AbstractTumor angiogenesis, the formation of new blood vessels from existing blood vessels in and around the tumor stroma, is orchestrated by multiple biological factors in the tumor microenvironment, including tumor stromal cells, extracellular matrices, and secreted growth factors. Here, we present the processes to define and optimize the biological conditions for robust interactions between tumor spheroids and engineered blood vessels in a microfluidic organ-on-a-chip device in vitro. Within the device, vascular lumen formation and vessel sprouting in human umbilical vein endothelial cell based engineered blood vessels are observed in a variety of extracellular matrices (collagen, Matrigel, and fibrin), containing metastatic breast tumor cells (MDA-MB-231), and tumor stromal cells (mesenchymal stem cells, and human lung fibroblasts) to show the crosstalk between the tumor spheroids and the perfused blood vasculatures in vitro.