Identification of liver cancer stem cell-like clones causing treatment resistance by cancer evolution

• Project/Area Number: 19H03637
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 53010:Gastroenterology-related
• Research Institution: University of Fukui
• Principal Investigator: Nakamoto Yasunari 福井大学, 学術研究院医学系部門, 教授 (40293352)
• Co-Investigator(Kenkyū-buntansha): 平松 活志 福井大学, 学術研究院医学系部門(附属病院部), 准教授 (30646849) ; 野阪 拓人 福井大学, 学術研究院医学系部門, 助教 (70748441) ; 山下 太郎 金沢大学, 附属病院, 准教授 (90377432)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,160,000 (Direct Cost: ¥13,200,000、Indirect Cost: ¥3,960,000); Fiscal Year 2021: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2020: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2019: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
• Keywords: 肝細胞がん / がん幹細胞 / 循環腫瘍細胞 / シングルセル解析 / 創薬シーズ

Outline of Research at the Start

進行がんの治療薬に対する耐性には、がん進化現象が関与することが示唆されている。がん進化を引き起こす肝がん幹細胞様クローンを同定するために、消化器がんの中でも細胞不均一性に富む肝細胞がん患者について、治療耐性から転移を誘発するがん幹細胞様クローンに着目して探索する。具体的には、分子標的薬や化学療法の治療中に肝内・肝外転移を生じた患者において、独自の液体生検技術で血液中の循環腫瘍細胞（CTC）を採取する。得られたCTCに対してシングルセル分子解析を実施しがん幹細胞様クローンを同定する。さらに独創的な 機能評価系でがん幹細胞性の責任遺伝子を見出し新たな創薬シーズとして提案する。

Outline of Final Research Achievements

The aim was to identify cancer stem cell-like clones that induce treatment resistance in patients with advanced-stage hepatocellular carcinoma (HCC). To this end, circulating tumor cells (CTCs) were collected from the peripheral blood of patients undergoing anticancer treatment by liquid biopsy, and the characteristics of CTC cells showing stem cell characteristics were examined by a new single-cell analysis method. CTC stem cell markers (SOX2, MYC, NANOG) were significantly elevated and pathway analysis showed enhanced MAPK and TGF-β signaling pathways in patients who were refractory to Atezolizumab + Bevacizumab immune combination therapy. Thus, it was considered to be a significant result in considering the characteristics of cancer stem cell-like clones that induce treatment resistance in HCC patients.

Academic Significance and Societal Importance of the Research Achievements

進行がんの薬物治療において完治することは稀である。この原因は、腫瘍を形成するがん細胞に不均一性があるために、大部分の細胞に有効な薬剤であっても一部の細胞は抵抗性を示して生き残るためである。そして、生き残った細胞はがん幹細胞様クローンとして血液中に放出され、循環腫瘍細胞（CTC）となりがん転移を形成する。
本研究でのがん幹細胞様クローンの検出は、最新治療に抵抗性となった進行期肝がん患者の血液から独自の解析技術でCTCを採取し、遺伝子・タンパクレベルで特徴を分析した。これより、治療抵抗性の原因となるがん幹細胞様クローンの特性が明らかとなり、進行がんに対する新たな治療開発に繋がることが期待される。

Research Products

• [Journal Article] Liver-related events after direct-acting antiviral therapy in patients with hepatitis C virus-associated cirrhosis (2022)
  • Author(s): Tahata Y, Hikita H, Mochida S, Enomoto N, Kawada N, Kurosaki M, Ido A, Miki D, Yoshiji H, Takikawa Y, Sakamori R, Hiasa Y, Nakao K, Kato N, Ueno Y, Yatsuhashi H, Itoh Y, Tateishi R, Suda G, Takami T, Nakamoto Y, Asahina Y, et al.
  • Journal Title: Journal of Gastroenterology Volume: 57 Issue: 2 Pages: 120-132
  • DOI: 10.1007/s00535-021-01845-5
  • Peer Reviewed
• [Journal Article] Circulating tumor cells detected with a microcavity array predict clinical outcome in hepatocellular carcinoma (2021)
  • Author(s): Takahashi Kazuto、Ofuji Kazuya、Hiramatsu Katsushi、Nosaka Takuto、Naito Tatsushi、Matsuda Hidetaka、Endo Katsuya、Higuchi Masayuki、Ohtani Masahiro、Nemoto Tomoyuki、Nakamoto Yasunari
  • Journal Title: Cancer Medicine Volume: 10 Issue: 7 Pages: 2300-2309
  • DOI: 10.1002/cam4.3790
  • Peer Reviewed / Open Access
• [Journal Article] Serum laminin γ2 monomer as a novel diagnostic and predictive biomarker for hepatocellular carcinoma. (2021)
  • Author(s): T Yamashita, N Koshikawa, T Shimakami, T Terashima, M Nakagawa, K Nio, R Horii, N Iida, K Kawaguchi, K Arai, Y Sakai, T Yamashita, E Mizukoshi, M Honda, A Kitao, S Kobayashi, S Takahara, Y Imai, K Yoshimura, T Murayama, Y Nakamoto, E Yoshida, T Yoshimura, M Seiki, S Kaneko.
  • Journal Title: Hepatology Volume: - Issue: 2 Pages: 760-775
  • DOI: 10.1002/hep.31758
  • Peer Reviewed / Open Access
• [Journal Article] Regulatory function of interferon‐inducible 44‐like for hepatitis B virus covalently closed circular DNA in primary human hepatocytes (2021)
  • Author(s): Nosaka Takuto、Naito Tatsushi、Murata Yosuke、Matsuda Hidetaka、Ohtani Masahiro、Hiramatsu Katsushi、Nishizawa Tsutomu、Okamoto Hiroaki、Nakamoto Yasunari
  • Journal Title: Hepatology Research Volume: 52 Issue: 2 Pages: 141-152
  • DOI: 10.1111/hepr.13722
  • Peer Reviewed / Open Access
• [Journal Article] Sofosbuvir plus velpatasvir treatment for hepatitis C virus in patients with decompensated cirrhosis: a Japanese real-world multicenter study (2020)
  • Author(s): Tahata Yuki、Hikita Hayato、Matsuura Kentaro、Takehara Tetsuo, et al.
  • Journal Title: Journal of Gastroenterology Volume: 56 Issue: 1 Pages: 67-77
  • DOI: 10.1007/s00535-020-01733-4
  • Peer Reviewed / Open Access
• [Journal Article] Daily Usage of Proton Pump Inhibitors May Reduce the Severity of Critical Upper Gastrointestinal Bleeding in Elderly Patients (2020)
  • Author(s): Matsuda Hidetaka、Nosaka Takuto、Akazawa Yu、Saito Yasushi、Ozaki Yoshihiko、Takahashi Kazuto、Naito Tatsushi、Ofuji Kazuya、Ohtani Masahiro、Hiramatsu Katsushi、Nakamoto Yasunari
  • Journal Title: Gastroenterology Research and Practice Volume: 2020 Pages: 1-8
  • DOI: 10.1155/2020/7168621
  • Peer Reviewed / Open Access
• [Journal Article] Safety and Long-Term Outcome of Intratumoral Injection of OK432-Stimulated Dendritic Cells for Hepatocellular Carcinomas After Radiofrequency Ablation (2020)
  • Author(s): Kitahara Masaaki、Mizukoshi Eishiro、Terashima Takeshi、Nakagawa Hidetoshi、Horii Rika、Iida Noriho、Arai Kuniaki、Yamashita Taro、Sakai Yoshio、Yamashita Tatsuya、Honda Masao、Nakamoto Yasunari、Kaneko Shuichi
  • Journal Title: Translational Oncology Volume: 13 Issue: 7 Pages: 100777-100777
  • DOI: 10.1016/j.tranon.2020.100777
  • Peer Reviewed / Open Access
• [Journal Article] Bipolar-current needle-knife with a water jet function (Jet B-knife) shortens the procedure time of endoscopic submucosal dissection for colorectal tumors. (2020)
  • Author(s): Hiramatsu K, Naito T, Akazawa Y, Saito Y, Nosaka T, Takahashi K, Ofuji K, Matsuda H, Ohtani M, Matsuda M, Sakai A, Nakamoto Y.
  • Journal Title: Surg Endosc. Volume: - Issue: 7 Pages: 3600-3606
  • DOI: 10.1007/s00464-020-07832-w
  • Peer Reviewed / Open Access
• [Journal Article] Usefulness of serum microRNA as a predictive marker of recurrence and prognosis in biliary tract cancer after radical surgery. (2019)
  • Author(s): Akazawa, Y., （4名略）, Ochiya, T., Nakamoto, Y., Nakatsura, T.
  • Journal Title: Sci Rep. Volume: 9(1) Issue: 1 Pages: 5925-5925
  • DOI: 10.1038/s41598-019-42392-7
  • Peer Reviewed / Open Access
• [Journal Article] Reactivation of intraabdominal tuberculous lymphadenopathy after drug-eluting beads transcatheter arterial chemoembolization in a patient with hepatocellular carcinoma. (2019)
  • Author(s): Murata Y, Hiramatsu K, Yoshida Y, Akazawa Y, Saito Y, Nosaka T, Ozaki Y, Hayama R, Takahashi K, Naito T, Ofuji K, Matsuda H, Ohtani M, Nemoto T, Nakamoto Y.
  • Journal Title: Clin J Gastroenterol. Volume: 12 Issue: 1 Pages: 76-81
  • DOI: 10.1007/s12328-018-0894-9
  • Peer Reviewed / Open Access
• [Journal Article] Higher human lymphocyte antigen class I expression in early-stage cancer cells leads to high sensitivity for cytotoxic T lymphocytes. (2019)
  • Author(s): Akazawa Y, Nobuoka D, Takahashi M, Yoshikawa T, Shimomura M, Mizuno S, Fujiwara T, Nakamoto Y, Nakatsura T.
  • Journal Title: Cancer Sci. Volume: 110 Issue: 6 Pages: 1842-1852
  • DOI: 10.1111/cas.14022
  • Peer Reviewed / Open Access
• [Journal Article] Molecular signature of hepatitis?B virus regulation by interferon‐γ in primary human hepatocytes (2019)
  • Author(s): Nosaka Takuto、Naito Tatsushi、Matsuda Hidetaka、Ohtani Masahiro、Hiramatsu Katsushi、Nemoto Tomoyuki、Nishizawa Tsutomu、Okamoto Hiroaki、Nakamoto Yasunari
  • Journal Title: Hepatology Research Volume: 50 Issue: 3 Pages: 292-302
  • DOI: 10.1111/hepr.13450
  • Peer Reviewed / Open Access
• [Presentation] Baseline FIB4 Index, Albumin, and ALBI Scores Predict Hypervascularization in Preneoplastic Nodules of Hepatocellular Carcinoma after HCV Eradication. (2021)
  • Author(s): Tanaka T, Murata Y, Nosaka T, Takahashi K, Naito T, Ofuji K, Matsuda H, Ohtani M, Hiramatsu K, Nakamoto Y.
  • Organizer: AASLD The Liver Meeting 2021
  • Int'l Joint Research
• [Presentation] Comparison of Circulating Tumor Cells in Hepatocellular Carcinoma Patients Treated by Atezolizumab-Bevacizumab and Transcatheter Arterial Chemoembolization. (2021)
  • Author(s): Murata Y, Nosaka T, Naito T, Takahashi K, Ofuji K, Matsuda H, Ohtani M, Hiramatsu K, Nakamoto Y.
  • Organizer: AASLD The Liver Meeting 2021
  • Int'l Joint Research
• [Presentation] Leukotrienes Derived from Tumor-Infiltrating Macrophages Promote Hepatocellular Carcinoma Progression. (2021)
  • Author(s): Nosaka T, Murata Y, Naito T, Takahashi K, Ofuji K, Matsuda H, Ohtani M, Hiramatsu K, Nakamoto Y.
  • Organizer: AASLD The Liver Meeting 2021
  • Int'l Joint Research
• [Presentation] Morphological Analysis of M2 Tumor-Associated Macrophage Subsets in Patients with Hepatocellular Carcinoma using Machine Learning Algorithm. (2021)
  • Author(s): Hiramatsu K, Nosaka T, Yamamoto A, Saito Y, Takahashi K, Naito T, Ofuji K, Matsuda H, Ohtani M, Nakamoto Y.
  • Organizer: AASLD The Liver Meeting 2021
  • Int'l Joint Research
• [Presentation] Analysis of Mucosa-Associated Dysbiosis Related to Portal Hypertension and Liver Fibrosis in Hepatitis C Patients. (2021)
  • Author(s): Yohei Midori, Katsushi Hiramatsu, Takuto Nosaka, Kazuto Takahashi, Tatsushi Naito, Kazuya Ofuji, Hidetaka Matsuda, Masahiro Ohtani and Yasunari Nakamoto.
  • Organizer: AASLD The Liver Meeting 2021
  • Int'l Joint Research
• [Presentation] Analysis of Immune Microenvironment of Hepatocellular Carcinoma after Hepatitis C Virus Eradication. (2020)
  • Author(s): Hiramatsu K, Nosaka T, Saito Y, Takahashi K, Naito T, Ofuji K, Matsuda H, Ohtani M, Nakamoto Y.
  • Organizer: AASLD THE LIVER MEETING 2020
  • Int'l Joint Research
• [Presentation] Signal Changes in Preneoplastic Nodules on DWI of Gd-EOB-DTPA MRI in Patients at Risk for Hypervascular Hepatocellular Carcinoma. (2020)
  • Author(s): Tanaka T, Murata Y, Nosaka T, Takahashi K, Naito T, Ofuji K, Matsuda H, Ohtani M, Hiramatsu K, Nakamoto Y.
  • Organizer: AASLD THE LIVER MEETING 2020
  • Int'l Joint Research
• [Presentation] Circulating Tumor Cells Detected with Microcavity Array Predict Clinical Outcome in Patients with Hepatocellular Carcinoma. (2020)
  • Author(s): Takahashi K, Ofuji K, Hiramatsu K, Nosaka T, Naito T, Matsuda H, Ohtani M, Nakamoto Y.
  • Organizer: AASLD THE LIVER MEETING 2020
  • Int'l Joint Research
• [Presentation] Sensitive detection of circulating tumor cells in patients with chronic liver disease and hepatocellular carcinoma using a microcavity array. (2019)
  • Author(s): Takahashi K, Ofuji K, Hiramatsu K, Nosaka T, Naito T, Matsuda H, Ohtani M, Nemoto T, Nakamoto Y.
  • Organizer: The Asian Pacific Association for the Study of the Liver single Topic Conference(APASL STC Tokyo 2019)
  • Int'l Joint Research