Optimization of Methylcellulose-Based 3D Culture Reveals Microenvironment-Dependent Regulation of ERα Activity in Luminal Breast Cancer Cells
Objective: Breast cancer (BC) is the most frequently diagnosed cancer in women, with the luminal subtype representing most cases. Luminal tumors depend on the ERα-FOXA1-GATA3 transcriptional network, which is influenced by the tumor microenvironment. Although three-dimensional (3D) culture systems better mimic in vivo conditions than 2D models, luminal breast cancer cells often fail to form stable spheroids. This study aimed to optimize 3D culture conditions and examine how microenvironmental architecture influences the ERα–FOXA1–GATA3 network.Material and Methods: We cultured MCF7 and T47D cells under standard conditions to establish 3D models. We seeded cells on Petri dishes containing media supplemented with different concentrations of methylcellulose (MC) to promote spheroid formation. We monitored morphology microscopically. We analyzed the expression levels of ESR1, FOXA1, and GATA3 in 2D and 3D cultures by RT-qPCR. We evaluated ERα chromatin binding at selected target regions by ChIP-qPCR. We assessed statistical significance using Student's t-test.Results: Optimization of MC-based 3D culture conditions showed that medium viscosity affects both spheroid morphology and transcriptional regulation. Spheroids formed under 10% MC displayed increased expression of ESR1, FOXA1, and GATA3 compared with 2D cultures. CRISPR/Cas9-mediated knockout of ESR1 and FOXA1 disrupted spheroid organization. ChIP-qPCR analysis revealed viscosity-dependent changes in ERα chromatin binding, demonstrating that spheroid compactness influences ERα transcriptional activity. Data represent mean ± SD from three independent experiments.Conclusion: These findings indicate that the physical properties of the microenvironment influence ESR1 expression and ERα activity. Optimization of 3D culture conditions is therefore important for accurately studying ERα signaling in luminal breast cancer models.