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.