The use of vascularized spheroids to evaluate the in vitro activity of antiangiogenic drugs.

Abstract

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Angiogenesis is a key aspect of cancer progression and patient survival. We have produced three-dimensional spheroid co-cultures of breast cancer and melanoma cell lines with microvascular endothelial cells (MVECs). Numerous obstacles had to be overcome in the development of these models, which are homogeneous and reproducible in both size and shape. Different methods of producing spheroids were explored, ranging from a hanging drop model to methylcellulose to agar matrix models. Additionally, nutritional requirements for all co-cultured cell-types had to be satisfied, leading to a comparison of various media. In these models, lumen-like structures made up of MVECs penetrate the tumor spheroid, thereby creating a model of angiogenesis. Whereas numerous models for angiogenesis exist, few allow for the direct analysis of MVEC-cancer cell interactions. Our model of vascularized spheroids makes it possible to examine how cell lines of differing phenotypes modulate MVEC-mediated lumen formation. Another advantage of our model is based on its spherical shape, which leads to radial gradients in nutrient exposure, hypoxia, and drug concentrations. Such gradients produce hypoxic and non-hypoxic populations that mimic in vivo tumor growth. These models were developed in part to test our hypothesis that spheroids derived from tumor cell lines expressing low levels of Thrombospondin-1 (TSP-1) would have greater endothelial cell invasion than those with high TSP-1. TSP-1 is the natural inhibitor of neovascularization and tumorigenesis in healthy tissue. It activates the CD36 receptor on endothelial cells, which can induce apoptosis. TSP-1 expression by various tumor cell lines was characterized using western blotting and immunohistochemistry. Endothelial cell invasion was measured by staining MVECs with CD31 and CD105 reagents, and the cells were enumerated using light microscopy at a 200x field. The cytotoxic activities of anti-angiogenesis and chemotherapeutic agents on vascularized tumor spheroids are also evaluated to determine if TSP-1 levels modulated the apoptotic response of MVECs to therapy.