Ewing sarcoma (EwS) is a rare pediatric malignancy which develops in a physiologically stiff bone environment. Conventional in vitro models fail to faithfully recapitulate the complexity of this microenvironment, showing limits in translational relevance of preclinical drug screening.

In this study, we present a novel three-dimensional (3D) in vitro EwS model using extrusion-bioprinting technology with a collagen-based bioink. Rheological and morphometric analyses were performed to establish a printability framework, optimizing bioink formulations and cell densities for effective biofabrication. Alamar Blue assay was used to identify optimal cell densities for the evaluation of cell viability and growth in short- and long-term studies. The optimized condition (collagen 1:10 ratio; 1.4x10⁶ cells/mL TC-71 EwS cell line) enabled reproducible biofabrication of structurally stable constructs with improved viscoelastic properties and shape fidelity. The 3D tumour microenvironment was modulated by hydrogel viscoelasticity, which supported sustained cell growth and remodelling activities as indicated by increased expression of MMP-9 and MMP-13. Under these conditions, viable multicellular spheroids formed within the hydrogel by day 3 and exhibited sustained growth through day 14, enabling both short- and long-term studies. The immunohistochemical analysis confirmed homogenous cell distribution, and together with the gene expression analysis recapitulated key tumor features. As proof of concept, treatment with talazoparib, a potent poly-ADP ribose- polymerase (PARP) inhibitor (PARPi), led to reduced proliferation (Ki-67 labeling) and activation of the DNA damage response (ɣH2AX expression) in the 3D EwS model, demonstrating the feasibility of this platform for drug screening. Comparative analysis with a conventional 2D model further illustrates the enhanced physiological relevance of the 3D environment. Notably, the analysis of Ki-67 expression revealed that the 3D model exhibited slower proliferation kinetics compared to the 2D culture, with significantly lower Ki-67 levels at day 3 and comparable levels to the 2D control by day 10. Furthermore, treatment with 20 nM talazoparib in the 3D model significantly reduced Ki-67 expression and increased γH2AX positivity by day 10, indicating a delayed DNA damage response relative to the rapid accumulation of DNA damage observed in the 2D cultures. Overall, this study provides a reproducible and biologically relevant 3D bioprinted EwS model, supporting its application as a predictive preclinical tool for therapeutic evaluation.