Fig. 5: Significant enrichment of LRRC15⁺ CAFs following immunotherapy in nonresponsive ESCC.

a UMAP plot showing 13,612 CAFs colored and labeled by distinct cell clusters based on all enrolled samples (n = 23). b Bubble plot showing differential expression of marker genes for various CAF clusters. c Proportion of iCAF-CFD, iCAF-CXCL12, iCAF-PLA2G2A and tCAF-MMP11 in all CAFs. ^**p < 0.01 of Wilcoxon test. Post-treatment samples from responder (Post-R), n = 6; Post-treatment samples from non-responder (Post-NR), n = 10. For each boxplot, the center line represents the median, the box indicates the upper and lower quartiles, and the whiskers show minima and maxima. Detailed information of each sample in provided in the Source Data. d Scatterplot showing a significant correlation between post-treatment cell proportion of CD4⁺ Treg-TNFRSF4 in CD4⁺ T cells with that of tCAF-MMP11 in CAFs. Spearman-correlation test. Post-treatment samples, n = 16. e UMAP plot showing the distribution of FAP, MMP11, LRRC15 and LGALS9 in CAFs. f Differential expression of selected genes in tCAF-MMP11. ^***p ^<0.001 of two-sided Wilcoxon test. g The differential mRNA expression level of LRRC15 among distinct timepoints and efficacy groups based on the external validation cohort. ^***p < 0.001 of Wilcoxon test. Pre-treatment samples of responders (Pre-R, n = 32) and non-responders (Pre-NR, n = 22); Post-R (matched with Pre-R, n = 32) and Post-NR (matched with Pre-NR, n = 22). The box indicates median ± interquartile range; whiskers represent minima and maxima. Source data are provided as a Source Data file. h The difference in infiltration score of tCAF-MMP11 among distinct timepoints and efficacy groups of the validation cohort. ^***p < 0.001 of Wilcoxon test. Pre-R, n = 32; Pre-NR, n = 22; Post-R, n = 32; Post-NR, n = 22. The box indicates median ± interquartile range; whiskers represent minima and maxima. Detailed infiltration score of each sample in provided in the Source Data. i Scatterplot of correlation between the infiltration level of CD4⁺ Treg-TNFRSF4 with that of tCAF-MMP11 in pre^-treatment samples. Spearman-correlation test. Pre-treatment samples, n = 54. j Scatterplot showing a significant correlation between the infiltration score of CD4⁺ Treg-TNFRSF4 with that of tCAF-MMP11 in post-treatment tissues. Spearman-correlation test. Post-treatment samples, n = 54. k Representative images of multiplex IHC staining for α-SMA⁺LRRC15⁺ CAFs and CD4⁺FOXP3⁺ T cells from post-treatment tumors of responders and non-responders. Post-R, n = 12; Post-NR, n = 15. Scale bar: 10 μm. l Cell density of LRRC15⁺ CAFs, CD4⁺FOXP3⁺ cells, CD4⁺ cells, and CD4⁺FOXP3^- cells in post-treatment samples from responders (n = 12) and non-responders (n = 15). ^*p < 0.05, ^***p < 0.001 of two-sided t-test. m Cell number of CD4⁺FOXP3⁺ cells at different distances from LRRC15⁺ CAFs. n Comparison of average distance from CD4⁺FOXP3⁺ cells to LRRC15⁺ CAFs between post-treatment responders and non^-responders. ^**p < 0.01 of two-sided t-test. Post-R, n = 12; Post-NR, n ⁼ 15.