From 2D to 3D Bioprinted In Vitro Breast Cancer Model: A Comparative Study of Proliferation, Tissue Structure, and mTOR Signaling.

Three-dimensional (3D) bioprinting offers a suitable in vitro preclinical model system to reduce or replace animal experiments; however, published studies are difficult to compare. In this study, we characterized growth dynamics, tissue architecture, and mammalian target of rapamycin (mTOR) pathway activity in a 3D bioprinted breast carcinoma model of T47D cell line and compared these features with conventional two-dimensional (2D) monolayer cultures. Tissue-mimetic structures (TMSs) were generated by 3D bioprinting and analyzed for cell viability, proliferation, autophagy, and apoptosis, as well as the expression of cell-cell and cell-extracellular matrix (ECM) adhesion proteins. In addition, mTOR pathway activity and responsiveness to mTOR inhibitors (rapamycin and ipatasertib) and chemotherapeutic agents (cisplatin) were assessed. The bioprinted TMSs remained viable for up to 3 weeks and developed a tissue-like architecture characterized by heterogeneous marker expression (β-catenin, E-cadherin, N-cadherin, fibronectin, and syndecan) and complex cellular organization. Compared with 2D monolayer cultures, 3D TMSs exhibited reduced mTOR signaling activity, which led to significantly decreased sensitivity to mTOR inhibition. These findings indicate that 3D bioprinted breast cancer models recapitulate key structural and signaling features of in situ tumors more accurately than 2D systems, highlighting their potential value for preclinical drug testing and mechanistic studies.