Treatment of human cells with the anti-cancer drug cisplatin results in the caspase-dependent release of adduct-containing cell-free DNA

Cell-free DNA (cfDNA) found in biofluids is increasingly being used in the diagnosis and treatment of a variety of disease states, including cancer. Though DNA is known to be susceptible to damage by many different chemotherapeutic compounds and genotoxic agents, the fact that cfDNA may be damaged and contain DNA adducts associated with specific exposures has not previously been considered to any significant extent. Here, using differential centrifugation of culture medium from cells treated with the anti-cancer drug cisplatin, we show that DNA containing cisplatin adducts is readily detectable in the extracellular milieu and is enriched in fractions known to contain small extracellular vesicles and cfDNA. However, our data indicates that this damaged cfDNA is non-vesicular in nature and likely represents fragments of chromatin. Dose and time course experiments suggest that the release of cfDNA containing cisplatin-DNA adducts is correlated with the activation of apoptotic signaling. Indeed, the generation of cisplatin-damaged cfDNA is exacerbated by the loss of nucleotide excision repair and is abrogated by caspase inhibition. Finally, we show that native cisplatin-damaged cfDNA, but not purified, protein-free cfDNA, can be taken up by cells by phagocytosis to result in the presence of cisplatin-DNA adduct-containing DNA in non-cisplatin-treated cells. These results indicate that tumors from patients undergoing cisplatin-based chemotherapy may shed damaged cfDNA that could have additional biological effects in bystander cells, which could both impact chemotherapeutic responses and lead to improved treatments and diagnostic tools for monitoring therapeutic efficacy.