ヒト肉腫幹細胞特異抗原に対する分子免疫応答制御

2013 Fiscal Year Research-status Report

• Project/Area Number: 25462344
• Research Institution: Sapporo Medical University
• Principal Investigator: 塚原 智英 札幌医科大学, 医学部, 助教 (20404634)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: 肉腫幹細胞 / 自家CTLクローン / 抗原 / SP / 人工CTL

Research Abstract

悪性線維性組織球腫(MFH)細胞株MFH2003Hoechst染色を行って分離したSP細胞を抗原として，患者MFH2003末梢血単核球(PBMC)とmixed lymphocyte-tumor cell culture (MLTC)を４回行い，限界希釈によって得たSP細胞特異的CTLクローンTc-4c6よりRNAを抽出し，5'RACE法によりT細胞受容体(TCR)α鎖β鎖cDNAの可変領域塩基配列を決定した．そしてこれらのcDNAをプラスミドpRSVにサブクローニングを行って，電気穿孔法によりTCR欠損マウスハイブリドーマ58α-β-細胞に遺伝子導入して，選択薬剤によりTCR発現ハイブリドーマクローン２種(a5b, a7b)を得た．これらのハイブリドーマクローンはCD3eを発現しており，TCRαβ両鎖がアセンブルされて発現していると考えられた．

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

CTLクローンより適切なTCRα鎖β鎖を得てサブクローニングすることができた．自家CTLクローンに認識される肉腫幹細胞抗原の同定に向けて有益な結果であった．

Strategy for Future Research Activity

今後はTCR分子の発現レベルをさらに増強して，SP細胞を強く認識するCTLハイブリドーマクローンを得ることを目指す．そして腫瘍細胞の抗原提示HLA分子を決定して，自家CTLクローンに認識される肉腫幹細胞抗原遺伝子のクローニングにつなげる．

Research Products

• [Journal Article] Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells. (2014)
  • Author(s): Morita R, Nishizawa S, Torigoe T, Takahashi A, Tamura Y, Tsukahara T, Kanaseki T, Sokolovskaya A, Kochin V, Kondo T, Hashino S, Asaka M, Hara I, Hirohashi Y, Sato N.
  • Journal Title: Cancer Sci Volume: 105 Pages: 389–395
  • DOI: 10.1111/cas.12362.
  • Peer Reviewed
• [Journal Article] High expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells in the novel epithelioid sarcoma cell line ESX and is related to poor prognosis of soft tissue sarcoma. (2013)
  • Author(s): Emori M, Tsukahara T, Murase M, Kano M, Murata K, Takahashi A, Kubo T, Asanuma H, Yasuda K, Kochin V, Kaya M, Nagoya S, Nishio J, Iwasaki H, Sonoda T, Hasegawa T, Torigoe T, Wada T, Yamashita T, Sato N.
  • Journal Title: PLoS One Volume: 8 Pages: e84187
  • DOI: 10.1371/journal.pone.0084187. eCollection 2013.
  • Peer Reviewed
• [Journal Article] Ectopically expressed variant form of sperm mitochondria-associated cysteine-rich protein augments tumorigenicity of the stem cell population of lung adenocarcinoma cells. (2013)
  • Author(s): Takahashi A, Hirohashi Y, Torigoe T, Tamura Y, Tsukahara T, Kanaseki T, Kochin V, Saijo H, Kubo T, Nakatsugawa M, Asanuma H, Hasegawa T, Kondo T, Sato N.
  • Journal Title: PLoS One Volume: 8 Pages: e69095
  • DOI: 10.1371/journal.pone.0069095. eCollection 2013.
  • Peer Reviewed
• [Journal Article] Small proline-rich protein-1B is overexpressed in human oral squamous cell cancer stem-like cells and is related to their growth through activation of MAP kinase signal. (2013)
  • Author(s): Michifuri Y, Hirohashi Y, Torigoe T, Miyazaki A, Fujino J, Tamura Y, Tsukahara T, Kanaseki T, Kobayashi J, Sasaki T, Takahashi A, Nakamori K, Yamaguchi A, Hiratsuka H, Sato N.
  • Journal Title: Biochem Biophys Res Commun Volume: 439 Pages: 96-102
  • DOI: 10.1016/j.bbrc.2013.08.021.
  • Peer Reviewed
• [Presentation] Development of the functional probe against an autologous CTL-defined sarcoma stem cell-associated antigen (2013)
  • Author(s): 塚原智英
  • Organizer: 日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 20131003-20131005
• [Presentation] Development of peptide vaccination therapy targeting apoptosis regulator protein PBF for patients with osteosarcoma. (2013)
  • Author(s): 塚原智英
  • Organizer: International Congress of Immunology
  • Place of Presentation: Milan, Italy
  • Year and Date: 20130822-20130827
• [Presentation] Co-existence of HLA class I expression and CD8+ T cell infiltration is associated with favorable clinical outcomes in Ewing’s sarcoma family of tumors (ESFT). (2013)
  • Author(s): Yabe H, Tsukahara T, Kawaguchi S, Torigoe T, Sato N, Morioka H, Yabe H.
  • Organizer: International Congress of Immunology
  • Place of Presentation: Milan, Italy
  • Year and Date: 20130822-20130827