Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related death in the world.Unfortunately, no effective therapy in clinic has been established to significantly improve the overall 5-year survival rate of the NSCLC patients.This might be mainly due to the lack of more accurate models that could highly recapitulate those in vivo biological or molecular events.To address this issue, here we developed a more pathologically-relevant, three-dimensional (3D)culture model of human lung cancer cells through encapsulating the immortalized human NSCLC cells (A549 cells) within alginate-calcium(AC) gel microsphere.We hypothesized that A549 cells growing in the 3D microenvironment provided by AC microsphere would not only generated tumor cultures that more closely resemble the in vivo tumor morphology, but also contribute to producing a more defined NSCLC culture model that potentially enhances the molecularly targeted drug evaluation.Our results showed that A549 cells encapsulated within AC microspheres proliferated and generated cell aggregates during cultivation.These aggregates displayed good viability evidenced by CCK-8 assay and live/dead staining.Histological analysis further revealed cell distribution and cell junction formation within the aggregates.A significantly increased expression of EGF, EGFR,VEGF-D genes was observed in the 3D cultures, which was consistent with their protein expression level assayed by western-blotting.Moreover, Compared to the 2D cultures, increased IC50 values were found in the encapsulated A549 cells, indicating their enhanced drug resistant potential during the 3D cultivation.Their capability of responding to the targeted medicine (gifitinib) was also decreased in comparison to that of the 2D cultures.This culture system of human NSCLC cells effectively represent an improved culture model of human lung cancer in vitro and would potentially provide a robust tool for drug evaluation.