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<h3>Background</h3> Neutrophil Extracellular Traps (NETs) are a form of cell-free DNA (cfDNA) that very often arises from intratumoral neutrophils as part of an inflammatory or antitumor immune response. Excessive NET formation has been linked to increased tumor growth, metastasis, the awakening of dormant metastases, and hypercoagulability. NETs can also shield tumor cells, induce CD8+ T cell dysfunction, and promote the activity of regulatory T cells (Tregs). Several human cancers, and gastrointestinal cancers (e.g., CRC) in particular, have high levels of neutrophil infiltration and NETs, which contribute to an immunosuppressive, protumor microenvironment (TME), leading to poor response to immunotherapies. In CRC, where the majority of patients (~85%) are microsatellite stable (MSS), mismatch repair proficient (MMRp), ICIs have shown very little clinical activity. There are several reasons for this, but a major issue is an immunosuppressive TME, to which NETs contribute significantly. We show that DNase I treatment can improve anti-CTLA-4 therapy in a MSS/MMRp CRC model, by targeting NETs. <h3>Methods</h3> Eight-week-old BALB/c mice were inoculated with 3*10<sup>5</sup> CT26 CRC cells using two models: subcutaneously (sc, primary tumor model) or intraperitoneally (ip, metastatic model). When the average tumor volume reached 100 mm<sup>3</sup>, the mice were treated with either daily (qd) or biweekly (biw) ip injections of PBS control, anti-CTLA-4, or DNase I (administered either ip or iv), or a combination of both agents, for three weeks. Response was monitored by measuring tumor volume biweekly (mm<sup>3</sup>) and survival in the sc, primary tumor model, and by survival in the ip, metastatic model. Blood was collected at several time points for pharmacodynamic analysis. <h3>Results</h3> DNase I significantly improved the efficacy of anti-CTLA-4 immune checkpoint blockade in both primary tumor and ip metastasis models. In the sc primary tumor model, more than half the mice treated with the DNase I/anti-CTLA-4 combination had complete responses (CRs), with no evidence of residual tumor at the end of the study. Furthermore, when these CR mice were rechallenged with CT26 tumor cells, we observed no evidence of tumor growth. <h3>Conclusions</h3> These data show the beneficial effects of targeting NETs with systemic DNase I in models of primary tumor and metastatic CRC, improving the efficacy of CTLA-4 immune checkpoint blockade. Importantly, mice successfully cleared of primary tumors with the DNase I/anti-CTLA-4 combination were subsequently immune from tumor rechallenge, suggesting that DNase I may not only improve ICI responses in MSS/MMRp CRC, but can enhance the duration of response to ICIs.