RAIN-Droplet: A Novel 3-D in vitro Angiogenesis Model

Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3-D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed as both outgrowths from endothelial cells on the surface of the droplets but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof-of-principle for the utility of the model showing significant inhibition of sprout formation (p<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay we also demonstrate a novel dose dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3-D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (p<0.05) more endothelial sprouts compared to controls over a 48-hour period. Higher doses of sorafenib (5 μM) resulted in a significant (p<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3-D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib which may impact the clinical application and dosing regimen of that drug.