GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE334nnn/GSE334585/suppl/GSE334585_all_samples_FeatureCount_count.txt.gzftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE334nnn/GSE334585/primaryOK200TranscriptomicsMus musculusExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE334585GEOGSEfalseiNOS is a key mediator of anti-PD-1 melanoma therapy responseBackground: Inducible nitric oxide synthase (iNOS) and its product nitric oxide (NO) were historically linked to poor melanoma outcomes, yet recent evidence shows NO supports anti-tumor immunity. This study examines how iNOS shapes anti–PD-1 efficacy, particularly through interferon signaling. Methods: B16 D5 melanoma tumors were implanted in wild-type (WT) and iNOS knockout (KO) mice to compare tumor growth and response to anti–PD-1 therapy. Flow cytometry, apoptosis assays, and RNA sequencing assessed NO production, PD-L1 expression, and interferon-related gene activation. In vitro, melanoma cells were treated with NO donors (DETA NONOate, SNAP) to assess proliferation and apoptosis. Peripheral blood mononuclear cells from 27 melanoma patients receiving anti–PD-1 therapy were analyzed with multiparameter flow cytometry to correlate NO-associated immune subsets with progression-free survival (PFS). Results: Tumors grew significantly faster in iNOS KO mice, and anti–PD-1 therapy had no effect, demonstrating that iNOS-derived NO contributes to treatment efficacy. NO donors inhibited melanoma proliferation and induced apoptosis in vitro. Transcriptomic analysis showed anti–PD 1 upregulated interferon pathway genes (STAT1, IRF1, IFNB1) in WT but not iNOS KO mice. In patients, a NO-producing dendritic cell subset (DAF FM⁺CD11c⁺) was associated with improved PFS (hazard ratio 0.453; 95% CI=0.270-0.992; p = 0.048), indicating a NO-dependent enhancement of interferon-driven immune activity.2026/07/15GSE334585GSM9791337GSM9791315GSM9791316GSM9791338GSM9791339GSM9791317GSM9791318GSM9791319GSM9791340GSM9791341GSM9791342GSM9791320GSM9791343GSM9791321GSM9791322GSM9791344GSM9791345GSM9791323GSM9791346GSM9791324GSM9791325GSM9791347GSM9791348GSM9791326GSM9791349GSM9791327GSM9791328GSM9791306GSM9791329GSM9791307GSM9791308GSM9791309GSM9791350GSM9791351GSM9791330GSM9791352GSM9791353GSM9791331GSM9791310GSM9791332GSM9791333GSM9791311GSM9791334GSM9791312GSM9791313GSM9791335GSM9791336GSM979131424247334585Mus musculus[42440610]