Biological, Clinical, and Functional Implications of Cell-Free DNA Topology in Cancer
Cells can release their DNA into the extracellular space and circulation by active mechanisms or as a product of cell death. In oncology, this cell-free DNA (cfDNA) has emerged as a promising source of cancer biomarkers because it contains actionable information about a patient’s disease and can be readily obtained via a simple blood draw. In addition to its use in biomarker applications, cfDNA may also function as a mediator of pro-inflammatory signaling in the tumour microenvironment. Despite its mounting importance both clinically and physiologically, the biological features of cfDNA remain poorly understood. In particular, the structural relationship between cfDNA and other biological macromolecules – its topology – has not been thoroughly investigated. This lack of knowledge has limited the implementation of cfDNA to its full potential as a cancer biomarker, while also hampering our understanding of how cfDNA influences intercellular communication and tumour immunity. To address this gap, I first conducted a fundamental and comprehensive characterization of cfDNA topology in pre-clinical cancer models. I implemented a novel DNA-targeted immunoprecipitation assay to demonstrate that distinct topological subsets comprising membrane-protected or accessible cfDNA had unique sub-cellular origins and mechanisms of active release. I then applied these methods to clinical cohorts and found that topological characterization enhanced the diagnostic and prognostic capabilities of mitochondrial cfDNA. Based on these findings, I proposed a series of experiments to elucidate the in vivo origins of distinct cfDNA topological subsets and discussed important considerations for future clinical studies evaluating cfDNA topology as a liquid biopsy analyte. Next, I examined the impact of topology on the ability of tumour-derived cfDNA to stimulate DNA-sensing pathways in immune cells. Through rigorous mechanistic investigations, I observed pro-inflammatory macrophage signaling in response to accessible cfDNA released from tumour cells undergoing secondary necrosis. From this in vitro model, I outlined subsequent experimental steps to determine the fate of cfDNA-induced inflammation in the tumour microenvironment, and I considered the impact of cfDNA immunogenicity on the successful implementation of DNA-sensor-targeting cancer immunotherapies. Altogether, the knowledge gained from these studies contributes to a refined model of cfDNA biology and structure, which can be leveraged both for cancer liquid biopsy applications and to bolster our overall understanding of tumour-immune interactions as they relate to patient responses to immunotherapy.
Cell-Free DNA, cGAS-STING, Liquid Biopsy, Tumour Immunity
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