Bioactive Sphingolipids of Cordyceps sinensis Modulate Tumor Immunity Through the SphK1 ‐Mediated Cer/ S1P Axis

ABSTRACT Cordyceps sinensis is a traditional medicinal fungus known for its immunomodulatory properties. Its bioactive sphingolipids (SPLs) exhibit antitumor potential, though their mechanisms remain poorly understood. This study aimed to identify the active SPLs from C. sinensis and investigate their synergistic effects with αPD‐1 (anti‐programmed death‐1 antibody) therapy. SPLs were isolated from C. sinensis via UPLC–MS/MS‐guided purification. The antitumor efficacy and immunomodulatory synergy with αPD‐1 were assessed using a Lewis lung carcinoma (LLC) tumor‐bearing mouse model and in vitro co‐culture systems. Mechanisms involving sphingosine kinase 1 (SphK1) were explored through cellular thermal shift assays, enzymatic activity tests, surface plasmon resonance, molecular docking, and molecular dynamics simulations. The combination of SPLs and αPD‐1 enhanced antitumor immunity by promoting CD8 + T‐cell infiltration and suppressing PD‐L1 expression in the tumor microenvironment. Metabolomic and transcriptomic analyses revealed that SPLs shifted the sphingolipid balance by targeting the SphK1‐mediated ceramide/sphingosine‐1‐phosphate (Cer/S1P) axis, promoting antitumor immunity. A novel ceramide, cordysinamide A, was identified as a key bioactive constituent and shown to bind directly to SphK1 (IC₅₀ = 28.45 μM; KD = 14.4 μM), stabilizing its structure and inhibiting S1P production. This shift increased IL‐2 and IFN‐γ levels and sensitized tumors to αPD‐1 treatment. This study identifies C. sinensis ‐derived SPLs as key bioactive components that overcome αPD‐1 resistance by targeting the SphK1‐mediated Cer/S1P balance. Our findings propose a natural product‐based strategy to change immunosuppressive metabolism in non‐small cell lung cancer.