Serum Metabolomic Profiling for OSCC Stages
We also investigated whether serum metabolomic pattern differences exist not only between HC and OSCC but also among different stages (from stage T1 to stage T4). The OPLS-DA model visualized the distribution of the OSCC cases in the development cohort (Fig. 4A ). It can be seen the OSCC at T1 and T2 stages can be ideally separated from these samples at the T3 and T4 stages. Unfortunately, there was no obvious separation between T1 and T2 or T3 and T4. The variables that made a high contribution to this T1/T2 and T3/T4 separation were searched according to their variable importance on projection (VIP) values. The variables with VIPs large than 1.5 were picked (Table S8 ). After removing redundant ions, the top 50 metabolites were annotated and their relative contents in serum were displayed in a heatmap. It was worth noting that 4 out of the top 10 metabolites discovered in the univariate analysis (DG(34:2), oleamide, LPC(20:4), and nonanoyl carnitine) also contributed to this T1/T2 and T3/T4 stages discrimination (Fig. 4B ). As shown in Fig. 4C, the relative contents of these metabolites in the OSCC group (T1-T4) were completely different from those in the HC group. Furthermore, they also showed increasing or decreasing trends from T1 to T4. Unfortunately, none of these top 50 metabolites showed statistical significance among the four stages by the analysis of one-way variance (ANOVA).