Directional Migration of MDA-MB-231 Cells Under O₂/pH Gradients

We hypothesized that cancer cells actively migrate toward intratumor microvessels, guided by tissue gradients of metabolic substrates (such as O₂) and/or metabolites (such as CO₂/H⁺). To test this hypothesis, we developed an in vitro model in which cellular energy metabolism establishes gradients of O₂/nutrient/metabolite in monolayer cells cultured in a conventional culture dish. When gradients of O₂ ranging from 3% to ~0% were produced, MDA-MB-231 cells located at 300, 500 and 1500 μm downstream in the gradient demonstrated significant directional migrations (Rayleigh z test). We also found a similar directionality in cell migration at the same location even when the initial O₂ level in the O₂ gradient was raised from 3% to 21%. Interestingly, such directionalities were no longer demonstrated when the cell density was lowered from 1.8 × 10⁶ to 0.9 × 10⁶ cells/ml. In the former, the magnitude of the extracellular pH gradient in regions 300 and 500 μm downstream in the gradient was significantly larger. Thus, the direction of cell migrations appeared to depend on the gradient of extracellular pH rather than on O₂.