In situ Validation of the Serum Metabolite Markers
The top 10 characteristic metabolites discovered in serum were further analyzed at the tissue level. Frozen cryosections of intact OSCC tumor tissues were prepared for DESI-MSI analysis. Then the spatial distribution of target metabolites across the resected OSCC tissues were mapped and compared. The in situ validation by DESI-MSI revealed that 8 out of top 10 metabolites showed the same trends of change with OSCC in tissue and serum. (Figure S1 ). Taking lysoPC(20:4) and PC(34:6) as examples, the former one declined in the cancer region compared to the negative contrast region whereas the latter one was elevated in the cancer region and can specifically delineate the cancer margin. The low expression of lysoPC(20:4) and high expression of PC(34:6) in the cancer region were consistent with their trends in the serum cohort analysis (Figs. 2D, 2E, and 2F ).