Journal Article

Cytosolic nucleic acid sensors and interferon beta-1 activation drive radiation-induced anti-tumour immune effects in human pancreatic cancer cells

Röhrer, Anna, Widder, Joachim, Walch, Lena, Slade, Dea, Kleinwächter, Ava, Stasny, Katharina, Kneringer, Alexandra, Postl, Andreas, Franke, Vedran, Kerschbaum-Gruber, Sylvia, Dias, Ana Beatriz, Georg, Dietmar, Akalin, Altuna, Appel, Lisa-Marie, Kratzer, Bernhard, Pickl, Winfried F., Benedum, Johannes, Popova, Katerina, Barna, Sandra, Horvat, Filip

2024

Overview

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines. Human PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation. We demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons. Our findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Acids / Adaptor Proteins, Signal Transducing / Adenocarcinoma / Cancer therapies / Carcinoma, Pancreatic Ductal - immunology / Carcinoma, Pancreatic Ductal - radiotherapy / Carcinoma, Pancreatic Ductal - therapy / Cell cycle / Cell death / Cell Line, Tumor / Chemoresistance / Chemotherapy / CRISPR / Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase / Cytosol - metabolism / Cytotoxicity / Double-stranded RNA / Enzyme-linked immunosorbent assay / Flow cytometry / Galectin-1 / Humans / Immune checkpoint inhibitors / Immune response / Immunity (Disease) / Immunofluorescence / Immunogenicity / Immunomodulation / Immunostimulation / Immunotherapy / Inflammation / Interferon / Interferon-beta - metabolism / Localization / Major histocompatibility complex / MAVS / Medical prognosis / Membrane Proteins - genetics / Membrane Proteins - metabolism / Mitochondrial DNA / Nucleotidyltransferases - genetics / Nucleotidyltransferases - metabolism / Pancreatic cancer / Pancreatic Neoplasms - immunology / Pancreatic Neoplasms - radiotherapy / Phosphorylation / Photons / Protons / radiation / Radiation therapy / Signal Transduction / STING / STING Protein / Tumor necrosis factor-TNF / Tumors / Western blotting / X-rays