Framework for evaluating in vitro vasculogenic mimicry using structural and metabolic parameters

Vasculogenic mimicry (VM) refers to the ability of non-endothelial cells to form fluid-conducting, vessel-like structures independently of endothelial cells. In cancer, VM is associated with tumor aggressiveness and poor prognosis, although similar structures may also be formed by non-cancerous cells. Here, we systematically screened more than 23 human and murine cell lines, including diverse cancer types, fibroblasts, and primary-tumor-derived cells, identifying cell types capable or incapable of VM formation. Using confocal microscopy and 3D reconstruction, we define in vitro VM as the formation of lumen-containing tubular structures by non-endothelial cells, distinguishing this phenomenon from simple cellular clustering or alignment. Importantly, cell types capable of confirmed VM in vitro corresponded closely with previous findings in mouse xenograft models. We further provide a practical protocol for screening VM capacity and demonstrate that cell density, glucose concentration, serum availability, and matrix stiffness are critical environmental determinants of VM formation.