Tisch Cancer Institute 1 article published in JoVE Bioengineering 2D and 3D Matrices to Study Linear Invadosome Formation and Activity Julie Di Martino*1,2, Elodie Henriet*1, Zakaria Ezzoukhry1,3,4, Chandrani Mondal2, Jose Javier Bravo-Cordero2, Violaine Moreau1, Frederic Saltel1 1INSERM U1053, 2Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute, 3Faculté de Médecine, Casablanca, Université Mohammed 6 des Sciences de la Santé (UM6SS), 4Laboratoire National de Référence This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.