Fig. 3

5-FU-resistant CRC cell-derived CCL20 promotes recruitment of Tregs. a GO analysis showed the top 10 gene functions that were mostly different among the CCL20 high and low expression based on TCGA data. b Relative expression of immune system-related genes in tumor tissues from 104 CRC patients with high and low CCL20 expression, stratified by the median, was analyzed by qPCR. c The relationship between the expression of CCL20 and FOXP3 in CRC tissues was detected by immunohistochemistry (200 ×; P1: low expression, P2: high expression). d The percentage of CD3⁺CD4⁺, CD4⁺FOXP3⁺ and CD4⁺FOXP3⁻ cells from TILs and paired PBMCs of 26 CRC patients was analyzed by flow cytometry. e The percentage of CCR6⁺ cells in CD3⁺CD4⁺, CD4⁺FOXP3⁺, and CD4⁺FOXP3⁻ cells from TILs and PBMCs of 26 CRC patients was analyzed by flow cytometry. f Migration of purified CD4⁺CD25⁺ Tregs from healthy donors co-cultured with recombinant human CCL20 or the supernatants of SW620 cells before and after treatment with CCL20 inhibitor was analyzed by Transwell assay. The migration index was calculated by dividing the number of cells that migrated in the indicated groups by the number migrating in control groups (n = 3). g Migration of purified CD4⁺ cells from healthy donors co-cultured with the supernatants of SW620 cells before and after treatment with 5-FU was analyzed by Transwell assay. h Migration of purified CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ cells from healthy donors co-cultured with the supernatants of SW620 cells treated with 5-FU was analyzed by Transwell assay (n = 3). i Migration of SW620 cells co-cultured with 5-FU and/or anti-CCL20 antibody was analyzed by Transwell assay (n = 3). j The percentage of FOXP3⁺ cells in the tumor tissues of CRC patients (n = 12) with or without neoadjuvant therapy was detected by immunohistochemistry. *P < 0.05, **P < 0.01, ***P < 0.001, NS- non-significant