Diosmetin alleviates the immunosuppressive tumor microenvironment in esophageal squamous cell carcinoma by dually inhibiting angiogenesis and promoting CD8T cell cytotoxicity.

[BACKGROUND] The prognosis for patients with esophageal squamous cell carcinoma (ESCC) is poor, mainly due to the immunosuppressive tumor microenvironment (TME). However, the underlying mechanism and strategies to reverse this immunosuppressive TME remain to be clarified.

[PURPOSE] This study aimed to identify key factors contributing to ESCC immunosuppressive TME, to investigate the regulatory role of diosmetin (DIOS), and to explore its potential in enhancing the anti-PD-1 therapy efficacy.

[METHODS] Bioinformatics analysis identified the key factors affecting the ESCC immune microenvironment. Cell proliferation, transwell migration, invasion, tube formation, spheroid sprouting and Chick chorioallantoic membrane (CAM) assays evaluated the effect of DIOS on angiogenesis. Transcriptomic sequencing clarified the mechanism of DIOS in ESCC cells and HUVECs. Molecular docking, Cellular thermal shift assay (CETSA), pull down and Surface plasmon resonance (SPR) assays detected the binding of DIOS to target proteins. Finally, the combined effect of DIOS and anti-PD-1 antibody was explored in vivo.

[RESULTS] Angiogenesis and CD8T cell suppression were key contributors to ESCC immunosuppression. DIOS suppressed angiogenesis in ESCC cells via the AURKB/AKT/STAT4/PDGFC axis and in HUVECs stimulated by ESCC CM via the JAK1/STAT1/PDGFC pathway. Clinically, high PDGFC was associated with poor prognosis and limited immune checkpoint blockade efficacy in ESCC patients. Meanwhile, DIOS promoted both T cell adhesion and migration, as well as the killing capacity of CD8⁺T cells. Furthermore, anti-CD8 antibody attenuated the anti-tumor effect of DIOS. Notably, the combination of DIOS with anti-PD-1 antibody inhibited the growth of ESCC without causing significant kidney toxicity.

[CONCLUSION] This study demonstrates for the first time that the natural compound DIOS dually suppresses tumor angiogenesis and enhances CD8⁺T cell function. And the combination of DIOS with anti-PD-1 antibody enhanced anti-tumor efficacy in ESCC. These findings provide a novel strategy for developing low-toxicity immunotherapy combinations based on natural products.