Chapter 24
Isolation and Identification of EMT Subtypes
Robert J. Norgard and Ben Z. Stanger
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.
Key words Epithelial-to-mesenchymal transition, EMT, Plasticity, Partial EMT, Subtypes, E-cadherin, Flow cytometry-activated cell sorting, Magnetic-activated cell sorting
1 Introduction
In response to harmful stimuli, normal tissues take protective measures to repair resulting damage. Cellular plasticity—which refers to
the ability of cells to undergo significant phenotypic and functional
changes—is one such protective measure. When aberrantly activated in cancer cells, however, plasticity can contribute to tumor
initiation, progression, and resistance to therapy [1]. In
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_24,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
The authors declare no potential conflicts of interest.
315
Isolation and Identification of EMT Subtypes
Robert J. Norgard and Ben Z. Stanger
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.
Key words Epithelial-to-mesenchymal transition, EMT, Plasticity, Partial EMT, Subtypes, E-cadherin, Flow cytometry-activated cell sorting, Magnetic-activated cell sorting
1 Introduction
In response to harmful stimuli, normal tissues take protective measures to repair resulting damage. Cellular plasticity—which refers to
the ability of cells to undergo significant phenotypic and functional
changes—is one such protective measure. When aberrantly activated in cancer cells, however, plasticity can contribute to tumor
initiation, progression, and resistance to therapy [1]. In
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_24,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
The authors declare no potential conflicts of interest.
315