Analyzing the Roles of Rho GTPases in Cancer Cell Migration with a Live Cell Imaging 3D-Morphology-Based Assay

Rho GTPases are master regulators of cytoskeleton dynamics and therefore regulate cell motility. Rho GTPases, as well as their regulators and effectors, are often deregulated in cancers and thus contribute to tumor progression to metastasis. Cancer progression involves multiple steps, including invasion of the surrounding tissues. Several methods to investigate the invasion of tumors cells in 3D matrices in vitro have been developed. In this chapter we describe a 3D-based morphology assay that can be used for medium-throughput microscopy-based screening to identify regulators of cancer cell invasion. We use this method coupled to RNAi to investigate how Rho GTPases affect prostate cancer cell morphology in 3D Matrigel.