Instituto de Medicina Molecular 1 article published in JoVE Cancer Research A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies Ana C. Martins Cavaco1,2, Johannes A. Eble1 1Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 2Instituto de Medicina Molecular, Lisbon The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.