Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, the identification of biological signatures that assists in the prognostic outcomes for OSCC patients would be of great clinical significance. Here we show by histopathology-guided discovery proteomics differentially expressed proteins between invasive tumor front (ITF) and inner tumor in neoplastic islands and tumor stroma. Immunohistochemistry analysis indicates low expression level of cystatin-B in the ITF of neoplastic islands as an independent marker for local recurrence. Selected reaction monitoring mass spectrometry of saliva proteins combined with machine learning methods demonstrate the most relevant prognostic signature to distinguish patients with and without lymph node metastasis. Our results identify a robust prognostic signature, which may enhance prognostic decisions in OSCC and better guide appropriate treatment and reduction of tumor recurrence or lymph node metastasis.

Saliva samples from 40 OSCC patients (Supplementary Table 23-25) were selected to monitor peptides derived from CSTB, NDRG1, LTA4H, PGK1, COL6A1, ITGAV and MB based on DDA analysis. The samples were divided into two groups: those without lymph node metastasis (N0) and those with lymph node metastasis (N+).

The saliva samples were centrifuged at 1,500 g for 5 min at 4°C to pellet the debris. The resulting supernatant was collected and quantified by the Bradford assay (Bio-Rad, Hercules, CA, USA). A volume corresponding to 10 µg of total protein was used for sample preparation, and the sample volumes were adjusted. Ten micrograms of total protein were denatured in urea buffer (at a final concentration of 100 mM Tris-HCl pH 7.5, 8 M urea, 2 M thiourea, 5 mM EDTA, 1 mM PMSF, 1 mM DTT) containing Protease Inhibitor Cocktail Complete Mini Tablets (Roche, Auckland New Zealand). The samples were sonicated for 10 min and then centrifuged at 10,000 g for 5 min. The supernatant was collected, and the proteins were reduced with DDT and alkylated with iodoacetamide. Proteins were digested overnight at 37°C using 1.8 µg of trypsin.