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Protocol to induce and assess cGAS-STING pathway activation in vitro.

ABSTRACT: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in several cellular processes including pathogen recognition and inflammatory responses. We describe a protocol to activate the cGAS-STING pathway in murine cells using nucleic acids transfection. We describe how to prepare the nucleic acid probes and validate activation of the pathway by western blot and gene expression analysis. The protocol can be applied to investigate cGAS-STING signaling in both murine and human cell lines. For complete details on the use and execution of this protocol, please refer to Vila et al. (2022).

SUBMITTER: Chamma H

PROVIDER: S-EPMC9117926 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Protocol to induce and assess cGAS-STING pathway activation <i>in vitro</i>.

Chamma Hanane H Guha Soumyabrata S Laguette Nadine N Vila Isabelle K IK

STAR protocols 20220514 2

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in several cellular processes including pathogen recognition and inflammatory responses. We describe a protocol to activate the cGAS-STING pathway in murine cells using nucleic acids transfection. We describe how to prepare the nucleic acid probes and validate activation of the pathway by western blot and gene expression analysis. The protocol can be applied to investigate cGAS-STING signaling ...[more]

PMID: 35600929

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Project description:BackgroundThe absence of representative Epstein-Barr virus-associated intrahepatic cholangiocarcinoma (EBVaICC) cell lines has limited our understanding of the molecular and immunological characteristics of this cancer subtype.MethodsWe reviewed patients with metastatic cholangiocarcinoma at Sun Yat-sen University Cancer Center from January 2015 to August 2023. Among them, 22 patients with EBVaICC and 66 patients with non-EBVaICC who received anti-PD1 treatment were included. Additionally, 2 EBV-positive ICC cell lines, RBE-EBV and HuH28-EBV, were developed through cell-to-cell infection. Stable EBV infection and responsiveness to viral reactivation were confirmed. Transcriptomic and bioinformatics analyses were performed, and in vitro experiments examined the immune effects of EBV-positive ICC. Key immune-related genes and cytokines were validated by reverse transcription quantitative polymerase chain reaction and ELISA in cell lines and patient plasma samples.ResultsIn this study, we found that patients with EBVaICC showed enhanced immune responses and improved overall and progression-free survival compared to patients with non-EBVaICC. We first successfully established and validated 2 EBV-positive ICC cell lines (RBE-EBV and HuH28-EBV). These cell lines were confirmed for stable EBV infection and displayed responsiveness to viral reactivation, making them suitable for future studies. Transcriptomic analyses and in vitro studies revealed that EBV activated the cGAS-STING pathway, resulting in MHC-I upregulation and CXCL10 secretion in ICC cells, which collectively enhanced CD8+ T cell chemotaxis and cytotoxicity. Furthermore, ELISA analysis showed higher plasma levels of CXCL10 and IFN-γ in patients with EBVaICC, suggesting a potential role for EBV in enhancing immunotherapy sensitivity in this subtype.ConclusionsThe established EBV-positive ICC cell lines revealed enhanced immunogenicity driven by cGAS-STING pathway activation, providing valuable models for future research and insights into the mechanisms of improved immunotherapy sensitivity in EBVaICC.

Dataset Information

Protocol to induce and assess cGAS-STING pathway activation in vitro.

Publications

Protocol to induce and assess cGAS-STING pathway activation <i>in vitro</i>.

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