Integrative Transcriptomic Analysis Reveals a Multiphasic Epithelial–Mesenchymal Spectrum in Cancer and Non-tumorigenic Cells

Abstract

Epithelial-mesenchymal transition (EMT), the conversion between rigid epithelial cells and motile mesenchymal cells, is a reversible cellular process involved in tumorigenesis, metastasis, and chemoresistance. Numerous studies have found that several types of tumor cells show a high degree of cell-to-cell heterogeneity in terms of their gene expression signatures and cellular phenotypes related to EMT. Recently, the prevalence and importance of partial or intermediate EMT states have been reported. It is unclear, however, whether there is a general pattern of cancer cell distribution in terms of the overall expression of epithelial-related genes and mesenchymal-related genes, and how this distribution is related to EMT process in normal cells. In this study, we performed integrative transcriptomic analysis that combines cancer cell transcriptomes, time course data of EMT in non-tumorigenic epithelial cells, and epithelial cells with perturbations of key EMT factors. Our statistical analysis shows that cancer cells are widely distributed in the EMT spectrum, and the majority of these cells can be described by an EMT path that connects the epithelial and the mesenchymal states via a hybrid expression region in which both epithelial genes and mesenchymal genes are highly expressed overall. We found that key patterns of this EMT path are observed in EMT progression in non-tumorigenic cells and that transcription factor ZEB1 plays a key role in defining this EMT path via diverse gene regulatory circuits connecting to epithelial genes. We performed Gene Set Variation Analysis to show that the cancer cells at hybrid EMT states also possess hybrid cellular phenotypes with both high migratory and high proliferative potentials. Our results reveal critical patterns of cancer cells in the EMT spectrum and their relationship to the EMT process in normal cells, and provide insights into the mechanistic basis of cancer cell heterogeneity and plasticity.

Comparative Study of Transcriptomics-Based Scoring Metrics for the Epithelial-Hybrid-Mesenchymal Spectrum

“…Various attempts to characterize the spectrum of EMT at molecular and/or morphological levels have been made recently, enabled by latest developments in multiplex imaging, single-cell RNAseq and inducible systems (Mandal et al, 2016;Pastushenko et al, 2018;Stylianou et al, 2018;Cook and Vanderhyden, 2019;Devaraj and Bose, 2019;Karacosta et al, 2019;Wang W. et al, 2019;Watanabe et al, 2019;Lam et al, 2020). These approaches have highlighted the dynamical nature of EMT in driving cancer progression in patients (Jolly and Celia-Terrassa, 2019), and the heterogeneity in EMT status in cell lines and patient samples (Panchy et al, 2020;Shen et al, 2020). Further, various approaches to quantify the EMT spectrum of samples based on different signatures of tumor types have been made (Foroutan et al, 2017;Puram et al, 2017).…”

Hypoxia, partial EMT and collective migration: Emerging culprits in metastasis

“…Multiple single-cell studies have used different epithelial and mesenchymal markers to identify the relative abundance of hybrid E/M cells in cancer cell lines, for example, SNAI/E-cadherin in lung cancer cell lines (A549, LT73, and H460) [ 92 ] and in clear cell renal cell carcinoma cells [ 93 ], SNAI/E-cadherin/occludin in colorectal cancer [ 94 ], epithelial surface marker CD24 and mesenchymal marker CD44 in breast cancer cell line HMLER [ 78 ], and vimentin and keratin intermediate filaments in breast cancer cell lines [ 95 ]. In addition, hybrid E/M phenotype is also observed in circulating tumor cells (CTCs) [ 96 , 97 ], in vivo mouse models [ 46 ], lymph node progression of NSCLC patients [ 98 ] and in brain metastases and patient samples [ 99 , 100 ]. Moreover, the relative abundance of E-cadherin and vimentin in a cell population is used to classify cell lines into epithelial, mesenchymal and hybrid E/M phenotypes in many cancer types [ 44 , 101 , 102 ].…”

A plausible accelerating function of intermediate states in cancer metastasis

“…We have discussed the scenario with only one path in which epithelial cells proceed through a step-wise transition of intermediate states to become mesenchymal cells. Recently, parallel EMT paths were reported [46,47]. Here, we consider multiple paths in parallel ( Fig 3A) (Fig 3A).…”