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IFN-γ応答を一細胞レベルで定量するバイオセンサーの開発

田中, 泰生 京都大学 DOI:10.14989/doctor.k24046

2022.03.23

概要

免疫細胞が分泌する IFN (Interferon)-γ は腫瘍免疫における重要なエフェクター分子のひとつであり、がん細胞に作用して多角的な効果を発揮する。しかし腫瘍微小環境における IFN-γ の時空間的な広がりに関する情報は未だ明らかになっておらず、IFN-γ 応答を一細胞の分解能で可視化するツールも成熟していない。本研究ではがん細胞における IFN-γ 応答を可視化する新規ツール開発を目指した。STAT1 が IFN-γ に応答してリン酸化し、ホモ二量体を形成して核内へ移行することに着目し、STAT1 の cDNA に蛍光タンパク質を付加した融合タンパク質を細胞に導入した。その結果、導入した STAT1 バイオセンサーが IFN-γ に応答して核内へ移る様子が観察され、定量的評価が可能となった。

また、IFN-γ に応答して核内へ移行した STAT1 が下流因子の転写を開始することに着目し、IFN-γ 応答プロモーター活性を生物発光及び蛍光で検出する IFN-γ センシングプローブ(ISP)を作製した。ISP は IFN-γ 応答プロモーターと PGK 安定発現プロモーターを双方向に組み合わせた構造をとる。IFN-γ シグナルに応答して赤色蛍光タンパク質 mCherry と生物発光酵素 Akaluc を発現し、IFN-γ 応答プロモーター活性を生物発光及び蛍光で検出することができる。加えて、PGK プロモーター下でシアン色蛍光タンパク質 Turquoise-GL を安定的に発現し、ゲノム内挿入サイトの影響を補正することが可能である。5 種類の IFN-γ 応答プロモーターを比較した結果、GAS (Gamma-interferon activation site)及び ISRE (interferon-stimulatedresponse element)が IFN-γ に応答したプロモーター活性を示すことがわかった。これらを組み込んだ ISP を 4 種類の細胞に導入して IFN-γ 刺激を in vitro で与えたところ、すべての細胞種で ISP の活性上昇が見られ、かつその上昇度合いには IFNγ に対する容量依存性が見られた。また、これら 4 種類の細胞を IFN-γ で刺激した際、IFN-γ に対する感受性及び ISP の活性度合いが細胞種により異なることがわかった。

作製した STAT1 バイオセンサー及び ISP-GAS 発現がん細胞の皮下への同種移植モデルを利用することにより、生体マウスにおける移植がんの IFN-γ 応答可視化を試みた。STAT1 バイオセンサーを発現する Braf V600E 細胞を皮下担がんしたところ、担がん後 5 日の時点で STAT1 バイオセンサーが核内に集積している様子が観察された。ISP-GAS を発現する B16F10 細胞を皮下担がんしたところ皮下担がん後7 日の時点で IFN-γ 依存的な ISP-GAS 活性上昇を発光イメージングにより認めた。またこのとき、二光子顕微鏡観察及びフローサイトメトリーによる蛍光検出によりがんの IFN-γ 応答を一細胞レベルで検出することに成功した。

以上のことから、STAT1 バイオセンサーは IFN-γ 初期応答を、ISP-GAS は IFNγ 二次的応答を可視化するバイオセンサーであることが示され、特に双方向性プロモーターを導入した ISP-GAS は、バイオセンサーのゲノム内挿入サイトの影響を克服しうる新規ツールとして有用であることが示された。

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参考文献

Adra, C.N., Boer, P.H., and McBurney, M.W. 1987. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene, 60: 65-74.

Altan-Bonnet, G. and Mukherjee, R. 2019. Cytokine-mediated communication: a quantitative appraisal of immune complexity. Nat. Rev. Immunol., 19: 205-217.

Baba, S., Cho, S.Y., Ye, Z., Cheng, L., Engles, J.M., and Wahl, R.L. 2007. How reproducible is bioluminescent imaging of tumor cell growth? Single time point versus the dynamic measurement approach. Mol. Imaging, 6: 315-322.

Bardehle, S., Kruger, M., Buggenthin, F., Schwausch, J., Ninkovic, J., Clevers, H., Snippert, H.J., Theis, F.J., Meyer-Luehmann, M., Bechmann, I., Dimou, L., and Gotz, M. 2013. Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation. Nat. Neurosci., 16: 580-586.

Beatty, G.L. and Paterson, Y. 2000. IFN-gamma can promote tumor evasion of the immune system in vivo by down-regulating cellular levels of an endogenous tumor antigen. J. Immunol., 165: 5502-5508.

Bonavita, E., Bromley, C.P., Jonsson, G., Pelly, V.S., Sahoo, S., Walwyn-Brown, K., Mensurado, S., Moeini, A., Flanagan, E., Bell, C.R., Chiang, S.C., Chikkanna-Gowda, C.P., Rogers, N., Silva-Santos, B., Jaillon, S., Mantovani, A., Reis e Sousa, C., Guerra, N., Davis, D.M., and Zelenay, S. 2020. Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.Immunity, 53: 1215-1229 e1218.

Bromberg, J.F., Horvath, C.M., Wen, Z.L., Schreiber, R.D., and Darnell, J.E. 1996.Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc. Natl. Acad. Sci. U. S. A., 93: 7673-7678.

Castro, F., Cardoso, A.P., Goncalves, R.M., Serre, K., and Oliveira, M.J. 2018. Interferon- Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front.Immunol., 9: 847.

Chin, Y.E., Kitagawa, M., Kuida, K., Flavell, R.A., and Fu, X.Y. 1997. Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol. Cell. Biol., 17: 5328-5337.

Chin, Y.E., Kitagawa, M., Su, W.C., You, Z.H., Iwamoto, Y., and Fu, X.Y. 1996. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science, 272: 719-722.

Cranfill, P.J., Sell, B.R., Baird, M.A., Allen, J.R., Lavagnino, Z., de Gruiter, H.M., Kremers, G.J., Davidson, M.W., Ustione, A., and Piston, D.W. 2016. Quantitative assessment of fluorescent proteins. Nat Methods, 13: 557-562.

Detjen, K.M., Farwig, K., Welzel, M., Wiedenmann, B., and Rosewicz, S. 2001. Interferon gamma inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis. Gut, 49: 251-262.

Dighe, A.S., Richards, E., Old, L.J., and Schreiber, R.D. 1994. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors. Immunity, 1: 447-456.

Dong, H., Strome, S.E., Salomao, D.R., Tamura, H., Hirano, F., Flies, D.B., Roche, P.C., Lu,J., Zhu, G., Tamada, K., Lennon, V.A., Celis, E., and Chen, L. 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat.Med., 8: 793-800.

Freeman, G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki, T., Byrne, M.C., Horton, H.F., Fouser, L., Carter, L., Ling, V., Bowman, M.R., Carreno, B.M., Collins, M., Wood, C.R., and Honjo, T. 2000.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med., 192: 1027-1034.

Fulda, S. and Debatin, K.M. 2002. IFNgamma sensitizes for apoptosis by upregulating caspase-8 expression through the Stat1 pathway. Oncogene, 21: 2295-2308.

Garcia-Diaz, A., Shin, D.S., Moreno, B.H., Saco, J., Escuin-Ordinas, H., Rodriguez, G.A., Zaretsky, J.M., Sun, L., Hugo, W., Wang, X., Parisi, G., Saus, C.P., Torrejon, D.Y., Graeber, T.G., Comin-Anduix, B., Hu-Lieskovan, S., Damoiseaux, R., Lo, R.S., and Ribas, A. 2017. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD- L2 Expression. Cell Rep., 19: 1189-1201.

Gerber, S.A., Sedlacek, A.L., Cron, K.R., Murphy, S.P., Frelinger, J.G., and Lord, E.M. 2013.IFN-γ mediates the antitumor effects of radiation therapy in a murine colon tumor.Am. J. Pathol., 182: 2345-2354.

Han, F., Liang, P., Wang, F., Zeng, L., and Zhang, B. 2011. Automated analysis of time-lapse imaging of nuclear translocation by retrospective strategy and its application to STAT1 in HeLa cells. PLoS One, 6: e27454.

Han, F., Luo, Y., Ge, N., and Xu, J. 2008. Construction of fluorescence resonance energy transfer vectors and their application in study of structure and function of signal transducers and activators of transcription 1. Acta Biochim Biophys Sin (Shanghai), 40: 934-942.

Hoekstra, M.E., Bornes, L., Dijkgraaf, F.E., Philips, D., Pardieck, I.N., Toebes, M., Thommen, D.S., van Rheenen, J., and Schumacher, T.N.M. 2020. Long-distance modulation of bystander tumor cells by CD8(+) T cell-secreted IFNgamma. Nat Cancer, 1: 291-301.

Huse, M., Lillemeier, B.F., Kuhns, M.S., Chen, D.S., and Davis, M.M. 2006. T cells use two directionally distinct pathways for cytokine secretion. Nat. Immunol., 7: 247-255.

Ivashkiv, L.B. 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat. Rev. Immunol., 18: 545-558.

Ivashkiv, L.B. and Donlin, L.T. 2014. Regulation of type I interferon responses. Nat. Rev.Immunol., 14: 36-49.

Iwano, S., Sugiyama, M., Hama, H., Watakabe, A., Hasegawa, N., Kuchimaru, T., Tanaka, K.Z., Takahashi, M., Ishida, Y., Hata, J., Shimozono, S., Namiki, K., Fukano, T., Kiyama, M., Okano, H., Kizaka-Kondoh, S., McHugh, T.J., Yamamori, T., Hioki, H., Maki, S., and Miyawaki, A. 2018. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science, 359: 935-939.

Kalderon, D., Roberts, B.L., Richardson, W.D., and Smith, A.E. 1984. A short amino acid sequence able to specify nuclear location. Cell, 39: 499-509.

Kaplan, D.H., Shankaran, V., Dighe, A.S., Stockert, E., Aguet, M., Old, L.J., and Schreiber,R.D. 1998. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. U. S. A., 95: 7556-7561.

Key, J. and Leary, J.F. 2014. Nanoparticles for multimodal in vivo imaging in nanomedicine.Int J Nanomedicine, 9: 711-726.

Khiar, S., Lucas-Hourani, M., Nisole, S., Smith, N., Helynck, O., Bourgine, M., Ruffie, C., Herbeuval, J.P., Munier-Lehmann, H., Tangy, F., and Vidalain, P.O. 2017.Identification of a small molecule that primes the type I interferon response to cytosolic DNA. Sci. Rep., 7: 2561.

Kienast, Y., von Baumgarten, L., Fuhrmann, M., Klinkert, W.E., Goldbrunner, R., Herms, J., and Winkler, F. 2010. Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med., 16: 116-122.

Klein, R., Ruttkowski, B., Knapp, E., Salmons, B., Gunzburg, W.H., and Hohenadl, C. 2006. WPRE-mediated enhancement of gene expression is promoter and cell line specific. Gene, 372: 153-161.

Komatsu, N., Aoki, K., Yamada, M., Yukinaga, H., Fujita, Y., Kamioka, Y., and Matsuda, M.2011. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell, 22: 4647-4656.

Komatsu, N., Terai, K., Imanishi, A., Kamioka, Y., Sumiyama, K., Jin, T., Okada, Y., Nagai, T., and Matsuda, M. 2018. A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging. Sci. Rep., 8: 8984.

Konishi, Y., Ichise, H., Watabe, T., Oki, C., Tsukiji, S., Hamazaki, Y., Murakawa, Y., Takaori- Kondo, A., Terai, K., and Matsuda, M. 2021. Intravital Imaging Identifies the VEGF- TXA2 Axis as a Critical Promoter of PGE2 Secretion from Tumor Cells and Immune Evasion. Cancer Res., 81: 4124-4132.

Koster, M. and Hauser, H. 1999. Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins. Eur. J. Biochem., 260: 137-144.

Mazzolini, G., Narvaiza, I., Martinez-Cruz, L.A., Arina, A., Barajas, M., Galofre, J.C., Qian, C., Mato, J.M., Prieto, J., and Melero, I. 2003. Pancreatic cancer escape variants that evade immunogene therapy through loss of sensitivity to IFNgamma-induced apoptosis. Gene Ther., 10: 1067-1078.

Meunier, M.C., Delisle, J.S., Bergeron, J., Rineau, V., Baron, C., and Perreault, C. 2005. T cells targeted against a single minor histocompatibility antigen can cure solid tumors. Nat. Med., 11: 1222-1229.

Miyoshi, H., Blomer, U., Takahashi, M., Gage, F.H., and Verma, I.M. 1998. Development of a self-inactivating lentivirus vector. J. Virol., 72: 8150-8157.

O'Shea, J.J. and Plenge, R. 2012. JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity, 36: 542-550.

Okada, Y., Watanabe, T., Shoji, T., Taguchi, K., Ogo, N., and Asai, A. 2018. Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi. Sci. Rep., 8: 2385.

Pellegrini, S. and Schindler, C. 1993. Early events in signalling by interferons. Trends Biochem. Sci., 18: 338-342.

Reynolds, C.J., Chong, D.L.W., Li, Y., Black, S.L., Cutler, A., Webster, Z., Manji, J., Altmann, D.M., and Boyton, R.J. 2019. Bioluminescent Reporting of In Vivo IFN- gamma Immune Responses during Infection and Autoimmunity. J. Immunol., 202:2502-2510.

Ritsma, L., Steller, E.J., Ellenbroek, S.I., Kranenburg, O., Borel Rinkes, I.H., and van Rheenen, J. 2013. Surgical implantation of an abdominal imaging window for intravital microscopy. Nat. Protoc., 8: 583-594.

Romei, M.G. and Boxer, S.G. 2019. Split Green Fluorescent Proteins: Scope, Limitations, and Outlook. Annu Rev Biophys, 48: 19-44.

Salas, A., Hernandez-Rocha, C., Duijvestein, M., Faubion, W., McGovern, D., Vermeire, S., Vetrano, S., and Vande Casteele, N. 2020. JAK-STAT pathway targeting for the treatment of inflammatory bowel disease. Nat. Rev. Gastroenterol. Hepatol., 17: 323- 337.

Samsonov, A., Zenser, N., Zhang, F., Zhang, H., Fetter, J., and Malkov, D. 2013. Tagging of genomic STAT3 and STAT1 with fluorescent proteins and insertion of a luciferase reporter in the cyclin D1 gene provides a modified A549 cell line to screen for selective STAT3 inhibitors. PLoS One, 8: e68391.

Sanderson, N.S., Puntel, M., Kroeger, K.M., Bondale, N.S., Swerdlow, M., Iranmanesh, N., Yagita, H., Ibrahim, A., Castro, M.G., and Lowenstein, P.R. 2012. Cytotoxic immunological synapses do not restrict the action of interferon-gamma to antigenic target cells. Proc. Natl. Acad. Sci. U. S. A., 109: 7835-7840.

Schiessl, I.M. and Castrop, H. 2016. Deep insights: intravital imaging with two-photon microscopy. Pflugers Arch., 468: 1505-1516.

Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol., 22: 1567-1572.

Stringer, C., Wang, T., Michaelos, M., and Pachitariu, M. 2021. Cellpose: a generalist algorithm for cellular segmentation. Nat Methods, 18: 100-106.

Takeda, K., Nakayama, M., Hayakawa, Y., Kojima, Y., Ikeda, H., Imai, N., Ogasawara, K., Okumura, K., Thomas, D.M., and Smyth, M.J. 2017. IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting. Nat Commun, 8: 14607.

Tanaka, N., Kawakami, T., and Taniguchi, T. 1993. Recognition DNA sequences of interferon regulatory factor 1 (IRF-1) and IRF-2, regulators of cell growth and the interferon system. Mol. Cell. Biol., 13: 4531-4538.

Taniguchi, K., Petersson, M., Hoglund, P., Kiessling, R., Klein, G., and Karre, K. 1987.

Interferon gamma induces lung colonization by intravenously inoculated B16 melanoma cells in parallel with enhanced expression of class I major histocompatibility complex antigens. Proc. Natl. Acad. Sci. U. S. A., 84: 3405-3409.

Thibaut, R., Bost, P., Milo, I., Cazaux, M., Lemaitre, F., Garcia, Z., Amit, I., Breart, B., Cornuot, C., Schwikowski, B., and Bousso, P. 2020. Bystander IFN-gamma activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment. Nat Cancer, 1: 302-314.

Trubiani, O., Bosco, D., and Di Primio, R. 1994. Interferon-gamma (IFN-gamma) induces programmed cell death in differentiated human leukemic B cell lines. Exp. Cell Res., 215: 23-27.

Uccellini, M.B. and Garcia-Sastre, A. 2018. ISRE-Reporter Mouse Reveals High Basal and Induced Type I IFN Responses in Inflammatory Monocytes. Cell Rep., 25: 2784-2796 e2783.

Watanabe, T., Seki, T., Fukano, T., Sakaue-Sawano, A., Karasawa, S., Kubota, M., Kurokawa, H., Inoue, K., Akatsuka, J., and Miyawaki, A. 2017. Genetic visualization of protein interactions harnessing liquid phase transitions. Sci. Rep., 7: 46380.

Weihua, X., Kolla, V., and Kalvakolanu, D.V. 1997. Interferon gamma-induced transcription of the murine ISGF3gamma (p48) gene is mediated by novel factors. Proc. Natl.Acad. Sci. U. S. A., 94: 103-108.

Yamauchi, F., Kamioka, Y., Yano, T., and Matsuda, M. 2016. In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability. Cancer Res., 76: 5266-5276.

Yusa, K., Rad, R., Takeda, J., and Bradley, A. 2009. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods, 6: 363-369.

Zelenay, S., van der Veen, A.G., Bottcher, J.P., Snelgrove, K.J., Rogers, N., Acton, S.E., Chakravarty, P., Girotti, M.R., Marais, R., Quezada, S.A., Sahai, E., and Reis e Sousa,C. 2015. Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity.Cell, 162: 1257-1270.

Zoller, M. 1988. IFN-treatment of B16-F1 versus B16-F10: relative impact on non-adaptive and T-cell-mediated immune defense in metastatic spread. Clin. Exp. Metastasis, 6: 411-429.

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