骨肉腫幹細胞モデルと微小環境のクロストークの解明とそれらを標的にした治療戦略

• Project/Area Number: 21K09317
• 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: Kanazawa University
• Principal Investigator: 武内 章彦 金沢大学, 附属病院, 助教 (70512218)
• Co-Investigator(Kenkyū-buntansha): 山本 靖彦 金沢大学, 医学系, 教授 (20313637)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2023: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2021: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: 骨肉腫 / がん幹細胞 / エクソソーム / 微小環境

Outline of Research at the Start

骨肉腫幹細胞モデルが、微小環境を形成する細胞にどのように作用しているかの解析を進めるとともに、mir34a、Wnt5aそれぞれの作用も解析する。また、免疫系保持マウスにマウス骨肉腫細胞を移植し、合成mir34aとWnt5a阻害剤および免疫チェックポイント阻害剤(ICI) やマルチキナーゼ阻害剤(MKI)で治療し、微小環境を含めた効果を解析する。さらに、臨床検体において、微小環境を形成している細胞と臨床経過・予後との関連があるかを明らかにすることで、新たな病態解明および治療ターゲットの創出を行うことが研究目的である

Outline of Annual Research Achievements

本研究では、これまでの解析で見出したRAGEによって誘導される骨肉腫幹細胞モデルで、miR34aとWnt5aが重要である点から、骨肉腫幹細胞の微小環境に対する作用、miR34aとWnt5aの微小環境に与える影響をin-vitroで解析し、さらにマウス骨肉腫細胞を免疫保持マウスに移植し、微小環境を標的とした治療薬（免疫チェックポイント阻害薬、マルチキナーゼ阻害薬）と合成miR34aやWnt5a阻害薬を併用することで、がん幹細胞と微小環境を標的とした治療効果を解析し新たな治療戦略創出へと繋げることを目的としている。
2022年度は以下の解析を行った。
1. マウス骨肉腫細胞株であるDunnでマウス型RAGEと内在性分泌型RAGEが発現していることを遺伝子発現解析（RT-PCR）にて確認した。また、高転移株であるLM8よりもDunnの方がマウス型RAGEと内在性分泌型RAGEの発現が高く、Dunnのスフェロイド形成能がLM8よりも高いという結果であった。2. Dunnにヒト型RAGE遺伝子とmockを導入し、安定発現していることを確認した。3.LM8-RAGE、LM8-Mockをマウスに移植し、LM8-RAGEはLM8-Mockよりも移植部位での腫瘍形成脳が高かった。4. 骨肉腫組織（手術検体）におけるa miR-34の発現の解析をIn-situ-hybridizationで症例数を増やして行い、陽性率と臨床経過や原発巣と転移巣での発現率に相関はない印象であったが、発現している部位が症例ごとに腫瘍内と周囲組織で異なっていた。さらにRAGEの染色も追加して、RAGEの発現とmiR-34aの発現の相関を解析している。

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

2022年度に、骨肉腫幹細胞とmockのエクソソームの網羅的解析を計画していたが、高転移株のLM8の親株のDunn骨肉腫細胞株にRAGE遺伝子を導入することと、LM8とDunnのRAGE高発現細胞のin vitroのマウス移植の解析をする予定としたため実施しなかった。また、微小環境を形成する細胞との共培養も上記のために2023年度に行う予定とした。

Strategy for Future Research Activity

2023年度は、LM8-RAGE細胞株とDunn-RAGE細胞株を用いて、vitroでは,miR-34a, Wnt5aの発現解析、エクソソームの網羅的解析、微小環境を形成する細胞との共培養解析を行い、マウスに移植し腫瘍形成・転移能の評価と微小環境を標的とした治療薬（免疫チェックポイント阻害薬、マルチキナーゼ阻害薬）による抗腫瘍効果の解析を予定している。また、骨肉腫の手術検体におけるRAGEとmir-34aの発現を二重染色で解析予定である。

Research Products

• [Journal Article] Primary Osteosarcoma in Patients Over 75 Years of Age: A Case Series and Literature Review (2022)
  • Author(s): ARAKI YOSHIHIRO、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、HIGUCHI TAKASHI、ABE KENSAKU、TANIGUCHI YUTA、YONEZAWA HIROTAKA、MORINAGA SEI、ASANO YOHEI、NOJIMA TAKAYUKI、TSUCHIYA HIROYUKI
  • Journal Title: In Vivo Volume: 36 Issue: 6 Pages: 2899-2909
  • DOI: 10.21873/invivo.13031
  • Peer Reviewed
• [Journal Article] Pretreatment Neutrophil Count and Platelet-lymphocyte Ratio as Predictors of Metastasis in Patients With Osteosarcoma (2022)
  • Author(s): ARAKI YOSHIHIRO、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、HIGUCHI TAKASHI、ABE KENSAKU、TANIGUCHI YUTA、YONEZAWA HIROTAKA、MORINAGA SEI、ASANO YOHEI、TSUCHIYA HIROYUKI
  • Journal Title: Anticancer Research Volume: 42 Issue: 2 Pages: 1081-1089
  • DOI: 10.21873/anticanres.15570
  • Peer Reviewed
• [Journal Article] Clinical Outcomes of Soft Tissue Sarcoma around the Elbow Joint: A Retrospective Single Institution Study (2022)
  • Author(s): Azurin Edelyn S.、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Tada Kaoru、Miwa Shinji、Igarashi Kentaro、Higuchi Takashi、Yonezawa Hirotaka、Morinaga Sei、Asano Yohei、Saito Shiro、Tsuchiya Hiroyuki
  • Journal Title: Sarcoma Volume: 2022 Pages: 1-8
  • DOI: 10.1155/2022/1087726
  • Peer Reviewed / Open Access
• [Journal Article] Monostotic Fibrous Dysplasia in the Femur Strongly Expressing RANKL With Concomitant Osteoporotic Vertebral Compression Fracture: A Case Report (2022)
  • Author(s): AZURIN EDELYN S.、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、HIGUCHI TAKASHI、YONEZAWA HIROTAKA、MORINAGA SEI、ASANO YOHEI、SAITO SHIRO、TSUCHIYA HIROYUKI
  • Journal Title: Cancer Diagnosis & Prognosis Volume: 2 Issue: 1 Pages: 93-100
  • DOI: 10.21873/cdp.10082
  • Peer Reviewed / Open Access
• [Journal Article] Clinical Outcomes of Limb-sparing Tumor Surgery With Vascular Reconstruction for Bone and Soft-tissue Tumors (2022)
  • Author(s): ASANO YOHEI、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、TANIGUCHI YUTA、MORINAGA SEI、TADA KAORU、NOJIMA TAKAYUKI、TSUCHIYA HIROYUKI
  • Journal Title: Anticancer Research Volume: 42 Issue: 9 Pages: 4619-4626
  • DOI: 10.21873/anticanres.15966
  • Peer Reviewed
• [Journal Article] The Therapeutic Effect and Clinical Outcome of Immune Checkpoint Inhibitors on Bone Metastasis in Advanced Non-Small-Cell Lung Cancer (2022)
  • Author(s): Asano Yohei、Yamamoto Norio、Demura Satoru、Hayashi Katsuhiro、Takeuchi Akihiko、Kato Satoshi、Miwa Shinji、Igarashi Kentaro、Higuchi Takashi、Yonezawa Hirotaka、Araki Yoshihiro、Morinaga Sei、Saito Shiro、Sone Takashi、Kasahara Kazuo、Tsuchiya Hiroyuki
  • Journal Title: Frontiers in Oncology Volume: 12 Pages: 871675-871675
  • DOI: 10.3389/fonc.2022.871675
  • Peer Reviewed / Open Access
• [Journal Article] Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation (2022)
  • Author(s): Hayashi Kenjiro、Sato Koichi、Ochi Seishi、Kawano Shuhei、Munesue Seiichi、Harashima Ai、Oshima Yu、Kimura Kumi、Kyoi Takashi、Yamamoto Yasuhiko
  • Journal Title: International Journal of Molecular Sciences Volume: 23 Issue: 10 Pages: 5757-5757
  • DOI: 10.3390/ijms23105757
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Soluble receptor for advanced glycation end products protects from ischemia- and reperfusion-induced acute kidney injury (2022)
  • Author(s): Miyagawa Taro、Iwata Yasunori、Oshima Megumi、Ogura Hisayuki、Sato Koichi、Nakagawa Shiori、Yamamura Yuta、Kamikawa Yasutaka、Miyake Taito、Kitajima Shinji、Toyama Tadashi、Hara Akinori、Sakai Norihiko、Shimizu Miho、Furuichi Kengo、Munesue Seiichi、Yamamoto Yasuhiko、Kaneko Shuichi、Wada Takashi
  • Journal Title: Biology Open Volume: 11 Issue: 1 Pages: 1-8
  • DOI: 10.1242/bio.058852
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A scoring system combining clinical, radiological, and histopathological examinations for differential diagnosis between lipoma and atypical lipomatous tumor/well-differentiated liposarcoma (2022)
  • Author(s): Asano Yohei、Miwa Shinji、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Igarashi Kentaro、Yonezawa Hirotaka、Araki Yoshihiro、Morinaga Sei、Nojima Takayuki、Ikeda Hiroko、Tsuchiya Hiroyuki
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 237-237
  • DOI: 10.1038/s41598-021-04004-1
  • Peer Reviewed / Open Access
• [Journal Article] Therapeutic Targets and Emerging Treatments in Advanced Chondrosarcoma (2022)
  • Author(s): Miwa Shinji、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Igarashi Kentaro、Tsuchiya Hiroyuki
  • Journal Title: International Journal of Molecular Sciences Volume: 23 Issue: 3 Pages: 1096-1096
  • DOI: 10.3390/ijms23031096
  • Peer Reviewed / Open Access
• [Journal Article] Primary total knee arthroplasty assisted by computed tomography-free navigation for secondary knee osteoarthritis following massive calcium phosphate cement packing for distal femoral giant-cell bone tumor treatment: a case report (2022)
  • Author(s): Takeuchi A, Yamamoto N, Ohmori T, Hayashi K, Miwa S, Igarashi K, Higuchi T, Abe K, Yonezawa H, Morinaga S, Araki Y, Asano Y, Saito S, Tsuchiya H.
  • Journal Title: BMC Musculoskeletal Disorders Volume: 22 Issue: 1 Pages: 170-170
  • DOI: 10.1186/s12891-022-05131-0
  • Peer Reviewed / Open Access
• [Journal Article] Impact of Tumor Growth Speed of Primary Lesions on the Clinical Outcomes of Appendicular Skeletal Metastases (2021)
  • Author(s): ASANO YOHEI、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、YONEZAWA HIROTAKA、ARAKI YOSHIHIRO、MORINAGA SEI、SAITO SHIRO、NOJIMA TAKAYUKI、TSUCHIYA HIROYUKI
  • Journal Title: Anticancer Research Volume: 42 Issue: 1 Pages: 229-236
  • DOI: 10.21873/anticanres.15477
  • Peer Reviewed
• [Journal Article] Clinical characteristics of patients with undergoing unplanned excisions of malignant soft tissue tumors (2021)
  • Author(s): Morinaga Sei、Miwa Shinji、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Igarashi Kentaro、Tada Kaoru、Langit Mickhael B、Yonezawa Hirotaka、Araki Yoshihiro、Asano Yohei、Tsuchiya Hiroyuki
  • Journal Title: Journal of Orthopaedic Surgery Volume: 29 Issue: 3 Pages: 1-12
  • DOI: 10.1177/23094990211057597
  • Peer Reviewed / Open Access
• [Journal Article] A Radiological Scoring System for Differentiation between Enchondroma and Chondrosarcoma (2021)
  • Author(s): Miwa Shinji、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Igarashi Kentaro、Tada Kaoru、Yonezawa Hirotaka、Morinaga Sei、Araki Yoshihiro、Asano Yohei、Saito Shiro、Okuda Miho、Taki Junichi、Ikeda Hiroko、Nojima Takayuki、Tsuchiya Hiroyuki
  • Journal Title: Cancers Volume: 13 Issue: 14 Pages: 3558-3558
  • DOI: 10.3390/cancers13143558
  • Peer Reviewed / Open Access
• [Journal Article] Efficacy of perioperative chemotherapy for synovial sarcoma: a retrospective analysis of a Nationwide database in Japan (2021)
  • Author(s): Xu Gang、Aiba Hisaki、Yamamoto Norio、Hayashi Katsuhiro、Takeuchi Akihiko、Miwa Shinji、Higuchi Takashi、Abe Kensaku、Taniguchi Yuta、Araki Yoshihiro、Saito Shiro、Yoshimura Kenichi、Murakami Hideki、Tsuchiya Hiroyuki、Kawai Akira
  • Journal Title: BMC Cancer Volume: 21 Issue: 1 Pages: 773-773
  • DOI: 10.1186/s12885-021-08485-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Delayed Initiation of Treatment Is Associated With Metastasis of Malignant Bone Tumor (2021)
  • Author(s): ARAKI YOSHIHIRO、YAMAMOTO NORIO、HAYASHI KATSUHIRO、TAKEUCHI AKIHIKO、MIWA SHINJI、IGARASHI KENTARO、HIGUCHI TAKASHI、ABE KENSAKU、TANIGUCHI YUTA、YONEZAWA HIROTAKA、MORINAGA SEI、ASANO YOHEI、TSUCHIYA HIROYUKI
  • Journal Title: Anticancer Research Volume: 41 Issue: 6 Pages: 2993-2999
  • DOI: 10.21873/anticanres.15081
  • Peer Reviewed
• [Journal Article] Osteosarcoma-Derived Small Extracellular Vesicles Enhance Tumor Metastasis and Suppress Osteoclastogenesis by miR-146a-5p (2021)
  • Author(s): Yoshihiro Araki, Hisaki Aiba, Takeshi Yoshida, Norio Yamamoto, Katsuhiro Hayashi, Akihiko Takeuchi, Shinji Miwa, Kentaro Igarashi, Tuan D. Nguyen, Kiyo-aki Ishii, Takayuki Nojima, Satoru Takahashi, Hideki Murakami, Hiroyuki Tsuchiya and Rikinari Hanayama
  • Journal Title: Frontiers in Oncology Volume: 11 Pages: 667109-667109
  • DOI: 10.3389/fonc.2021.667109
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Associations of PD-L1, PD-L2, and HLA class I expression with responses to immunotherapy in patients with advanced sarcoma: post hoc analysis of a phase 1/2 trial (2021)
  • Author(s): Miwa S.、Nojima T.、Alomesen A. A.、Ikeda H.、Yamamoto N.、Nishida H.、Hayashi K.、Takeuchi A.、Igarashi K.、Higuchi T.、Yonezawa H.、Araki Y.、Morinaga S.、Asano Y.、Tsuchiya H.
  • Journal Title: Clinical and Translational Oncology Volume: 23 Issue: 8 Pages: 1620-1629
  • DOI: 10.1007/s12094-021-02559-z
  • Peer Reviewed / Open Access
• [Presentation] Over 7-years follow-up of tumor-devitalized frozen autografts of long bones for the patients with malignant bone tumors (2021)
  • Author(s): Akihiko Takeuchi, Norio Yamamoto, Katsuhiro Hayashi, Shinji Miwa, Kentaro Igarashi, Yuta Taniguchi, Yoshihiro Araki, Hirotaka Yonezawa, Sei Morinaga, Hiroyuki Tsuchiya
  • Organizer: The 13th Asia Pacific Musculoskeletal Tumor Society Meeting
  • Int'l Joint Research
• [Presentation] Randomized placebo-contorolled double blind phase II study of zaltoprofen for patient with diffuse-type and unresectable localized tenosynovial giant cell tumors (2021)
  • Author(s): Akihiko Takeuchi,Makoto Endo, Akira Kawai, Yoshihiro Nishida, Ryu Terauchi, Akihiko Matsumine, Hisaki Aiba, Tomoki Nakamura, Susumu Tandai, Toshifumi Ozaki, Manabu Hoshi, Kenichi Yoshimura, Akihiro Nomura,Toshinori Murayama, Hiroyuki Tsuchiya
  • Organizer: The Connective Tissue Oncology Society Annual Meeting
  • Int'l Joint Research