title: A dual-color fluorescence imaging-based system for the dissection of antiangiogenic and chemotherapeutic activity of molecules.
abstract: 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 anti-angiogenics 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.
authors.affiliation: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Shiladitya
authors.initials: S
authors.lastname: Sengupta
authors.forename: Tanyel
authors.initials: T
authors.lastname: Kiziltepe
authors.forename: Ram
authors.initials: R
authors.lastname: Sasisekharan
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001632:Bibenzyls / Q000494:pharmacology; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004317:Doxorubicin / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005453:Fluorescence; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000098:blood supply / Q000188:drug therapy / Q000473:pathology; D009369:Neoplasms / Q000098:blood supply / Q000188:drug therapy / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000188:drug therapy*; D010793:Phthalazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D013267:Stilbenes / Q000494:pharmacology; D013792:Thalidomide / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D001632:Bibenzyls; D010793:Phthalazines; D011725:Pyridines; D013267:Stilbenes; D042461:Vascular Endothelial Growth Factor A; D013792:Thalidomide; C404768:vatalanib; D017228:Hepatocyte Growth Factor; C040105:combretastatin; D004317:Doxorubicin