Impact of silk-derived sericin on the formation and metformin sensitivity of breast cancer-fibroblast heterotypic spheroids

The development of physiologically relevant three-dimensional (3D) tumor models is essential for accurately mimicking the tumor microenvironment (TME) and improving the predictive value of in vitro drug testing. This study evaluated the effect of culture media supplementation with sericin, a silk-derived glycoprotein, in the formation of heterotypic spheroids based on MCF-7 breast cancer cells and normal NHDF. Spheroids were assembled under orbital agitation with sericin concentrations ranging from 0.025 to 0.2% and cultured for 14 days. Computational fluid dynamics simulations indicate low shear stress (<0.1 dynes/cm 2 ), supporting stable spheroid formation. Morphometric analysis and 3D reconstruction (AnaSP/ReViSP) revealed that intermediate sericin levels improved spheroid compactness and sphericity, whereas higher supplementation with sericin reduced structural homogeneity. Moreover, considering the validation of supplementation effect of drug response, metformin was used as antitumoral drug model. Metformin had an IC₅₀ of 19.44 mM in 2D MCF-7 cells but was less effective in 3D spheroids. Sericin increased spheroid resistance, with ≥40% viability at 3× IC₅₀ versus ~15% in controls, reflecting enhanced compactness and reduced drug penetration. These findings demonstrate that sericin-enriched media promote compact, viable spheroids with improved physiological relevance. Sericin is thus a promising culture media supplement for constructing 3D tumor models suitable for high-throughput drug screening and personalized oncology. Future studies should focus on molecular profiling, drug diffusion kinetics, and integration with microfluidic systems to further enhance translational potential.