Fig. 5. CTCs recruit Tregs via CCL5 expression to generate an immunosuppressive and pro-tumorigenic microenvironment.

a Expression of CCL5 in HCC cell lines with different metastatic potential. b The numbers of migrated Tregs cocultured with supernatant medium of Huh7 or MHCC97H cells without treatment (left panel), and treated with antihuman CCL5-neutralizing antibody or IgG (right panel). c Histogram showing relative mRNA expression of CCL5 in Hepa1–6 cells with CCL5 knockdown or control. d Immunofluorescence images of CCL5 expression in Hepa1–6 cells with CCL5 knockdown or control. Scale bar is 10 µm. e CTC clearance curves in blood after injection with 5 × 10⁶ Hepa1–6 cells into the tail veins of C57BL/6J mice (n = 30 per condition, five mice per time point). f Histograms showing the percentage of circulating Tregs in mice from CCL5 knockdown (n = 5) and vector control groups (n = 5). g Scatter plots showing the numbers of lung (upper) and liver metastases (lower) developed under conditions described in e (n = 5 per group). h Histograms showing the numbers of Tregs (upper) and granzyme B⁺ cytotoxic T lymphocytes (lower) in liver metastases under conditions described in e. Five independent microscopic field, representing the densest lymphocytic infiltrates, were selected for one liver metastatic tumor each mouse. i The comparison of mice Treg migration toward CTCs isolated from vector and shCCL5 Hepa1–6 orthotopic models. CCL5-neutralizing antibody was added to the coculture system to determine the effect on Treg migration (n = 5 per group). Comparisons were calculated by two-tailed Student’s t test. Data are mean ± SD of three (b, c) and five (e–g, i) biological replicates, and are representative of two independent experiments. *** represents P < 0.001. The exact P values at the time point of 12 h in e: vector + IgG vs shCCL5 + IgG, P = 3.49 × 10⁻⁹; vector + IgG vs vector + anti-CD25ab, P = 1.84 × 10⁻⁹.