Fig. 4: The effect of CCL5 derived from CAFs on HCC metastasis.

A The migration and invasion abilities of Huh7 and Hep3B cells treated with hCCL5 (20 ng/ml and 100 ng/ml) were assessed by transwell assay. Scale bar: 50 μm. B The expression of EMT markers in Huh7 and Hep3B cells treated with hCCL5 (20 ng/ml and 100 ng/ml) was examined by western blotting. C Transwell assay was used to access the metastasis ability of Huh7 and Hep3B cells underwent CAF-CM incubated with a CCL5 neutralizing antibody or CCR3/CCR5 antagonists. Scale bar: 50 μm. D The protein level of EMT markers in Huh7 and Hep3B cells pretreated by the CAF-CM, a CCL5 neutralizing antibody or CCR3/CCR5 antagonists was accessed by western blotting. E NOD/SCID mice were injected subcutaneously on the flanks with Huh7 cells, Huh7 cells mixed with CAFs, Huh7 cells mixed with CCL5 knockdown (shCCL5) CAFs or CCR3/5 knockdown (shCCR3/5) Huh7 cells mixed with CAFs (n = 5). Bouin’s liquid staining showed the pulmonary metastatic nodules (red arrows) from the subcutaneous xenograft tumors of mice (upper); H&E staining showed tumor distribution (red arrows) in the lung (lower). Scale bar: 100 μm. N = 5 per group. Mean ± SD, **p < 0.01, ***p < 0.001, compared with the tumor control group. ##p < 0.01, compared with the CAF-CM or Huh7+CAF group.