Establishment of an artificial osteosarcoma stem cell line by transduction of defined factors with with iPS cell technology

• Project/Area Number: 18K09031
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 56020:Orthopedics-related
• Research Institution: Kobe University
• Principal Investigator: Kawamoto Teruya 神戸大学, 医学研究科, 特命講師 (30420558)
• Co-Investigator(Kenkyū-buntansha): 原 仁美 神戸大学, 医学部附属病院, 助教 (40437489) ; 深瀬 直政 神戸大学, 医学部附属病院, 医員 (60579324) ; 秋末 敏宏 神戸大学, 保健学研究科, 教授 (90379363) ; 青井 貴之 神戸大学, 科学技術イノベーション研究科, 教授 (00546997) ; 川上 洋平 神戸大学, 医学研究科, 医学研究員 (50626570)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: 骨肉腫 / がん幹細胞 / iPS細胞 / 肉腫幹細胞 / iPS / stemness

Outline of Final Research Achievements

This study aimed to generate the cancer stem cell (CSC)-like cells from human osteosarcoma cells by introducing reprogramming factors, and to reveal the obtained cell characteristics.
We could obtain the osteosarcoma stem cells by transduction of OCT3/4-KLF4-SOX2 genes into a human osteosarcoma cell line MG63. mRNA expression of reported CSC markers significantly increased in the cells. In the cells, WST-8 assays revealed that significantly lower cell proliferation and higher chemo-resistance were observed. After osteogenic induction, strongly positive staining of Alizarin red S and ALP was also observed. In vivo experiments, tumors from the cells were significantly larger in size, and strong immunopositivity of Ki-67, Osteocalcin and HIF1-α was observed. We could generate the osteosarcoma stem cells by introducing reprogramming factors. This method could be useful to elucidate the molecular mechanisms and the effective therapeutic targets in human osteosarcoma.

Academic Significance and Societal Importance of the Research Achievements

本研究は，希少かつ治療困難な骨肉腫において，iPS細胞作製に用いられる技術を応用することで骨肉腫幹細胞を得ることに成功した初めての研究である．この成功によって骨肉腫においてもがん幹細胞研究を遂行することが可能となる革新的な成果であり，学術的意義が非常に高い．また得られた細胞の特性を解析することは，骨肉腫における幹細胞の機能を解明することに繋がり，骨肉腫の新たな病態理解や治療標的分子の同定とそれらに基づく新規治療開発を行うことが期待出来る。とくに新規治療開発は限られた治療法しか存在しない骨肉腫の臨床において，非常に有用な成果であり，社会的意義も非常に高いものである．

Research Products

• [Journal Article] Surgical outcomes of metastatic bone tumors in the extremities (Surgical outcomes of bone metastases) (2021)
  • Author(s): Hara H、Sakai Y、Kawamoto T、Fukase N、Kawakami Y、Takemori T、Fujiwara S、Kitayama K、Yahiro S、Miyamoto T、Kakutani K、Niikura T、Miyawaki D、Okada T、Sakashita A、Imamura Y、Sasaki R、Kizawa Y、Minami H、Matsumoto T、Matsushita T、Kuroda R、Akisue T
  • Journal Title: Journal of Bone Oncology Volume: 27 Pages: 100352-100352
  • DOI: 10.1016/j.jbo.2021.100352
  • NAID: 120007038644
  • Peer Reviewed / Open Access
• [Journal Article] Clinical Outcome of the Patients With Brain Metastasis from Soft Tissue Sarcomas (2021)
  • Author(s): Takemori T, Kawamoto T, Morishita M, Hara H, Fukase N, Kawakami Y, Fujiwara S, Kitayama K, Yahiro S, Miyamoto T, Fujimoto T, Fujita I, Mifune Y, Hoshino Y, Kakutani K, Matsumoto T, Matsushita T, Niikura T, Kuroda R, Akisue T
  • Journal Title: Anticancer Research Volume: 41 Issue: 2 Pages: 1027-1034
  • DOI: 10.21873/anticanres.14858
  • Peer Reviewed
• [Journal Article] Acquisition of cancer stem cell properties in osteosarcoma cells by defined factors (2020)
  • Author(s): Fujiwara S, Kawamoto T, Kawakami Y, Koterazawa Y, Hara H, Takemori T, Kitayama K, Yahiro S, Kakutani K, Matsumoto T, Matsushita T, Niikura T, Koyanagi-Aoi M, Aoi T, Kuroda R, Akisue T
  • Journal Title: Stem Cell Research & Therapy Volume: 11 Issue: 1
  • DOI: 10.1186/s13287-020-01944-9
  • NAID: 120006895443
  • Peer Reviewed / Open Access
• [Journal Article] Prognostic influence of the treatment approach for pulmonary metastasis in patients with soft tissue sarcoma (2020)
  • Author(s): Kawamoto Teruya、Hara Hitomi、Morishita Masayuki、Fukase Naomasa、Kawakami Yohei、Takemori Toshiyuki、Fujiwara Shuichi、Kitayama Kazumichi、Yahiro Shunsuke、Miyamoto Tomohiro、Fujimoto Takuya、Fujita Ikuo、Kakutani Kenichiro、Matsumoto Tomoyuki、Matsushita Takehiko、Niikura Takahiro、Kuroda Ryosuke、Akisue Toshihiro
  • Journal Title: Clinical & Experimental Metastasis Volume: 37 Issue: 4 Pages: 509-517
  • DOI: 10.1007/s10585-020-10038-y
  • NAID: 120006873085
  • Peer Reviewed
• [Presentation] Identification Of Associated MicroRNAs In Bone Destruction By Breast Cancer Metastasis. (2021)
  • Author(s): Kitayama K, Kawamoto T, Hara H, Fukase N, Kawakami Y, Takemori T, Fujiwara S, Yahiro S, Miyamoto T, Kuroda R, Akisue T.
  • Organizer: 67th Annual Meeting of the Orthopaedic Research Society
  • Int'l Joint Research
• [Presentation] Identification of Dormant Cells in Ewing sarcoma. (2021)
  • Author(s): Yahiro S, Kawamoto T, Hara H, Kawakami Y, Takemori T, Fujiwara S, Kitayama K, Miyamoto T, Aoi T, Koyanagi-Aoi T, Kuroda R, Akisue T.
  • Organizer: 67th Annual Meeting of the Orthopaedic Research Society
  • Int'l Joint Research
• [Presentation] A New Method to Generate Osteosarcoma Cells with Cancer Stem Cell Properties by Reprogramming Factors. (2020)
  • Author(s): Fujiwara S, Kawamoto T, Hara H, Kawakami Y, Kitayama K, Yahiro S, Aoi T, Kuroda R, Akisue T.
  • Organizer: 66th Annual Meeting of the Orthopaedic Research Society
  • Int'l Joint Research
• [Presentation] Identification Of Associated MicroRNAs In Bone Destruction By Breast Cancer Metastasis. (2020)
  • Author(s): Kitayama K, Kawamoto T, Hara H, Fukase N, Kawakami Y, Takemori T, Fujiwara S, Yahiro S, Kuroda R, Akisue T.
  • Organizer: 66th Annual Meeting of the Orthopaedic Research Society
  • Int'l Joint Research
• [Presentation] 骨肉腫細胞における特定因子導入を用いたがん幹細胞特性の獲得 (2020)
  • Author(s): 藤原周一, 河本旭哉, 川上洋平, 小寺澤康文, 原仁美, 竹森俊幸, 北山和道, 八尋俊輔, 青井貴之, 黒田良祐, 秋末敏宏.
  • Organizer: 第53回日本整形外科骨・軟部腫瘍学術集会
• [Presentation] がん骨転移における骨破壊に寄与するmiRNAの探索 (2020)
  • Author(s): 北山和道, 河本旭哉, 原仁美, 深瀬直政, 川上洋平, 竹森俊幸, 藤原周一, 八尋俊輔, 黒田良祐, 秋末敏宏.
  • Organizer: 第53回日本整形外科骨・軟部腫瘍学術集会
• [Presentation] 骨肉腫細胞における特定因子導入を用いたがん幹細胞特性の獲得 (2020)
  • Author(s): 藤原周一, 河本旭哉, 川上洋平, 小寺澤康文, 原仁美, 竹森俊幸, 北山和道, 八尋俊輔, 青井貴之, 黒田良祐, 秋末敏宏.
  • Organizer: 第35回日本整形外科学会基礎学術集会
• [Presentation] 乳癌骨転移における骨破壊に寄与するmiRNAの探索 (2020)
  • Author(s): 北山和道, 河本旭哉, 原仁美, 深瀬直政, 川上洋平, 竹森俊幸, 藤原周一, 八尋俊輔, 黒田良祐, 秋末敏宏.
  • Organizer: 第35回日本整形外科学会基礎学術集会
• [Presentation] Ewing肉腫におけるcellular dormancyの同定とその特性の探索 (2020)
  • Author(s): 八尋俊輔, 河本旭哉, 川上洋平, 原仁美, 竹森俊幸, 藤原周一, 北山和道, 宮本智弘, 青井貴之, 黒田良祐, 秋末敏宏.
  • Organizer: 第35回日本整形外科学会基礎学術集会
• [Presentation] Antitumor effect of LAT1 inhibition on clear cell sarcoma cells. (2020)
  • Author(s): Morishita M, Fujimoto T, Kawamoto T, Akisue T, Fujita I, Takemori T, Fujiwara S, Kitayama K, Yahiro S, Miyamoto T, Kawakami Y, Fukase N, Hara H, Kuroda R.
  • Organizer: 2020 The Connective Tissue Oncology Society Annual Meeting
  • Int'l Joint Research
• [Presentation] A New Method to Generate Osteosarcoma Cells with Cancer Stem Cell Properties by Reprogramming Factors (2020)
  • Author(s): Shuichi Fujiwara, Teruya Kawamoto, Hitomi Hara, Yohei Kawakami, Kazumichi Kitayama, Shunsuke Yahiro, Yasufumi Koterazawa, Toshihiro Akisue, Takashi Aoi, Ryosuke Kuroda
  • Organizer: ORS 2020 Annual Meeting
• [Presentation] リプログラミング因子を用いた人工骨肉腫幹細胞株の樹立 (2019)
  • Author(s): 藤原 周一、河本 旭哉、川上 洋平、小寺澤 康文、原 仁美、深瀬 直政、竹森 俊幸、北山 和道、秋末 敏宏、青井 貴之、黒田 良祐
  • Organizer: 第2回日本サルコーマ治療研究学会学術集会
• [Presentation] リプログラミング因子を用いた人工骨肉腫幹細胞株の樹立 (2019)
  • Author(s): 藤原 周一、河本 旭哉、川上 洋平、小寺澤 康文、原 仁美、深瀬 直政、竹森 俊幸、北山 和道、秋末 敏宏、青井 貴之、黒田 良祐
  • Organizer: 第18回日本再生医療学会総会
• [Presentation] リプログラミング因子を用いた人工骨肉腫幹細胞株の樹立 (2019)
  • Author(s): 藤原 周一、河本 旭哉、川上 洋平、小寺澤 康文、原 仁美、深瀬 直政、竹森 俊幸、北山 和道、秋末 敏宏、青井 貴之、黒田 良祐
  • Organizer: 第52回日本整形外科骨・軟部腫瘍学術集会
• [Presentation] Development of Induced Osteosarcoma Stem Cells Using Induced Pluripotent Stem (iPs) Cells Production Technology (2019)
  • Author(s): Shuichi Fujiwara, Teruya Kawamoto, Naomasa Fukase, Hitomi Hara, Yohei Kawakami, Toshiyuki Takemori, Kazumichi Kitayama, Takashi Aoi, Ryosuke Kuroda, Toshihiro Akisue
  • Organizer: ORS 2019 Annual Meeting
  • Int'l Joint Research
• [Presentation] リプログラミング因子を用いた人工骨肉腫幹細胞株の樹立 (2019)
  • Author(s): 藤原周一、河本旭哉、川上洋平、小寺澤康文、原仁美、深瀬直政、竹森俊幸、北山和道、秋末敏宏、青井貴之、黒田良祐
  • Organizer: 第2回日本サルコーマ治療研究学会学術集会
• [Presentation] iPS作製技術を用いた人工骨肉腫幹細胞株の樹立 (2018)
  • Author(s): 藤原周一、河本旭哉、青井貴之、小寺澤康文、原仁美、深瀬直政、蒲田悦子、竹森俊幸、秋末敏宏、黒田良祐
  • Organizer: 第33回日本整形外科学会 基礎学術集会