Esophageal cancer cells produce TGF-β to activate stromal normal fibroblasts. Tumor stromal fibroblasts become transdifferentiated into myofibroblasts that secrete VEGF which in turn induces endothelial cells migration and the formation of a micro-capillary network.

(A) Control GFP-tagged fibroblasts (green) expressed very little a-smooth muscle actin. (B–D) Fibroblasts co-cultured with the ESCC lines (TE1, TE11, TE8, TE10 and TE12) for 48 hours stain strongly for stress fibers of α-smooth muscle actin (α-SMA) (red). (E) Western blot analysis showing that incubation of fibroblasts with conditioned media (CM) from ESCC lines for 47 hrs leads to increased α-SMA expression in esophageal fibroblasts. Blots were stripped and reprobed with anti-β-actin as a loading control. (F) Conditioned media form human esophageal keratinocytes does not induce α-smooth muscle actin expression in esophageal fibroblasts. Images; scale bar = 100 µm.

(A) TGF-β inhibition has little effect upon fibroblast-induced vascular network formation. Fibroblasts (FEF3; 1.5 × 10⁵ cells/ml) were incubated with HMVECs in the absence or presence of one of two TGF-β inhibitors (SB505124 or GW788388, both 1 µM) for 7 days. Cultures were stained for HMVECs (red: CD31), fibroblasts (GFP, green) and nuclei (DAPI, blue). (B, C) TGF-β inhibition completely blocks ESCC-induced vascular network formation. Addition of either SB505124 or GW788388 (both 1 µM) led to complete inhibition of vascular network formation. (D) Bar graph shows mean data for vascular network formation in the absence and presence of either SB505124 or GW788388. All representative images are shown as 3-color merges, and original monochrome CD31 (white) are inset. Scale bar = 200 µm.

(A) Stimulation of esophageal fibroblasts with either TGF-β through co-culture with ESCCs leads to enhanced VEGF release in 2D adherent culture. Fibroblasts (FEF3) in monoculture or FEF3 and ESCCs (TE1) at the ratio 1:1 in co-culture were cultured in the absence or presence of SB505124 (1 µM) for 48 hr. Supernatants were harvested and quantified for VEGF expression using a specific ELISA. (B) Stimulation of esophageal fibroblasts with either TGF-β or ESCC conditioned media leads to enhanced VEGF release in 3D culture. Mono and co-cultures of esophageal fibroblasts were grown in a 3D collagen in the absence or presence of SB505124 for 48 hr. (C) The VEGF inhibitor GW654652 (1 µM) inhibits vascular network formation. (D) Bar graph shows mean data for vascular network formation in the absence and presence of GW654652. All representative images are shown as 3-color merges, monochrome images of CD31 are inset. Scale bar = 200 µm.

Immunofluorescence staining shows GFP-tagged FEF3 esophageal fibroblasts (Green), HMVECs (CD31: Red) and total nuclei (DAPI: Blue). CD31 staining as the inset. Incubation of the HMVECs with each of three ESCC lines (TE1, TE8, TE11) was not associated with any vascular network formation (A). Co-culture of the HMVECs with human esophageal fibroblasts (FEF3) led to increased vascular network formation (B). The addition of ESCCs to the fibroblast/endothelial cell co-culture markedly increased the organization of the vascular networks (C). Increasing the culture time to 14 days dramatically enhanced the organization of the vascular network. (D) ESCC number was also found to increase the degree of vascular network formation. (E) The extent of network formation under each of the culture conditions was scored, with the addition of increasing numbers of ESCC cells being found to significantly increase the level of vascular network formation. Images; scale bar = 200 µm.

(A) The TGF-β receptor kinase inhibitor SB505124 blocks TGF-β induced fibroblast transdifferentiation. Fibroblasts were cultured in the presence of TGF-β1 (1 ng/ml) and increasing concentrations of SB505124 (0.01 – 10 µM) for 48 hr followed by blotting for α-SMA expression. (B) SB505124 inhibits α-smooth muscle actin overexpression in fibroblasts stimulated by ESCC-conditioned medium (CM; from TE1 cells). Fibroblasts were cultured with CM as described in the methods in the absence or presence of SB505124 (1µM) for 48 h. (C) SB505124 inhibits phosphorylation of SMAD2 induced by ESCC-conditioned media. Fibroblasts were cultured with TGF-β1 (1ng/ml) or conditioned medium as indicated and treated with SB505124 (1 µM) for 1 h. (D) TGF-β inhibitors have little effect upon the proliferation of ESCCs, fibroblasts or HMVECs. Fibroblasts (FEF3), HMVECs and esophageal cancer cell lines (TE) were treated with increasing concentrations of TGFβRI inhibitors (either SB505124 or GW788388) for 72 hr before being subject to the MTT assay. The results were evaluated as a percentage of control absorbance.

(A) ELISA assay showing secretion of total TGF-β1 and β2 by esophageal cancer cell lines (TE). TE cells were cultured for 48 h and secreted TGF was measured by ELISA. (B) Exogenous TGF-β1 induces α-smooth muscle actin (α-SMA) expression in fibroblasts and stimulates TGF-related signaling pathways. Exogenous rhTGF-β1 (48 hr) induces α-SMA expression in esophageal fibroblasts. (B) Exogenous rhTGF-β1 induces SMAD2 phosphoryation in human esophageal fibroblasts. (C) Both exogenous rhTGF-β1 and ESCC conditioned media induce SMAD3 activation in esophageal fibroblasts. Fibroblasts were treated with either rhTGF-β1 (1 ng/ml) or by conditioned medium of TE1 for 1 hr. Images show p-SMAD3 up-regulation and its subsequent nuclear translocation (green), cell morphology is indicated by phalloidin (Red) and nuclei by DAPI (Blue). (D) Exogenous TGF-β1 markedly enhances vascular network formation in the absence of ESCC. Fibroblasts and HMVECs were co-cultured in the presence of TGF-β1 (1 ng/ml), for 7 days. Vascular network formation was stained for CD31 (red), fibroblasts (GFP, green) and nuclei (DAPI, blue). Inset shows CD31 staining alone. Graph shows the significantly (P<0.005) increased numbers of micro-capillary networks per field treated by TGF-β1. (E) Western blot showing expression of TGFβRII expression in a panel of ESCC lines, fibroblasts and primary human esophageal keratinocytes (EPC2). (F) Exogenous TGF-β1 (72 hrs) reduces the growth of human esophageal keratinocytes (EPC2) but not ESCC lines. Cell proliferation was measured by the MTT assay. Images; scale bar = 50 µm. (E); scale bar = 200 µm.