀発表者賞
   

  受賞者: 久保田 晋平
• 2014年09月 International symposium "Homeodynamics in Clocks, Sleep and Metabolism" Tokyo Translational Therapeutics Meeting Best Poster Award
   

  受賞者: 久保田 晋平

共同研究・競争的資金等の研究課題

• 微小炎症ネットワークの解明に資する全身全細胞解析技術の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 久保田 晋平
• がんの三次元病理解析基盤の確立

  日本学術振興会：科学研究費助成事業

  研究期間 : 2020年04月 -2022年03月 

  代表者 : 久保田 晋平

   

  本研究においてはがん病態を分子から個体レベルまで統合して理解するためにマウス全身に局在するがん細胞の可視化技術の開発に取り組み、個体レベルでの網羅的1細胞解析の基盤技術を構築した。その結果、様々な臓器への転移を個体・臓器レベルで3次元観察することが可能となり、がんの転移に関与するサイトカインであるTGF-betaが、がん微小環境を変えることでがんの転移を促進するという、がん微小環境の新たなリモデリング機構を見出しました (Kubota et al., Commun. Biol., 2021)。
• 全身全細胞解析技術を用いた微小炎症制御機構の解明

  公益財団法人 日本応用酵素協会：2022 年度 成人病の病因・病態の解明に関する研究助成(TMFC)

  研究期間 : 2022年
• 治療抵抗性獲得機序の解明に資する全身がん病態可視化技術の開発

  国際科学技術財団：

  研究期間 : 2018年04月 -2019年03月 

  代表者 : 久保田 晋平