A dual-color fluorescence imaging-based system for the dissection of antiangiogenic and chemotherapeutic activity of molecules

We have developed a simple yet sensitive dual color fluorescence-based technique for dissecting the tumor-neovascularization relationship and evaluated the susceptibility of each component to therapeutic interventions. Green fluorescent protein (GFP)-expressing melanoma cells were cocultured with endothelial cells on different three-dimensional (3-D) matrices and exposed to multiple growth factors and molecules with established anti-angiogenic or anticancer activities. Cells were fixed and stained with propidium iodide, imaged using a confocal microscope, and stereologically analyzed. Three-dimensionality of the system was tested by depth-coding and pseudocolor 3-D reconstruction in the z-axis. Selective ablation of the tumor cells was affected by the anthracycline antibiotic doxorubicin. Treatment with vascular endothelial growth factor ( VEGF) and hepatocyte growth factor (HGF) promoted the neovascular responses on matrigel and collagen-1 matrices. VEGF-induced angiogenesis was inhibited after treatment with combretastatin and thalidomide. In contrast, HGF exerted a protective effect against these antiangiogenics in a matrigel matrix. However, this effect was lost when the matrix was substituted with collagen, suggesting that the extracellular matrix impinges on cellular function, possibly through an Akt-mediated mechanism. The VEGF-receptor antagonist PTK787 also selectively ablated the VEGF-induced angiogenic effect without inhibiting the HGF-induced response, demonstrating the sensitivity of the system to detect modulation of distinct signal cascades. The current model encompasses the possibility of studying tumor-angiogenesis- matrix interaction on the same platform, expanding the rapid screening of novel molecules in a simulated clinicopathological setting.