Metformin sensitizes esophageal squamous cell carcinoma to Vγ9Vδ2 T cell-mediated cytotoxicity by upregulating BTN3A1 and BTN2A1

Abstact Metformin, a first-line anti-diabetic agent, exhibits broad-spectrum antitumor properties, though its underlying immunomodulatory mechanisms remain incompletely characterized. Here, we demonstrate that metformin significantly upregulates BTN3A1 and BTN2A1 expression on esophageal cancer cells in an AMPK-dependent manner, thereby sensitizing them to Vγ9Vδ2 T cell-mediated cytotoxicity. This molecular priming enhanced tumor immunogenicity, leading to synergistic tumor cell killing in vitro and potent suppression of tumor growth in xenograft models. Mechanistically, metformin-induced BTN3A1/BTN2A1 upregulation promoted Vγ9Vδ2 T cell activation, and Granzyme B-mediated apoptosis in tumor tissues. The combination therapy demonstrated excellent tolerability without observable systemic toxicity. Moreover, Integrating GEPIA3 database and clinical specimen analyses, we find that BTN3A1 and BTN2A1 are highly but heterogeneously expressed in esophageal cancer tissues, and that metformin‑mediated upregulation may restore sensitivity to Vγ9Vδ2 T cell immunotherapy particularly in patients with low baseline expression—uncovering a novel immunomodulatory function of metformin that provides a compelling rationale for its repurposing as a combinatorial agent against immunologically cold tumors such as esophageal carcinoma.