Background
Small extracellular vesicles (sEVs) release by cancer cells promotes immune evasion, in part by exporting cytoplasmic DNA (cyDNA). However, how inhibition of sEV release impacts intracellular DNA sensing and immune recognition is not fully understood. Here, we investigated the effects of blocking sEV release on cyDNA accumulation, cGAS activation, ULBP1 expression, and NK cell cyto- toxicity in acute myeloid leukemia (AML) and hepatocellular carcinoma (HCC) cells.
Methods
AML (MV4-11, OCI-AML3) and HCC (HepG2, Huh-7) cells were treated with the farnesyltransferase inhibitor Manumycin A and sphingomyelinase inhibitor GW4869, with or without DNA-damaging agents (cytarabine, doxorubicin, or hydroxyurea), to inhibit sEV release. Rab27a-knockout cells served as a genetic control. sEVs were isolated by size exclusion chromatography and characterized per MISEV2024 guidelines. Cytoplasmic DNA was assessed by dsDNA staining and quantification after subcellular fractionation.
Results
Pharmacological inhibition of sEV release and Rab27a knockout markedly reduced sEV release in both AML and HCC cells, as confirmed by western blot and CytofFLEX nano. DNA-damaging agents increased EV-DNA content in sEVs. Inhibition of sEV release led to substantial cytoplasmic DNA accumulation and robust activation of cGAS and IRF3 signaling, evidenced by increased IRF3-S386 phospho- rylation and pIRF3 (S396) nuclear foci. Enhanced γH2A.X and TREX1 expression were detected in both cancer types. Surface ULBP1 levels were significantly upregulated following sEV release inhibition and DNA damage, correlating with increased NK cell-mediated cytotoxic- ity. RNA-seq analysis revealed upregulation of interferon-stimulated genes and immune activation pathways under these conditions.
Conclusion
Inhibiting sEV release induces cyDNA accumulation, cGAS pathway activation, ULBP1 expression, and enhanced NK cell cyto- toxicity. These findings identify sEV release as a key mechanism limiting immunogenic DNA sensing and suggest targeting sEV pathways could potentiate cancer immunotherapy.
Keywords
Small extracellular vesicles, Cancer immunogenicity, EV-DNA, cGAS pathway, Cytoplasmic DNA sensing
Funding/Acknowledgments
Stiftung Universitätsmedizin Essen, Deutsche Kinderkrebsstiftung (DKS 2018.17), and Deutsche Jose Car- reras Leukämie-Stiftung (DJCLS 14 R/2023).
