Spatial transcriptomics reveals tumor niche heterogeneity in advanced gastric cancer and suggests a tumor-promoting role for KRT6B

Figure 5 Molecular and functional profiling of a KRT6B-enriched tumor nest (N9) in P4. A: Spatial location of N9 (red with outlined boundary) in P4; B: Gene set variation analysis heatmap of tumor-associated niches (N4, N9, N2, and N5), showing enrichment patterns in N9, including cell-cycle/proliferation, metabolic, and immune/inflammatory programs. Red indicates relative upregulation and blue indicates relative downregulation, and color intensity reflects row-scaled enrichment scores; C: Gene set enrichment analysis comparing N9 with N5/N4/N2, showing significantly upregulated (left) and downregulated (right) gene sets in N9, including proliferation-related, metabolism-related, immune-response-related, and hypoxia-related pathways. The X-axis represents GeneRatio, the dot size indicates the number of enriched genes, and the color denotes adjusted P values; D: Volcano plot of differentially expressed genes between N9 and N5, with representative upregulated genes including MMP7, LCN2, UBD, and KRT6B. Red, significantly upregulated genes; blue, significantly downregulated genes; gray, non-significant genes; E: Gene Ontology biological process enrichment of genes upregulated in N9. Dot size represents the number of enriched genes, and color indicates adjusted P values; F: Spatial map of dominant cell types in the region surrounding the N9 niche, showing that fibroblasts are enriched along the left and upper margins of N9; G: Bubble plots of significant inferred ligand-receptor interactions between N9 tumor epithelial cells and fibroblasts. Left: N9 to fibroblasts; right: Fibroblasts to N9. Dot size indicates statistical significance (small: 0.01 < P < 0.05; large: P < 0.01), and color denotes communication probability; H: Spatial heatmap of KRT6B expression in P4, showing high-expression regions overlapping the N9 tumor nest. Color scale indicates relative expression intensity (low to high); I: Immunofluorescence staining of gastric cancer tissue sections: Nuclei (DAPI, blue), epithelial cells (EPCAM, green), and KRT6B (red). In the panoramic view (top), KRT6B signal overlaps with EPCAM in tumor nests and at the invasive front (scale bar, 500 μm). In the magnified view (bottom), a representative tumor nest shows pronounced KRT6B signal overlapping EPCAM, with little to no detectable KRT6B in surrounding stroma (scale bar, 100 μm).