Identification of altered endogenous metabolites
In order to evaluate the underlying mechanism of action of the dual mTOR
inhibitor PP242, the LS174T xenograft mouse model was treated with PP242
for three weeks, and plasma and tumor samples were analyzed via
metabolomics and lipidomics approaches. The representative base peak
intensity (BPI) chromatograms of the plasma and tumor tissues for
metabolomics and lipidomics studies in both positive and negative ion
modes are displayed in Supplementary Figs. 2 and 3. To clearly visualize
the metabolic differences among the NC, XC, and PP242-treated groups in
plasma and the XC and PP242-treated groups in tumor tissues,
multivariate statistical analysis was used to analyze the processed mass
spectrometric data. PLS-DA score plots of plasma and tumor tissue
metabolomics and lipidomics are shown in Figs. 4 and 5. In the plasma
metabolomics and lipidomics analyses, the xenograft groups (XC and
PP242-treated) were clearly separated from the NC group, indicating
metabolic differences due to tumor formation, but the XC and
PP242-treated groups slightly overlapped (specifically in positive
metabolomics; Fig. 4). In addition, the PLS-DA score plots of tumor
tissues displayed a clear separation between the XC and PP242-treated
groups, which clearly indicates that metabolism in tissue was altered
due to the inhibition of mTOR by PP242 and that these metabolic changes
were clearer in tissue than in plasma. The R2 and
Q2 values for plasma metabolomics and lipidomics were
in the ranges of 0.43 to 0.61 and 0.1 to 0.20, respectively, whereas
those for tumor tissue were in the ranges of 0.82 to 0.99 and 0.04 to
0.87, respectively. The overall R2 and
Q2 values showed that the model was reliable and had
good predictability. The obtained low Q2 value could
be because of the partial overlap of the XC and PP242 groups.
In the metabolomics and lipidomics profiling of plasma, comparisons were
carried out among the NC, XC and PP242 groups. The xenograft groups (XC
and PP242-treated) were compared with the NC group to investigate the
metabolic differences that occurred due to tumor formation and how much
was recovered after PP242 treatment. Initially, a total of 49
significantly altered metabolites were identified. Of these, 22
metabolites were finally selected and used for further analyses by
considering a VIP value >1.0 and a p value (obtained by
Student’s t test ) <0.05. Detailed information on the
identified metabolites in plasma is listed in Table 1. A heat map was
also used to visualize the change pattern and is displayed in Fig. 6.
The identified metabolites were mostly limited to glycerophospholipids,
fatty acids and a few organic compounds. Compared to the NC group, the
overall change patterns in metabolites in both xenograft groups were
similar. Interestingly, none of the identified metabolites were
significantly altered in the XC group compared with the PP242-treated
group, and there was no consistency in the alteration pattern of all
metabolite classes. Consequently, a potential effect of PP242 was not
observed in plasma. However, when we observed the tumor volume and
weight (Fig. 2), the changes were significant after treatment with
PP242, indicating an obvious therapeutic effect of PP242. Hence, in
order to investigate the underlying meaning of the PP242 effect in tumor
size reduction, we further analyzed tumor tissues.
In tumor tissue, compared to the XC group, a total of 93 significantly
altered metabolites were identified in the PP242-treated group via
metabolomics and lipidomics analyses. Considering a VIP
>1.0 and a p value <0.05, 59 metabolites were
ultimately selected and used for further analysis (Table 2). A wide
range of metabolites, including a few other organic compounds, fatty
acyls, glycerophospholipids, glycerolipids and sphingolipids, were
significantly altered after PP242 treatment. The metabolites related to
energy production (lactate, aspartic acid, fatty acids and carnitines)
and cell growth and proliferation (glycerophospholipids) were mainly
affected. We observed that the levels of all carnitines were decreased,
and fatty acids, which are the precursors of various lipid molecules and
are involved in energy generation, were increased due to the inhibition
of mTOR by PP242. Other significantly altered metabolite classes were
also identified, including amino acids (L-methionine), carboxylic acids
and derivatives (creatine and L-aspartic acid), hydroxyl acids and
derivatives (L-lactic acid), purine nucleotides (inosine), pyrimidine
nucleotides (uridine 5’-monophosphate) and phenylpropanoic acids
(phenyllactic acid). All glycerophospholipids, including
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI) and phosphatidylserine (PS), showed
significant downregulation in the PP242-treated group compared to the XC
group. However, only one identified LysoPC (LysoPC(18:2)) was
significantly altered and showed upregulation after mTOR inhibition. The
level of glycerolipid DG(36:2) was significantly upregulated, and the
sphingolipid SM(34:1) level was significantly downregulated. A heat map
is displayed in Fig. 7 to display the direct variation of each
differential metabolite.