CYP4X1 Inhibition by Flavonoid CH625 Normalizes Glioma Vasculature through Reprogramming TAMs via CB2 and EGFR-STAT3 Axis

Tumor-associated macrophages (TAMs) are pivotal effector cells in angiogenesis. Here, we tested whether CYP4X1 inhibition in TAMs by flavonoid CH625 prolongs survival and normalizes glioma vasculature. CH625 was selected against the CYP4X1 3D model by virtual screening and showed inhibitory activity on the CYP4X1 catalytic production of 14,15-EET-EA in the M2-polarized human peripheral blood mononuclear cells (IC₅₀ = 16.5 μM). CH625 improved survival and reduced tumor burden in the C6 and GL261 glioma intracranial and subcutaneous model. In addition, CH625 normalized vasculature (evidenced by a decrease in microvessel density and HIF-1α expression and an increase in tumor perfusion, pericyte coverage, and efficacy of temozolomide therapy) accompanied with the decreased secretion of 14,15-EET-EA, VEGF, and TGF-β in the TAMs. Furthermore, CH625 attenuated vascular abnormalization and immunosuppression induced by coimplantation of GL261 cells with CYP4X1^high macrophages. In vitro TAM polarization away from the M2 phenotype by CH625 inhibited proliferation and migration of endothelial cells, enhanced pericyte migration and T cell proliferation, and decreased VEGF and TGF-β production accompanied with the downregulation of CB2 and EGFR-dependent downstream STAT3 expression. These effects were reversed by overexpression of CYP4X1 and STAT3 or exogenous addition of 14,15-EET-EA, VEGF, TGF-β, EGF, and CB2 inhibitor AM630. These results suggest that CYP4X1 inhibition in TAMs by CH625 prolongs survival and normalizes tumor vasculature in glioma via CB2 and EGFR-STAT3 axis and may serve as a novel therapeutic strategy for human glioma.