Cell-Free DNA Release Kinetics and Fragmentation Reflect Treatment Response and Resistance In Vitro

Background: Circulating tumor DNA (ctDNA) has emerged as a clinically valuable biomarker for cancer detection, treatment monitoring, and minimal residual disease assessment. Despite its growing clinical utility, the biological mechanisms governing ctDNA release remain incompletely understood. The objective of this study was to investigate how chemotherapy-induced cytotoxicity and chemoresistance influence the kinetics and fragmentation patterns of cfDNA released by cancer cells in vitro. Methods: Human lung adenocarcinoma (A549) and esophageal adenocarcinoma (FLO-1, OE19) cell lines were used to investigate cell-free DNA (cfDNA) release and fragmentation in vitro. Cancer cells were treated with chemotherapy (cisplatin and 5-fluorouracil), and cfDNA released into the culture medium was quantified as total cfDNA by Qubit fluorometry and mutation-specific cfDNA by droplet digital PCR (ddPCR). A cisplatin-resistant OE19 model was generated to directly compare cfDNA release kinetics between chemosensitive and chemoresistant cells. Fragment size distributions were determined and evaluated alongside cell death mechanisms assessed by flow cytometry. Results: cfDNA release correlated positively with viable tumor cell number across all cell lines. Chemotherapy increased per-cell cfDNA release in all cell lines. Chemosensitive OE19 cells exhibited significantly higher cfDNA release compared to chemoresistant OE19 cells following cisplatin exposure, with distinct temporal release kinetics. Additionally, chemotherapy treatment induced a shift toward the release of larger DNA fragments in both chemosensitive and resistant cells. This was accompanied by changes in PI-positive and Annexin V/PI double-positive cell populations, suggesting altered cell death processes following cisplatin treatment. Conclusions: Chemotherapy-induced cytotoxicity significantly influences cfDNA release kinetics and fragmentation patterns, with distinct effects in chemosensitive and chemoresistant cancer cells. These findings provide mechanistic insight into tumor-derived DNA release biology and may have important implications for the interpretation, timing, and standardization of liquid biopsy testing during treatment. Furthermore, they establish in vitro cancer models as a valuable platform for studying cfDNA dynamics and informing preclinical therapeutic development.