遺伝子改変ヒトiPS細胞由来マクロファージを用いた悪性黒色腫に対する免疫療法

2013 Fiscal Year Research-status Report

• Project/Area Number: 25870545
• Research Category: Grant-in-Aid for Young Scientists (B)
• Research Institution: Kumamoto University
• Principal Investigator: 福島 聡 熊本大学, 医学部附属病院, 講師 (50398210)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: iPS細胞 / 免疫療法 / 悪性黒色腫 / 細胞治療 / 腫瘍抗原 / 腫瘍マーカー / microRNA / マクロファージ

Research Abstract

免疫不全マウス（SCID）においてヒトメラノーマ細胞を用いた抗腫瘍実験を行うために、まず治療に用いる細胞を作成した。ヒトiPS細胞をOP9フィーダー細胞上でミエロイド系細胞に分化誘導し、C-MYC遺伝子をレンチウイルスを用いて導入、不死化した。この細胞はM-CSF依存性ひ無限に増殖し、マクロファージ様の表面マーカー及び機能を持っている。以下この細胞をiPS細胞由来ミエロイド細胞ライン（iPS-ML）と呼称する。iPS-MLはそのままでは抗腫瘍効果は限定的であるため、抗腫瘍作用を持つ分子の遺伝子改変を行う必要があった。メラノーマにおいては、治療としてインターフェロンα（IFN-α）およびβ（IFN-β）が用いられていることから、これらを遺伝子導入した細胞を作成した。SCIDマウスにヒトメラノーマを腹膜播種させ、iPS-ML-IFNα、iPS-ML-IFN-βにて治療実験を行ったが、いずれも、コントロール群に対して、極めて強い腫瘍抑制効果を示した。抗腫瘍効果のメカニズムを検討するためにiPS-ML-IFNα、iPS-ML-IFN-βおよびコントロールのiPS-MLを用いて、腫瘍貪食能、サイトカイン産生能、表面マーカーの検討を行った。以上の結果については、論文作成中である。また新規メラノーマ抗原の探索として、FOXM1について検討したところ、メラノーマ細胞株、患者組織において高発現していた。また細胞株における細胞増殖実験、およびMAPK経路との関与についても検討し、論文を投稿中である。一方、新規腫瘍マーカーとしての血清microRNAについては、複数のmicroRNAを用いることで、転移の検出ができることを見出し論文報告した。さらに、皮膚組織を用いて、mir-146aのSNPが健常者に比べてメラノーマ患者では、有意にCGフェノタイプが多いことを見出し発表した。

Current Status of Research Progress

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

Reason

当初の計画で予定していたCSPG4のscFvの遺伝子導入については、共同研究者であるピッツバーグ大とMTAは締結しているが、まだ先方において、準備中とのことでscFvを入手できていない。しかし、上述のようにCSPG4の遺伝子改変を行うまでもなく、IFN-αおよびIFN-βで十分な抗腫瘍効果が得られており、研究全体として進行に問題はない。
新規がん抗原の探索については、FOXM1について成果を得てすでに論文投稿中である。また、新規腫瘍マーカーとしてのmicroRNAについても、上述のように論文発表することができた。さらに血清中のEGFRやレプチンレセプターがそれぞれ腫瘍マーカーとなりうることを論文報告した。以上より、おおむね順調に進展していると考えている。

Strategy for Future Research Activity

iPS-MLの研究については、さらにその遊走能等について抗腫瘍効果のメカニズム解明を進めていく。さらに、IL-15など他の遺伝子改変についても施行予定である。iPS-MLはM-CSF存在下ではマクロファージ様であるが、GM-CSFとIL-4存在下では樹状細胞(DC)様となる。今後は、iPS-ML由来DCについても抗腫瘍実験を進めていく予定である。また、将来の臨床応用にむけては、安全性や副作用の検討が必須である。その点についてもマウスを用いて検討してく予定としている。
また新規メラノーマ抗原としては次にCDCA1についての検討を行う予定としている。さらに新規腫瘍マーカーとしては、最近microRNAが患者の毛髪からも検出可能という知見を得たので、メラノーマ患者でも検討して、さらに非侵襲的なマーカーの確立にむけて研究を進めて予定である。

Expenditure Plans for the Next FY Research Funding

物品費に予算のほとんどを使用したが、端数として379円の残高が生じた。
379円の残高については、次年度に物品費の一部として使用する予定である。

Research Products

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• [Journal Article] Analysis of expression pattern of serum microRNA levels in patients with psoriasis. (2014)
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• [Journal Article] Sensitive detection of melanoma metastasis utilizing circulating microRNA expression profiles (2014)
  • Author(s): Sensitive detection of melanoma metastasis using circulating microRNA expression profiles. Shiiyama R, Fukushima S, Jinnin M, Yamashita J, Miyashita A, Nakahara S, Kogi A, Aoi J, Masuguchi S, Inoue Y, Ihn H.
  • Journal Title: Melanoma Res Volume: in press Pages: -
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• [Journal Article] Clinical evaluation of moisturizers with physiological analysis of stratum corneum TARC and TSLP. (2014)
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• [Journal Article] Lymphocytic inflammation with CD123 positive cells is a prognostic factor of lupus erythematosus profundus. (2014)
  • Author(s): Miyashita A, Fukushima S, Makino T, Yoshino Y, Yamashita J, Honda N, Aoi J, Jinnin M, Inoue Y, Ihn H.
  • Journal Title: Acta Derm Venereol Volume: in press Pages: -
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• [Journal Article] Case of disseminated cutaneous Mycobacterium chelonae infection mimicking cutaneous vasculitis. (2014)
  • Author(s): Ichihara A, Jinnin M, Fukushima S, Inoue Y, Ihn H.
  • Journal Title: J Dermatol Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] miR-424 levels in hair shaft are increased in psoriasis patients. (2014)
  • Author(s): Tsuru Y, Jinnin M, Ichihara A, Fujisawa A, Moriya C, Sakai K, Fukushima S, Ihn H.
  • Journal Title: J Dermatol Volume: in press Pages: -
  • DOI: 10.1111/1346-8138.12460.
  • Peer Reviewed
• [Journal Article] A case of psoriasis accompanied by arthritis after delivery. (2014)
  • Author(s): Kanemaru H, Jinnin M, Asao K, Ichihara A, Makino K, Kajihara I, Fujisawa A, Fukushima S, Ihn H.
  • Journal Title: Bioscience trends Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] Clinical significance of serum VE-cadherin concentrations in inflammatory skin diseases. (2014)
  • Author(s): Kuriyama H, Jinnin M, Kanemaru H, Ichihara A, Fujisawa A, Moriya C, Fukushima S, Ihn H.
  • Journal Title: Ann Dermatol Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] MIRSNP rs2910164 of miR-146a is associated with the muscle involvement in polymyositis/dermatomyositis. (2014)
  • Author(s): Okada Y, Jinnin M, Makino T, Kajihara I, Makino K, Honda N, Nakayama W, Inoue K, Fukushima S, Ihn H.
  • Journal Title: Int J Dermatol Volume: in press Pages: -
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• [Journal Article] Decreased IL-20 expression in scleroderma skin contributes to cutaneous fibrosis. (2014)
  • Author(s): Kudo H, Jinnin M, Asano Y, Trojanowska M, Nakayama W, Inoue K, Honda N, Kajihara I, Makino K, Fukushima S, Ihn H.
  • Journal Title: Arthritis Rheumatol Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] Serum levels of leptin receptor in patients with systemic sclerosis. (2014)
  • Author(s): Ohyoshi Y, Makino T, Nakayama W, Fukushima S, Inoue Y, Jinnin M, Ihn H.
  • Journal Title: Rheumatol Int Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] A case of psoriasis accompanied by arthritis after delivery. (2014)
  • Author(s): Kanemaru H, Jinnin M, Asao A, Ichihara A, Makino K, Kajihara I, Fujisawa A, Fukushima S, Ihn H.
  • Journal Title: J Dermatol Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] Up-regulation of miR-18a contributes to the epidermal necrolysis in severe drug eruptions. (2014)
  • Author(s): Ichihara A, Wang Z, Jinnin M, Izono Y, Shimozono N, Yamane K, Fujisawa A, Moriya C, Fukushima S, Inoue Y, Ihn H.
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• [Journal Article] Angiopoietin-like Protein 2 Accelerates Carcinogenesis by Activating Chronic Inflammation and Oxidative Stress. (2014)
  • Author(s): Aoi J, Endo M, Kadomatsu T, Miyata K, Ogata A, Horiguchi H, Odagiri H, Masuda T, Fukushima S, Jinnin M, Hirakawa S, Sawa T, Akaike T, Ihn H, Oike Y.
  • Journal Title: Mol Cancer Res Volume: 12 Pages: 239-49
  • DOI: 10.1158/1541-7786.MCR-13-0336
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• [Journal Article] Successful treatment of skin fistulas in systemic sclerosis patients with the combination of topical negative pressure therapy and split-thickness skin grafting. (2014)
  • Author(s): Kajihara I, Jinnin M, Yamada S, Ichihara A, Makino T, Igata T, Masuguchi S, Fukushima S, Ihn H.
  • Journal Title: Mod Rheumatol Volume: 24 Pages: 374-6
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• [Journal Article] Dots/ globules on dermoscopy in nail-apparatus melanoma. (2014)
  • Author(s): Inoue Y, Menzies SW, Fukushima S, Nishi-Kogushi H, Miyashita A, Masuguchi S, Muchemwa F, Kageshita T, Ihn H.
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• [Journal Article] Detection of hair root miR-19a as a novel diagnostic marker for psoriasis. (2014)
  • Author(s): Hirao H, Jinnin M, Ichihara A, Fujisawa A, Makino K, Kajihara I, Sakai K, Fukushima S, Inoue Y, Ihn H.
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• [Journal Article] Serum levels of leptin receptor in patients with malignant melanoma as a new tumor marker. (2013)
  • Author(s): Mizutani H, Fukushima S, Masuguchi S, Yamashita J, Miyashita A, Nakahara S, Aoi J, Inoue Y, Jinnin M, Ihn H.
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• [Journal Article] The rs2910164 G>C polymorphism in microRNA-146a is associated with the incidence of malignant melanoma. (2013)
  • Author(s): Yamashita J, Iwakiri T, Fukushima S, Jinnin M, Miyashita A, Hamasaki T, Makino T, Aoi J, Masuguchi S, Inoue Y, Ihn H.
  • Journal Title: Melanoma Res Volume: 23 Pages: 13-20
  • Peer Reviewed
• [Journal Article] Serum epidermal growth factor receptor levels in patients with malignant melanoma. (2013)
  • Author(s): Kanemaru H, Fukushima S, Yamashita J, Jinnin M, Sakai K, Masuguchi M, Aoi J, Makino T, Inoue Y, Ihn H.
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• [Journal Article] Role of c-Jun N-termainal kinase isoforms in the cellular activity of melanoma cell lines. (2013)
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  • DOI: 10.1111/ced.12102.
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• [Journal Article] Association of linear IgA bullous disease with ulcerative colitis: a case of successful treatment with infliximab. (2013)
  • Author(s): Yamada S, Makino T, Jinnin M, Sakai K, Fukushima S, Inoue Y, Ihn H.
  • Journal Title: Dermatology. Volume: 227 Pages: 295-8
  • DOI: 10.1159/000355354.
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• [Journal Article] Hair miR-29a levels are decreased in patients with scleroderma. (2013)
  • Author(s): Takemoto R, Jinnin M, Wang Z, Kudo H, Inoue K, Nakayama W, Ichihara A, Igata T, Kajihara I, Fukushima S, Ihn H.
  • Journal Title: Exp Dermatol Volume: 22 Pages: 832-3
  • DOI: 10.1111/exd.12245.
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• [Journal Article] Detection of hair-microRNAs as the novel potent biomarker: Evaluation of the usefulness for the diagnosis of scleroderma. (2013)
  • Author(s): Wang Z, Jinnin M, Kudo H, Inoue K, Nakayama W, Honda N, Makino K, Kajihara I, Fukushima S, Inoue Y, Ihn H.
  • Journal Title: J Dermatol Sci Volume: 72 Pages: 134-41
  • DOI: 10.1016/j.jdermsci.2013.06.018.
  • Peer Reviewed
• [Journal Article] miR-150 down-regulation contributes to the constitutive type I collagen overexpression in scleroderma dermal fibroblasts via the induction of integrin β3. (2013)
  • Author(s): Honda N, Jinnin M, Kira-Etoh T, Makino K, Kajihara I, Makino T, Fukushima S, Inoue Y, Okamoto Y, Hasegawa M, Fujimoto M, Ihn H.
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• [Journal Article] The miR-146a rs2910164G/C polymorphism is associated with telangiectasia in systemic sclerosis. (2013)
  • Author(s): Sakoguchi A, Jinnin M, Makino T, Kajihara I, Makino K, Honda N, Nakayama W, Inoue K, Fukushima S, Ihn H.
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• [Journal Article] microRNA-7 down-regulation mediates excess collagen expression in localized scleroderma. (2013)
  • Author(s): Etoh M, Jinnin M, Makino K, Yamane K, Nakayama W, Aoi J, Honda N, Kajihara I, Makino T, Fukushima S, Ihn H.
  • Journal Title: Arch Dermatol Res Volume: 305 Pages: 9-15
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• [Journal Article] Down-regulation of miR-124/ -214 in cutaneous squamous cell carcinoma mediates abnormal cell proliferation via the induction of ERK. (2013)
  • Author(s): Yamane K, Jinnin M, Etoh T, Kobayashi Y, Shimozono N, Fukushima S, Masuguchi S, Maruo K, Inoue Y, Ishihara T, Aoi J, Oike Y, Ihn H.
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• [Journal Article] Intravenous immunoglobulin treatment recovers the down-regulatied levels of Th1 cytokines in the sera and skin of scleroderma patients. (2013)
  • Author(s): Kudo H, Jinnin M, Yamane K, Makino T, Kajihara I, Makino K, Honda N, Nakayama W, Fukushima S, Ihn H.
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• [Journal Article] Diccoidin domain receptor 2-micro RNA 196a -mediated negative feedback against excess type I collagen expression is impaired in scleroderma dermal fibroblasts. (2013)
  • Author(s): Makino K, Jinnin M, Aoi J, Hirano A, Kajihara I, Makino T, Sakai K, Fukushima S, Inoue Y, Ihn H.
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• [Journal Article] Serum miR-21 levels in patients with dermatomyositis. (2013)
  • Author(s): Shimada S, Jinnin M, Ogata A, Makino T, Kajihara I, Makino K, Honda N, Nakayama W, Inoue K, Fukushima S, Ihn H.
  • Journal Title: Clin Exp Rheumatol Volume: 31 Pages: 161-2
  • Peer Reviewed
• [Journal Article] Hidradenocarcinoma showing prominent mucinous and squamous differentiation and associated with pgetoid cells. (2013)
  • Author(s): Honda Y, Tanigawa H, Harada M, Fukushima S, Masuguchi S, Ishihara T, Ihn H, Iyama K.
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• [Journal Article] Case of paraneoplastic pemphigus with follicular lymphoma treated with rituximab (2013)
  • Author(s): Aoi J, Makino K, Sakai K, Masuguchi S, Fukushima S, Jinnin M, Inoue Y, Koga H, Hashimoto T, Ihn H.
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• [Journal Article] he down-regulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma. (2013)
  • Author(s): Makino K, Jinnin M, Hirano A, Yamane K, Eto M, Kusano T, Honda N, Kajihara K, Makino T, Sakai K, Masuguchi S, Fukushima S, Ihn H.
  • Journal Title: J Immunol, 190: 3905-3915, 2013. Volume: 190 Pages: 3905-15
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• [Journal Article] Down-regulation of miR-223 contributes to the formation of Gottron’s eruption in dermatomyositis via the induction of PKCε. (2013)
  • Author(s): Inoue K, Jinnin M, Yamane K, Makino T, Kajihara I, Makino K, Honda N, Nakayama W, Fukushima S, Ihn H.
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  • Peer Reviewed
• [Journal Article] Decreased miR-7 expression in the skin and sera of patients with dermatomyositis. (2013)
  • Author(s): Oshikawa Y, Jinnin M, Makino T, Kajihara I, Makino K,Honda N, Nakayama W, Inoue K, Fukushima S, Ihn H.
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• [Journal Article] Expression analysis of multiple microRNAs in each patient with scleroderma. (2013)
  • Author(s): Koba S, Jinnin M, Inoue K, Nakayama W, Honda N, Makino K, Kajihara I, Makino T, Fukushima S, Ihn H.
  • Journal Title: Exp Dermatol Volume: 22 Pages: 489-91
  • DOI: 10.1111/exd.12173.
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• [Journal Article] Scleroderma dermal fibroblasts overexpress vascular endothelial growth factor due to autocrine transforming growth factor β signaling. (2013)
  • Author(s): Kajihara I, Jinnin M, Honda N, Makino K, Makino T, Masuguchi S, Sakai K, Fukushima S, Inoue Y, Ihn H.
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• [Journal Article] Geographical flushing of the children’s face: A new clinical entity. (2013)
  • Author(s): Jinnin M, Fukushima S, Inoue Y, Ihn H.
  • Journal Title: Case Rep Dermatol Med Volume: - Pages: -
  • DOI: 10.1155/2013/802130
  • Peer Reviewed
• [Journal Article] Serum levels of tenascin-C in collagen diseases. (2013)
  • Author(s): Inoue K, Jinnin M, Hara Y, Makino K, Kajihara I, Makino T, Sakai K, Fukushima S, Inoue Y, Ihn H.
  • Journal Title: J DermatoL Volume: 40 Pages: 715-9
  • DOI: 10.1111/1346-8138.12218.
  • Peer Reviewed
• [Presentation] メラノーマに対する遺伝子改変ヒトiPS細胞由来ミエロイドラインによる免 疫細胞療法 (2014)
  • Author(s): 福島聡、宮下梓、千住覚、尹浩信
  • Organizer: 第35回日本炎症・再生医学会
  • Place of Presentation: 万国津梁館
  • Year and Date: 20140701-20140704
• [Presentation] The rs2910164 G>C polymorphism in microRNA-146a is associated with the pathogenesis of malignant melanoma (2013)
  • Author(s): Satoshi Fukushima, Junji Yammashita, Takuma Iwakiri, Masatoshi Jinnin, Azusa Miyashita, Jun Aoi, Hisashi Kanemaru, Katsunari Makino, Asako Ichihara, Yuji Inoue, Hironobu Ihn
  • Organizer: International Investigative Dermatology 2013,
  • Place of Presentation: Edinburgh, Scotland
  • Year and Date: 20130508-20130511
• [Book] 皮膚悪性腫瘍「悪性黒色腫の治療 免疫療法 樹状細胞療法」 (2013)
  • Author(s): 福島聡
  • Total Pages: 843
  • Publisher: 日本臨床社