Topology of cell-free DNA from tumour cells influences its immunogenicity in macrophages and dendritic cells in vitro

Abstract

Abstract Activation of DNA-sensing pathways can induce an anti-tumour immune response, but the mechanisms by which tumour-derived cell-free DNA (cfDNA) activates pro-inflammatory signaling is not well-understood. In particular, the topological features of cfDNA that contribute to DNA sensor activation in phagocytes are unknown. As such, we investigated how tumour-derived cfDNA topology influences DNA sensor activation in macrophages and dendritic cells. We treated a murine colon adenocarcinoma cell line (MC38) with chemotherapy (topotecan or staurosporine) for varying durations to induce cfDNA release. We then harvested cfDNA, separated the protected (membrane-bound) and accessible (non-membranous) fractions, and quantified nuclear and mitochondrial cfDNA abundance. The cfDNA fractions were then added to murine RAW 264.7 macrophages or bone marrow-derived dendritic cells (BMDCs), and a panel of inflammatory markers was measured by RT-qPCR. We found that accessible cfDNA from MC38 cells treated with chemotherapy for 48h increased Il6expression in RAW 264.7, but other inflammatory markers were not elevated. Interestingly, both nuclear and mitochondrial cfDNA abundance directly correlated with Il6expression. Surprisingly, cfDNA did not induce pro-inflammatory signalling in BMDCs. These results demonstrate that tumour-derived cfDNA immunogenicity depends on its mechanistic origins, abundance, and the immune cell type to which it is exposed. We are currently examining the impact of other cfDNA biophysical properties on macrophage activation, as well as elucidating the DNA sensing pathways responsible for inducing Il6expression. Our findings will help uncover the role of cfDNA in mediating anti-tumour immunity. E.Z. Malkin is supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through CIHR. S.V. Bratman is supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Dr. Mariano Elia Chair in Head and Neck Cancer Research at University Health Network and the University of Toronto.