In vitro angiogenesis induced by tumor-endothelial cell co-culture in bilayered, collagen I hydrogel bioengineered tumors.

Although successful remission has been achieved when cancer is diagnosed and treated during its earliest stages of development, a tumor that has established neovascularization poses a significantly greater risk of mortality. The inability to recapitulate the complexities of a maturing in vivo tumor microenvironment in an in vitro setting has frustrated attempts to identify and test anti-angiogenesis therapies that are effective at permanently halting cancer progression. We have established an in vitro tumor angiogenesis model driven solely by paracrine signaling between MDA-MB-231 breast cancer cells and telomerase-immortalized human microvascular endothelial (TIME) cells co-cultured in a spatially relevant manner. The bilayered bioengineered tumor model consists of TIME cells cultured as an endothelium on the surface of an acellular collagen I hydrogel under which MDA-MB-231 cells are cultured in a separate collagen I hydrogel. Results showed that TIME cells co-cultured with the MDA-MB-231 cells demonstrated a significant increase in cell number, rapidly developed an elongated morphology, and invasively sprouted into the underlying acellular collagen I layer. Comparatively, bioengineered tumors cultured with less aggressive MCF7 breast cancer cells did not elicit an angiogenic response. Angiogenic sprouting was demonstrated by the formation of a complex capillary-like tubule network beneath the surface of a confluent endothelial monolayer with lumen formation and anastomosing branches. In vitro angiogenesis was dependent on vascular endothelial growth factor secretion, matrix concentration, and duration of co-culture. Basic fibroblast growth factor supplemented to the co-cultures augmented angiogenic sprouting. The development of improved preclinical tumor angiogenesis models, such as the one presented here, is critical for accurate evaluation and refinement of anti-angiogenesis therapies.