Fig. 4

ESCC cells induce fibrocyte precursor expansion through FGF2. A. Relative expression of FGF2 in primary ESCCs (n = 40) and adjacent normal tissues (n = 40) were detected by qPCR. Horizontal lines represent the median. The whiskers extend to the highest values excluding outliers and extremes. ***, P < 0.001. B. FGF2 levels in serum samples from ESCC patients (n = 19) and healthy donors (n = 10) were measured by ELISA. Horizontal lines represent the median. The whiskers extend to the highest values excluding outliers and extremes. **, P < 0.01. C. Serum FGF2 levels were monitored by ELISA at a series of indicated time points after tumor implantation with ESCC cells or tumor resection. The data are represented as mean ± SD on 5 independent samples. **, P < 0.01; ***, P < 0.001. D. FCM was used to detect the proportions of fibrocyte precursors (FGFR2⁺CD34⁺ cells) and circulating fibrocytes (FGFR2⁺Col I⁺ cells) in nude mice treated with 3 different doses of FGF2 recombinant protein. The data are represented as mean ± SD on 4 independent samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E. FCM was used to detect the proportions of fibrocyte precursors and circulating fibrocytes in nude mice treated with 80 pg FGF2 recombinant protein and increasing concentrations of FGF2 neutralizing antibody. The data are represented as mean ± SD on 4 independent samples. *, P < 0.05; **, P < 0.01. F. Representative IHC images of FGFR2⁺ cells in the KYSE30 xenografts obtained from the nude mice treated with FGF2 neutralizing antibody, control IgG or PBS. The immunostaining area of FGFR2 was summarized in the right panel. The data are represented as mean ± SD in 4 mice. ***, P < 0.001. G. The therapeutic effect of FGF2 neutralizing antibody was evaluated on KYSE30 tumor-bearing mice. Mice were treated intravenously everyday with FGF2 neutralizing antibody (4 μg/kg), control IgG or PBS. Xenografts were harvested at day 20 (left panel). Tumor size and tumor weight was summarized in the middle panel and right panel. The data are represented as mean ± SD in 4 mice. *, P < 0.05. Abbreviations: ESCC, esophageal squamous cell carcinoma; qPCR, semi-quantitative PCR; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry