Fig. 3

ESCC cells trigger differentiation of fibrocytes into mature CAFs through direct cell–cell contact. A. Expression of indicated marker genes in Bsd resistant-Fbcs direct contacted with KYSE30 in vivo [(i.v.) + Bsd] were determined by qPCR and summarized in heat map. The cells cultured with normal culture medium (+ Medium) or medium containing Bsd (+ Bsd) were used as controls. The experiment was repeated three times. B. Expression of indicated marker genes in Bsd resistant-Fbcs direct co-cultured with KYSE30 [(KYSE30) + Bsd] were determined by qPCR and summarized in heat map. The cells cultured with normal culture medium (+ Medium) or medium containing Bsd (+ Bsd) or direct co-cultured with NE1 [(NE1) + Bsd] were used as controls. The experiment was repeated three times. Schematic diagrams in A-B showing the experimental procedures of differentiation assays. Color intensity of heat map represents log10(Relative expression) within each measurement. C. The effect of normal culture medium (+ Medium), CM from Fbcs (+ Fbc-CM) or CM from iCAFs (+ iCAF-CM) on the growth of ESCC cells (KYSE30 and EC9706) was compared by XTT assay. The data are represented as mean ± SD on 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. D. Representative images showing the invasion of ESCC cells (KYSE30 and EC9706) cultured in the normal culture medium, Fbc-CM or iCAF-CM. Number of invaded cells was quantified in the lower panel. The data are represented as mean ± SD on 3 independent experiments. **, P < 0.01; ***, P < 0.001. E. Representative images of xenograft tumors (generated by KYSE30 and EC9706) harvested from nude mice with Fbc or PBS i.v. injection. Tumor growth curves are summarized in the bottom panel. The data are represented as mean ± SD in 4 mice. *, P < 0.05; **, P < 0.01. Abbreviations: CAF, cancer-associated fibroblast; Bsd, blasticidin; Fbc, fibrocyte; qPCR, semi-quantitative PCR; CM, conditioned medium; iCAF, induced-cancer-associated fibroblast