American Chemical Society (ACS) Acta Medica Okayama 1043-1802 37 3 2026 A Cysteine-Specific Cationization Strategy for Versatile Antibody Production against Intrinsically Disordered Proteins 580 589 EN Ryui Sakaguchi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ai Miyamoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rikako Kutsuma Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takeru Mori Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Daichi Nakashima Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mirei Masui Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tomoko Honjo Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Midori Futami Department of Bioscience, Faculty of Life Science, Okayama University of Science Mariko Morii Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshiyuki Oshiki Division of Applied Chemistry, Graduate School of Natural Science and Technology, Okayama University Junichiro Futami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Several autoantigens relevant to the immune system, especially those targeted by autoantibodies induced by antitumor responses, tend to be rich in disordered regions and are prone to aggregation. This inherent instability presents significant challenges for the production, purification, and analysis of autoantigens in laboratory settings. Cysteine-specific cationization can effectively solubilize and purify these challenging proteins, allowing the isolation of full-length water-soluble antigens in their denatured state. The purified antigens enable accurate multiplex autoantibody assays using a suspension Luminex bead array platform. However, well-validated positive control antibodies are essential to ensuring precise clinical diagnosis. In this study, we prepared and characterized a panel of control antibodies by immunizing rabbits with cysteine-specific S-cationized antigens. The resulting antibodies predominantly recognized linear epitopes and were highly effective as quality control reagents in autoantibody array assays. Additionally, these antibodies maintained their ability to bind to their native, unmodified intracellular counterparts, highlighting the usefulness of this approach for producing antibodies against intrinsically disordered proteins. Although a modest immune response against the S-cationized modification site was observed, it remained minimal and did not affect the usefulness of the antibodies for assay validation. We propose this versatile cysteine-specific cationization platform for managing unstable proteins rich in disordered regions, supporting antigen production for diagnostics, and antibody development for research and validation purposes. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells 1215 1227 EN Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Tetta Takahashi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Toshiki Ochi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Kazumi Sagayama Organization for Research and Innovation Strategy, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector’s shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 15 1 2025 Autoantibody spark response predicts treatment outcome in patients receiving chemoradiation followed by durvalumab therapy 27502 EN Takeru Mori Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mio Kitagawa Department of Radiology, Sapporo Medical University School of Medicine Tomokazu Hasegawa Department of Radiology, Sapporo Medical University School of Medicine Masanori Someya Department of Radiology, Sapporo Medical University School of Medicine Takaaki Tsuchiya Department of Radiology, Sapporo Medical University School of Medicine Toshio Gocho Department of Radiology, Sapporo Medical University School of Medicine Tomoko Honjo Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mirei Date Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mariko Morii Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ai Miyamoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Junichiro Futami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University The PACIFIC regimen, comprising chemoradiotherapy (CRT) followed by maintenance with the immune checkpoint inhibitor (ICI) durvalumab, has become the standard of care for patients with unresectable non-small cell lung cancer (NSCLC). Although ICI is used to prevent recurrence by targeting residual microtumors, biomarkers capable of monitoring immune activity during this phase remain lacking. Here, we evaluated whether temporal changes in serum autoantibody levels can predict treatment efficacy. This retrospective study included 20 patients with unresectable stage II or III NSCLC who received the PACIFIC regimen. Serum autoantibodies against 130 antigens were quantified before CRT, after CRT, and two weeks after the first ICI dose. The primary outcome was progression-free survival (PFS), and its association with autoantibody dynamics was examined. We observed an immediate and strong autoantibody response (spark response [SR]) after ICI initiation in patients with favorable treatment outcomes. Patients with SR and programmed death ligand 1 (PD-L1) expression ≥ 50% showed better PFS (two-year PFS; 72.9% vs. 18.2%, p = 0.0021). These findings suggest that serial monitoring of serum autoantibodies can provide a noninvasive approach to assess immune activity and predict treatment outcomes in patients receiving CRT or ICI therapy. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis 1371307 EN Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Kazumi Ninomiya Faculty of Science and Technology, Division of Molecular Science, Gunma University Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-beta 1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-beta 1 hastens the invasive outgrowth of TNBC cells at the molecular level.<br> Methods LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively.<br> Results Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-kappa B (NF-kappa B). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-beta activated kinase 1 (TAK1) were required for the activation of NF-kappa B through I kappa beta kinase kinase (IKK alpha/beta) phosphorylation.<br> Conclusion Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-beta 1-TRAF4-TAK1-IKK alpha/beta-I kappa beta alpha-NF-kappa B-MMP9, is crucial for TNBC cell invasiveness. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0946-2716 101 4 2023 Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells 431 447 EN Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Léna Audebert Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Youyi Chen Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Chikako Yoshizawa Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masami Watanabe Department of Urology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3—namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface 1371342 EN Tetta Takahashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshito Zamami Department of Pharmacy, Okayama University Hospital Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain.<br> <br> Methods: Cell invasion was assessed using a transwell-based assay, protein–protein interactions by an immunoprecipitation–Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography.<br> <br> Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion.<br> <br> Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 19 2022 Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model 11035 EN Tsukasa Shigehiro Research Institute for Biomedical Sciences, Tokyo University of Science Maho Ueno Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Mayumi Kijihira Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Ryotaro Takahashi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Chiho Umemura Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Eman A. Taha Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Chisaki Kurosaka Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Megumi Asayama Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Hiroshi Murakami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshimasa Nakamura Graduate School of Environmental and Life Science, Okayama University Junichiro Futami Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Junko Masuda Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-gamma production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-gamma expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103(+)CD11b(+) dendritic cells (DCs) into the mLN, along with increased (1 -> 3)-beta-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103(+)CD11b(+) DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 18 2022 Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells 10300 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidenori Wake Department of Pharmacology, Kindai University Faculty of Medicine Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University I. Made Winarsa Ruma Faculty of Medicine, Udayana University Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Faculty of Medicine, Udayana University Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis. No potential conflict of interest relevant to this article was reported.

FRONTIERS MEDIA SA Acta Medica Okayama 2234-943X 12 2022 Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading 869393 EN Ai Miyamoto Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mirei Masui Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-targeted Therapy, Okayama University Kazuhiro Kakimi Department of Immunotherapeutics, The University of Tokyo Hospital Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4425 13 2 2022 Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice 285 EN Ruizhi Xue Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Wenfeng Lin Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jingkai Sun Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Ochiai Laboratory of Veterinary Hygiene, Nippon Veterinary and Life Science University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Zhengyan Tang Department of Urology, Xiangya Hospital, Central South University Peng Huang Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasutomo Nasu Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 +/- 8.12% vs. 23.26 +/- 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 +/- 0.69 vs. 8.27 +/- 0.87, x10(6)), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 +/- 1.55% vs. 72.50 +/- 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0021-924X 170 3 2021 Unusual aggregation property of recombinantly expressed cancer-testis antigens in mammalian cells 435 443 EN Hannaneh Ahmadi Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kohei Shogen Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Kana Fujita Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazuhiro Kakimi Department of Immunotherapeutics, The University of Tokyo Hospital Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein–protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 15560864 14 12 2019 Serum Antibody Against NY-ESO-1 and XAGE1 Antigens Potentially Predicts Clinical Responses to Anti–Programmed Cell Death-1 Therapy in NSCLC 2071 2083 EN Yoshihiro Ohue Department of Respiratory Medicine, Kawasaki Medical School Koji Kurose Department of Respiratory Medicine, Kawasaki Medical School Takahiro Karasaki Department of Thoracic Surgery, The University of Tokyo Midori Isobe Department of Respiratory Medicine, Kawasaki Medical School Takaaki Yamaoka Department of Respiratory Medicine, Kawasaki Medical School Junichiro Futami Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Isao Irei Department of Pathology, Kawasaki Medical School Takeshi Masuda Department of Respiratory Internal Medicine, Hiroshima University Hospital Masaaki Fukuda Department of Respiratory Medicine, The Japanese Red Cross Nagasaki Genbaku Hospital Akitoshi Kinoshita Department of Respiratory Medicine, Nagasaki Prefecture Shimabara Hospital Hirokazu Matsushita Department of Immunotherapeutics, Graduate School of Medicine, The University of Tokyo Katsuhiko Shimizu Department of General Thoracic Surgery, Kawasaki Medical School Masao Nakata Department of General Thoracic Surgery, Kawasaki Medical School Noboru Hattori Department of Respiratory Internal Medicine, Hiroshima University Hospital Hiroyuki Yamaguchi Department of Respiratory Medicine, Nagasaki University Graduate School of Biomedical Sciences Minoru Fukuda Department of Respiratory Medicine, Nagasaki University Graduate School of Biomedical Sciences Ryohei Nozawa Faculty of Health and Welfare Services Administration, Kawasaki University of Medical Welfare Kazuhiro Kakimi Department of Immunotherapeutics, Graduate School of Medicine, The University of Tokyo Mikio Oka Department of Immuno-Oncology, Kawasaki Medical School Introduction: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits.<br/> Methods: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing.<br/> Results: In the discovery cohort (n = 13), six antibody-positive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8+ T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004).<br/> Conclusions: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1522-8002 21 7 2019 Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness 627 640 EN Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Yosuke Mitsui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences I Made Winarsa Ruma Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroki Sato Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuta Tomida Department of Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichi Soh Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miyoko Kubo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endy Widya Putranto Department of Pediatrics, Dr. Sardjito Hospital/Faculty of Medicine, Universitas Gadjah Mada Takashi Murakami Department of Microbiology, Faculty of Medicine, Saitama Medical University Ming Liu Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Toshihiko Hibino Department of Dermatology, Tokyo Medical University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes. No potential conflict of interest relevant to this article was reported.

シーエムシー Acta Medica Okayama 09106545 33 5 2016 腫瘍免疫応答の活性化を測定する抗体検査技術の開発 19 24 EN Junichiro Futami がん免疫治療や関連の医薬品開発において、腫瘍に対する免疫応答のレベルを簡便な血液検査で定量評価ができる診断技術が必要だ。がんに対する免疫応答が亢進している際には、血液中に様々な抗がん抗原抗体が増加する。この抗体価の定量評価には、独自開発の変性タンパク質の可溶化技術の活用が強力な手段となる。 No potential conflict of interest relevant to this article was reported.

Spandidos Publications Ltd. Acta Medica Okayama 1107-3756 32 4 2013 Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 938 944 EN Endy Widya Putranto Hitoshi Murata Ken-Ichi Yamamoto Ken Kataoka Hidenori Yamada Jun-Ichiro Futami Masakiyo Sakaguchi Nam-Ho Huh The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 118 3 2007 内臓脂肪組織に由来するセリンプロテアーゼ阻害剤：Vaspin の同定肥満状態でインスリン感受性を高める新規アディポサイトカイン 215 220 EN No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1999 Study on Human Pancreatic-field name="type" Ribonucleases and Application to the Development of Humanized Immunotoxin EN No potential conflict of interest relevant to this article was reported.