Fig. 6: Single-cell and spatial transcriptomic features of endothelium subsets of GC with varying TLS status.

a Sub-clustering of all endothelial cells generated 6 subsets (Endo1 to Endo6). b Heatmap showing the expression of the top 30 DEGs in 6 endothelial cell subsets. c Heatmap showing the relative cell abundance of 6 endothelial cell subsets in GC with varying TLS status (****p < 0.0001). Relative enrichment of epithelial subsets across TLS distributions in GC was calculated using the Ro/e ratio. Significance was assessed by two-sided Fisher’s exact test, with permutation tests for selected comparisons and FDR-adjusted P values. Source data are provided as a Source data file. d Enriched GO term for Endo1 cluster. GO enrichment was performed using Fisher’s exact test, and P values were adjusted for multiple testing using the Benjamini–Hochberg method (FDR < 0.05). e H&E staining and multiplex immunostaining showing the spatial localization of HEVs and CXCL13⁺CD8⁺ TLC in GCs with varying TLS status. f Bubble plot showing the intercellular interactions between endothelial cells, mCAF1 with TLC, and BLC. g Dot plot showing the expression of VCAM1, ACRK1, and SELP in Endo1 subset cells accumulated in GC with varying TLS status. Bubble size represents communication probability, and color denotes P value (two-sided Wilcoxon rank-sum test, P < 0.05). h H&E staining and ST-seq analysis showing the spatial distribution of TLS, CXCL13⁺CD8⁺ TLC, and Endo1/HEV in GC with varying TLS status. i ST-seq analysis showing the colocalization of ligand CXCL13 secreted by CXCL13⁺ TLC and the receptor ACKR1 secreted by Endo1/HEV in GC with varying TLS status. j CXCL13 stimulation upregulates VCAM1 and ICAM1 expression in ACKR1-overexpressing endothelial cells (ACKR1^OE EC) (n = 4). The left panel illustrates the schematic of the functional assay. The right panel shows gene expression levels of VCAM1 (top) and ICAM1 (bottom) in ACKR1^OE EC treated with PBS (control), 50 ng/mL CXCL13, 5 ng/mL TNF, or a combination of 50 ng/mL CXCL13 and 5 ng/mL TNF. Source data are provided as a Source data file. k Transwell experiment showing the functional impact of CXCL13-ACKR1 axis on TLC migration (n = 4). The left panel illustrates the schematic of the co-culture assay. The middle panel shows the image of T cells. P value was calculated by the Kruskal–Wallis test followed by Dunn’s multiple comparison test. The box indicates median ± interquartile range, and whiskers represent minima and maxima. Source data are provided as a Source data file.