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Bystander IFN- γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment

ABSTRACT: The cytokine IFN-g produced by tumor-reactive T cells is a key effector molecule with well characterized pleiotropic effects during anti-tumor immune responses. While IFN-g production is a transient event targeted at the immunological synapse, how IFN-g acts in time and space within the tumor microenvironment has remained elusive. Specifically, the link between duration of exposure to the cytokine and cellular response is poorly understood. Using RNA sequencing, we show that tumor cell phenotypic alterations require several hours of exposure to IFN-g, most likely due to a positive feedback loop mediated by STAT1 and IRF8.

ORGANISM(S): Mus musculus

PROVIDER: GSE140191 | GEO | 2020/02/03

REPOSITORIES: GEO

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Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.

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Bystander IFN- γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment

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