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Multicellular tumour spheroids are an important model system to investigate the response of tumour cells to radio- and chemotherapy. They share more properties with the original tumor than cells cultured as 2D monolayers do, which helps distinguish the intrinsic properties of monolayer cells from those induced during cell aggregation in 3D spheroids. The paper investigates some metabolic aspects of small tumour spheroids of breast cancer and their originating MCF-7 cells, grown as monolayer, by means of high resolution 1H NMR spectroscopy and MR microimaging before and after gamma irradiation. The spectra of spheroids were characterized by higher intensity of mobile lipids (ML), mostly neutral lipids, and glutamine (Gln) signals with respect to their monolayer cells counterpart, mainly owing to the lower oxygen supply in spheroids.Morphological changes of small spheroids after gamma-ray irradiation, such as loss of their regular shape, were observed by MR microimaging. Lipid signal intensity increased after irradiation, as evidenced in both MR localized spectra of the single spheroid and in high-resolution NMR spectra of spheroid suspensions. Furthermore, the intense Gln signal from spectra of irradiated spheroids remained unchanged, while the low Gln signal observed in monolayer cells increased after irradiation. Similar results were observed in cells grown in hypoxic conditions. The different behavior of Gln in 2D monolayers and in 3D spheroids supports the hypothesis that a lower oxygen supply induces both an upregulation of glutamine synthetase and a downregulation of glutaminases with the consequent increase in Gln content, as already observed under hypoxic conditions.The data herein indicate that 1H NMR spectroscopy can be a useful tool for monitoring cell response to different constraints. The use of spheroid suspensions seems to be a feasible alternative to localized spectroscopy since similar effects were found after radiation treatment.