Metformin enhances, whereas acetaminophen attenuates, γδT cell antitumor efficacy and shapes stem-like escape in breast cancer

Background: Cellular immunotherapies utilizing γδT cells hold immense promise for breast cancer as an MHC-independent approach; however, their clinical efficacy is profoundly dictated by the tumor microenvironment and systemic metabolic contexts. While cancer patients routinely consume widely available pharmacological agents such as Metformin (MET) and Acetaminophen (APAP), their inadvertent modulatory impacts on γδT cell-mediated cytotoxicity and therapy-induced tumor evasion remain fundamentally unexplored. Methods: We established an in vitro co-culture model utilizing human MCF-7 breast cancer cells and ex vivo expanded primary human γδT cells. Multiparametric flow cytometry and RT-qPCR were employed to interrogate the modulatory effects of MET and APAP on targeted cytotoxicity, apoptotic trajectories, cell cycle dysregulation, the dynamic enrichment of cancer stem cells (CSCs; CD44+CD24-), and the expression profiles of immune checkpoints. Results: Activated γδT cells exerted robust baseline cytotoxicity, primarily driving target cells into late apoptosis. Strikingly, pharmacological interventions yielded divergent immunomodulatory outcomes. MET synergistically potentiated γδT cell-mediated apoptosis and accelerated tumor clearance. Conversely, APAP profoundly abrogated immune-mediated killing, shifting the death modality toward necrosis and inducing a stalled, pre-lethal sub-G1 cell cycle accumulation. Notably, while γδT cells efficiently eliminated bulk tumor populations, immune-mediated pressure paradoxically enriched the CSC fraction, indicative of aggressive phenotypic plasticity. MET further exacerbated this CSC selection despite maximizing overall cytotoxicity. Furthermore, the surviving stem-like fraction exhibited a marked post-transcriptional upregulation of the immune checkpoint B7-H3 (CD276), revealing a potent adaptive resistance mechanism. Conclusions: Our findings unveil a critical translational dichotomy: MET acts as a synergistic immunometabolic adjuvant, whereas concurrent APAP exposure may inadvertently compromise γδT cell efficacy. Furthermore, therapy-induced CSC enrichment coupled with B7-H3 upregulation highlights a distinct stem-like immune evasion strategy, underscoring the necessity of combining cellular therapies with B7-H3 blockade to eradicate residual tumor plasticity and prevent relapse.