BackgroundMetformin slows tumor growth and progression in vitro, and in combination with chemoradiotherapy, resulted in high overall survival in patients with head and neck cancer squamous cell carcinoma (HNSCC) in our phase 1 clinical trial (NCT02325401). Metformin is also postulated to activate an antitumor immune response. Here, we investigate immunologic effects of metformin on natural killer (NK) and natural killer T cells, including results from two phase I open-label studies in patients with HNSCC treated with metformin (NCT02325401,NCT02083692).MethodsPeripheral blood was collected before and after metformin treatment or from newly diagnosed patients with HNSCC. Peripheral immune cell phenotypes were evaluated using flow cytometry, cytokine expression by ELISA and/or IsoLight, and NK cell-mediated cytotoxicity was determined with a flow-based NK cell cytotoxicity assay (NKCA). Patient tumor immune infiltration before and after metformin treatment was analyzed with immunofluorescence. NK cells were treated with either vehicle or metformin and analyzed by RNA sequencing (RNA-seq). NK cells were then treated with inhibitors of significant pathways determined by RNA-seq and analyzed by NKCA, ELISA, and western blot analyses.ResultsIncreased peripheral NK cell activated populations were observed in patients treated with metformin. NK cell tumor infiltration was enhanced in patients with HNSCC treated with metformin preoperatively. Metformin increased antitumorigenic cytokines ex vivo, including significant increases in perforin. Metformin increased HNSCC NK cell cytotoxicity and inhibited the CXCL1 pathway while stimulating the STAT1 pathway within HNSCC NK cells. Exogenous CXCL1 prevented metformin-enhanced NK cell-mediated cytotoxicity. Metformin-mediated NK cell cytotoxicity was found to be AMP-activated protein kinase independent, but dependent on both mechanistic target of rapamycin and pSTAT1.ConclusionsOur data identifies a new role for metformin-mediated immune antitumorigenic function through NK cell-mediated cytotoxicity and downregulation of CXCL1 in HNSCC. These findings will inform future immunomodulating therapies in HNSCC.
coverage. There were no significant differences for the mandible max dose, cord max dose, OAR pharynx mean dose, or the right parotid mean dose. Conclusion: In this retrospective study, there was minimal dose variation to target volumes and OAR structures when substituting artifact with soft tissue density HUs. There were significant differences in terms of maximum dose and dose to the uninvolved oral cavity, which suggests that ignoring artifacts in CT images during planning may result in greater hot spots. While small, these differences are worthy of further study as they may at least partially contribute to increased rates of empirically observed mucosal toxicity in the vicinity of dental artifacts.
6037 Background: Metformin is a biguanide, widely used oral hypoglycemic agent. Metformin has also shown to inhibit tumor growth and progression in a wide variety of cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). Metformin activates AMP protein kinase (AMPK) related pathways leading to inactivation of mammalian target of rapamycin (mTOR) and suppression of its downstream effectors. In addition, metformin is postulated to alter immune regulation in the tumor microenvironment leading to increased tumor cell killing. Here, we report our findings on the impact of metformin on T cells, NK cells and cytokines from patient peripheral blood mononuclear cells (PBMCs) from a phase I open-label single site dose escalation study combining metformin and chemoradiation (CRT) in HNSCC (NCT02325401). Methods: In this study, we evaluated the immune cell phenotypes and cytokine profiles of peripheral blood in patients before and after metformin treatment on trial by using flow cytometry and cytokine magnetic bead assays (Luminex). Cytokine profiles were further studied in co-culture experiments combining PBMCs, HNSCC cell lines, and metformin. Results: Patients who received metformin developed expanded NK cell populations, increased NKG2D expression, and a shift in their CD8+ T-cell memory phenotypes. Patient serum ELISA examination revealed increased anti-tumorigenic cytokine profiles. Metformin treatment of HNSCC cell lines in vitro as well as HNSCC PBMCs ex vivo resulted in downregulation of STAT3 compared to healthy controls. Downregulation of STAT3 may be a potential mechanism in which metformin stimulates NK cells. Conclusions: Here we show evidence that metformin treatment has a direct effect on the innate immune system in patients with HNSCC, inducing an anti-tumorigenic immune response suggesting that metformin continues to be a good candidate to yield improved clinical outcomes in patients with advanced stage HNSCC. Clinical trial information: NCT02325401.
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