Metabolome analysis
The metabolome could directly reflect the physiological characteristics of cells, and the minor changes in gene transcription and protein expression would cause significant differences in the metabolome (Klein & Heinzle, 2012). In this study, we analyzed 330 metabolites, including carbohydrates, amino acids, lipids, nucleotides, organic phosphates, sterols, coenzymes of tumor cells cultured under both 2D monolayer cultures and 3D MTSs before and after the 16 μM 5-FU treatment. The PCA result showed the significant differences among groups and good repeatability in groups, which proved that the following data analysis could be supported (Figure. S6A ). Table 2 shows 107 and 76 differential metabolites under 2D monolayer cultures and 3D MTS before and after the 5-FU treatment, respectively. With the 5-FU treatment, the metabolites involved in the ABC transport system of cells were significantly different between 2D monolayer cells and 3D MTSs (Figure. S6B , Figure. S6C ). The effects of 5-FU on sugar metabolism and amino acid metabolism in 3D MTSs were less pronounced than in monolayer cell culture. Under the 5-FU treatment, the intermediates of TCA cycle (including citrate, malate and fumarate) and of alanine, aspartate and glutamate metabolism were all decreased in 2D monolayer cells, while there were no significant differences in 3D MTSs, except for the increase of asparagine. Similarly, the effect of 5-FU on the nucleotide metabolism of MTSs was also relatively weakened. 5-FU disrupts the de novo nucleotide synthesis pathway by targeting TYMS (Longley et al., 2003). Under the 5-FU treatment, the nucleotide synthesis pathway of 2D monolayer cells was inhibited with the increase of 5-phospho-ribulose, while purine nucleotide synthesis was blocked with the increase of purine synthesis precursors inosine and hypoxanthine and the decrease of inosine monophosphate (IMP) and adenosine monophosphate (AMP). It was also found that the precursor orotate to uracil synthesis was decreased in 2D monolayer cells with the 5-FU treatment. Under the 5-FU treatment, we find the decrease of inosine, xanthine and orotate, and the increase of uridine. After 5-FU treatment, the pentose phosphate pathway (PPP) changed significantly. Under 2D culture conditions, gluconolactone and gluconic acid were significantly increased, while 6-phosphogluconic acid was significantly decreased, possibly due to the increased production of ribulose 5-phosphate (Figure. S6B ). This process is accompanied by the production of NADPH (Cairns, Harris, & Mak, 2011), which contributes to the elimination of ROS induced by 5-FU (Figure. 3D ). In 3D MTSs, we also observed an increase in ribulose 5-phosphate, which resulted in a significant decrease in gluconolactone and gluconic acid (Figure. S6B ).
In the control conditions, there were 99 different metabolites between 2D and MTSs while 91 different metabolites were detected under the condition of the 5-FU treatment. We selected 50 differential metabolites which exerted greater impacts on tumor cell growth, metabolism, and drug resistance, and then their expression levels were hierarchically clustered between 2D monolayer cultures and 3D MTSs under both control and 5-FU conditions (Figure. 6A ). KEGG enrichment was performed to examine the differences in metabolic pathways of tumor cells under different culture conditions (Figure. 6B ). The ABC transport system played an important role in metabolite transport and was associated with the multidrug resistance (Nunes, Costa, Barros, de Melo-Diogo, & Correia, 2019). We found that the metabolites involved in the ABC transport system, were significantly different between 2D monolayer cultures and 3D MTSs regardless of the 5-FU treatment, which may indicate the differences in drug transport capacity. The carbon metabolism pathways performed significant differences in 2D monolayer cultures and 3D MTSs. Under control conditions, compared with monolayer cells, the TCA cycle intermediates including citric acid, cis-aconitic acid, succinic acid, fumaric acid, malic acid were decreased in 3D MTSs, which was consistent with the significant increase in cellular non-mitochondrial respiration and the down-regulated protein expression related to mitochondrial activity (Figure. 3C ,Table S3 and Table S4 ). However, we did not observe significant differences in the intermediate metabolites of the glycolysis pathway between 2D monolayer cultures and 3D MTSs. This indicated that the flux control may be governed by other regulation mechanisms, e.g., phosphorylation-mediated reprogramming of glycolytic activity (Ruprecht et al., 2017). We found that there were no significant differences in glutamine and glutamate between 2D monolayer cultures and 3D MTSs, while with 5-FU treatment, glutamine and glutamate significantly increased in 3D MTSs (Figure. 6A ). Glutamine participated in the TCA cycle by converting to glutamate and then further to α-ketoglutarate, which maintained the TCA cycle in 3D MTSs under the 5-FU treatment, resulting in no significant difference in the TCA cycle between 2D culture and 3D MTSs after 5-FU treatment. Meanwhile, asparagine significantly decreased under both conditions in the 3D MTSs, which led to the decrease of aspartate involved in the TCA cycle. Furthermore, compared with 2D culture, gluconolactone, gluconic acid and ribulose 5-phosphate involved in the PPP were significantly increased in 3D MTSs under the control condition; under the 5-FU treatment, only the significant increase of ribulose 5-phosphate was found in 3D MTSs.