Fig. 3: Comparative proteomics analysis of primary tumor and lymph node sites for malignant and non-malignant cells.

a Experimental design to define the differential protein profile of the malignant and non-malignant populations between the tumors and lymph nodes (matched malignant samples from primary tumor and lymph nodes, n = 11 samples per site; matched non-malignant samples from primary tumor and lymph nodes, n = 27 samples per site). Created with BioRender.com. b Frequency of downregulated or upregulated proteins between tumors and lymph nodes in malignant and non-malignant populations (malignant vs. non-malignant; p ≤ 0.0001; two-sided Chi-square test). ****p ≤ 0.0001. c Hierarchical clustering when considering the proteomic profile of malignant and non-malignant cells from primary tumor and lymph nodes (n = 2478 and 2360 proteins, respectively). Clustering was performed using the Ward method with Euclidean distance. *Samples that do not follow the main clustering pattern. d Top-5 GO biological processes enriched for proteins that were differentially abundant between lymph nodes and primary sites in malignant and non-malignant samples (Enrichment FDR ≤ 0.05; hypergeometric test followed by FDR correction). *Actin-based cell movement processes modulated in malignant cells through the deregulation of nine proteins. These proteins were selected for verification. Box plots representing the abundance of transcripts and proteins that are involved in actin-based cell movement using RT-qPCR (e) and PRM-MS (f), respectively (primary tumor malignant samples, n = 9 vs. lymph node matched malignant samples, n = 9; two-sided Wilcoxon signed-rank test for PRM-MS and two-sided Mann-Whitney test for RT-qPCR). The boxplots depict the 25–75% interquartile range (IQR) (box limits), with the median shown as a central line; whiskers indicate the minimum and maximum values. *p ≤ 0.05. g EMT scores of HNSCC malignant cells considering the 76-gene signature proposed by Byers et al.³³ in the discovery datasets (primary tumor malignant samples, n = 11 vs. lymph node (metastasis) matched malignant samples, n = 11; p = 0.0391; two-sided Wilcoxon signed-rank test). *p ≤ 0.05. The left-sided data are mean ± standard deviation and the right image represent individual samples. Box plots representing the abundance of EMT markers at protein- (h) and peptide-levels (i) in malignant cells using PRM-MS (primary tumor malignant samples, n = 9 vs. lymph node matched malignant samples, n = 9; two-sided Wilcoxon signed-rank test or two-sided paired Student’s t-test). Only statistically significant results are shown for peptides. Boxplots depict the 25–75% interquartile range (IQR) (box limits), with the median shown as a central line; whiskers indicate the minimum and maximum values. *p ≤ 0.05; **p ≤ 0.01. HNSCC head and neck squamous cell carcinoma, EMT epithelial-mesenchymal transition. Source data are provided as a Source Data file.