Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models

Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell–cell and cell–matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease–matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.