Isolation and Identification of EMT Subtypes.

Abstract

Metastasis and chemoresistance, the most lethal features of cancer progression, are strongly associated with a form of cellular plasticity known as the epithelial-to-mesenchymal transition (EMT). Carcinoma cells undergoing EMT lose their epithelial morphology and become more mobile, allowing them to invade and migrate more efficiently. This shift is also associated with a change in vulnerability to chemotherapeutic agents. Importantly, EMT does not involve a single mechanism, but rather encompasses a spectrum of phenotypes with differing degrees of epithelial and mesenchymal characteristics. These hybrid/partial epithelial-mesenchymal states are associated with other important aspects of tumor biology, such as distinct modes of cellular invasion and drug resistance, illustrating the need to further characterize this phenomenon in tumor cells. Although simple in theory, the identification of tumor cells that have undergone EMT in vivo has proven difficult due to their high similarity to other mesenchymal cells that populate tumor stroma, such as cancer-associated fibroblasts. This protocol describes two methods for isolating epithelial and EMT cancer cell populations from primary murine tumors and cultured cancer cells to identify different EMT subtypes. These populations can then be used for several applications, including, but not limited to, functional studies of motility or invasion, gene expression analysis (RNA sequencing and RT-qPCR), DNA sequencing, epigenetic analysis, tumor subtyping, western blotting, immunohistochemistry, etc. Finally, we describe a flow cytometry-based approach to identify and study tumors cells that are undergoing partial EMT.