Impact of dual inhibition of iNOS and COX2 on antitumor immunity and response to immune checkpoint blockade in hepatocellular carcinoma.

3153 Background: Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide. Although immune checkpoint blockade (ICB) benefits a subset of patients, efficacy is often limited by an immunosuppressive tumor microenvironment. Inducible nitric oxide synthase (iNOS/NOS2) and cyclooxygenase-2 (COX2) are inflammation-associated enzymes implicated in immune regulation. We hypothesized that inhibition of these pathways enhances antitumor immunity and improves responsiveness to immune checkpoint therapy in HCC. Methods: Tumor tissues from 75 patients with resected hepatocellular carcinoma (HCC) were analyzed by immunohistochemistry for NOS2, COX2, and CD8 to assess expression levels and spatial correlations. In parallel, an orthotopic murine HCC model was established by intrahepatic injection of 7.5 × 10⁵ RIL-175 cells (n = 6–10 mice per group). Mice were treated with vehicle control, selective NOS2 inhibition (1400W), COX2 inhibition (celecoxib), anti–PD-L1, or combination regimens including triple therapy. After two weeks of treatment, tumors were harvested for immune profiling, including flow cytometry, spatial imaging, plasma cytokine analysis, and CITE-seq. In vitro, splenocytes isolated from B6 mice were treated with prostaglandin E2 (PGE2) or the nitric oxide donor DETA-NONOate, and expression of CD226 and TIGIT was quantified by flow cytometry. Results: High intratumoral NOS2 expression was associated with inferior overall survival in resected HCC patients (HR 3.65, p=0.0011), with further worsening observed in tumors co-expressing NOS2 and COX2 (HR 9.31, p<0.0001). In murine HCC, combined iNOS and COX2 inhibition significantly reduced tumor burden and promoted intratumoral accumulation of CD8⁺ and CD4⁺ T cells, accompanied by increased B-cell recruitment and elevated circulating IL-2 and IFN-γ. Triple therapy with dual enzyme inhibition plus anti–PD-L1 resulted in superior tumor control and prolonged survival. Single-cell transcriptomic analysis comparing control, dual inhibition, and triple-treatment groups revealed a progressive enhancement of CD8 + T-cell activation states, characterized by increased expression of activation-associated markers and reduced expression of inhibitory receptors. Dual and triple therapy induced upregulation of CD226 and downregulation of TIGIT in CD8⁺ T cells, reflecting enhanced antitumor effector function. In vitro, nitric oxide and prostaglandin E2 directly impaired CD8⁺ T-cell function, as evidenced by reduced CD226 and increased TIGIT expression. Conclusions: iNOS and COX2 cooperatively establish an immunosuppressive inflammatory program in HCC that limits CD8⁺ T-cell–mediated antitumor immunity and response to checkpoint blockade. Dual targeting of these pathways represents a promising strategy to enhance immunotherapy efficacy in HCC.