Development of reduced graphene oxide-incorporated gelatin–polymannose hydrogel scaffolds as a 3D cell culture model for cancer research

= 3). Live staining assays confirmed progressive cell migration and high cell viability. Confocal microscopy images further confirmed elevated ROS generation within the 3D scaffold, indicating the establishment of a hypoxia-like tumor microenvironment. Gene expression analysis showed elevated levels of HIF-1α, MMP2, β-catenin, and CDH1 in 3D cultures compared with 2D cultures. These findings were further supported by Western blot analysis, which showed increased expression of HIF-1α, MMP-2, β-catenin, and E-cadherin. Notably, the molecular expression profile of the Gel:OPM:rGO scaffold closely resembled that of Matrigel. Drug response was evaluated using doxorubicin (Dox) as a model anticancer drug. A549 cells cultured in the Gel:OPM:rGO scaffold and Matrigel exhibited higher inhibitory concentration of Dox when compared to the 2D culture. Our results suggest that the Gel:OPM:rGO scaffold is better suited for A549 cell culture and may serve as a preclinical 3D lung cancer model for drug testing.