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

Figure 1 Cellular composition and spatial architecture in advanced gastric cancer. A: Hematoxylin and eosin-stained full-thickness advanced gastric cancer tissue sections from samples P2 and P4 (left). Spatial maps of detected gene counts (right); B: Spatial mapping of predicted major cell types in P2 and P4; C: Spatial distribution maps of major cell types in P2 and P4. Color intensity indicates the deconvolution-predicted proportion of each cell type in each spot.

Figure 2 Spatial niches identification and characterization using BANKSY. A: UMAP of integrated P2 and P4 spatial transcriptomic data. Left: Spots colored by sample (P2, red; P4, blue). Right: Spots colored by BANKSY-defined niche identities (N0-N12); B: Spatial maps of BANKSY-defined niches (N0-N12) in P2 (left) and P4 (right); C: Bar plots showing the number of spots assigned to each first-weight cell type within each niche in P2 (top) and P4 (bottom). Colors denote different cell types; D: Spatial localization of each niche in P2 (top) and P4 (bottom), with red indicating spatial regions assigned to each niche.

Figure 3 Spatial functional profiling of distinct niches in advanced gastric cancer. A: Spatial distribution of niches N5, N4, and N2 in P2 (red shading with outlined boundaries), showing a continuous distribution from the mucosa to the submucosa and muscularis propria; B: Violin plots showing expression of canonical tumor epithelial markers (EPCAM, KRT18, KRT8, and KRT19) across niches in P2 (red) and P4 (blue); C: Four gene-expression trends identified by Mfuzz based on expression profiles across N5, N4, and N2; D and E: Top enriched Gene Ontology biological process terms for Trend 3 and Trend 4. Dot size represents the number of input genes, and color indicates the adjusted P value; F: Violin plots showing expression of immune-related genes (CD8A, CD4, and CXCL9) in N2, N4, and N5; G: Violin plots showing expression of stemness-associated genes (CD44, ALDH1A1, PROM1, LGR5, and SOX9) in N2, N4, and N5. Statistical significance: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 4 Niche-specific intercellular communication in sample P2. A: Heatmaps showing the overall interaction strengths among major cell types within N5; B: Bubble plots showing significant inferred ligand-receptor interactions targeting tumor epithelial cells (TECs) in N5. Dot size indicates the significance level, and color indicates communication probability; C: Spatial interaction networks of the MDK signaling pathway in N5. The background indicates the spatial distribution of major cell types within each niche; nodes denote cell types, and edges indicate inferred ligand-receptor interactions; D: Heatmaps showing the overall interaction strengths among major cell types within N4; E: Bubble plots showing significant inferred ligand-receptor interactions targeting TECs in N4. Dot size indicates the significance level, and color indicates communication probability; F: Spatial interaction networks of the MDK signaling pathway in N4. The background indicates the spatial distribution of major cell types within each niche; nodes denote cell types, and edges indicate inferred ligand-receptor interactions; G: Heatmaps showing the overall interaction strengths among major cell types within N2; H: Bubble plots showing significant inferred ligand-receptor interactions targeting TECs in N2. Dot size indicates the significance level, and color indicates communication probability; I: Spatial interaction networks of the MDK signaling pathway in N2. The background indicates the spatial distribution of major cell types within each niche; nodes denote cell types, and edges indicate inferred ligand-receptor interactions.

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).

Figure 6 KRT6B is upregulated in gastric cancer, correlates with poor prognosis, and promotes malignant phenotypes in vitro. A: Representative immunohistochemical staining images showing KRT6B expression in gastric cancer tissues and matched adjacent non-tumor tissues (scale bar, 200 μm); B: Quantification of KRT6B immunohistochemistry scores (H-score) in paired tumor and adjacent non-tumor tissues; C: Analysis of UCSC Xena-integrated TCGA-STAD and GTEx transcriptomic data showing significantly increased KRT6B mRNA expression in gastric cancer tissues compared with normal gastric tissues; D: Kaplan-Meier overall survival analysis of patients stratified by KRT6B expression level; E: Western blot validation of stable KRT6B overexpression in AGS and SNU-601 cells (GAPDH as a loading control); F and G: CCK-8 assays showing increased proliferative capacity in KRT6B-overexpressing AGS (F) and SNU-601 (G) cells; H and I: Representative flow cytometry histograms of EdU incorporation in AGS (H) and SNU-601 (I) cells. A no-EdU control was used to define the APC-positive gate; J and K: Quantification of EdU-positive cell proportions in AGS (J) and SNU-601 (K) cells; L and M: Representative Transwell invasion assay images of AGS (L) and SNU-601 (M) cells; N and O: Quantification of invading cell numbers in AGS (N) and SNU-601 (O) cells; P and Q: Representative wound-healing assay images (0 hour and 24 hours) of AGS (P) and SNU-601 (Q) cells; R and S: Quantification of wound closure rates in AGS (R) and SNU-601 (S) cells. Data are presented as mean ± SD from independent experiments. NS: Not significant; ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.