A U‐shaped Microfluidic Platform for Culturing Vascularized Colorectal Cancer Spheroids Reveals Tumor–Endothelial Crosstalk and Invasion‐Associated Remodeling

ABSTRACT Current tumor‐on‐chip platforms force trade‐offs between reproducible single‐spheroid positioning, size control, and symmetric full‐surface vascularization. To bridge this gap, we developed a closed U‐shaped microfluidic device that traps a single pre‐formed colorectal cancer (CRC) spheroid at a defined position, uniformly embedded in a fibrin matrix containing GFP‐HUVECs to enable volumetric tumor–endothelial interactions and longitudinal imaging. Optimized HT29 spheroids generated a measurable pro‐angiogenic secretory profile. A dedicated angiogenesis assay showed that VEGF gradients contribute to multicellular sprouting from endothelial monolayers, while interstitial endothelium may further enhance this response. In tumor–vascular co‐cultures, spheroids were associated with hyperbranched vascular remodeling, with increased junction density, branching, and vessel length relative to endothelial‐only controls. Extracellular VEGF levels were markedly reduced in vascularized constructs, consistent with endothelial ligand binding, uptake, and/or sequestration. Reciprocally, vascularized co‐culture was accompanied by tumor surface protrusions, anisotropic growth, disruption of epithelial junction organization, and upregulation of mesenchymal‐associated markers. By overcoming the limitations of heterogeneous self‐assembling models and asymmetric open‐well designs, this platform provides reproducible quantitative readouts of CRC–vascular dynamics and a tractable framework for studying early tumor–vascular remodeling.