Publications by authors named "Taiji Yamazoe"

21 Publications

  • Page 1 of 1

MYC levels regulate metastatic heterogeneity in pancreatic adenocarcinoma.

Cancer Discov 2021 Sep 22. Epub 2021 Sep 22.

Ontario Institute for Cancer Research.

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1826DOI Listing
September 2021

Cluster of Differentiation 44 Promotes Liver Fibrosis and Serves as a Biomarker in Congestive Hepatopathy.

Hepatol Commun 2021 Aug 8;5(8):1437-1447. Epub 2021 Apr 8.

Research Center for Hepatitis and Immunology National Center for Global Health and Medicine Ichikawa Japan.

Congestive hepatopathy (CH) with chronic passive congestion is characterized by the progression of liver fibrosis without prominent inflammation and hepatocellular damage. Currently, the lack of reliable biomarkers for liver fibrosis in CH often precludes the clinical management of patients with CH. To explore fibrosis biomarkers, we performed proteome analysis on serum exosomes isolated from patients with CH after the Fontan procedure. Exosomal cluster of differentiation (CD)44 levels were increased in patients with CH compared to healthy volunteers and was accompanied by increases in serum levels of soluble CD44 and CD44 expression in the liver. To address the roles of CD44 in CH, we established a mouse model of chronic liver congestion by partial inferior vena cava ligation (pIVCL) that mimics CH by fibrosis progression with less inflammation and cellular damage. In the pIVCL mice, enhanced CD44 expression in hepatic stellate cells (HSCs) and deposition of its ligand hyaluronan were observed in the liver. Blood levels of soluble CD44 were correlated with liver fibrosis. The blockade of CD44 with specific antibody inhibited liver fibrosis in pIVCL mice and was accompanied by a reduction in S100 calcium-binding protein A4 expression following activation of HSCs. Chronic liver congestion promotes fibrosis through CD44. This identifies CD44 as a novel biomarker and therapeutic target of liver fibrosis in patients with CH.
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http://dx.doi.org/10.1002/hep4.1721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8369942PMC
August 2021

Calcium signaling induces a partial EMT.

EMBO Rep 2021 Sep 29;22(9):e51872. Epub 2021 Jul 29.

Abramson Family Cancer Research Institute and Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.
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http://dx.doi.org/10.15252/embr.202051872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419705PMC
September 2021

Phenotypic Characterization by Single-Cell Mass Cytometry of Human Intrahepatic and Peripheral NK Cells in Patients with Hepatocellular Carcinoma.

Cells 2021 Jun 14;10(6). Epub 2021 Jun 14.

Department of Liver Diseases, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan.

Overall response rates of systemic therapies against advanced hepatocellular carcinoma (HCC) remain unsatisfactory. Thus, searching for new immunotherapy targets is indispensable. NK cells are crucial effectors and regulators in the tumor microenvironment and a determinant of responsiveness to checkpoint inhibitors. We revealed the landscape of NK cell phenotypes in HCC patients to find potential immunotherapy targets. Using single cell mass cytometry, we analyzed 32 surface markers on CD56 and CD56 NK cells, which included Sialic acid-binding immunoglobulin-type lectins (Siglecs). We compared peripheral NK cells between HCC patients and healthy volunteers. We also compared NK cells, in terms of their localizations, on an individual patient bases between peripheral and intrahepatic NK cells from cancerous and noncancerous liver tissues. In the HCC patient periphery, CD160CD56 NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49aCD56 NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56 NK cells were significantly correlated to other NK-related markers in HCC patients, which suggested that CD160 and CD49a were signature molecules. CD49a CX3CR1 Siglec-10 NK cells had accumulated in HCC tissues. Considering further functional analyses, CD160, CD49a, CX3CR1, and Siglec-10 on CD56 NK cells may be targets for immunotherapies of HCC patients.
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http://dx.doi.org/10.3390/cells10061495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231799PMC
June 2021

Myostatin as a fibroblast-activating factor impacts on postoperative outcome in patients with hepatocellular carcinoma.

Hepatol Res 2021 Jul 25;51(7):803-812. Epub 2021 May 25.

Department of Liver Disease, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan.

Aim: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis.

Methods: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin.

Results: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm /m , and the median body mass index was 23.4 kg/m . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm /m ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin.

Conclusions: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production.
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http://dx.doi.org/10.1111/hepr.13667DOI Listing
July 2021

Mutant p53 regulates Survivin to foster lung metastasis.

Genes Dev 2021 04 18;35(7-8):528-541. Epub 2021 Mar 18.

Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York 10032, USA.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. (homologous to in mice) is a common hot spot mutation. How metastasis is regulated by p53 in ESCC remains to be investigated. To investigate p53-mediated molecular mechanisms, we used a carcinogen-induced approach in mice to model ESCC. In the primary tumor cell lines, we depleted Trp53 (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP- axis as a potential mediator of -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by , increases in the presence of Trp53 Furthermore, depletion of Survivin specifically decreases Trp53-driven lung metastasis. Mechanistically, Trp53 but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.
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http://dx.doi.org/10.1101/gad.340505.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015716PMC
April 2021

Blood angiopoietin-2 predicts liver angiogenesis and fibrosis in hepatitis C patients.

BMC Gastroenterol 2021 Feb 8;21(1):55. Epub 2021 Feb 8.

The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa, Chiba, 272-8516, Japan.

Background: Pathological angiogenesis is involved in the development of hepatocellular carcinoma. In patients with chronic hepatitis C (CHC), the level of angiogenic factor angiopoietin (ANGP)-2 is reported to be increased in the blood, correlating with fibrosis. In this study, we aimed to clarify whether blood ANGP-2 is useful as a biomarker for liver angiogenesis and fibrosis in CHC patients and to further reveal the relationship between such pathology in a carbon tetrachloride (CCl)-treated liver fibrosis mouse model.

Methods: Plasma levels of ANGP-2, expression of a liver sinusoidal endothelial cell (LSEC) marker (CD31), collagen deposition (Sirius Red staining) in the liver, clinical fibrosis markers (Mac-2 binding protein glycosylation isomer, virtual touch quantification, and liver stiffness measurement), and liver function (albumin bilirubin score) were examined in CHC patients. To determine the effects of an anti-angiogenic agent on liver fibrosis in vivo, sorafenib was administered to the CCl-treated mice (BALB/c male).

Results: The plasma levels of ANGP-2 were increased in CHC patients compared to healthy volunteers and decreased by the eradication of hepatitis C with direct-acting antivirals. In addition, plasma ANGP-2 levels were correlated with CD31 expression, collagen deposition, clinical fibrosis markers, and liver function. Sorafenib inhibited liver angiogenesis and fibrosis in the CCl-treated mice and was accompanied by decreased ANGP-2 expression in LSECs.

Conclusions: ANGP-2 may serve as a useful biomarker for liver angiogenesis and fibrosis in CHC patients. In addition, angiogenesis and fibrosis may be closely related.
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http://dx.doi.org/10.1186/s12876-021-01633-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871374PMC
February 2021

Hepatocyte ploidy and pathological mutations in hepatocellular carcinoma: impact on oncogenesis and therapeutics.

Glob Health Med 2020 Oct;2(5):273-281

Department of Liver Disease, Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan.

Hepatocellular carcinoma (HCC) occurs in the chronic liver inflammation such as viral hepatitis, alcoholic and non-alcoholic steatohepatitis. While anti-viral treatment has been significantly improved, the prevalence of HCC remains high and treatment is still challenging. The continuation of hepatocyte death, inflammation, and fibrosis leads to the accumulation of gene alterations, which may trigger carcinogenesis. Hepatocytes are a unique cell type having more than one complete set of 23 chromosomes, termed polyploidy. Due to gene redundancy, hepatocytes may tolerate lethal mutations. Next generation sequencing technology has revealed gene alterations in HCC related to telomere maintenance, Wnt/β-catenin pathway, p53 cell-cycle pathway, epigenetic modifiers, oxidative stress pathway, PI3K/AKT/mTOR, and RAS/RAF/MAPK pathway with or without a chromosomal instability. Some type of driver gene mutations accumulates in hepatocytes and breaks the orchestration of excessive copies of chromosomes, which may lead to unfavorable gene expressions and fuel tumorigenesis. Recently, molecular targeted drugs, developed with the aim of interfering with these signaling pathways, are being used for HCC patients in the clinics. Therefore, a deeper understanding of hepatocyte ploidy and genetic or epigenetic alterations is indispensable for the establishment of novel therapeutic strategies against HCC.
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http://dx.doi.org/10.35772/ghm.2020.01089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731061PMC
October 2020

Impact of Immune Reconstitution-Induced Hepatic Flare on Hepatitis B Surface Antigen Loss in Hepatitis B Virus/Human Immunodeficiency Virus-1 Coinfected Patients.

J Infect Dis 2021 Jun;223(12):2080-2089

AIDS Clinical Center, National Center for Global Health and Medicine, Tokyo, Japan.

Background: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss.

Methods: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection.

Results: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF.

Conclusions: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.
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http://dx.doi.org/10.1093/infdis/jiaa662DOI Listing
June 2021

Global Regulation of the Histone Mark H3K36me2 Underlies Epithelial Plasticity and Metastatic Progression.

Cancer Discov 2020 06 18;10(6):854-871. Epub 2020 Mar 18.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFICANCE: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269857PMC
June 2020

Tumor Cell-Intrinsic USP22 Suppresses Antitumor Immunity in Pancreatic Cancer.

Cancer Immunol Res 2020 03 23;8(3):282-291. Epub 2019 Dec 23.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Although immune checkpoint blockade (ICB) improves clinical outcome in several types of malignancies, pancreatic ductal adenocarcinoma (PDA) remains refractory to this therapy. Preclinical studies have demonstrated that the relative abundance of suppressive myeloid cells versus cytotoxic T cells determines the efficacy of combination immunotherapies, which include ICB. Here, we evaluated the role of the ubiquitin-specific protease 22 (USP22) as a regulator of the immune tumor microenvironment (TME) in PDA. We report that deletion of USP22 in pancreatic tumor cells reduced the infiltration of myeloid cells and promoted the infiltration of T cells and natural killer (NK) cells, leading to an improved response to combination immunotherapy. We also showed that ablation of tumor cell-intrinsic USP22 suppressed metastasis of pancreatic tumor cells in a T-cell-dependent manner. Finally, we provide evidence that USP22 exerted its effects on the immune TME by reshaping the cancer cell transcriptome through its association with the deubiquitylase module of the SAGA/STAGA transcriptional coactivator complex. These results indicated that USP22 regulates immune infiltration and immunotherapy sensitivity in preclinical models of pancreatic cancer.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173406PMC
March 2020

iPreP is a three-dimensional nanofibrillar cellulose hydrogel platform for long-term ex vivo preservation of human islets.

JCI Insight 2019 11 1;4(21). Epub 2019 Nov 1.

Gastroenterology Division, Department of Medicine.

Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel-based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.
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http://dx.doi.org/10.1172/jci.insight.124644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948755PMC
November 2019

Tumor cell-intrinsic EPHA2 suppresses anti-tumor immunity by regulating PTGS2 (COX-2).

J Clin Invest 2019 06 4;129(9):3594-3609. Epub 2019 Jun 4.

Department of Medicine.

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.
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http://dx.doi.org/10.1172/JCI127755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715369PMC
June 2019

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy.

Immunity 2018 07 26;49(1):178-193.e7. Epub 2018 Jun 26.

Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address:

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8 T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.
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http://dx.doi.org/10.1016/j.immuni.2018.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707727PMC
July 2018

EMT Subtype Influences Epithelial Plasticity and Mode of Cell Migration.

Dev Cell 2018 06;45(6):681-695.e4

Department of Medicine, Gastroenterology Division, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, 512 BRB II/III, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Epithelial-mesenchymal transition (EMT) is strongly implicated in tumor cell invasion and metastasis. EMT is thought to be regulated primarily at the transcriptional level through the repressive activity of EMT transcription factors. However, these classical mechanisms have been parsed out almost exclusively in vitro, leaving questions about the programs driving EMT in physiological contexts. Here, using a lineage-labeled mouse model of pancreatic ductal adenocarcinoma to study EMT in vivo, we found that most tumors lose their epithelial phenotype through an alternative program involving protein internalization rather than transcriptional repression, resulting in a "partial EMT" phenotype. Carcinoma cells utilizing this program migrate as clusters, contrasting with the single-cell migration pattern associated with traditionally defined EMT mechanisms. Moreover, many breast and colorectal cancer cell lines utilize this alternative program to undergo EMT. Collectively, these results suggest that carcinoma cells have different ways of losing their epithelial program, resulting in distinct modes of invasion and dissemination.
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http://dx.doi.org/10.1016/j.devcel.2018.05.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014628PMC
June 2018

Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

Methods Mol Biol 2016 ;1357:475-83

Program for Leading Graduate Schools "HIGO (Health life science; Interdisciplinary and Glocal Oriented) Program", Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto, 860-0811, Japan.

The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.
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http://dx.doi.org/10.1007/7651_2014_138DOI Listing
October 2016

Generation of familial amyloidotic polyneuropathy-specific induced pluripotent stem cells.

Stem Cell Res 2014 Mar 27;12(2):574-83. Epub 2014 Jan 27.

Department of Neurology, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; Department of Diagnostic Medicine, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan. Electronic address:

Familial amyloidotic polyneuropathy (FAP) is a hereditary amyloidosis induced by amyloidogenic transthyretin (ATTR). Because most transthyretin (TTR) in serum is synthesized by the liver, liver transplantation (LT) is today the only treatment available to halt the progression of FAP, even though LT is associated with several problems. Despite the urgent need to develop alternatives to LT, the detailed pathogenesis of FAP is still unknown; also, no model fully represents the relevant processes in patients with FAP. The induction of induced pluripotent stem (iPS) cells has allowed development of pluripotent cells specific for patients and has led to useful models of human diseases. Because of the need for a tool to elucidate the molecular pathogenesis of FAP, in this study we sought to establish heterozygous ATTR mutant iPS cells, and were successful, by using a Sendai virus vector mixture containing four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to reprogram dermal fibroblasts derived from FAP patients. Moreover, FAP-specific iPS cells had the potential to differentiate into hepatocyte-like cells and indeed expressed ATTR. FAP-specific iPS cells demonstrated the possibility of serving as a pathological tool that will contribute to understanding the pathogenesis of FAP and development of FAP treatments.
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http://dx.doi.org/10.1016/j.scr.2014.01.004DOI Listing
March 2014

VMAT2 identified as a regulator of late-stage β-cell differentiation.

Nat Chem Biol 2014 Feb 15;10(2):141-8. Epub 2013 Dec 15.

1] Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan. [2] Program for Leading Graduate Schools, Health Life Science Interdisciplinary and Global Oriented (HIGO) Program, Kumamoto University, Kumamoto, Japan.

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy.
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http://dx.doi.org/10.1038/nchembio.1410DOI Listing
February 2014

A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells.

J Cell Sci 2013 Dec 7;126(Pt 23):5391-9. Epub 2013 Oct 7.

Division of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.
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http://dx.doi.org/10.1242/jcs.129767DOI Listing
December 2013

Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation.

Stem Cell Res 2013 Mar 27;10(2):179-94. Epub 2012 Nov 27.

Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.

Although progresses in developing differentiation procedures have been achieved, it remains challenging to generate hES/iPS cell-derived mature hepatocytes. We performed knock-in of a monomeric Kusabira orange (mKO1) cassette in the albumin (ALB) gene, in human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells, with the use of the helper-dependent adenovirus vector (HDAdV). Upon induction into the hepatic lineages, these knock-in hES/iPS cells differentiated into cells that displayed several known hepatic functions. The mKO1 knock-in (ALB/mKo1) hES/hiPS cells were used to visualize hepatic differentiation in vitro. mKO1 reporter expression recapitulated endogenous ALB transcriptional activity. ALB/mKo1 [Hi] population isolated by flow cytometry was confirmed to be enriched with ALB mRNA. Expression profile analyses revealed that characteristic hepatocyte genes and genes related to drug metabolism and many aspects of liver function were highly enriched in the ALB/mKo1 [Hi] population. Our data demonstrate that ALB/mKo1 knock-in hES/iPS cells are valuable resources for monitoring in vitro hepatic differentiation, isolation and analyses of hES and hiPS cells-derived hepatic cells that actively transcribing ALB. These knock-in hES/iPS cell lines could provide further insights into the mechanism of hepatic differentiation and molecular signatures of the hepatic cells derived from hES/iPS cells.
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http://dx.doi.org/10.1016/j.scr.2012.11.003DOI Listing
March 2013

Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum.

PLoS One 2011 26;6(8):e24228. Epub 2011 Aug 26.

Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo, Kumamoto, Japan.

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024228PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162614PMC
February 2012
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