The Essential Role of Fibroblasts in Esophageal Squamous Cell Carcinoma–Induced Angiogenesis

Background & Aims: Esophageal squamous cell carcinoma (ESCC) is known to be a highly angiogenic tumor. Here, we investigated the role of the stromal fibroblasts in the ESCC-induced angiogenic response using a novel 3-dimensional model. Methods: A novel assay was developed where cocultures of ESCC and esophageal fibroblasts induced human microvascular endothelial cell (HMVEC) vascular network formation in a 3-dimensional collagen gel. Biochemical studies showed that the ESCC-induced activation of the fibroblasts was required to induce vascular network formation via a transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF)-dependent pathway. Results: Conditioned media from a panel of 4 ESCC lines transdifferentiated normal esophageal fibroblasts into myofibroblasts via TGF-β signaling. The presence of fibroblasts was essential for efficient HMVEC network formation, and the addition of ESCC cells to these cultures greatly enhanced the angiogenic process. The role of TGF-β in this process was shown by the complete inhibition of network formation following TGF-β inhibitor treatment. Finally, we showed that ESCC-derived TGF-β regulates angiogenesis through the release of VEGF from the fibroblasts and that the VEGF release was blocked following TGF-β inhibition. Conclusions: This study shows the essential role of fibroblasts in the ESCC angiogenic-induced response and suggests that the pharmacologic targeting of the TGF-β signaling axis could be of therapeutic benefit in this deadly disease.

Abbreviations used in this paper: CAF, carcinoma-associated fibroblast, DAPI, 4′,6-diamidino-2-phenylindole, DMEM, Dulbecco's modified Eagle medium, ELISA, enzyme-linked immunosorbent assay, ESCC, esophageal squamous cell carcinoma, FBS, fetal bovine serum, GFP, green fluorescent protein, HMVEC, human microvascular endothelial cell, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, α-SMA, α–smooth muscle actin, TGF, transforming growth factor, 3D, 3-dimensional, VEGF, vascular endothelial growth factor