4-Methylumbelliferone Modulates CAIX to Mitigate Hypoxia-Driven Dysregulation and Enhance PD-1 Immunotherapy in Lung Cancer

Hypoxia is a hallmark of solid tumors, driving metabolic reprogramming and immune evasion. In lung cancer, hypoxia-induced activation of carbonic anhydrase IX (CAIX) promotes lactate accumulation and extracellular acidification, fostering an immunosuppressive tumor microenvironment (TME). Analysis of public datasets revealed that patients with high CAIX expression exhibited significantly reduced median survival (<i>p</i> < 0.001). Moreover, CAIX correlated with HIF-1α, PD-L1, and immunosuppressant molecules, linking hypoxia-driven metabolic alterations with immune dysfunction. Here, we evaluated the capacity of 4-methylumbelliferone (4Mu) to counteract these effects and enhance antitumor immunity. In vitro, hypoxia increased CAIX and monocarboxylate transporter -4 (MCT4) expression in lung carcinoma cells, elevated lactate release, and reduced extracellular pH while promoting an M2-like macrophage profile and impairing antigen-specific splenocyte proliferation (<i>p</i> < 0.01). Treatment with 4Mu downregulated CAIX expression, restored extracellular pH, decreased lactate secretion, and rescued lymphocyte proliferation (<i>p</i> < 0.01). In vivo, 4Mu reduced CAIX expression, shifted macrophage polarization toward a pro-inflammatory phenotype, and enhanced CD8+ T cell infiltration. 4Mu was safe and well tolerated, and notably, combined with anti-PD-1 therapy, it synergistically inhibited tumor growth and increased both CD4+ and CD8+ T cell infiltration. These findings support 4Mu as a metabolic modulator capable of mitigating CAIX-driven acidosis and improving the efficacy of immunotherapy in lung cancer.