Metabolic pathways of cancer cells depend on the concentrations of nutrients in their micro-environment. However, they can vary also between monolayer cultures of cancer cells and tumour spheroids. Here we examined whether the absence of glucose, pyruvate and glutamine increases the sensitivity of MDA-MB-231 cells to metabolic drug metformin using two in vitro cell models (monolayer culture and tumour spheroids). To evaluate the effects of nutrient depletion in more detail, we tested the

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... of metformin in commonly used media (DMEM, MEM and RPMI-1640) that differ mainly in the concentrations of amino acids. We used MTS, Hoechst and propidium iodide assay to determine cell number, viability and survival, respectively. We evaluated the effects of metformin on the size of tumour spheroids and determined cell survival by calcein and propidium iodide staining. Finally, we observed the effects of metformin in nutrient depleted conditions on the phosphorylation of AMP-activated protein kinase using Western blotting. Our main finding is that the effects of metformin on MDA-MB-231 cells depend on in vitro cell model used (monolayer culture vs. tumour spheroids). While metformin did not have any major effect on proliferation of MDA-MB-231 cells grown in complete cell culture media in a monolayer culture, it disintegrated tumour spheroids in MEM and RPMI-1640 medium. The effects of metformin on tumour spheroids were most pronounced in MEM, which is deficient of several non-essential amino acids. Glutamine depletion had no effect on the sensitivity of MDA-MB-231 cells to metformin in all tested conditions, whereas pyruvate depletion sensitized MDA-MB-231 cells to metformin in a monolayer culture only in MEM. Taken together, our results show that media formulation as well as in vitro cell model (monolayer culture vs. tumour spheroids) must be considered, when we evaluate the effects of metformin on MDA-MB-231 cells as a function of nutrient availability.