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Three-dimensional (3D) cell culture models have been proposed as alternatives for initial drug screening to increase drug development efficiency. In addition to their ability to reproduce key aspects of the tumor architecture and microenvironment, 3D cell culture models may help decrease the use of laboratory animals in drug testing, in accordance with the 3R principles (Replacement, Reduction, Refinement). This review aims to help researchers making a transition from two-dimensional (2D) to 3D cell culture models for drug screening, by discussing the impact of 3D models on cancer research, their advantages, limitations, and compatibility with high-throughput screenings. It also outlines the relevance of available readouts provided by such models as well as the importance of incorporating key microenvironmental cues towards improving the predictive value of drug efficacy and safety.

Today, innovative three-dimensional (3D) cell culture models have been proposed as viable and biomimetic alternatives for initial drug screening, allowing the improvement of the efficiency of drug development. These models are gaining popularity, given their ability to reproduce key aspects of the tumor microenvironment, concerning the 3D tumor architecture as well as the interactions of tumor cells with the extracellular matrix and surrounding non-tumor cells. The development of accurate 3D models may become beneficial to decrease the use of laboratory animals in scientific research, in accordance with the European Union’s regulation on the 3R rule (Replacement, Reduction, Refinement). This …