Abstract 1694: Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells

Abstract Introduction: Detection of circulating free DNA (cfDNA) is a promising tool to monitor and predict tumor progression and treatment efficacy. Our group has shown the clinical relevance of cfDNA as a biomarker of tumor development and progression in several cancer models, including colorectal carcinoma and melanoma. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions. However, the source and mechanism of cfDNA release in cancer cells are still unclear. Indeed, while some data state that cfDNA is derived from apoptosis, other studies report it is mainly a product of cellular secretions. To better understand cfDNA release and its correlation with tumor biology, this study aimed to assess cfDNA kinetics under cell death, cell cycle arrest, and senescence by using an in vitro cancer model. Methods: Colorectal (HTC116 and HT29), lung (A549), and melanoma (MP41 and OMM2.5) cancer cells underwent cytotoxic conditions by drugs: TRIAL/APO2L, Roscovitine, and valproic acid (VAP), as well as environmental stress by heat and irradiation. Cell death and cell cycle were evaluated by FACS. β-galactosidase staining assessed senescence. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit and evaluated by digital droplet PCR through hot spot mutations (Mut), and a mitochondrial gene (mt-cfDNA). Isolated cfDNA was visualized by fragment size using Bioanalyzer 2100. Results: Mut and mt-cfDNA increased during cytotoxic conditions in a dose-dependent manner. Cells under apoptosis released greater mut and mt-cfDNA compared to necrotic and untreated cells. Similarly, cell cycle disturbances by VAP were associated with an increase in Mut and mt-cfDNA. Moreover, β-galactosidase positive cells (5D post-irradiation) showed lower Mut and mt-cfDNA levels than control conditions. Electropherogram images showed that cytotoxic conditions alter fragment size distribution. While fragments about 100-200 bp were observed in apoptosis, necrosis showed more abundant fragments about >1000 bp. Conclusions: The release of cfDNA is influenced by cytotoxic and stress conditions. Knowing the biology of cfDNA can help understand its role in cancer development and how best to utilize this marker clinically, especially post cytotoxic agents that induce different cell death mechanisms. These findings have major implications in the interpretation of ctDNA in liquid biopsy as a biomarker of treatment response and tumor progression. Citation Format: Prisca Bustamante, Thupten Tsering, Julia Valdemarin Burnier. Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1694.