DiscussionCurrent models for evaluating breast cancer progression lack a comprehensive, physiological approach to modeling the complex tumor microenvironment. There has been much evidence supporting the use of tumor spheroids to mimic tumor physiology; they exhibit cell-cell bond formation, comparable morphology, elevated cell survival and proliferation in the outer cell layers, reduced proliferation rates in the intermediate layers, and a hypoxic core. While the tumor spheroid provides a physiological tumor model, other cell types within the surrounding tumor microenvironment are essential for tumor behavior and subsequent cancer progression. The tissue vasculature provides a critical component for tumor progression given the metabolic requirements of a growing tumor, and by incorporating endothelial tubules with the tumor spheroid, we are able to model interactions between vascular networks and growing tumors. At the same time, there is also an important stromal component involved in cancer progression where stromal cells have been shown to promote cell proliferation, dissemination, and drug resistance during cancer development. By using extracellular matrix proteins, we are able to promote the proper physiology for each of these cell types in triple cell coculture and compartmentalize their activities. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Tumor spheroids are formed using low adhesion microwells and deposited into fully formed HUVEC tubular networks, and the hMSCs are then added within a hydrogel overlay matrix. Cellular interactions and dissemination are monitored via fluorescence microscopy and cell proliferation of the breast cancer cells is monitored using a fluorescence plate reader. The breast cancer spheroids exhibit cell-cell interactions with endothelial tubules and stromal cells, and there is an increase in cell proliferation and invasion over standard spheroid monoculture model.