The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Tumor progression and metastasis are critically dependent on the tumor stroma consisting of fibrous connective tissue, newly formed blood vessels, extracellular matrix, and recruited inflammatory cells. Tumor stromal fibroblasts (activated fibroblasts or myofibroblasts) acquire a specific phenotype that differentiates them from normal resting fibroblasts. They express smooth muscle actin (SMA), and isolated myofibroblasts have higher proliferation rates than their normal counterparts.1 The importance of stromal mesenchymal interactions with adjacent epithelial cells during development is well characterized. Proliferation of mesenchymal cells is tightly controlled by soluble paracrine factors. Conversely, differentiated fibroblasts do not proliferate and acquire a quiescent phenotype.2 In contrast, the complex paracrine regulatory mechanisms between the mesenchymal tumor stroma compartment and the tumor compartment are far from being understood. Coinjection of activated fibroblasts and tumor cells has been shown to enhance the invasiveness of colon tumors.3 Tumor-associated fibroblasts affect migration and invasion of tumor cells by secreting extracellular matrix proteins, matrix metalloproteinases, and growth factors.4,5 Paracrine-acting factors affecting tumor and stromal cell interactions include fibroblast growth factors, hepatocyte growth factor, and transforming growth factor-␤. Correspondingly, elevated plasma levels of transforming growth factor-␤ have been shown to predict early metastasis.