Nihon University School of Dentistry 1 article published in JoVE Medicine Three-dimensional Co-culture Model for Tumor-stromal Interaction Masafumi Horie1,2, Akira Saito1,3, Yoko Yamaguchi4, Mitsuhiro Ohshima5, Takahide Nagase1 1Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 2Department of Clinical Laboratory, Graduate School of Medicine, The University of Tokyo, 3Division for Health Service Promotion, The University of Tokyo, 4Department of Biochemistry, Nihon University School of Dentistry, 5Department of Biochemistry, Ohu University School of Pharmaceutical Sciences Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.