Fig. 2

FGFR2⁺ CAFs are HSC-derived fibrocytes. A. Cell-type identification of FGFR2⁺ cells in peripheral blood from ESCC tumor-bearing mice. Leukocytes obtained at indicated time points after tumor implantation or resection were double-stained for FGFR2 in combination with indicated cell surface markers and analyzed by FCM. The data are represented as mean ± SD on 3 independent samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B. Quantification of circulating fibrocytes in ESCC patients (n = 17) and healthy donors (n = 8) by FCM. Fibrocytes were identified by double-staining for FGFR2 and indicated fibrocyte markers. Horizontal lines represent the median. The whiskers extend to the highest values excluding outliers and extremes. ***, P < 0.001. C. The proportions of FGFR2⁺ fibrocyte progenitors (FGFR2⁺CD34⁺ or FGFR2⁺CD45⁺ cells) in BM were measured by FCM (upper panel) at 4 weeks after s.c. injecting KYSE30 or PBS. The data are represented as mean ± SD on 3 independent samples. **, P < 0.01. FGFR2⁺ fibrocyte precursors in the same samples were also detected by IF (lower panel). Double positive (FGFR2⁺CD34⁺ or FGFR2⁺CD45⁺) cells are indicated by yellow arrows. Selected regions are magnified. D. Luciferase-expressing human fibrocytes (hFbcs) were subjected to the in vivo chemotaxis assays in tumor-bearing mice (#5 and #6; s.c. injected with GFP-expressing KYSE30) or control mice (#1 and #2; s.c. injected with PBS). hFbcs distribution (upper panel) and tumor burden (lower panel) were determined at 24 h after i.v. injection of hFbcs. Tumor-bearing mice with PBS i.v. injection (#3 and #4) were used to discard non-specific background signals. Three mice will be evaluated for each condition. E. IHC staining demonstrated the presence of FGFR2⁺ cells or exogenous cells (Luciferase⁺ cells) in the xenografts obtained from the nude mice in in vivo chemotaxis assays. Abbreviations: HSC, hematopoietic stem cell; FCM, flow cytometry; hFbc, human fibrocyte; IHC, immunohistochemistry; IF, immunofluorescence; SD, standard deviation