Metabolic alterations and immune evasion are emerging hallmarks of cancer. Metformin and 2-deoxy-D-glucose (2DG) are metabolic drugs with several promising anti-cancer and immunomodulatory effects. Their combination induces detachment of viable MDA-MB-231 breast cancer cells retaining proliferation capacity in vitro. Anchorage-independent growth of cancer cells in vitro is correlated to metastasis formation in vivo and requires mitochondrial biogenesis, but the metabolic changes involved in detachment and the effect of metformin and 2DG on mitochondria are unknown. We used liquid chromatography-mass spectrometry metabolomics to identify alterations in metformin and 2DG treated MDA-MB-231 cells. High concentration of 2DG and the combination of metformin and low concentration of 2DG (metformin+2DG) altered the metabolic profile similarly to metformin. These alterations could not explain detachment, which was actually induced by suppressed protein N-glycosylation by 2DG and its potentiation by metformin. The detached cells were softer and, surprisingly, metabolically closer to control than their attached counterparts. They exhibited altered NADPH, glutamine and fatty acid metabolism characteristic for anchorage-independent growth. Metformin+2DG and high 2DG increased mitochondrial mass in breast cancer cells by activating mitochondrial biogenesis and increasing mitochondrial size, but not their number. 2DG and metformin+2DG induced endoplasmic reticulum stress which could trigger mitochondrial biogenesis together with energy stress. Adenosine monophosphate activated protein kinase activation was also involved but not sufficient for mitochondrial biogenesis. Overall, metformin and 2DG induce metabolic and mitochondrial adaptations enabling breast cancer cell survival in detachment and energy stress. In parallel, 2DG and metformin+2DG reduced programmed death ligand 1 (PD-L1) expression in MDA-MB-231 cells by suppressing N-glycosylation and reduced programmed death 1 (PD-1) expression in Jurkat cells. Metformin, high 2DG and metformin+2DG inhibited Jurkat cell proliferation, activation and interleukin 2 secretion by reducing adenosine triphosphate production, but low 2DG preserved interleukin 2 and boosted interferon γ secretion. Low glucose concentration potentiated the effect of metformin, while supra-physiological glucose levels or exogenous pyruvate had no effect. While metabolic drugs partially inhibit Jurkat function, specific combinations could improve anti-tumor immunity via PD-1/PD-L1 axis inhibition.
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