Introduction:Two-dimensional cell cultures have contributed substantially to lung cancer research, but 3D cultures are gaining attention as a new, more efficient, and effective research model. A model reproducing the 3D characteristics and tumor microenvironment of the lungsin vivo, including the co-existence of healthy alveolar cells with lung cancer cells, is ideal. Here, we describe the creation of a successfulex vivolung cancer model based on bioengineered lungs formed by decellularization and recellularization.Methods:Human cancer cells were directly implanted into a bioengineered rat lung, which was created with a decellularized rat lung scaffold reseeded with epithelial cells, endothelial cells and adipose-derived stem cells. Four human lung cancer cell lines (A549, PC-9, H1299, and PC-6) were applied to demonstrate forming cancer nodules on recellularized lungs and histopathological assessment were made among these models. MUC-1 expression analysis, RNA-seq analysis and drug response test were performed to demonstrate the superiority of this cancer model.Results:The morphology and MUC-1 expression of the model were like those of lung cancerin vivo. RNA sequencing revealed an elevated expression of genes related to epithelial-mesenchymal transition, hypoxia, and TNF-α signaling via NF-κB; but suppression of cell cycle-related genes including E2F. Drug response assays showed that gefitinib suppressed PC-9 cell proliferation equally well in the 3D lung cancer model as in 2D culture dishes, albeit over a smaller volume of cells, suggesting that fluctuations in gefitinib resistance genes such as JUN may affect drug sensitivity.Conclusions:A novelex vivolung cancer model was closely reproduced the 3D structure and microenvironment of the actual lungs, highlighting its possible use as a platform for lung cancer research and pathophysiological studies.