Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts

This study elucidates mechanical and metabolic differences among cancer-associated fibroblasts (CAFs) derived from oral squamous cell carcinoma (OSCC) specimens at distinct malignancy stages. Four CAF lines (CAFS1, CAFS2, SCC17F, and MO-1000) were isolated, cultured as 3D spheroids, and compared with non-cancer fibroblasts. The authors demonstrated that CAFs exhibit lower mitochondrial respiration and increased glycolytic capacity (indicative of a ‘reverse Warburg effect’) and that spheroid stiffness correlates with tumor differentiation state. Ultrastructural and trypsin resistance assays further revealed ECM density and cohesion differences between CAF subtypes. Collectively, 3D spheroid stiffness and metabolic parameters are proposed as definitive indices for CAF subtype characterization and tumor aggressiveness.