Synthetic and tissue-derived models for studying rigidity effects on invadopodia activity.

Invasion by cancer cells through the extracellular matrix (ECM) of tissues is a critical step in cancer progression and metastasis. Actin-rich subcellular protrusions known as invadopodia are thought to facilitate this process by localizing proteinases which degrade the ECM and allow for cancer cell penetration. We have shown in vitro that invadopodia activity is regulated by the rigidity of the ECM, which suggests that matrix remodeling in vivo may also be regulated by the mechanical properties of tissues. In order to study rigidity effects on invadopodia activity in a controlled manner, we have developed assays in which we have conjugated degradable fluorescent matrix molecules to tunable synthetic substrates. In addition, we have also utilized ex vivo tissue-derived substrates to corroborate our findings. In this chapter, we present detailed protocols describing the synthesis and preparation of our synthetic substrates, polyacrylamide gels and polyurethane elastomers, for use in these matrix degradation assays as well as the steps required to utilize our tissue-derived substrates.