Dynamic 3D imaging system detects vasculogenic mimicry and screens inhibitors in EGFR-mutant lung cancer

Vasculogenic mimicry (VM), wherein tumor cells form vessel-like structures independent of endothelial cells, drives metastasis and therapeutic resistance. Current in vitro VM research relies on conventional two-dimensional imaging, which fails to capture complex three-dimensional VM architecture, limiting anti-VM therapeutic development. We established a novel dynamic 3D imaging platform utilizing light sheet fluorescence microscopy for real-time visualization of physiologically relevant VM structures. Non-small cell lung cancer cell lines with different EGFR mutation statuses were cultured in three-dimensional matrices and monitored continuously. Structural characteristics including tubule diameter, lumen formation, and network complexity were quantified. Following validation of physiological 3D VM formation, we developed a high-content screening platform to assess anti-VM therapeutics, including foslinanib and osimertinib, individually and in combination. Our 3D VM platform revealed patent tubules and hollow lumens (25-50 μm diameter) approximating physiological 3D VM architecture, fundamentally distinct from planar network patterns seen with conventional 2D images. EGFR-mutant cells consistently formed complex 3D VM structures with functional lumens, whereas wild-type cell lines examined did not develop comparable three-dimensional architecture. In an EGFR L858R model, the foslinanib-osimertinib combination produced synergistic disruption of 3D VM structures; in other EGFR-mutant lines, the combination showed additive rather than synergistic effects. These findings indicate that combination activity is context-dependent and linked to specific EGFR genotypes. This 3D imaging platform provides a physiologically relevant, quantitative method for studying VM and screening anti-VM therapeutics, addressing a key limitation of conventional 2D assays. The association between EGFR mutation status and 3D VM formation offers insight into resistance mechanisms and identifies candidate combination strategies for further investigation, including the potential contribution of co-occurring genetic alterations. As a preclinical, hypothesis-generating study, these findings warrant validation in vivo and in clinical settings.