A Dual Promoter System to Monitor IFN-γ Signaling <i>in vivo</i> at Single-cell Resolution

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  • Tanaka Taisei
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University
  • Konishi Yoshinobu
    Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School
  • Ichise Hiroshi
    Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
  • Tsukiji Shinya
    Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology
  • Matsuda Michiyuki
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University Institute for Integrated Cell-Material Sciences, Kyoto University
  • Terai Kenta
    Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University

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  • A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution

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Abstract

<p>IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.</p><p>Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system</p>

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