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

Taisei Tanaka,Michiyuki Matsuda,Shinya Tsukiji,Kenta Terai,Yoshinobu Konishi,Hiroshi Ichise

doi:10.1247/csf.21052

Abstract

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.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

Highlights

IFN-γ is a cytokine secreted by immune cells and has pleiotropic effects on anti-tumor immune response (Dighe et al, 1994; Kaplan et al, 1998)
To generate the interferon sensing probe (ISP) (IFN-γ sensing probe) constructs, cDNA encoding mCherry-Akaluc (Iwano et al, 2018) was inserted into the pPB piggyBac transposon vector (Yusa et al, 2009), and the 3-phosphoglycerate kinase (PGK) promoter-driven Turquoise-GL-nuclear localization signal (NLS) gene cassette was inserted in the opposite direction with a stuffer sequence upstream of it (Adra et al, 1987)
Fluorescent images were acquired with two different detector channels using the following filters and mirrors: an infrared (IR)-cut filter, BA685RIF-3 (Olympus), two dichroic mirrors, DM505 and DM570 (Olympus), and three emission filters, FF01-425/30 (Semrock) for the second harmonic generation channel (SHG), BA460-500 (Olympus) for TurquoiseGL, and BA575-630 (Olympus) for mCherry

Summary

Introduction

IFN-γ is a cytokine secreted by immune cells and has pleiotropic effects on anti-tumor immune response (Dighe et al, 1994; Kaplan et al, 1998). Visualization of the IFN-γ production in vivo is helpful to understand its temporal changes and tissue specificity (Reynolds et al, 2019) For this purpose, JAK-STAT signaling pathway can be a versatile indicator to visualize the response of cancer cells to IFN-γ. We combined a dual promoter system and an IFN-γ signal-responsive elements to create a probe that could compensate for the integration site effect and the copy number of the biosensor gene. Using this probe, we visualized the IFN-γ signaling in transplanted cancer in vivo

Material and Methods

Results and Discussion

Objective lens