Cancer biology and drug discovery are heavily dependent on conventional 2D cell culture systems. However, a 2D culture is significantly limited by its ability to reflect 'true biology' of tumor in vivo. Three-dimensional (3D) in vitro cell culture models have been introduced to aid cancer drug discovery by better modeling the tumor microenvironment. Here, decellularized lung scaffolds cultured with MCF-7 cancer cells were bioengineered as a platform to study tumor development and anti-cancer drug evaluation. Excellent cell compatibility of decellularized lung scaffolds promoted cell growth and proliferation. Multicellular tumor cell spheroids (tumoriods) were formed and enlarged exclusively in decellularized lung scaffolds over time. The expression of breast cancer biomarkers (BRCA1 and HER2) in MCF-7 cells significantly increased in the lung matrix compared to those cultured in 2D systems. Insufficient oxygen and nutrient diffusion into the internal surface of lung scaffolds resulted in intracellular hypoxia, quantified by a significant upregulation of HIF-1α protein expression compared to that of cell monolayers. Higher survival rates after exposure to 5-FU were observed in lung scaffolds (52.04%) compared to that in 2D systems (18.39%) on day 3 of culture. Overall, this new breast tumor system provides a promising platform to study breast cancer progression and develop new targeted therapeutic strategies.