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Kawamura H.,Kansai Medical University | Takahashi M.,Kansai Medical University | Uemura Y.,Exploratory Oncology Research | Asano H.,Kyoto Prefectural University of Medicine | And 4 more authors.
Stem Cells | Year: 2015

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage-CD45-CD271+SSEA-4+ fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34+) as well as CD34-negative (CD34-) HSCs was important for the support/maintenance of the CD34+/- HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. Stem Cells 2015;33:1554-1565 © 2014 AlphaMed Press.


Nakatsuka R.,Kansai Medical University | Matsuoka Y.,Kansai Medical University | Uemura Y.,Exploratory Oncology Research | Sumide K.,Kansai Medical University | And 5 more authors.
Cell Transplantation | Year: 2015

It is well documented that specialized mesenchymal stem/stromal cells (MSCs) constitute the hematopoietic stem cell (HSC) niche in the bone marrow (BM), and these MSCs support/maintain the HSCs in an undifferentiated state. A number of studies have demonstrated that BM-derived MSCs (BM-MSCs) can support HSCs in vitro. However, it remains unclear whether nonhematopoietic tissue-derived MSC-like cells, such as dental pulp stem cells (DPSCs), have the ability to support HSCs. In this study, we prospectively isolated DPSCs from mouse mandibular incisors by fluorescence-activated cell sorting (FACS) using BM-MSC markers, such as PDGFRα and Sca-1. The PDGFRα and Sca-1 double-positive DPSCs and BM-MSCs showed similar morphologies and expression patterns of MSC markers. The ability of the DPSCs to support hematopoietic stem/progenitor cells (HSPCs) was then analyzed by an in vitro coculture system. Moreover, their HSC-supporting activity was evaluated by in vivo xenotransplantation assays using NOD/Shi-scid/IL-2Rγc null (NOG) mice. Interestingly, the DPSCs supported human cord blood (CB)-derived CD34-positive (CD34+), as well as CD34 negative (CD34-), HSCs. The supporting activities of DPSCs for human CB-derived CD34+ and CD34- HSCs were comparable to those of BM-MSCs. The results of the present study demonstrated, for the first time, that prospectively isolated murine PDGFRα and Sca-1 double-positive DPSCs could support primitive human CD34+ and CD34- HSCs in vitro. © 2015 Cognizant Comm. Corp.


Sawada Y.,Exploratory Oncology Research | Sawada Y.,Yokohama City University | Yoshikawa T.,Exploratory Oncology Research | Ofuji K.,Exploratory Oncology Research | And 21 more authors.
OncoImmunology | Year: 2016

The recurrence rates of Hepatocellular carcinoma (HCC) are high, necessitating novel and effective adjuvant therapies. Therefore, we conducted a phase II study of glypican-3 (GPC3) peptide vaccine as an adjuvant therapy for HCC patients. Forty-one patients with initial HCC who had undergone surgery or radiofrequency ablation (RFA) were analyzed in this phase II, open-label, single-arm trial. Ten vaccinations were performed for 1 y after curative treatment. We also investigated case-control subjects, where selected patients treated surgically during the same period were analyzed. The expression of GPC3 in the available primary tumors was determined by immunohistochemical analysis. Six patients received RFA therapy while 35 received surgery. The recurrence rate tended to be lower in the 35 patients treated with surgery plus vaccination compared to 33 patients who underwent surgery alone (28.6% vs. 54.3% and 39.4% vs. 54.5% at 1 and 2 y, respectively; p = 0.346, 0.983). Twenty-five patients treated with surgery and vaccination had GPC3-positive tumors; the recurrence rate in this group was significantly lower compared to that in 21 GPC3-positive patients who received surgery only (24% vs. 48% and 52.4% vs. 61.9% at 1 and 2 y, respectively; p = 0.047, 0.387). The GPC3 peptide vaccine improved the 1-y recurrence rate in patients with GPC3-positive tumors. This study demonstrated that GPC3 expression by the primary tumor may be used as a biomarker in a putative larger randomized clinical trial to determine the efficacy of the GPC3-derived peptide vaccine. © 2016, © Yu Sawada, Toshiaki Yoshikawa, Kazuya Ofuji, Mayuko Yoshimura, Nobuhiro Tsuchiya, Mari Takahashi, Daisuke Nobuoka, Naoto Gotohda, Shinichiro Takahashi, Yuichiro Kato, Masaru Konishi, Taira Kinoshita, Masafumi Ikeda, Kohei Nakachi, Naoya Yamazaki, Shoichi Mizuno, Tadatoshi Takayama, Kenji Yamao, Katsuhiko Uesaka, Junji Furuse, Itaru Endo, and Tetsuya Nakatsura.


Ando M.,Aichi Cancer Center Hospital | Yamauchi H.,St Lukes International Hospital | Aogi K.,National Hospital Organization | Shimizu S.,Kanagawa Cancer Center | And 9 more authors.
Breast Cancer Research and Treatment | Year: 2014

Addition of carboplatin to neoadjuvant chemotherapy in HER2-negative breast cancer may improve pathological complete response (pCR) rates. We evaluated the efficacy and safety of carboplatin and weekly paclitaxel (wPTX) followed by cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) as neoadjuvant chemotherapy for HER2-negative breast cancer. Patients with stage II/IIIA HER2-negative breast cancer were randomly assigned to preoperatively receive CP-CEF (four 3-week cycles of carboplatin [area under the curve 5 mg/mL/min, day 1] and wPTX [80 mg/m2, day 1, 8, 15] followed by four 3-week cycles of CEF [500/100/500 mg/m2] or P-CEF (four cycles of wPTX followed by four cycles of CEF). The primary objective was pCR rate. Of 181 eligible patients, 89 were randomly assigned to the CP-CEF and 92 to the P-CEF. Two patients in each arm refused to receive neoadjuvant chemotherapy. Overall 88 patients in the CP-CEF and 91 patients in the P-CEF were assessable for efficacy and safety. The pCR rate in the CP-CEF was significantly higher than that in the P-CEF (31.8 vs. 17.6 %, one-sided P = 0.01). Among patients with triple-negative breast cancer, the pCR rate in the CP-CEF was significantly higher than that in the P-CEF [61.2 (23/37) vs. 26.3 % (10/38), P = 0.003]. Grade 3-4 neutropenia was observed in the CP-CEF more frequently than in the P-CEF (65.9 vs. 38.5 %). Adding carboplatin to neoadjuvant wPTX followed by CEF for HER2-negative breast cancer improved the pCR rate and exacerbated hematotoxicity. © 2014 Springer Science+Business Media New York.


Kitayama S.,Kyoto University | Zhang R.,Aichi Cancer Center Research Institute | Zhang R.,Exploratory Oncology Research | Liu T.-Y.,Aichi Cancer Center Research Institute | And 16 more authors.
Stem Cell Reports | Year: 2016

Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer. © 2016 The Authors.


Kuboki Y.,National Cancer Center Hospital East | Kuboki Y.,Exploratory Oncology Research | Kuboki Y.,Keio University | Yamashita S.,National Cancer Center Research Institute | And 9 more authors.
Annals of Oncology | Year: 2016

Background: In advanced gastric cancer (AGC), most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Recently, next-generation sequencing (NGS) allowed us to comprehensively profile the tumor gene status. This study aimed to elucidate the profiling between gene alterations and protein expression in AGC to aid in future clinical trials on AGC. Patients and methods: Formalin-fixed, paraffin-embedded tumor samples from 121 stage III/IV gastric cancer patients were examined for protein expression of tyrosine kinase receptors (RTKs; ERBB2, EGFR, c-MET, and FGFR2) using immunohistochemistry (IHC). Furthermore, 409 cancer-related genes were sequenced to detect mutations and copy number variations using NGS. Results: Most ERBB2 overexpression (IHC 3+) cases (80.0%) had ERBB2 amplification and did not have other RTK amplification or oncogene mutations. However, one-fourth of MET overexpression cases (25.0%) had ERBB2 alterations. EGFR and FGFR2 overexpression cases had ERBB2 alterations or other gene alterations such as KRAS or PIK3CA. On the other hand, most of the four RTK amplification cases (88.2%) were mutually exclusive with each amplification. However, RTK amplification did not simply correlate with protein overexpression, whereas cases with RTK high-level amplification had protein overexpression and rarely showed other co-existing gene alterations. Conclusion: AGC involves a complicated arrangement of protein expression and gene alterations. Comprehensive analyses of NGS and IHC will be necessary to design the optimal therapy for treating the appropriate population of patients in future clinical trials. © The Author 2015.


Tsuchiya N.,Yokohama City University | Tsuchiya N.,Exploratory Oncology Research | Sawada Y.,Yokohama City University | Endo I.,Yokohama City University | And 3 more authors.
World Journal of Gastroenterology | Year: 2015

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Although the prognosis of patients with HCC is generally poor, the 5-year survival rate is > 70% if patients are diagnosed at an early stage. However, early diagnosis of HCC is complicated by the coexistence of inflammation and cirrhosis. Thus, novel biomarkers for the early diagnosis of HCC are required. Currently, the diagnosis of HCC without pathological correlation is achieved by analyzing serum α.fetoprotein levels combined with imaging techniques. Advances in genomics and proteomics platforms and biomarker assay techniques over the last decade have resulted in the identification of numerous novel biomarkers and have improved the diagnosis of HCC. The most promising biomarkers, such as glypican-3, osteopontin, Golgi protein-73 and nucleic acids including microRNAs, are most likely to become clinically validated in the near future. These biomarkers are not only useful for early diagnosis of HCC, but also provide insight into the mechanisms driving oncogenesis. In addition, such molecular insight creates the basis for the development of potentially more effective treatment strategies. In this article, we provide an overview of the biomarkers that are currently used for the early diagnosis of HCC. © 2015 Baishideng Publishing Group Inc. All rights reserved.


Iwama T.,Exploratory Oncology Research | Uchida T.,Saitama University | Sawada Y.,Exploratory Oncology Research | Sawada Y.,Yokohama City University | And 12 more authors.
Biochemical and Biophysical Research Communications | Year: 2016

Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer. © 2015 The Authors . Published by Elsevier Inc.


Hashimoto H.,National Cancer Center Research Institute | Ueda R.,National Cancer Center Research Institute | Narumi K.,National Cancer Center Research Institute | Heike Y.,Exploratory Oncology Research | And 2 more authors.
Cancer Gene Therapy | Year: 2014

Type I interferon (IFN) is a pleiotropic cytokine regulating the cancer cell death and immune response. IFN-α can, as we have also reported, effectively induce an antitumor immunity by the activation of tumor-specific T cells and maturation of dendritic cells in various animal models. Unknown, however, is how the type I IFN alters the immunotolerant microenvironment in the tumors. Here, we found that intratumoral IFN-α gene transfer significantly decreased the frequency of regulatory T cells (Tregs) per CD4 + T cells in tumors. The concentration of a Treg-inhibitory cytokine, interleukin (IL)-6, was correlated with the IFN-α expression level in tumors, and intratumoral CD11c + cells produced IL-6 in response to IFN-α stimulation. To confirm the role of IL-6 in the suppression of Tregs in tumors, an anti-IL-6 receptor antibody was administered in IFN-α-treated mice. The antibody increased the frequency of Tregs in the tumors, and attenuated systemic tumor-specific immunity induced by IFN-α. Furthermore, the IFN-α-mediated IL-6 production increased the frequency of Th17 cells in the tumors, which may be one of the mechanisms for the reduction of Tregs. The study demonstrated that IFN-α gene delivery creates an environment strongly supporting the enhancement of antitumor immunity through the suppression of Tregs. © 2014 Nature America, Inc. All rights reserved.

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