Three-dimensional environments that mimic in vivo microenvironments promote native epithelial cell polarity and differentiation by enabling cell-matrix interactions. When tumor cells were embedded as spherical cell aggregates (spheroids) in such microenvironments, particularly in semi-solid extracellular matrices, further the intimate cell-cell adhesion architecture as well as direct cell-matrix interactions can be efficiently recapitulated, including the presence of nutritional and metabolic gradients. The complexity of these spheroids can be further increased by the use of intercalating stromal cells. Here, we describe a simple and rapid method in which tumor and stromal cells are placed in hanging drop culture to generate homogenous and complex spheroids before embedding them in laminin−/collagen IV-rich basement membrane extracts (Matrigel), which in turn can be used as model system for cancer progression and to evaluate the efficacy of anticancer drugs, especially after radiation treatment.