Three-dimensional vascular microenvironments uncover endothelial plasticity during TGF-β2-driven vascular remodeling

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-β (TGF-β) family. TGF-β family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-β signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-β signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-β2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-β2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-β and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.