Metastasis and immunosuppression promoted by mtDNA and PD‐L1 in extracellular vesicles are reversed by WGP β‐glucan in oral squamous cell carcinoma

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D‐loop and programmed cell death‐ligand 1 (PD‐L1) in extracellular vesicles (EVs), and attenuate T‐cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) β‐glucan in oral squamous cell (OSCC) patients. Arecoline‐induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D‐loop were analyzed in OSCC cell lines. mtDNA D‐loop, PD‐L1, IFN‐γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D‐loop, PD‐L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D‐loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D‐loop leakage and PD‐L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP β‐glucan could elevate CD4(+) and CD8(+) T‐cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D‐loop and PD‐L1, and in turn elicits immune suppression. However, WGP β‐glucan potentially enhances dual effects on T‐cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.