Cell-Free DNA (cfDNA) Regulates Metabolic Remodeling in the ES-2 Ovarian Carcinoma Cell Line, Influencing Cell Proliferation, Quiescence, and Chemoresistance in a Cell-of-Origin-Specific Manner

Abstract

Funding Information: This work benefited from access to CERMAX, ITQB-NOVA, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/SAICT/2016. Funding Information: The institutions are funded by Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), to MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and the Associated Laboratory LS4FUTURE (LA/P/0087/2020). No Self s.r.l. grant funded the NOVA Medical School and Instituto Portugu\u00EAs de Oncologia de Lisboa, Francisco Gentil, to support the development of Isabel Lemos PhD project. Isabel Lemos was funded by an FCT individual Ph.D. fellowship (UI/BD/154203/2022). Catarina Freitas-Dias was funded by the No Self project. Ana Hip\u00F3lito was funded by a FCT individual Ph.D. fellowship (SFRH/BD/148441/2019). Luis G. Gon\u00E7alves was financed by a FCT contract according to DL57/2016, (SFRH/BPD/111100/2015). This work benefited from access to CERMAX, ITQB-NOVA, Oeiras, Portugal with equipment funded by FCT, project AAC 01/SAICT/2016. Publisher Copyright: © 2025 by the authors.Background: The cell-free DNA (cfDNA) is an extracellular fragmented DNA found in body fluids in physiological and pathophysiological contexts. In cancer, cfDNA has been pointed out as a marker for disease diagnosis, staging, and prognosis; however, little is known about its biological role. Methods: The role of cfDNA released by ES-2 ovarian cancer cells was investigated, along with the impact of glucose bioavailability and culture duration in the cfDNA-induced phenotype. The effect of cfDNA on ES-2 cell proliferation was evaluated by proliferation curves, and cell migration was assessed through wound healing. We explored the impact of different cfDNA variants on ES-2 cells’ metabolic profile using nuclear magnetic resonance (NMR) spectroscopy and cisplatin resistance through flow cytometry. Moreover, we assessed the protein levels of DNA-sensitive Toll-like receptor 9 (TLR9) by immunofluorescence and its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study demonstrated that despite inducing similar effects, different variants of cfDNA promote different effects on cells derived from the ES-2 cell line. We observed instant reactions of adopting the metabolic profile that brings back the cell functioning of more favorable culture conditions supporting proliferation and resembling the cell of origin of the cfDNA variant, as observed in unselected ES-2 cells. However, as a long-term selective factor, certain cfDNA variants induced quiescence that favors the chemoresistance of a subset of cancer cells. Conclusions: Therefore, different tumoral microenvironments may generate cfDNA variants that will impact cancer cells differently, orchestrating the disease fate.publishersversionpublishe