シングルセル遺伝子発現解析/エピゲノム解析による骨巨細胞腫の発生・進展過程の解明

• Project/Area Number: 21K16648
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 56020:Orthopedics-related
• Research Institution: The University of Tokyo
• Principal Investigator: 津田 祐輔 東京大学, 医学部附属病院, 助教 (70896018)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2022: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2021: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
• Keywords: シングルセル解析 / 軟部明細胞肉腫 / 腫瘍免疫 / 腫瘍免疫環境 / シングルセル遺伝子発現解析 / 骨巨細胞腫 / エピゲノム解析

Outline of Research at the Start

骨巨細胞腫は骨を破壊しながら増大し患者の機能的予後に大きな影響を及ぼすが、その発生・進展機序の理解は不十分であり、治療法も外科的切除が困難な症例に関しては確立されていない。本研究では、骨巨細胞腫を構成する細胞を1細胞レベルで解析することで、骨巨細胞腫において、腫瘍内の構成細胞、遺伝子発現異常、エピゲノム異常を1細胞レベルで明らかにする。腫瘍細胞に高発現し破骨細胞を誘導するRANKLの阻害剤であるデノスマブを投与することで腫瘍内の不均一性や、腫瘍細胞、破骨細胞の遺伝子発現やエピゲノムがどのように変化するかを解明する。シングルセル遺伝子発現解析とシングルセルエピゲノム解析などの先端的な手法を駆使する。

Outline of Annual Research Achievements

明細胞肉腫の原発巣、リンパ節のそれぞれ1検体から組織を採取し、1細胞ごとにdropletに分離し、Reagent Kits v3（10x genomics社）を用いてライブラリ調整した。Hiseq 2500にてシークエンスし、Cell Ranger 6.0を用いて、細胞ごとの遺伝子発現情報を取得した。R 4.0.2, Seurat 4.0を用い、feature RNA 500以上,ミトコンドリア遺伝子占有率15%以下の質の低い細胞のデータを除外した。リガンド-受容体解析にはnichenetrを用いた。
原発巣では4631細胞、リンパ節転移部では2322細胞を解析対象とした。2検体を統合し解析を行い、10個のclusterに分けアノテーションを行ったところ、Tumor、T cell、NK cell、Macrophage/Monocyte、Endothelial cell、Fibroblast、B cellが同定された。腫瘍内免疫環境の解析を行うと、特にリンパ節内のCD4+細胞やCD8+細胞は、BATF+ FOXP3+ CTLA4+ CD4+ T cell (Regulatory T cell, Treg)やLAG3+ PD1+ CD8+ T cell(Exhausted T cell)が多く、免疫抑制的な腫瘍環境を形成していた。T regはTGFβ1やCXCL12によって分化誘導されることが知られており、リガンド-受容体解析においてMacrophageやEndothelial cellのTGFβ1リガンドとT細胞におけるTGFβ1受容体、FibroblastのCXCL12リガンドとT細胞のCXCR4受容体との結合が示唆された。
シングルセル解析によって、軟部明細胞肉腫内の細胞集団不均一性が明らかになり、免疫抑制的な環境が構築されていることが示唆された。

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

コロナウイルス感染症の蔓延により検体収集に遅れが生じた。

Strategy for Future Research Activity

検体数を増やし、シングルセル解析を行い、同様の所見が得られるかどうかを検討する。これらの検討から得られた結果をもとにして、遺伝子発現/エピゲノム異常をターゲットとした新規治療の可能性を探索する。新鮮腫瘍組織より樹立されたcell lineを使用して、治療標的となる遺伝子に関して遺伝子過剰発現・抑制実験により、増殖能をATP assayなどで測定し、コントロールの細胞株との比較を行う。また、足場非依存性増殖能を評価するためColony formationassay を行う。浸潤能を基底膜モデルキット、また、遊走能をScratch assayなどにより評価を行う。上記の結果より有望な治療標的が同定された場合には腫瘍細胞に対して薬剤を添加し評価を行う。ゼノグラフトモデルを作成して薬剤を投与し、腫瘍形成抑制効果を確認する。これらの結果をまとめて学会発表、論文作成を行う。

Research Products

• [Journal Article] Generating in vitro models of NTRK-fusion mesenchymal neoplasia as tools for investigating kinase oncogenic activation and response to targeted therapy. (2023)
  • Author(s): Vanoli F, Herviou L, Tsuda Y, Sung P, Xie Z, Fishinevich E, Min SS, Mallen W, de Wardin HT, Zhang Y, Jasin M, Antonescu CR.
  • Journal Title: Oncogenesis. Volume: - Issue: 1
  • DOI: 10.1038/s41389-023-00454-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Role of perioperative chemotherapy and radiotherapy for localized high-grade malignant peripheral nerve sheath tumor at the extremities and trunk wall: a population-based cohort study (2023)
  • Author(s): Kobayashi H, Zhang L, Okajima K, Hirai T, Tsuda Y, Ikegami M, Kawai A, Tanaka S.
  • Journal Title: Jpn J Clin Oncol. Volume: 53 Issue: 2 Pages: 138-145
  • DOI: 10.1093/jjco/hyac178
  • Peer Reviewed / Open Access
• [Journal Article] CORR Insights: What Proportion of Patients With Musculoskeletal Tumors Demonstrate Thromboelastographic Markers of Hypercoagulability? A Pilot Study (2023)
  • Author(s): Yusuke Tsuda
  • Journal Title: Clin Orthop Relat Res. Volume: 481 Issue: 3 Pages: 562-563
  • DOI: 10.1097/corr.0000000000002372
  • Peer Reviewed / Open Access
• [Journal Article] Dramatic response to entrectinib in a patient with malignant peripheral nerve sheath tumor harboring novel SNRNP70-NTRK3 fusion gene (2023)
  • Author(s): Kobayashi H, Makise N, Shinozaki-Ushiku A, Zhang L, Ishibashi Y, Ikegami M, Tsuda Y, Kohsaka S, Ushiku T, Oda K, Miyagawa K, Aburatani H, Mano H, Tanaka S.
  • Journal Title: Genes Chromosomes Cancer. Volume: 62 Issue: 1 Pages: 47-51
  • DOI: 10.1002/gcc.23089
  • Peer Reviewed
• [Journal Article] Atypical Neurofibromatous Neoplasm with Uncertain Biologic Potential in the Posterior Mediastinum of a Young Patient with Neurofibromatosis Type 1: A Case Report (2022)
  • Author(s): Miyamoto K, Kobayashi H, Zhang L, Tsuda Y, Makise N, Yasunaga Y, Ikemura M, Nakai Y, Shibata E, Ushiku T, Tanaka S.
  • Journal Title: Case Rep Oncol . Volume: 15 Issue: 3 Pages: 988-994
  • DOI: 10.1159/000526844
  • Peer Reviewed / Open Access
• [Journal Article] Preoperative arterial embolization and wound complications after resection of malignant bone tumor in the pelvis: a nationwide database study (2022)
  • Author(s): Zhang L, Michihata N, Matsui H, Fushimi K, Yasunaga H, Tsuda Y, Tanaka S, Kobayashi H.
  • Journal Title: Jpn J Clin Oncol. Volume: 52 Pages: 1176-1182
  • DOI: 10.1093/jjco/hyac100
  • Peer Reviewed / Open Access
• [Journal Article] Clinical characteristics of undifferentiated pleomorphic sarcoma of bone and the impact of adjuvant chemotherapy on the affected patients: a population-based cohort study. (2022)
  • Author(s): Kobayashi H, Zhang L, Hirai T, Tsuda Y, Ikegami M, Tanaka S.
  • Journal Title: Jpn J Clin Oncol Volume: hyac017 Issue: 6 Pages: 589-598
  • DOI: 10.1093/jjco/hyac017
  • Peer Reviewed
• [Journal Article] Role of adjuvant chemotherapy in patients with localized, undifferentiated pleomorphic sarcoma of soft tissue: a population-based cohort study. (2022)
  • Author(s): Kobayashi H, Zhang L, Hirai T, Tsuda Y, Ikegami M, Tanaka S.
  • Journal Title: Int J Clin Oncol Volume: 27 Issue: 4 Pages: 802-810
  • DOI: 10.1007/s10147-021-02102-8
  • Peer Reviewed
• [Journal Article] Impact of the national sarcoma guidelines on the prevalence and outcome of inadvertent excisions of soft tissue sarcomas: An observational study from a UK tertiary referral centre. (2022)
  • Author(s): Fujiwara T, Evans S, Stevenson J, Tsuda Y, Gregory J, Grimer R, Abudu A.
  • Journal Title: Eur J Surg Oncol Volume: Mar;48(3) Issue: 3 Pages: 533-540
  • DOI: 10.1016/j.ejso.2021.11.013
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Comparison of clinical features and outcomes of patients with leiomyosarcoma of bone and soft tissue: a population-based cohort study. (2022)
  • Author(s): Kobayashi H, Zhang L, Hirai T, Tsuda Y, Ikegami M, Tanaka S.
  • Journal Title: Jpn J Clin Oncol. Volume: 52 Issue: 2 Pages: 143-150
  • DOI: 10.1093/jjco/hyab176
  • Peer Reviewed
• [Journal Article] Clinical analysis of multimodal treatment for localized synovial sarcoma: A multicenter retrospective study. (2021)
  • Author(s): Hagiwara Yoko、Iwata Shintaro、Ogura Koichi、Kawai Akira、Susa Michiro、Morioka Hideo、Hiruma Toru、Tsuda Yusuke、Kawano Hirotaka、Yonemoto Tsukasa、Ishii Takeshi、Okazaki Ken.
  • Journal Title: Journal of Orthopaedic Science Volume: S0949-2658(21) Issue: 1 Pages: 00340-7
  • DOI: 10.1016/j.jos.2021.09.012
  • Peer Reviewed / Open Access
• [Journal Article] How Are Indeterminate Pulmonary Nodules at Diagnosis Associated with Survival in Patients with High-Grade Osteosarcoma? (2021)
  • Author(s): Tsoi KM, Lowe M, Tsuda Y, Lex JR, Fujiwara T, Almeer G, Gregory J, Stevenson J, Evans SE, Botchu R, Jeys LM.
  • Journal Title: Clin Orthop Relat Res. Volume: 479(2) Issue: 2 Pages: 298-308
  • DOI: 10.1097/corr.0000000000001491
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Epithelioid hemangioma of bone harboring FOS and FOSB gene rearrangements: A clinicopathologic and molecular study. (2021)
  • Author(s): Tsuda Y, Suurmeijer AJH, Sung YS, Zhang L, Healey JH, Antonescu CR.
  • Journal Title: Genes Chromosomes Cancer Volume: Jan;60(1) Issue: 1 Pages: 17-25
  • DOI: 10.1002/gcc.22898
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Impact of NICE guidelines on the survival of patients with soft-tissue sarcomas (2021)
  • Author(s): Fujiwara T, Grimer RJ, Evans S, Medellin Rincon MR, Tsuda Y, Le Nail LR, Abudu S.
  • Journal Title: Bone Joint J. Volume: 103-B(3) Issue: 3 Pages: 569-577
  • DOI: 10.1302/0301-620x.103b3.bjj-2020-0743.r1
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Pelvic reconstruction using an ice-cream cone prosthesis: correlation between the inserted length of the coned stem and surgical outcome. (2021)
  • Author(s): Fujiwara T, Stevenson J, Parry M, Le Nail LR, Tsuda Y, Grimer R, Jeys L.
  • Journal Title: Int J Clin Oncol. Volume: Jun;26(6) Issue: 6 Pages: 1139-1146
  • DOI: 10.1007/s10147-021-01882-3
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Prognostic factors and impact of surgery in patients with metastatic soft tissue sarcoma at diagnosis: A population-based cohort study (2021)
  • Author(s): Zhang Liuzhe、Akiyama Toru、Fukushima Takashi、Iwata Shintaro、Tsuda Yusuke、Takeshita Katsushi、Kawai Akira、Tanaka Sakae、Kobayashi Hiroshi
  • Journal Title: Japanese Journal of Clinical Oncology Volume: - Issue: 6 Pages: 918-926
  • DOI: 10.1093/jjco/hyab033
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Limb-salvage reconstruction following resection of pelvic bone sarcomas involving the acetabulum. (2021)
  • Author(s): Fujiwara T, Medellin Rincon MR, Sambri A, Tsuda Y, Clark R, Stevenson J, Parry MC, Grimer RJ, Jeys L.
  • Journal Title: Bone Joint J Volume: 103-B(4) Issue: 4 Pages: 795-803
  • DOI: 10.1302/0301-620x.103b4.bjj-2020-0665.r1
  • Peer Reviewed
• [Journal Article] Surgical outcomes of bone sarcoma of the foot (2021)
  • Author(s): Tsuda Y, Fujiwara T, Stevenson JD, Abudu A.
  • Journal Title: Jpn J Clin Oncol 2021 Volume: Oct 5;51(10) Issue: 10 Pages: 541-1546
  • DOI: 10.1093/jjco/hyab118
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Regional variation in the survival of patients with a soft-tissue sarcoma of the extremity and trunk wall under a centralized care system : what has been the impact of national policies in the UK? (2021)
  • Author(s): Fujiwara T, Evans S, Stevenson J, Tsuda Y, Gregory J, Grimer RJ, Abudu S.
  • Journal Title: Bone Joint J Volume: Sep;103-B(9) Issue: 9 Pages: 1541-1549
  • DOI: 10.1302/0301-620x.103b9.bjj-2020-2554.r1
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Hemangioma of the Rib Mimicking Chondrosarcoma: A Report of Two Cases and Literature Review. (2021)
  • Author(s): Tsuda Y, Kobayashi H, Makise N, Zhang L, Shinoda Y, Ushiku T, Tanaka S.
  • Journal Title: Case Rep Orthop. Volume: - Pages: 1-5
  • DOI: 10.1155/2021/9996380
  • Peer Reviewed
• [Journal Article] Extra-articular resection of the hip joint for pelvic sarcomas: Are there any oncological and functional risks compared with intra-articular resection? (2021)
  • Author(s): Fujiwara T, Tsuda Y, Stevenson J, Parry M, Jeys L.
  • Journal Title: J Bone Oncol Volume: Nov 1;31 Pages: 100401-100401
  • DOI: 10.1016/j.jbo.2021.100401
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Predictive factors of the 30-day mortality after surgery for spinal metastasis: Analysis of a nationwide database (2020)
  • Author(s): Sawada Ryoko、Yamana Hayato、Shinoda Yusuke、Tsuda Yusuke、Matsui Hiroki、Fushimi Kiyohide、Kobayashi Hiroshi、Matsubayashi Yoshitaka、Yasunaga Hideo、Tanaka Sakae、Haga Nobuhiko
  • Journal Title: Journal of Orthopaedic Science Volume: - Issue: 4 Pages: 666-671
  • DOI: 10.1016/j.jos.2020.07.015
  • Peer Reviewed / Open Access
• [Presentation] シングルセルRNAシークエンスによる軟部明細胞肉腫の腫瘍内免疫環境の解析 (2022)
  • Author(s): 津田祐輔
  • Organizer: 第55回日本整形外科学会骨軟部腫瘍学術集会
• [Presentation] シングルセルRNAシークエンスによる軟部明細胞肉腫の腫瘍内免疫環境の解析 (2021)
  • Author(s): 津田祐輔、岡田寛之、平井利英、宮原潤也、張劉哲、小俣康徳、田中栄、小林寛
  • Organizer: 第55回日本整形外科学会骨軟部腫瘍学術集会
• [Remarks] 東京大学医学部附属病院 整形外科・脊椎外科
  • URL: http://www.u-tokyo-ortho.jp/