Supplementary file 1_Reduced graphene oxide-incorporated gelatin-oxidized polymannose scaffold-based 3D Lung cancer model for mimicking the tumor microenvironment.docx

Metabolic profiling was performed by measuring glucose consumption and lactate production and the results were compared with 2D and Matrigel culture systems.</p>Results<p>Gene expression analysis revealed the upregulation of TME-associated genes, including the hypoxia marker VEGF-A, the extracellular matrix (ECM) remodeling marker MMP9, the epithelial–mesenchymal transition (EMT) marker VIM, the oncogenic marker ALK, and the growth factor receptor EGFR.

Western blot analysis showed increased expression of TME-related proteins in the Gel: OPM:rGO scaffold compared with conventional 2D culture. Major signaling pathways, including mTOR, PI3K/AKT, Wnt, c-Myc, STAT3, and TNF-α, were also modulated, indicating physiologically relevant cellular responses. Metabolic profiling in Gel: OPM:rGO culture supernatant demonstrated enhanced glucose consumption and lactate production, consistent with the Warburg effect and tumor-associated metabolic reprogramming.

Overall, gene and protein expression profiles in the Gel: OPM:rGO scaffold exhibited trends similar to those observed in Matrigel-based 3D culture.</p>Conclusion<p>Collectively, these findings demonstrate that the Gel: OPM:rGO scaffold is a robust and physiologically relevant 3D lung cancer model. As a potential 3D culture platform that complements Matrigel-based models, it provides a valuable in vitro platform for investigating tumor-associated cellular and molecular responses, cancer progression, and preclinical anticancer drug evaluation.</p>