Fig. 6

CXCL12/CXCR4 is involved in the recruitment of FGFR2⁺ fibrocytes to ESCC tumor mass. A. The blocking effect of CXCR4 neutralizing antibody on ESCC-induced fibrocyte migration was evaluated by in vitro chemotaxis assay. hFbcs were isolated from ESCC patients and treated by increasing concentrations of CXCR4 neutralizing antibody or control IgG for 24 h. Indicated cell lines ([KYSE30] or [EC9706]) were used as chemotactic stimulus. Representative images of hFbcs invaded through matrigel are shown in the left panel. Number of invaded hFbcs was quantified in the right panel. The data are represented as mean ± SD on 3 independent experiments. **, P < 0.01; ***, P < 0.001. B-C. The blocking effect of CXCR4 neutralizing antibody on the KYSE30-induced chemotaxis was evaluated by in vivo chemotaxis assay. 1 h after i.v. injection with Luciferase-expressing hFbcs, tumor-bearing mice (generated by GFP-expressing KYSE30) were treated with increasing doses of CXCR4 neutralizing antibody (#4, #5 and #6), control IgG (#3) or PBS (#2). hFbcs distribution (upper panel in B) and tumor burden (lower panel in B) were determined at 24 h after CXCR4 neutralizing antibody injection. Tumor-bearing mice treated with PBS (#1) were used to discard non-specific background signals. The relative radiance was summarized in C. The data are represented as mean ± SD on 4 independent experiments. *, P < 0.05; **, P < 0.01. D-E. Representative IHC images of exogenous cells (Luciferase⁺ cells) in KYSE30 xenografts obtained from the nude mice treated with CXCR4 neutralizing antibody, control IgG or PBS. The immunostaining area of Luciferase was summarized in E. The data are represented as mean ± SD in 4 mice. ***, P < 0.001. F. CXCL12-induced chemotaxis in hFbcs was measured using transwell system. Increasing concentrations of CXCL12 recombinant protein or PBS were used as chemotactic stimulus. Representative images of hFbcs invaded through matrigel are shown in the left panel. Number of invaded hFbcs was quantified in the right panel. The data are represented as mean ± SD on 3 independent experiments. **, P < 0.01; ***, P < 0.001. G. The blocking effect of CXCR4 neutralizing antibody on CXCL12-induced hFbc migration was evaluated by in vitro chemotaxis assay. hFbcs were treated by increasing concentrations of CXCR4 neutralizing antibody or control IgG for 12 h. CXCL12 were used as chemotactic stimulus. Representative images of hFbcs invaded through matrigel are shown in the left panel. Number of invaded hFbcs was quantified in the right panel. The data are represented as mean ± SD on 3 independent experiments. *, P < 0.05; ***, P < 0.001. Abbreviations: hFbc, human fibrocyte; ESCC, esophageal squamous cell carcinoma; i.v., intravenous