Targeting endothelial CYP3A5 in lung adenocarcinoma remodels the immunosuppressive microenvironment via VEGF inhibition: Mechanistic insights from Diosmetin

The tumor endothelium not only promotes angiogenesis but also actively fosters an immunosuppressive microenvironment in lung adenocarcinoma (LUAD). Cytochrome P450 enzymes, particularly CYP3A5, have emerged as potential regulators of endothelial function, yet their role in shaping tumor immunity remains unexplored. Diosmetin, a natural flavonoid, exhibits anti-tumor properties, but whether it acts by targeting endothelial CYP3A5 to reprogram the immune microenvironment is unknown. We integrated bioinformatic screening with single-cell and spatial transcriptomics to identify CYP3A5 as a key endothelial target in LUAD. Molecular docking, ChIP-qPCR, DNA pulldown, and luciferase assays validated transcriptional regulation by GATA6. Functional impacts on tumor-endothelial crosstalk and immune modulation were assessed using co-culture systems, ELISA, Western blot, enzyme activity assays, and immune profiling. In vivo efficacy was evaluated in a xenograft model. CYP3A5 was identified as a functional target associated with Diosmetin response, predominantly expressed in endothelial cells of minimally invasive LUAD under transcriptional control of GATA6. Single-cell and spatial analyses revealed enrichment of the VEGFA–FLT1 signaling axis between epithelial and endothelial compartments. Diosmetin inhibited CYP3A5 enzymatic activity without significantly altering its expression level, suggesting that CYP3A5 may represent a functional mediator of Diosmetin activity in LUAD. Further biochemical studies are required to confirm the direct physical interaction between Diosmetin and CYP3A5.Functionally, Diosmetin-treated endothelial cells impaired LUAD cell proliferation, migration, and invasion, and reduced secretion of immunosuppressive cytokines. In vivo, Diosmetin suppressed tumor growth and angiogenesis, and was associated with a shift in the immune microenvironment. Importantly, VEGF is known to inhibit T cell function and promote regulatory T cell infiltration; thus, by suppressing endothelial CYP3A5 activity and VEGF production, Diosmetin disrupts this immune-evasive circuit.These findings identify endothelial CYP3A5 as a potential functional vulnerability in LUAD and provide evidence that Diosmetin may suppress tumor progression through modulation of endothelial VEGF signaling. Further studies are required to validate immune-related effects in immune-competent models. Our study unveils a novel GATA6/CYP3A5/VEGF axis in LUAD-associated endothelium and demonstrates that targeting endothelial CYP3A5 enzymatic activity—exemplified by Diosmetin—modulates endothelial-mediated tumor microenvironment signaling. These findings provide a translational rationale for endothelial CYP3A5 as a dual-action target to inhibit angiogenesis and counteract VEGF-driven immune evasion in LUAD.