Country：Japan

Country：Japan

Country：Japan

Country：Japan

Language：English   Publishing type：Research paper (scientific journal)  

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

DOI： 10.1158/2159-8290.CD-24-0287

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Prognosis is improving with advances in multidisciplinary treatment strategies, but the development of new anticancer agents has not, and improvement in prognosis for patients with pulmonary metastases has stalled. In recent years, the tumor microenvironment (TME) has gained attention as a therapeutic target for cancer. The immune component of OS TME consists mainly of tumor-associated macrophages (TAMs). They exhibit remarkable plasticity, and their phenotype is influenced by the TME. In general, surface markers such as CD68 and CD80 show anti-tumor effects, while CD163 and CD204 show tumor-promoting effects. Surface markers have potential value as diagnostic and prognostic biomarkers. The cytokines and chemokines produced by TAMs promote tumor growth and metastasis. However, the role of TAMs in OS remains unclear to date. In this review, we describe the role of TAMs in OS by focusing on TAM surface markers and the TAM-produced cytokines and chemokines in the TME, and by comparing their behaviors in other carcinomas. We found contrary results from different studies. These findings highlight the urgency for further research in this field to improve the stalled OS prognosis percentages.

DOI： 10.3390/cancers16162801

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: The proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs. METHODS: We investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models. RESULTS: High infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs. CONCLUSION: Our data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.

DOI： 10.1038/s41416-024-02734-3

Web of Science

PubMed

Language：English  

DOI： 10.1038/s41419-024-06795-3

PubMed

Language：Japanese   Publisher：(株)羊土社  

がん関連線維芽細胞(CAF)はがん促進性の機能をもつと長く認識されてきた.一方で,CAFを除去あるいはその増殖を抑制するとがんの増悪が観察されたことから,CAFにはがん抑制性の機能があることも指摘されている.昨今の解析技術の発展により,CAFの多様性やその臨床的意義に関する理解は得られつつあるが,依然,がん抑制性CAFの本態は不明確である.本稿では,がん抑制性CAFに対する理解の現状と難しさについて,われわれの仮説も交えつつ概説したい.(著者抄録)

Language：Japanese   Publisher：(公社)愛知県医師会  

Language：Japanese   Publisher：(公社)愛知県医師会  

Language：English   Publisher：(一社)日本内分泌学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.cels.2024.01.007

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41419-024-06487-y

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Abstract

Desmoid tumors (DTs), also called desmoid‐type fibromatoses, are locally aggressive tumors of mesenchymal origin. In the present study, we developed a novel mouse model of DTs by inducing a local mutation in the Ctnnb1 gene, encoding β‐catenin in PDGFRA‐positive stromal cells, by subcutaneous injection of 4‐hydroxy‐tamoxifen. Tumors in this model resembled histologically clinical samples from DT patients and showed strong phosphorylation of nuclear SMAD2. Knockout of SMAD4 in the model significantly suppressed tumor growth. Proteomic analysis revealed that SMAD4 knockout reduced the level of Cysteine‐and‐Glycine‐Rich Protein 2 (CSRP2) in DTs, and treatment of DT‐derived cells with a TGF‐β receptor inhibitor reduced CSRP2 RNA levels. Knockdown of CSRP2 in DT cells significantly suppressed their proliferation. These results indicate that the TGF‐β/CSRP2 axis is a potential therapeutic target for DTs downstream of TGF‐β signaling.

DOI： 10.1111/cas.16037

Web of Science

PubMed

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publishing type：Research paper (scientific journal)  

Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin+ ) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin+ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin+ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin+ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin+ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin+ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin+ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.6211

Web of Science

PubMed

Language：English  

DOI： 10.1007/s12072-023-10581-2

Web of Science

PubMed

Language：English  

DOI： 10.1038/s41467-023-42774-6

Web of Science

PubMed

Language：English  

DOI： 10.3389/fendo.2023.1216424

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Background: Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. Methods: RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). Results: HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. Conclusion: Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs. [Figure not available: see fulltext.]

DOI： 10.1038/s41416-023-02406-8

Scopus

PubMed

Language：English   Publisher：(一社)日本癌学会  

Language：Japanese   Publisher：(公社)日本整形外科学会  

Language：Japanese   Publisher：(公社)日本整形外科学会  

Language：Japanese   Publisher：(一社)日本膵臓学会  

Language：English  

DOI： 10.1016/j.prp.2023.154443

Web of Science

PubMed

Language：English  

DOI： 10.1007/s00428-023-03513-w

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.

DOI： 10.1002/1873-3468.14592

Web of Science

PubMed

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Pancreatic cancer is notorious for its dismal prognosis. The enhanced permeability and retention (EPR) effect theory posits that nanomedicines (therapeutics in the size range of approximately 10-200 nm) selectively accumulate in tumors. Nanomedicine has thus been suggested to be the "magic bullet"-both effective and safe-to treat pancreatic cancer. However, the densely fibrotic tumor microenvironment of pancreatic cancer impedes nanomedicine delivery. The EPR effect is thus insufficient to achieve a significant therapeutic effect. Intratumoral fibrosis is chiefly driven by aberrantly activated fibroblasts and the extracellular matrix (ECM) components secreted. Fibroblast and ECM abnormalities offer various potential targets for therapeutic intervention. In this review, we detail the diverse strategies being tested to overcome the fibrotic barriers to nanomedicine in pancreatic cancer. Strategies that target the fibrotic tissue/process are discussed first, which are followed by strategies to optimize nanomedicine design. We provide an overview of how a deeper understanding, increasingly at single-cell resolution, of fibroblast biology is revealing the complex role of the fibrotic stroma in pancreatic cancer pathogenesis and consider the therapeutic implications. Finally, we discuss critical gaps in our understanding and how we might better formulate strategies to successfully overcome the fibrotic barriers in pancreatic cancer.

DOI： 10.3390/cancers15030724

Web of Science

PubMed

Language：Japanese   Publisher：(一社)日本膵臓学会  

膵癌の特徴として間質の線維化とそれに伴う組織硬化がある.組織硬化は間質内圧上昇と血管虚脱を誘導し,その結果抗癌剤の間質浸透と癌細胞への到達を阻害することが知られている.この間質の線維化に中心的な役割を果たすのが癌関連線維芽細胞(CAF)である.このため,これまで様々な方法でCAFをターゲットにする治療が試みられてきたが未だ成功していない.本研究では癌抑制性CAFのマーカーであり機能分子であるMeflinをCAFに発現誘導することで,膵癌における化学療法への感受性を改善させることを明らかにした.さらにスクリーニングにより合成レチノイドであるAM80がCAFにおけるMeflinの発現を効果的に誘導し,ドラッグデリバリーの改善を伴って化学療法に対する感受性を改善させることを明らかにした.筆者らは本結果を受けて切除不能膵癌に対するAM80と従来の抗癌剤の併用効果を検証する医師主導治験を行っている.(著者抄録)

Other Link： https://search.jamas.or.jp/default/link?pub_year=2023&ichushi_jid=J02025&link_issn=&doc_id=20230308380005&doc_link_id=10.2958%2Fsuizo.38.37&url=https%3A%2F%2Fdoi.org%2F10.2958%2Fsuizo.38.37&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The molecular pathogenesis of nonalcoholic steatohepatitis (NASH) includes a complex interaction of metabolic stress and inflammatory stimuli. Considering the therapeutic goals of NASH, it is important to determine whether the treatment can prevent the progression from NASH to hepatocellular carcinoma. Taxifolin, also known as dihydroquercetin, is a natural bioactive flavonoid with antioxidant and anti-inflammatory properties commonly found in various foods and health supplement products. In this study, we demonstrated that Taxifolin treatment markedly prevented the development of hepatic steatosis, chronic inflammation, and liver fibrosis in a murine model of NASH. Its mechanisms include a direct action on hepatocytes to inhibit lipid accumulation. Taxifolin also increased brown adipose tissue activity and suppressed body weight gain through at least two distinct pathways: direct action on brown adipocytes and indirect action via fibroblast growth factor 21 production in the liver. Notably, the Taxifolin treatment after NASH development could effectively prevent the development of liver tumors. Collectively, this study provides evidence that Taxifolin shows pleiotropic effects for the treatment of the NASH continuum. Our data also provide insight into the novel mechanisms of action of Taxifolin, which has been widely used as a health supplement with high safety.

DOI： 10.3390/nu15020350

Web of Science

PubMed

Publisher：Research Square Platform LLC  

Abstract

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remains unknown. In this study, we examined the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we found that directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. Elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further found that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

DOI： 10.21203/rs.3.rs-2314559/v1

Other Link： https://www.researchsquare.com/article/rs-2314559/v1.html

Language：English  

DOI： 10.3390/cancers14225688

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.

DOI： 10.1002/2211-5463.13468

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Thoracic Society  

Fibroblasts play a central role in the lung fibrotic process. Our recent study identified a novel subpopulation of lung fibroblasts expressing meflin, anti-fibrotic properties of which were confirmed by murine lung fibrosis model. Meflin expressing fibroblasts were resistant to fibrogenesis induced by transforming growth factor-β (TGF-β), but its underlying mechanisms remain unknown. In this study, evaluation of a silica-nanoparticles-induced lung fibrosis model confirmed the antifibrotic effect of meflin via the regulation of TGF-β signaling. We conducted comparative gene expression profiling in lung fibroblasts, which identified growth differentiation factor 10 (Gdf10) encoding bone morphogenic protein 3b (BMP3b) as the most down-regulated gene in meflin-deficient cells under the profibrotic condition with TGF-β. We hypothesized that BMP3b can be an effector molecule playing an anti-fibrotic role downstream of meflin. As suggested by single-cell transcriptomic data, restricted expressions of Gdf10 (Bmp3b) in stromal cells including fibroblasts were confirmed. We examined possible anti-fibrotic properties of BMP3b in lung fibroblasts and demonstrated that Bmp3b-null fibroblasts were more susceptible to TGF-β-induced fibrogenic changes. Furthermore, Bmp3b-null mice exhibited exaggerated lung fibrosis induced by silica-nanoparticles in vivo. We also demonstrated that treatment with recombinant BMP3B was effective against TGF-β-induced fibrogenesis in fibroblasts, especially in the suppression of excessive extracellular matrix production. These lines of evidence suggested that BMP3b is a novel humoral effector molecule regulated by meflin which exerts anti-fibrotic properties in lung fibroblasts. Supplementation of BMP3B could be a novel therapeutic strategy for fibrotic lung diseases.

DOI： 10.1165/rcmb.2021-0484OC

Web of Science

PubMed

Web of Science

Language：Japanese   Publisher：(株)羊土社  

ヒトのがんの間質の免疫細胞の浸潤、線維化、および腫瘍血管の増生の程度はがんの種類あるいは症例間で実に多様である。同一症例の標本でも視野を少し動かしただけで、同じがんとは思えないような組織像の違いを見せることもある。免疫チェックポイント阻害薬(ICI)の勃興によりがん治療の概念が変わり、それに伴いヒト免疫学も飛躍的に発展したと聞く。しかしながら、一部の特殊なゲノム異常を有するがんを除き、前述の腫瘍内(intratumor)および腫瘍間(intertumor)の多様性がICIの奏功予測や一律な奏効率の押し上げを困難なものにしていると推測される。本稿では、主に線維芽細胞と腫瘍血管に焦点をあて、これらが抗腫瘍免疫応答およびICIの効果に及ぼす影響、およびその知見に基づく治療戦略について概説したい。(著者抄録)

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English  

DOI： 10.18999/nagjms.84.3.484

Web of Science

PubMed

Language：Japanese   Publisher：医学図書出版(株)  

膵癌治療のボトルネックは、その豊富な間質に起因する薬剤送達(drug delivery)の障害であり、これを解決する治療法の開発が強く求められている。膵癌の豊富な間質の原因は、がん関連線維芽細胞(CAF)の増生であり、がんを促進する形質を有することから治療標的として注目されて久しい。しかしながら、線維芽細胞の活性化および増殖を阻害する薬剤を臨床応用する試みはうまくいっていない。それらの結果より近年では、線維芽細胞はがん促進性と抑制性の二つの極性(性質の連続的な差異)を有していること、さらに同細胞の極性はがん発育の文脈に沿って容易に変換されることの2点が原因であると考えられるようになった。われわれは最近、meflin陽性線維芽細胞が、がん抑制性線維芽細胞であり、その機能は従来の活性化線維芽細胞(筋線維芽細胞やCAF)とは異なっていること、および、線維芽細胞の極性を制御することで抗がん剤の送達性の向上が図れることをマウスモデルで示してきた。これらの知見をベースに、現在、われわれは線維芽細胞の極性の変化を誘導する薬剤の効果を検証する医師主導治験を行っている。(著者抄録)

Language：English   Publishing type：Research paper (scientific journal)  

Anticancer drugs and molecular targeted therapies are used for refractory desmoid-type fibromatosis (DF), but occasionally cause severe side effects. The purpose of this study was to identify an effective drug with fewer side effects against DF by drug repositioning, and evaluate its efficacy. FDA-approved drugs that inhibit the proliferation of DF cells harboring S45F mutations of CTNNB1 were screened. An identified drug was subjected to the investigation of apoptotic effects on DF cells with analysis of Caspase 3/7 activity. Expression of β-catenin was evaluated with western blot analysis, and immunofluorescence staining. Effects of the identified drug on in vivo DF were analyzed using Apc1638N mice. Auranofin was identified as a drug that effectively inhibits the proliferation of DF cells. Auranofin did not affect Caspase 3/7 activity compared to control. The expression level of β-catenin protein was not changed regardless of auranofin concentration. Auranofin effectively inhibited the development of tumorous tissues by both oral and intraperitoneal administration, particularly in male mice. Auranofin, an anti-rheumatic drug, was identified to have repositioning effects on DF. Since auranofin has been used for many years as an FDA-approved drug, it could be a promising drug with fewer side effects for DF.

DOI： 10.1038/s41598-022-15756-9

Web of Science

PubMed

Language：English  

A 42-year-old man with a history of acute myocarditis after streptococcal pharyngitis developed recurrent fulminant myocarditis. Endomyocardial biopsy revealed myocyte degeneration, interstitial edema, and neutrophil infiltration. The patient's cardiac function deteriorated rapidly, and he died despite mechanical circulatory support. Autopsy revealed neutrophil infiltration, interstitial edema, and micro-abscesses containing masses of streptococci and neutrophilic phagocytosis within the myocardium. The patient did not meet the diagnostic criteria for acute rheumatic fever; thus, he was diagnosed with non-rheumatic streptococcal myocarditis. Non-rheumatic streptococcal myocarditis rarely recurs, but it can be fulminant upon recurrence. Learning objective: We report a rare case of recurrent fulminant non-rheumatic streptococcal myocarditis. Endomyocardial biopsy and autopsy revealed neutrophil infiltration and micro-abscesses containing bacterial masses of streptococci and neutrophilic phagocytosis in the myocardium. The patient did not meet the diagnostic criteria for acute rheumatic fever; thus, he was diagnosed with non-rheumatic streptococcal myocarditis. Non-rheumatic streptococcal myocarditis rarely recurs, but it can be fulminant upon recurrence.

DOI： 10.1016/j.jccase.2022.02.004

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

A well-known feature of human pancreatic ductal adenocarcinoma (PDAC) is the extensive proliferation of cancer-associated fibroblasts (CAFs) and highly fibrotic stroma. Recent evidence, based mainly on single-cell analyses, has identified various subsets of CAFs in PDAC mouse models. However, we do not know how these CAF subsets are involved in the progression and drug resistance of human PDAC. Additionally, it remains unclear whether these diverse CAFs have distinct origins and are indicators of genuinely distinct CAF lineages or reflect different states of the same CAFs depending on the tumor microenvironment. Interestingly, recent preclinical studies have started to characterize the nature of cancer-restraining CAFs and have identified their markers Meflin and collagen type I alpha 1. These studies have led to the development of strategies to induce changes in CAF phenotypes using chemical reagents or recombinant viruses, and some of them have been tested in clinical studies. These strategies have the unique potential to convert the so-called bad stroma to good stroma and may also have therapeutic implications for non-cancer diseases such as fibrotic diseases. Together with recently developed sophisticated strategies that specifically target distinct CAF subsets via adoptive cell transfer therapy, vaccination, and antibody-drug conjugates, any future findings arising from these clinical efforts may expand our understanding of the significance of CAF diversity in human PDAC.

DOI： 10.3390/cancers14143315

Web of Science

PubMed

Authorship：Corresponding author   Language：English  

DOI： 10.1038/s41388-022-02336-4

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Although spindle cell lipoma (SCL) is a subtype of lipoma, the characteristics of SCL are observed in both lipomatous and non-lipomatous tumors. In this article, we present a case of SCL with ossification mimicking atypical lipomatous tumors/well-differentiated liposarcomas (ALTs/WDLs). Considering the findings of magnetic resonance imaging and needle biopsy, which exhibited ALTs/WDLs, marginal resection was performed. Histopathological findings demonstrated mature adipocytes and spindle cells without atypia and no malignant osteoid tissue in the ossified region. In addition, immunohistochemistry (IHC) showed positive staining for CD34, heterogeneous retinoblastoma protein deficiency, and negative staining for mouse double minute 2 homolog (MDM2) and cyclin-dependent kinase. Fluorescence in-situ hybridization showed negative amplification of MDM2. The final diagnosis of the tumor was established using IHC as an extremely rare SCL with ossification.

DOI： 10.1177/10668969211055798

Web of Science

PubMed

Language：English  

DOI： 10.1177/10668969211067764

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-β was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-β was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.

DOI： 10.1038/s41389-022-00403-9

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, nonnatural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.

DOI： 10.1038/s41388-022-02288-9

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：Japanese  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Ovarian cancer (OvCa), a lethal gynecological malignancy, disseminates to the peritoneum. Mesothelial cells (MCs) act as barriers in the abdominal cavity, preventing the adhesion of cancer cells. However, in patients with OvCa, they are transformed into cancer-associated mesothelial cells (CAMs) via mesenchymal transition and form a favorable microenvironment for tumors to promote metastasis. However, attempts for restoring CAMs to their original state have been limited. Here, we investigated whether inhibition of mesenchymal transition and restoration of MCs by vitamin D suppressed the OvCa dissemination in vitro and in vivo. The effect of vitamin D on the mutual association of MCs and OvCa cells was evaluated using in vitro coculture models and in vivo using a xenograft model. Vitamin D restored the CAMs, and thrombospondin-1 (component of the extracellular matrix that is clinically associated with poor prognosis and is highly expressed in peritoneally metastasized OvCa) was found to promote OvCa cell adhesion and proliferation. Mechanistically, TGF-β1 secreted from OvCa cells enhanced thrombospondin-1 expression in CAMs via Smad-dependent TGF-β signaling. Vitamin D inhibited mesenchymal transition in MCs and suppressed thrombospondin-1 expression via vitamin D receptor/Smad3 competition, contributing to the marked reduction in peritoneal dissemination in vivo. Importantly, vitamin D restored CAMs from a stabilized mesenchymal state to the epithelial state and normalized thrombospondin-1 expression in preclinical models that mimic cancerous peritonitis in vivo. MCs are key players in OvCa dissemination and peritoneal restoration and normalization of thrombospondin-1 expression by vitamin D may be a novel strategy for preventing OvCa dissemination.

DOI： 10.1016/j.matbio.2022.03.003

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, whose expression is upregulated in some types of malignant tumors. High levels of CD109 in tumor cells have been reported to correlate with poor prognosis; however, significance of CD109 stromal expression in human malignancy has not been elucidated. In this study, we investigated the tumorigenic properties of CD109 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis of 92 PDAC surgical specimens revealed that positive CD109 expression in tumor cells was significantly associated with poor prognosis (disease-free survival, p = 0.003; overall survival, p = 0.002), and was an independent prognostic factor (disease-free survival, p = 0.0173; overall survival, p = 0.0104) in PDAC. Furthermore, CD109 expression was detected in the stroma surrounding tumor cells, similar to that of α-smooth muscle actin, a histological marker of cancer-associated fibroblasts. The stromal CD109 expression significantly correlated with tumor progression in PDAC (TNM stage, p = 0.033; N factor, p = 0.024; lymphatic invasion, p = 0.028). In addition, combined assessment of CD109 in tumor cells and stroma could identify the better prognosis group of patients from the entire patient population. In MIA PaCa-2 PDAC cell line, we demonstrated the involvement of CD109 in tumor cell motility, but not in PANC-1. Taken together, CD109 not only in the tumor cells but also in the stroma is involved in the progression and prognosis of PDAC, and may serve as a useful prognostic marker in PDAC.

DOI： 10.1007/s00428-021-03230-2

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and has a unique metastatic route using ascites, known as the transcoelomic root. However, studies on ascites and contained cellular components have not yet been sufficiently clarified. In this review, we focus on the significance of accumulating ascites, contained EOC cells in the form of spheroids, and interaction with non-malignant host cells. To become resistant against anoikis, EOC cells form spheroids in ascites, where epithelial-to-mesenchymal transition stimulated by transforming growth factor-β can be a key pathway. As spheroids form, EOC cells are also gaining the ability to attach and invade the peritoneum to induce intraperitoneal metastasis, as well as resistance to conventional chemotherapy. Recently, accumulating evidence suggests that EOC spheroids in ascites are composed of not only cancer cells, but also non-malignant cells existing with higher abundance than EOC cells in ascites, including macrophages, mesothelial cells, and lymphocytes. Moreover, hetero-cellular spheroids are demonstrated to form more aggregated spheroids and have higher adhesion ability for the mesothelial layer. To improve the poor prognosis, we need to elucidate the mechanisms of spheroid formation and interactions with non-malignant cells in ascites that are a unique tumor microenvironment for EOC.

DOI： 10.3390/ijms23084383

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Perivascular mesenchymal cells (PMCs), which include pericytes, give rise to myofibroblasts that contribute to chronic kidney disease progression. Several PMC markers have been identified; however, PMC heterogeneity and functions are not fully understood. Here, we describe a novel subset of renal PMCs that express Meflin, a glycosylphosphatidylinositol-anchored protein that was recently identified as a marker of fibroblasts essential for cardiac tissue repair. Tracing the lineage of Meflin+ PMCs, which are found in perivascular and periglomerular areas and exhibit renin-producing potential, showed that they detach from the vasculature and proliferate under disease conditions. Although the contribution of Meflin+ PMCs to conventional α-SMA+ myofibroblasts is low, they give rise to fibroblasts with heterogeneous α-SMA expression patterns. Genetic ablation of Meflin+ PMCs in a renal fibrosis mouse model revealed their essential role in collagen production. Consistent with this, human biopsy samples showed that progressive renal diseases exhibit high Meflin expression. Furthermore, Meflin overexpression in kidney fibroblasts promoted bone morphogenetic protein 7 signals and suppressed myofibroblastic differentiation, implicating the roles of Meflin in suppressing tissue fibrosis. These findings demonstrate that Meflin marks a PMC subset that is functionally distinct from classic pericytes and myofibroblasts, highlighting the importance of elucidating PMC heterogeneity.

DOI： 10.1038/s41598-022-09331-5

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

DOI： 10.1111/pin.13198

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

DOI： 10.1053/j.gastro.2021.11.037

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cancer-associated fibroblasts (CAFs) are an integral component of the tumor microenvironment (TME). Most CAFs shape the TME toward an immunosuppressive milieu and attenuate the efficacy of immune checkpoint blockade (ICB) therapy. However, the detailed mechanism of how heterogeneous CAFs regulate tumor response to ICB therapy has not been defined. Here, we show that a recently defined CAF subset characterized by the expression of Meflin, a glycosylphosphatidylinositol-anchored protein marker of mesenchymal stromal/stem cells, is associated with survival and favorable therapeutic response to ICB monotherapy in patients with non-small cell lung cancer (NSCLC). The prevalence of Meflin-positive CAFs was positively correlated with CD4-positive T-cell infiltration and vascularization within non-small cell lung cancer tumors. Meflin deficiency and CAF-specific Meflin overexpression resulted in defective and enhanced ICB therapy responses in syngeneic tumors in mice, respectively. These findings suggest the presence of a CAF subset that promotes ICB therapy efficacy, which adds to our understanding of CAF functions and heterogeneity.

DOI： 10.26508/lsa.202101230

Web of Science

PubMed

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本内分泌学会  

Language：English   Publisher：(一社)日本内分泌学会  

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Cancer-associated fibroblasts (CAFs) are an important component of the tumour microenvironment. Recent studies revealed CAFs are heterogeneous and CAF subset(s) that suppress cancer progression (cancer-restraining CAFs [rCAFs]) must exist in addition to well-characterised cancer-promoting CAFs (pCAFs). However, the identity and specific markers of rCAFs are not yet reported. We recently identified Meflin as a specific marker of rCAFs in pancreatic and colon cancers. Our studies revealed that rCAFs may represent proliferating resident fibroblasts. Interestingly, a lineage tracing experiment showed Meflin-positive rCAFs differentiate into α-smooth muscle actin-positive and Meflin-negative CAFs, which are generally hypothesised as pCAFs, during cancer progression. Using a pharmacological approach, we identified AM80, a synthetic unnatural retinoid, as a reagent that effectively converts Meflin-negative pCAFs to Meflin-positive rCAFs. We aimed to investigate the efficacy of a combination of AM80 and gemcitabine (GEM) and nab-paclitaxel (nab-PTX) in patients with advanced pancreatic cancer. METHODS: The phase I part is a 3 + 3 design, open-label, and dose-finding study. The dose-limiting toxicity (DLT) of these combination therapies would be evaluated for 4 weeks. After the DLT evaluation period, if no disease progression is noted based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 or if the patient has no intolerable toxicity, administration of AM80 with GEM and nab-PTX would be continued for up to 24 weeks. The phase II part is an open-label, single-arm study. The maximum tolerated dose (MTD) of AM80 with GEM and nab-PTX, determined in phase I, would be administered until intolerable toxicity or disease progression occurs, up to a maximum of 24 weeks, to confirm efficacy and safety. The primary endpoints are frequency of DLT and MTD of AM80 with GEM and nab-PTX in the phase I part and response rate based on the RECIST in the phase II part. Given the historical control data, we hope that the response rate will be over 23% in phase II. DISCUSSION: Strategies to convert pCAFs into rCAFs have been developed in recent years. We hypothesised that AM80 would be a promising enhancer of chemosensitivity and drug distribution through CAF conversion in the stroma. TRIAL REGISTRATION: Clinicaltrial.gov: NCT05064618 , registered on 1 October 2021. jRCT: jRCT2041210056 , registered on 27 August 2021.

DOI： 10.1186/s12885-022-09272-2

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

The prognosis of elderly individuals with idiopathic pulmonary fibrosis (IPF) remains poor. Fibroblastic foci, in which aggregates of proliferating fibroblasts and myofibroblasts are involved, are the pathological hallmark lesions in IPF to represent focal areas of active fibrogenesis. Fibroblast heterogeneity in fibrotic lesions hampers the discovery of the pathogenesis of pulmonary fibrosis. Therefore, to determine of the pathogenesis of IPF, identification of functional fibroblasts is warranted. This study was aimed to determine the role of fibroblasts positive for meflin, identified as a potential marker for mesenchymal stromal cells, during the development of pulmonary fibrosis. We characterised meflin-positive cells in a single cell atlas established by single-cell RNA sequencing (scRNA-seq)-based profiling of 243 472 cells from 32 IPF lungs and 29 normal lung samples. scRNA-seq combined with in situ RNA hybridisation identified proliferating fibroblasts positive for meflin in fibroblastic foci, not dense fibrosis, of fibrotic lungs in IPF patients. We determined the role of fibroblasts positive for meflin using bleomycin (BLM)-induced pulmonary fibrosis. A BLM-induced lung fibrosis model for meflin-deficient mice showed that fibroblasts positive for meflin had anti-fibrotic property to prevent pulmonary fibrosis. Although transforming growth factor-β-induced fibrogenesis and cell senescence with senescence-associated secretory phenotype were exacerbated in fibroblasts via the repression or lack of meflin, these were inhibited in meflin-deficient fibroblasts with meflin reconstitution. These findings provide evidence to show the biological importance of meflin expression on fibroblasts and myofibroblasts in the active fibrotic region of pulmonary fibrosis.

DOI： 10.1183/13993003.03397-2020

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

DOI： 10.3389/fcell.2021.728383

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Recent comprehensive studies have revealed several molecular alterations that are frequently found in meningiomas. However, effective treatment reagents targeting specific molecular alterations have not yet been identified because of the limited number of representative research models of meningiomas. METHODS: We performed organoid cultures using meningioma cells and meningioma tumor tissues. Using immunohistochemistry and molecular analyses consisting of whole-exome sequencing, RNA-seq, and DNA methylation analyses, we compared the histological findings and molecular profiling of organoid models with those of parental tumors. Further, using these organoid models together with a public database of meningiomas, we explored molecular alterations, which are a potent treatment target for meningioma. RESULTS: We established 18 organoid models comprising of two malignant meningioma cells (HKBMM and IOMM-Lee), 10 benign meningiomas, four malignant meningiomas, and two solitary fibrous tumors (SFTs). The organoids exhibited consistent histological features and molecular profiles with those of the parental tumors. Using a public database, we identified that upregulated forkhead box M1 (FOXM1) was correlated with increased tumor proliferation. Overexpression of FOXM1 in benign meningioma organoids increased organoid proliferation; depletion of FOXM1 in malignant organoids decreased proliferation. Additionally, thiostrepton, a FOXM1 inhibitor combined with radiation therapy, significantly inhibited the proliferation of malignant meningioma organoid models. CONCLUSIONS: An organoid model for meningioma enabled us to elucidate the tumor biology of meningioma along with potent treatment targets for meningioma.

DOI： 10.1093/neuonc/noab155

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

PURPOSE: The integration process used as a pre-processing step in the reconstruction of differential phase-contrast X-ray CT (d-PCCT) causes the measurement noise to propagate throughout the projection image, which is leading to increased ring artifacts (RA) in the reconstructed image. It is difficult to eliminate the RA using conventional RA removal methods that were developed for the absorption-based CT field. We propose an effective method that can remove RA of d-PCCT images. METHODS: The proposed method uses Laplacian images reconstructed from second-derivative projections of d-PCCT. This method is based on a conditional generative adversarial network (cGAN), whose loss function is designed by adding the L1- and L2-norm to the original cGAN. The training data were taken from a numerical phantom generated by a d-PCCT imaging simulator. To validate the applicability of the trained network, we tested its RA removal effect on test data from numerical phantoms generated randomly and actual experimental data. RESULTS: The results of numerical validation using numerical phantoms showed that the proposed method improved the RA removal effect compared to conventional methods. In addition, image comparison by visual evaluation showed that only the proposed method was able to remove RA while preserving original structures in the actual biological d-PCCT images. CONCLUSION: We proposed a cGAN-based method for RA removal that exploits the physical properties of d-PCCT. The proposed method was able to completely remove RA from d-PCCT images on both simulated data and biological data. We believe that this method is useful for the observation of various types of biological soft tissue.

DOI： 10.1007/s11548-021-02500-3

Web of Science

PubMed

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Fibroblasts synthesise the extracellular matrix (ECM) such as collagen and elastin, the excessive accumulation of which can lead to fibrosis and organ dysfunction under pathological conditions. Cancer-associated fibroblasts (CAFs) are major constituents of the tumour microenvironment (TME) that accompany the desmoplastic reaction responsible for anti-cancer treatment resistance. Thus, it is important to dissect the roles of CAFs in the TME to develop new therapeutic strategies for refractory cancers. Recent progress in the studies of CAF biology suggests that the functions of CAFs are complicated and that they are composed of functionally distinct populations, including cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). We recently identified a new cell surface marker for rCAFs in pancreatic and colon cancers, designated as Meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue)/Islr (immunoglobulin super family containing leucine-rich repeat). Based on the distribution of Meflin/Islr-positive cells, we also considered it a specific candidate marker for mesenchymal stroma/stem cells. Meflin/Islr-positive CAFs have been shown to suppress cancer progression by being involved in regulating collagen structures and BMP signalling in the TME. This review describes the function of Meflin/Islr in cancer fibrosis as well as in cardiac and lung fibrosis and its potential in the development of new cancer therapeutics.

DOI： 10.3389/fcell.2021.749924

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

DOI： 10.3791/62630

Web of Science

PubMed

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：日本組織細胞化学会  

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. METHODS: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. FINDINGS: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. INTERPRETATION: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.

DOI： 10.1016/j.biopha.2021.111738

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

DOI： 10.1016/j.cub.2021.04.078

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.

DOI： 10.1111/gtc.12855

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

DOI： 10.1242/dev.195883

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties. RESULTS: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 μm; 3 μm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 μm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH2) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH2 induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH2 was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs. CONCLUSIONS: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.

DOI： 10.1186/s12989-021-00415-0

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

DOI： 10.1038/s41598-021-90209-3

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Immune-related adverse events induced by anti-programmed cell death-1 antibodies (PD-1-Ab), including destructive thyroiditis (thyroid-irAE), are thought to be caused by activated T cells. However, the T cell subsets that are directly responsible for damaging self-organs remain unclear. To clarify which T cell subsets are involved in the development of thyroid-irAE, a mouse model of thyroid-irAE was analyzed. PD-1-Ab administration 2.5 months after immunization with thyroglobulin caused destructive thyroiditis. Thyroiditis was completely prevented by previous depletion of CD4+ T cells and partially prevented by depleting CD8+ T cells. The frequencies of central and effector memory CD4+ T cell subsets and the secretion of interferon-γ after stimulation with thyroglobulin were increased in the cervical lymph nodes of mice with thyroid-irAE compared with controls. Histopathological analysis revealed infiltration of CD4+ T cells expressing granzyme B in thyroid glands and major histocompatibility complex class II expression on thyrocytes in mice with thyroid-irAE. Adoptive transfer of CD4+ T cells from cervical lymph nodes in mice with thyroid-irAE caused destruction of thyroid follicular architecture in the irradiated recipient mice. Flow cytometric analyses showed that the frequencies of central and effector memory CD4+ T cells expressing the cytotoxic marker CD27 were higher in peripheral blood mononuclear cells collected from patients with thyroid-irAE induced by PD-1-Ab versus those without. These data suggest a critical role for cytotoxic memory CD4+ T cells activated by PD-1-Ab in the pathogenesis of thyroid-irAE.

DOI： 10.1126/scitranslmed.abb7495

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Prognosis for advanced oral carcinoma remains poor. Oncolytic virotherapy uses replication-competent viruses to infect and kill only the tumor cells. However, it has been difficult to investigate the oncolytic activity of viruses against oral carcinomas in mouse models. This study established a mouse model of oral cancer and investigated the in vitro and in vivo anti-tumor effects of HF10, a highly attenuated, replication-competent herpes simplex virus (HSV)-1. Mouse tongue cancer was induced by injecting 4-nitroquinoline 1-oxide into the mouse tongue. The murine oral cancer cell line isolated from this tumor, named NMOC1, formed invasive carcinoma within a week when injected into mouse tongue. HF10 successfully infected, replicated, and spread in the cancer cells in vitro. HF10 was able to kill cancer cells isolated from human or mouse tongue tumor. HF10 injection into tongue carcinomas prolonged mouse survival without any side effects or weight loss. Intertumoral injection of GFP-expressing HF10 confirmed that viral spread was confined within the tumors. Immunohistochemical staining showed that HF10 induced infiltration of CD8-positive T cells around HSV-infected cells in the tumor mass, implying increased anti-tumor immunity. We successfully established an oral cancer cell line and showed that HF10 is a promising therapeutic agent for oral cancer.

DOI： 10.1016/j.omto.2020.12.007

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

DOI： 10.1053/j.gastro.2020.11.011

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Blood-brain barrier (BBB) dysfunction underlies the pathogenesis of many neurological diseases. Platelet-derived growth factor receptor-alpha (PDGFRα) induces hemorrhagic transformation (HT) downstream of tissue plasminogen activator in thrombolytic therapy of acute stroke. Thus, PDGFs are attractive therapeutic targets for BBB dysfunction. In the present study, we examined the role of PDGF signaling in the process of tissue remodeling after middle cerebral arterial occlusion (MCAO) in mice. Firstly, we found that imatinib increased lesion size after permanent MCAO in wild-type mice. Moreover, imatinib-induced HT only when administrated in the subacute phase of MCAO, but not in the acute phase. Secondly, we generated genetically mutated mice (C-KO mice) that showed decreased expression of perivascular PDGFRα. Additionally, transient MCAO experiments were performed in these mice. We found that the ischemic lesion size was not affected; however, the recruitment of PDGFRα/type I collagen-expressing perivascular cells was significantly downregulated, and HT and IgG leakage was augmented only in the subacute phase of stroke in C-KO mice. In both experiments, we found that the expression of tight junction proteins and PDGFRβ-expressing pericyte coverage was not significantly affected in imatinib-treated mice and in C-KO mice. The specific implication of PDGFRα signaling was suggestive of protective effects against BBB dysfunction during the subacute phase of stroke. Vascular TGF-β1 expression was downregulated in both imatinib-treated and C-KO mice, along with sustained levels of MMP9. Therefore, PDGFRα effects may be mediated by TGF-β1 which exerts potent protective effects in the BBB.

DOI： 10.1007/s10456-020-09742-w

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：Japanese  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-β binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-β activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-β signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.

DOI： 10.1111/cas.14673

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of β-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of β-catenin transcriptional activity. Consistent with this, wild-type Daple promoted β-catenin transcriptional activity, following dissociation of β-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated β-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.

DOI： 10.1016/j.bbrc.2020.08.066

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

The actin-binding protein Girdin is a hub protein that interacts with multiple proteins to regulate motility and Akt and trimeric G protein signaling in cancer cells. Girdin expression correlates with poor outcomes in multiple human cancers. However, those findings are not universal, as they depend on study conditions. Those data suggest that multiple aspects of Girdin function and its role in tumor cell responses to anticancer therapeutics must be reconsidered. In the present study, we found that Girdin is involved in DNA damage-induced cancer cell apoptosis. An esophageal cancer cell line that exhibited high Girdin expression showed a marked sensitivity to UV-mediated DNA damage compared to a line with low Girdin expression. When transcriptional activation of endogenous Girdin was mediated by an engineered CRISPR/Cas9 activation system, sensitivity to DNA damage increased in both stationary and migrating HeLa cancer cells. High Girdin expression was associated with dysregulated cell cycle progression and prolonged G1 and M phases. These features were accompanied by p53 activation, which conceivably increases cancer cell vulnerability to UV exposure. These data highlight the importance of understanding complex Girdin functions that influence cancer cell sensitivity to therapeutics.

DOI： 10.1111/cas.14637

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：Japanese  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Malignant mesothelioma is an aggressive neoplasm for which effective treatments are lacking. We often encounter mesothelioma cases with a profound desmoplastic reaction, suggesting the involvement of cancer‑associated fibroblasts (CAFs) in mesothelioma progression. While the roles of CAFs have been extensively studied in other tumors and have led to the view that the cancer stroma contains heterogeneous populations of CAFs, their roles in mesothelioma remain unknown. We previously showed that connective tissue growth factor (CTGF), a secreted protein, is produced by both mesothelioma cells and fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of α‑smooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and Elastica‑Masson staining. We also analyzed the expression of mesenchymal stromal cell‑ and fibroblast‑expressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancer‑restraining CAFs and differ from αSMA‑positive CAFs, by in situ hybridization. The extent of fibrosis and CTGF expression in mesothelioma cells did not correlate with patient prognosis. However, the expression of αSMA and CTGF, but not Meflin, in CAFs correlated with poor prognosis. The data suggest that CTGF+ CAFs are involved in mesothelioma progression and represent a potential molecular target for mesothelioma therapy.

DOI： 10.3892/or.2020.7669

Web of Science

PubMed

Language：Japanese  

Web of Science

Publishing type：Research paper (scientific journal)   Publisher：American Society of Clinical Oncology (ASCO)  

3118

Background: Tumor immunity is regulated by complex interactions between cancer and immune cells, which also involves other components of the tumor microenvironment (TME). Recently, cancer-associated fibroblasts (CAFs), a major constituent of the TME, have emerged as important regulators of tumor immunity. Specifically, for example, α-smooth muscle actin or leucine-rich repeat containing 15-positive CAFs have been shown to be crucial for the suppression of tumor immunity. However, a comprehensive picture of how other CAF subset(s) are involved in tumor immunity is still lacking. Here, we show the involvement of a CAF subset highly expressing Meflin, which was recently identified as a marker of cancer-restraining CAFs in pancreatic cancer (Mizutani et al., Cancer Res, 2019), in the response of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitors (ICIs). Methods: A sample cohort of 122 subjects with NSCLC who had received ICI monotherapy with nivolumab, pembrolizumab, or atezolizumab was identified at the Department of Respiratory Medicine at Nagoya University Hospital. We selected 92 eligible patients, collected formalin-fixed paraffin-embedded tumor tissues, and prepared 4–µm-thick slides for the analysis of Meflin expression by RNA-in situ hybridization assay, followed by the evaluation of treatment response of 88 patients using the iRECIST criteria. We assessed the number of Meflin-positive CAFs and divided the patients into Meflin-High (20% and more CAFs express Meflin) and -Low groups. The cut-off value was obtained by the ROC analysis. Primarily, objective response rate (ORR) was compared between Meflin-High and –Low groups. Overall survival (OS), and progression free survival (PFS) were also assessed. Results: Patients who started to receive ICIs till the end of March 2019 were enrolled and followed-up until the end of 2019. Analysis of the tumor tissues revealed that 24 (40.7%) of 59 Meflin-High patients responded to the ICI monotherapy. In contrast, none (0%) of 29 Meflin-Low patients showed any significant response (p-value: 0.0000174). Meflin-High groups showed statistically significant prolongations in both OS and PFS with the hazard ratios of 0.3114 [0.1591-0.6094] and 0.3997 [0.2290-0.6976], respectively. Conclusions: This retrospective observation indicated that the high infiltration of Meflin-positive CAFs may shape tumor-suppressive immune response and increase the sensitivity to ICIs, which differs from those of other CAF subsets.

DOI： 10.1200/jco.2020.38.15_suppl.3118

Language：English   Publishing type：Research paper (scientific journal)  

The roles of cancer-associated fibroblasts (CAF) in the progression of various types of cancers are well established. CAF promote cancer progression through pleiotropic mechanisms, including the secretion of soluble factors and extracellular matrix, physical interactions with cancer cells, and the regulation of angiogenesis, immunity and metabolism. Their contribution to therapeutic resistance is also well appreciated. Therefore, CAF have been considered as a therapeutic target in cancer. However, recent studies in autochthonous pancreatic cancer models suggest that specific subset(s) of CAF exhibit cancer-restraining roles, indicating that CAF are functionally and molecularly heterogeneous, which is supported by recent single-cell transcriptome analyses. While cancer-promoting CAF (pCAF) have been extensively studied, the nature and specific marker(s) of cancer-restraining CAF (rCAF) have remained uncharacterized. Interestingly, a recent study provided insight into the nature of rCAF and suggested that they may share molecular properties with pancreatic stellate cells (PSC) and mesenchymal stem/stromal cells (MSC). Complicating this finding is that PSC and MSC have been shown to promote the formation of a tumor-permissive and tumor-promoting environment in xenograft tumor models. However, these cells undergo significant transcriptional and epigenetic changes during ex vivo culture, which confounds the interpretation of experimental results based on the use of cultured cells. In this short review, we describe recent studies and hypotheses on the identity of rCAF and discuss their analogy to fibroblasts that suppress fibrosis in fibrotic diseases. Finally, we discuss how these findings can be exploited to develop novel anticancer therapies in the future.

DOI： 10.1111/cas.14346

Web of Science

PubMed

Language：Japanese  

Web of Science

Language：Japanese  

DOI： 10.1158/1538-7445.PANCA19-A53

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

DOI： 10.1161/CIRCULATIONAHA.119.041019

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

DOI： 10.1158/0008-5472.CAN-19-0454

Web of Science

PubMed

Language：Japanese  

DOI： 10.1183/13993003.congress-2019.PA2449

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

DOI： 10.1172/JCI124481

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

RATIONALE: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. OBJECTIVE: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. METHODS AND RESULTS: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. CONCLUSIONS: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

DOI： 10.1161/CIRCRESAHA.119.314806

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-β1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-β1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-β1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-β1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

DOI： 10.1016/j.bbrc.2019.05.058

Web of Science

PubMed

Language：Japanese  

DOI： 10.1158/1538-7445.AM2019-2029

Web of Science

Language：English  

DOI： 10.1016/j.bbrc.2019.03.167

Web of Science

PubMed

Language：English  

DOI： 10.1038/s41575-019-0115-0

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein and a member of the α2 -macroglobulin/C3,C4,C5 family of thioester-containing proteins first identified as being expressed on blood cells, including activated T cells and platelets, and a subset of CD34 + bone marrow cells containing megakaryocyte progenitors. Although CD109 carries the biallelic platelet-specific alloantigen Gov, the physiological functions or roles of CD109 in human disease remain largely unknown. It was recently demonstrated that CD109 is expressed in many malignant tumors, including various squamous cell carcinomas and adenocarcinomas, and plays a role as a multifunctional coreceptor. CD109 reportedly associates with transforming growth factor (TGF)-β receptors and negatively regulates TGF-β signaling in keratinocytes. Additionally, CD109 is potentially related to signal transducer and activator of transcription-3 signaling and aberrant cell proliferation. In this review, we describe recent evidence of CD109-specific significance in malignant tumors shown in mouse models and human tissues. Furthermore, we discuss the physiological functions of CD109 in vitro and in vivo, including results of phenotype analyses of CD109-deficient mice exhibiting epidermal hyperplasia and osteopenia.

DOI： 10.1111/pin.12798

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

RET finger protein (RFP) forms a complex with histone deacetylase 1, resulting in aberrant deacetylation of H3K27ac and dysregulation of cis-regulatory elements. We evaluated the modulatory effects of RFP knockdown on cis-regulatory elements, gene expression, and chemosensitivity to temozolomide both in glioblastoma cells and in an intracranial glioblastoma model. The combination of RFP knockdown and temozolomide treatment markedly suppressed the glioblastoma cell growth due to oxidative stress and aberrant cell cycle and increased survival time in mice with glioblastoma. ChIP-seq and RNA-seq revealed that RFP knockdown increased or decreased activity of numerous cis-regulatory elements that lie adjacent to genes that control functions such as apoptosis, mitosis, DNA replication, and cell cycle: FOXO1, TBP2, and PARPBP. This study suggests that RFP contributes to chemoresistance via aberrant deacetylation of histone H3 at K27, whereas dysregulation of RFP-associated cis-regulatory elements in glioma and RFP knockdown combined with temozolomide is an effective treatment strategy for lethal glioma.

DOI： 10.1016/j.celrep.2019.01.109

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41575-019-0115-0.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.celrep.2019.01.109.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/01443615.2018.1511971

Web of Science

PubMed

Web of Science

Web of Science

Web of Science

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Chemerin acts as a chemotactic factor for leukocyte populations expressing the G protein-coupled receptor CMKLR1 (ChemR23). It is also an adipocytokine involved in obesity and metabolic syndromes. Previous studies have demonstrated that chemerin promotes angiogenesis in vitro, although the precise mechanism has not been elucidated. In this study, we have investigated whether chemerin regulates angiogenic processes and validated the associated mechanisms. In this study, chemerin stimulated angiogenesis in mice, which was demonstrated using Matrigel plug implantation assay, mouse corneal models of angiogenesis, and ex vivo rat aortic ring assay. To explore the mechanisms by which chemerin induced angiogenesis, we examined the effects of chemerin in human umbilical vein endothelium cells (HUVECs). Chemerin stimulated the differentiation of HUVECs into capillary-like structures, promoted the proliferation of HUVECs, and functioned as a chemoattractant in migration assays. Chemerin induced the phosphorylation of Akt and p42/44 extracellular signal-regulated kinase (ERK) in HUVECs and chemerin promotes angiogenesis via Akt and ERK. SiRNA against the chemerin receptor CMKLR1 but not that against another chemerin receptor, CCRL2, completely inhibited the chemerin-induced migration and angiogenesis of HUVECs, which indicates that chemerin promotes the migration and angiogenic activities of HUVECs mainly through CMKLR1.

DOI： 10.14814/phy2.13962

Web of Science

PubMed

Language：English   Publisher：WILEY  

Web of Science

Web of Science

Web of Science

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin-binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E-cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells.

DOI： 10.1111/cas.13795

Web of Science

PubMed

Web of Science

J-GLOBAL

Language：English  

DOI： 10.1172/JCI97755

Web of Science

PubMed

DOI： 10.1158/1538-7445.AM2018-3160

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Osteoporosis is a global public health problem that is increasing along with an aging population. A major determinant of osteoporosis is high bone turnover, which results from osteoclast activation. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, a deficiency that leads to a psoriasis-like skin inflammation in mice. Although the expression of CD109 has been reported in mouse pre-osteoclast cells, its function in osteoclasts in vivo remains largely unknown. To investigate the physiological role of CD109 in bone metabolism, we analyzed bones from wild-type and CD109-deficient adult mice. Micro-computed tomography analysis of the femur (thigh bone) showed that bone volume was lower in CD109-deficient mice than in wild-type mice. Bone histomorphometric analysis showed not only a reduction in bone volume but also an increase in bone turnover in CD109-deficient mice as compared with wild-type mice. Additionally, we measured serum levels of several markers of bone turnover and found a significant increase in the N-terminal telopeptide of type I collagen, a bone resorption marker, as well as alkaline phosphatase, a bone formation marker, in CD109-deficient mice. These results indicate that CD109 deficiency induces a high-turnover, osteoporosis-like phenotype, which suggests that CD109 plays a role in bone metabolism in vivo.

DOI： 10.1111/gtc.12593

Web of Science

PubMed

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Linx is a member of the leucine-rich repeat and immunoglobulin family of membrane proteins which has critical roles in the development of the peripheral nervous system and forebrain connectivity. A previous study showed that Linx is expressed in projection neurons in the cortex and in cells that comprise the passage to the prethalamus that form the internal capsule, indicating the involvement of Linx in axon guidance and cell-cell communication. In this study, we found that Linx-deficient mice develop severe hydrocephalus and die perinatally by unknown mechanisms. Importantly, mice heterozygous for the linx gene exhibited defects in the development of the anterior commissure in addition to hydrocephalus, indicating haploinsufficiency of the linx gene in forebrain development. In N1E-115 neuroblastoma cells and primary cultured hippocampal neurons, Linx depletion led to impaired neurite extension and an increase in cell body size. Consistent with this, but of unknown significance, we found that Linx interacts with and upregulates the activity of Rho-kinase, a modulator of many cellular processes including cytoskeletal organization. These data suggest a role for Linx in the regulation of complex forebrain connectivity, and future identification of its extracellular ligand(s) will help clarify this function.

DOI： 10.1038/s41598-018-24064-0

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：(株)文光堂  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

DOI： 10.1016/j.bbrc.2018.03.016

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Autoimmune hypophysitis (AH) is thought to be an autoimmune disease characterized by lymphocytic infiltration of the pituitary gland. Among AH pathologies, lymphocytic infundibulo-neurohypophysitis (LINH) involves infiltration of the neurohypophysis and/or the hypothalamic infundibulum, causing central diabetes insipidus resulting from insufficiency of arginine vasopressin secretion. The pathophysiological and pathogenetic mechanisms underlying LINH are largely unknown. Clinically, differentiating LINH from other pituitary diseases accompanied by mass lesions, including tumours, has often been difficult, because of similar clinical manifestations. We recently reported that rabphilin-3A is an autoantigen and that anti-rabphilin-3A antibodies constitute a possible diagnostic marker for LINH. However, the involvement of rabphilin-3A in the pathogenesis of LINH remains to be elucidated. This study was undertaken to explore the role of rabphilin-3A in lymphocytic neurohypophysitis and to investigate the mechanism. We found that immunization of mice with rabphilin-3A led to neurohypophysitis. Lymphocytic infiltration was observed in the neurohypophysis and supraoptic nucleus 1 month after the first immunization. Mice immunized with rabphilin-3A showed an increase in the volume of urine that was hypotonic as compared with control mice. Administration of a cocktail of monoclonal anti-rabphilin-3A antibodies did not induce neurohypophysitis. However, abatacept, which is a chimeric protein that suppresses T-cell activation, decreased the number of T cells specific for rabphilin-3A in peripheral blood mononuclear cells (PBMCs). It ameliorated lymphocytic infiltration of CD3+ T cells in the neurohypophysis of mice that had been immunized with rabphilin-3A. Additionally, there was a linear association between the number of T cells specific for rabphilin-3A in PBMCs and the number of CD3+ T cells infiltrating the neurohypophysis. In conclusion, we suggest that rabphilin-3A is a pathogenic antigen, and that T cells specific for rabphilin-3A are involved in the pathogenesis of neurohypophysitis in mice. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp
Sons, Ltd.

DOI： 10.1002/path.5046

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing  

Embedding of tissue samples that maintains a desired orientation is critical for preparing sections suitable for diagnosis and study objectives. Methods to prepare tissue sections include: (i) paraffin embedding or snap-freezing followed by microtome or cryostat sectioning
and (ii) agarose embedding followed by cutting on a vibrating microslicer. Although these methods are useful for routine laboratory work, preparation of small and fragile tissues such as mouse organs, small human biopsy samples, and cultured floating spheres is difficult and requires special skills. In particular, tissue specimen orientation can be lost during embedding in molds and subsequent sectioning. Here, we developed a method using low melting temperature (LM) gelatin either alone or mixed with agarose to preliminarily embed collected tissues that are either prefixed or unfixed, followed by conventional fixation, paraffin embedding, freezing, and sectioning. The advantage of the method is that the LM gelatin and its mixture with agarose can be handled at room temperature but quickly hardens at 4°C, which allows embedding, trimming, and arranging of small and fragile tissues in a desired orientation and are compatible with traditional stainings. Thus, this method can have various laboratory applications and can be modified according to the needs of each laboratory.

DOI： 10.1111/pin.12652

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

DOI： 10.1371/journal.pbio.2005090

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.

DOI： 10.1111/cas.13448

Web of Science

PubMed

Web of Science

Web of Science

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Endocrine Society  

The molecular mechanism involved in the exocytosis of arginine vasopressin (AVP) is not fully known. Rabphilin-3A has been suggested as a novel autoantigen in infundibulo-neurohypophysitis (LINH), which leads to central diabetes insipidus through insufficient secretion of AVP. However, the role of rabphilin-3A in the pathogenesis of LINH remains unclear. Thus, the aim of the present study was to identify proteins binding rabphilin-3A in the posterior pituitary. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, cullin-associated NEDD8-dissociated protein 1 (CAND1) was identified as a rabphilin-3A-binding protein in the posterior pituitary. Co-immunoprecipitation assays indicated that CAND1 interacted endogenously with rabphilin-3A. In addition, immunohistochemistry experiments showed that CAND1 immunoreactivity was detected mainly in the posterior pituitary, intermediate lobe, and the supraoptic nucleus in the hypothalamus, and less in the anterior lobe, partially co-localizing with rabphilin-3A. Overexpression of CAND1 resulted in deubiquitylation of rabphilin-3A in PC12 cells. Moreover, overexpression of CAND1 in PC12 cells co-transfected with AVP enhanced both basal and KCl-stimulated AVP secretion. The findings indicate that CAND1 inhibits the ubiquitylation of rabphilin-3A and positively regulates AVP secretion. These data shed light on a novel potential mechanism involving rabphilin-3A in AVP secretion, and suggest a new role of CAND1 as a regulator of hormone or neurotransmitter secretion.

DOI： 10.1507/endocrj.EJ17-0399

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol-anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower-grade glioma (World Health Organization grade II/III) by clinicopathological and whole-genome sequencing analysis of tissues from human glioma. The importance of CD109-positive perivascular tumour cells was confirmed not only in human lower-grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109-positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright (C) 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4981

Web of Science

PubMed

Language：English   Publisher：OXFORD UNIV PRESS INC  

Web of Science

Web of Science

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF) flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP) proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveledassociating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

DOI： 10.1016/j.celrep.2017.06.089

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Glioblastomas (GBMs) are the most common and aggressive type of brain tumor. GBMs usually show hyperactivation of the PI3K-Akt pathway, a pro-tumorigenic signaling cascade that contributes to pathogenesis. Girdin, an actin-binding protein identified as a novel substrate of Akt, regulates the sprouting of axons and the migration of neural progenitor cells during early postnatal-stage neurogenesis in the hippocampus. Here, we show that Girdin is highly expressed in human glioblastoma (GBM). Stable Girdin knockdown in isolated GBM stem cells resulted in decreased expression of stem cell markers, including CD133, induced multilineage neural differentiation, and inhibited in vitro cell motility, ex vivo invasion, sphere-forming capacity and in vivo tumor formation. Furthermore, exogenous expression of the Akt-binding domain of Girdin, which competitively inhibits its Akt-mediated phosphorylation, diminished the expression of stem cell markers, SOX2 and nestin, and migration on the brain slice and induced the expression of neural differentiation markers glial fibrillary acidic protein/ΒIII Tubulin. Our results reveal that Girdin is required for GBM-initiating stem cells to sustain the stemness and invasive properties. © 2012 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2011.466

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status-specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.

DOI： 10.1369/0022155417702586

Web of Science

PubMed

Language：English   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/onc.2017.17

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs).
Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry.
BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide.
Our findings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

DOI： 10.1093/neuonc/now237

Web of Science

PubMed

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep22288

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/pin.12391

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publisher：(有)科学評論社  

Language：English   Publisher：日本組織細胞化学会  

Language：Japanese   Publisher：医学図書出版(株)  

膵癌治療のボトルネックは、その豊富な間質量(癌細胞以外の領域あるいは細胞)に起因する薬剤分布の障害であり、これを解決する治療法の開発が強く求められている。膵癌の豊富な間質の原因は、癌関連線維芽細胞の増生であり、問題はその多くが癌促進性の形質を有していることである。われわれは、最近、膵癌間質の癌関連線維芽細胞には癌促進性の細胞と癌抑制性の細胞の二種類が存在することを明らかにした。また、化合物ライブラリースクリーニングによって、急性前骨髄球性白血病(APL)に対してすでに薬事承認されているAM80(一般名:タミバロテン)が、癌関連線維芽細胞の形質を癌促進性から癌抑制性に転換することを見出した。さらに膵癌マウスモデルを用いた非臨床試験によって、AM80の投与が従来の化学療法への感受性を有意に上昇させることを明らかにした。われわれは、AM80と標準療法の併用が、年間3万人以上発症する同疾患の標準治療の一つとなることをめざし本年度より医師主導治験を行う予定である。(著者抄録)

Language：Japanese   Publisher：(有)科学評論社  

Language：Japanese   Publisher：(一社)日本病理学会  

J-GLOBAL

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：Japanese   Publisher：(一社)日本呼吸器学会  

Language：Japanese   Publisher：(一社)日本呼吸器学会  

Language：Japanese   Publisher：(株)羊土社  

がんの本質は言うまでもなく遺伝子変異を伴ったがん細胞の無秩序な増殖であるが、その治療を難しくしている理由の一つに間質(がん細胞以外の領域)の高度な増生がある。間質には線維芽細胞、同細胞が産生する細胞外基質、免疫系細胞、血管等が存在しており、膵がんに代表される難治がんでは間質量ががん細胞量を上回ることも多い。がん間質に増生する線維芽細胞は「がん関連線維芽細胞」とよばれており、多くの液性因子や細胞外基質等の産生により、がん細胞の増殖や浸潤能を促進することが知られている。一方、最近になり、がんを抑制する線維芽細胞が存在する可能性が示唆され、がん関連線維芽細胞は免疫系細胞と同様に多様性に富む細胞集団であることが判明しつつある。本稿ではがん関連線維芽細胞の多様性について、特にがん抑制性機能を有する線維芽細胞の本態について最近の知見を概説したい。(著者抄録)

Language：Japanese   Publisher：(一社)日本膵臓学会  

Language：Japanese   Publisher：(株)羊土社  

がんの組織標本をみると、がんは決してがん細胞の単なる集合ではなく、がん細胞以外の領域(間質)が非常に多いことがわかる。間質には線維芽細胞と同細胞が産生する細胞外基質、免疫系細胞、血管等が存在しており、膵がんに代表される難治がんでは、間質量ががん細胞量をはるかに上回ることも多い。細胞外基質の増生はがんを硬くし、そのことががん細胞のさらなる悪性化や血管の虚脱を誘導し、難治がんをさらに難治にさせる。このように、がんの本質の理解にはがん間質のメカノバイオロジーの理解が必須であり、本稿では特に間質の線維芽細胞に着目した最近の研究動向を概説したい。(著者抄録)

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：医歯薬出版(株)  

現在までに間葉系幹細胞(MSC)のマーカーとして多数の分子が報告されている。細胞マーカーという用語は、その細胞に特異的に発現する分子、分離のための細胞表面分子、細胞機能を規定する分子など、さまざまな意味で用いられ、状況と用途に応じて使い分けられる。これまでMSCマーカーとして報告された分子は多数あるが、いずれも特異性に問題があることが知られている。MSCが単一の細胞ではなく多様性に富む細胞集団であること、および採取する組織や培養条件によって特性が変わることもMSCマーカーの理解を難解なものにしている。また、MSCの機能を規定するマーカーの報告は少なく、その生体内機能や動態については不明な点も多い。本稿では、MSCマーカーとして知られる分子群について概略と問題点を述べ、次いで著者らが最近同定した新規MSCマーカーMeflinに関する検証結果と仮説を紹介し、生体内におけるMSCの生物学的意義について議論したい。(著者抄録)

Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2020&ichushi_jid=J00060&link_issn=&doc_id=20200309030001&doc_link_id=%2Faa7ayuma%2F2020%2F027210%2F002%2F0968-0974%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Faa7ayuma%2F2020%2F027210%2F002%2F0968-0974%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

J-GLOBAL

J-GLOBAL

Language：Japanese   Publisher：(NPO)日本肺癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：Japanese   Publisher：医歯薬出版(株)  

がんの生物学を考えるうえで、がん関連線維芽細胞(CAF)を理解することの重要性については他稿で解説されているとおり論をまたない。がんの種類によってはがん細胞よりも圧倒的に多い体積を占めているのが、CAFと同細胞が分泌する細胞外基質(ECM)である。CAFとECMはがんの促進因子であり、また最近では分子標的薬や免疫チェックポイント阻害薬の効果を阻害することも明らかになった。しかしCAFの研究が進むにつれ、最近では"がん抑制性CAF"の存在を仮定しないとうまく説明できない現象も徐々に明らかにされてきた。これは免疫細胞が多様性に富む集団であり、がんの進行を促進する細胞群と、逆に抑制する細胞群の両者が存在することと非常に似ている。本稿では長らく不明であった"がん抑制性CAF"の本態と、そのマーカー分子の候補について著者らの予備的データを含めて紹介し、CAFの性質を改変(初期化)することによる新たな治療法開発への道筋を議論したい。(著者抄録)

Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2019&ichushi_jid=J00060&link_issn=&doc_id=20190920030005&doc_link_id=%2Faa7ayuma%2F2019%2F027012%2F006%2F1133-1138%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Faa7ayuma%2F2019%2F027012%2F006%2F1133-1138%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：(公財)金原一郎記念医学医療振興財団  

Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2019&ichushi_jid=J00731&link_issn=&doc_id=20190814110011&doc_link_id=10.11477%2Fmf.2425201005&url=https%3A%2F%2Fdoi.org%2F10.11477%2Fmf.2425201005&type=%88%E3%8F%91.jp_%83I%81%5B%83%8B%83A%83N%83Z%83X&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00024_2.gif

Language：Japanese   Publisher：(一社)日本乳癌学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本内科学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本癌治療学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：English   Publisher：(一社)日本癌学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：English   Publisher：(一社)日本病理学会  

Language：Japanese   Publisher：(一社)日本病理学会  

Language：English   Publisher：日本癌学会  

J-GLOBAL

Application no：PCT/JP2012/079776