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.