Fig. 2

Boxplot depicting the gene expression across multiple types of regions of interest spatially profiled for both lymph node-positive and negative patients. A FN1 gene was highly upregulated in lymph node-positive segments averaged across multiple types of ROI where increased expression at the tumor margin is observed with a significant difference with normal epithelial segments (pairwise Wilcoxon test p-value < 0.05). B Gene expression of COL5A1, second highly upregulated in lymph node-positive cases averaged across multiple types of ROI where a significant difference in expression was observed between stromal end, and cells in the TME at a distance > 500 µm. (pairwise Wilcoxon test p-value < 0.05) There was also differences between tumor margins and tumor regions (pairwise Wilcoxon test p-value < 0.01). C Gene expression of HLA-DRB4 was highly downregulated in lymph node-positive segments averaged across multiple types of ROI where no statistical significance was observed for expression across multiple ROI types in both tumor end and stromal end (Kruskal Wallis p value > 0.05). D Expression of MSR1, the second highly downregulated gene in lymph node-positive segments averaged across multiple types of ROI where statistically significant differences in its expression was observed as the distance between tumor and immune cells increased (pairwise Wilcoxon test p-value < 0.05), within stromal end segments. E Volcano plot for the differentially expressed genes between lymph node-positive (n = 35) against the lymph node-negative (n = 52) spatial regions of interest. F Top 20 DAVID functional enrichment terms for GO BP and REACTOME pathways respectively of significant DEGs (p-value < 0.05) between lymph node-positives vs negatives. GO gene ontology; DEG differentially expressed gene, DAVID Database for Annotation, Visualization, and Integrated Discovery, BP biological processes