Biological Rationale for Circulating Cell-Free DNA Release

Abstract

The kinetics of circulating cell-free (cf)DNA release may provide a real-time assessment of induced cell death. However, there is limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. We utilized multiple in vitro and in vivo preclinical models to show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Using cfDNA from head and neck squamous cell carcinoma (HNSCC) cell lines and fibroblasts grown in culture, or as mouse xenografts, we identified treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that counteracts its release. Necrosis was the predominant cell death mechanism that consistently contributed to cfDNA release from HNSCC in response to ionizing radiation, while, surprisingly, apoptosis played a comparatively minor role in some tumours. Based on these results, we propose a generalizable model to explain cfDNA release from cells over time. These findings will have important implications for future studies evaluating the utility of measuring cfDNA kinetics in clinical settings.

Keywords: cell-free DNA, cfDNA, ctDNA, cell death, senescence, apoptosis, necrosis