Uncoupling of STING Accumulation and Activation upon Chromosome-Derived Extrachromosomal Circular DNA Formation

The innate immune system maintains cellular homeostasis by recognizing cytosolic nucleic acids. While the cGAS-STING pathway is well characterized in detecting non-self-DNA, its response to self-DNA originating from the host nucleus remains unclear. Extrachromosomal circular DNA (eccDNA), a form of self-DNA often enriched in cancer, has been implicated in immune modulation, yet its ability to activate cGAS-STING remains unresolved. Here, we developed a Cre-loxP-based eccDNA reporter system, where Cre-mediated eccDNA formation triggers expression of the fluorescent protein mCitrine, enabling time-resolved, single-cell analysis of chromosome-derived eccDNA dynamics and STING activation. Using an mRuby3-STING reporter, we found that low-dose plasmid transfection induced STING activation, as evidenced by modest STING accumulation and downstream cytokine induction. Cells carrying chromosome-derived cytosolic eccDNA also showed a modest, cGAS-dependent accumulation of STING. However, this accumulation was uncoupled from detectable STING-Ser366 phosphorylation or induction of downstream cytokines. These findings indicate that chromosome-derived eccDNA does not drive detectable STING signaling under the conditions tested, and that STING accumulation and its activation are mechanistically separable steps. This work provides a foundation for understanding the immunological roles of eccDNA in genome instability and cancer.