Integrating 3D Tumor Models and Microfluidics for Precise Metabolic Control

Abstract Understanding how metabolic deprivation shapes tumor behavior requires in vitro systems that faithfully reproduce millimeter-scale biochemical gradients. Here, we introduce MilliFlow3D, an open hydrogel-based microfluidic platform that generates controlled metabolic gradients and supports in situ spheroid formation, real-time imaging, and intact retrieval for spatial analyses. Gradients are generated by passive diffusion across a structured agarose microwell array positioned between two perfusion channels. Using a fluorescent tracer and numerical simulations, we show that MilliFlow3D establishes stable linear gradients with local metabolite levels matching those reported in avascular tumor regions. Using HCT116 colorectal cancer spheroids, we demonstrate that a L-glutamine gradient imposes graded effects on growth and proliferation: spheroid expansion decreases from high- to low-glutamine regions, and Ki-67–positive cells progressively shift toward the periphery under glutamine depletion. Thanks to the platform’s spatial accessibility, these phenotypic responses could in the future be coupled to molecular readouts, enabling spatial mapping of metabolic pathway activity along the gradient. Altogether, MilliFlow3D provides a robust and versatile platform to investigate how heterogeneous metabolic landscapes sculpt tumor behavior and to identify context-dependent metabolic vulnerabilities with high analytical precision.