Fig. 6

CCL20 blockade suppresses tumor progression and restores 5-FU sensitivity in CRC. a Graph showing the outline of HCT116 cell injection, 5-FU treatment, and CD4⁺ cell transfer in vivo. Groups received hypodermic injections of 5 × 10⁶ HCT116 cells (D-7). 5-FU (10 mg/kg/day, i.p.) treatment was started when the tumor volumes reached 250 mm³ (D0). Two days before the mice were sacrificed, human CD4⁺ cells (5 × 10⁶ cells) were transplanted by caudal vein (D2). b The percentage of CD4⁺FOXP3⁺ and CD4⁺FOXP3⁻ cells in xenografts with or without 5-FU treatment was analyzed by flow cytometry. c The percentage of CD4⁺FOXP3⁺ cells in xenografts and spleens with or without 5-FU treatment was analyzed by flow cytometry. d The percentage of CCR6⁺CD4⁺FOXP3⁺ cells in xenografts and in spleens with or without 5-FU treatment was analyzed by flow cytometry. e The expression of FOXO1, CEBPB, P-P65, and CCL20 in xenografts with or without 5-FU treatment was detected by immunohistochemistry (200 ×). f IRS of FOXO1, CEBPB, P-P65, and CCL20 in xenografts with or without 5-FU treatment as analyzed by immunohistochemistry was presented. g CRC tissues subjected to immunofluorescence for FOXP3 (red) and DAPI (blue). One representative micrograph is shown (200 ×). h Graph showing the outline of HCT116 cell injection, anti-CCL20 antibody usage, 5-FU treatment and Treg cell transfer in vivo. 5 × 10⁶ HCT116 cells were injected subcutaneously into the mice (D-7). Seven days after cell implantation, the anti-CCL20 antibody (1 mg/kg) or DMSO as a control, was administrated locally to the mice every 2 days for 2 weeks (D0, 2, 4, 6, 8, 10, 12). At day 6–12 after anti-CCL20 antibody administration, mice were treated daily with 5-FU (10 mg/kg/day, i.p.). At day 14 after anti-CCL20 antibody administration, human Tregs (5 × 10⁶ cells) were transplanted through the caudal vein (D14). After 17 days, the mice were sacrificed and tumors were isolated for further analysis. i Tumor volumes were measured from day 14 to day 31 after HCT116 and Treg cell implantation. The results are showed in the line chart. j Numbers of Treg cells in xenografts were calculated and analyzed. k The percentage of CCR6⁺CD4⁺FOXP3⁺ cells in xenografts and the spleens was analyzed by flow cytometry. *P < 0.05, **P < 0.01, NS- non-significant