Quantifying Cancer Epithelial-Mesenchymal Plasticity and its Association with Stemness and Immune Response

Abstract

Cancer cells can acquire a spectrum of stable hybrid epithelial/mesenchymal (E/M) states during epithelial-mesenchymal transition (EMT). Cells in these hybrid E/M phenotypes often combine epithelial and mesenchymal features and tend to migrate collectively commonly as small clusters. Such collectively migrating cancer cells play a pivotal role in seeding metastases and their presence in cancer patients indicates an adverse prognostic factor. Moreover, cancer cells in hybrid E/M phenotypes tend to be more associated with stemness which endows them with tumor-initiation ability and therapy resistance. Most recently, cells undergoing EMT have been shown to promote immune suppression for better survival. A systematic understanding of the emergence of hybrid E/M phenotypes and the connection of EMT with stemness and immune suppression would contribute to more effective therapeutic strategies. In this review, we first discuss recent efforts combining theoretical and experimental approaches to elucidate mechanisms underlying EMT multi-stability (i.e., the existence of multiple stable phenotypes during EMT) and the properties of hybrid E/M phenotypes. Following we discuss non-cell-autonomous regulation of EMT by cell cooperation and extracellular matrix. Afterwards, we discuss various metrics that can be used to quantify EMT spectrum. We further describe possible mechanisms underlying the formation of clusters of circulating tumor cells. Last but not least, we summarize recent systems biology analysis of the role of EMT in the acquisition of stemness and immune suppression.

Keywords: CTC clusters; EMT metrics; EMT spectrum; epithelial–mesenchymal transition; hybrid epithelial/mesenchymal phenotypes; immune suppression; stemness; systems biology.

Figure 1

An overview of epithelial–mesenchymal plasticity causes and consequences. Multiple signaling pathways such as TGF-$\beta$, HIF-1$\alpha$ and Notch have been implicated in driving epithelial–mesenchymal transition (EMT). All these pathways tend to converge to a core regulatory circuit which includes two EMT-inducing transcription factors (EMT-TFs), SNAIL and ZEB, and two microRNAs, miR-34 and miR-200. The core regulatory circuit exhibits multi-stable dynamics: multiple stable steady states for the same level of EMT-inducing signal. These stable steady states contain different levels of SNAIL/ZEB/miR-34/miR-200 and thus corresponding to different EMT-associated phenotypes. The multi-stable dynamics of the core regulatory circuit allow for transitions among different stable states which leads to epithelial–mesenchymal plasticity. Cancer epithelial–mesenchymal plasticity typically enhances metastasis, allowing for disparate forms of migration and dissemination. In addition, epithelial–mesenchymal plasticity has been implicated in the acquisition of stem cell-like properties and immune evasion.

Figure 2

Multiple mechanisms can lead to EMT-associated population heterogeneity. (A) During cell division, noise can lead to asymmetric partitioning of molecules among daughter cells. This may lead to the daughter cell exhibiting a phenotype different from that of the parent cell. Consequently, the fractions of cells exhibiting different phenotypes can change over time. (B) Cell–cell communication (left) mediated by, for example Notch-Delta-Jagged signaling, can lead to cells spontaneously acquiring “signal sender”, “signal receiver”, and “signal sender/receiver” phenotypes in a population (right: different colors correspond to different cell phenotypes). (C) The kinetics of gene regulation can vary in different cells due to stochasticity in kinetic parameters. Thus, each parameter can exhibit some variation in its value in a given isogenic population (left: each curve denotes the distribution of values of an individual parameter in a given population). This heterogeneity can lead to cells in the population acquiring different phenotypes in response to the same external cues (right: heatmap indicates the emergence of various phenotypes. Each row represents a cell and each column represents the expression level of one gene. Red color represents relatively high gene expression and blue color represents relatively low gene expression with white indicating intermediate expression levels).

Figure 3

Emergence of hybrid E/M phenotypes demonstrated by a combination of theoretical and experimental efforts. The cell lines non-small cell lung cancer (NSCLC) H1975 and human bladder cancer (HBC) RT4 exhibit a hybrid E/M phenotype at a single-cell level. Hybrid E/M phenotypes have also been characterized in vivo using mouse models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC).

Figure 4

Notch–Delta and Notch–Jagged signaling give rise to opposite cell patterning of EMT. (A) Coupling of Notch–Delta signaling with the core EMT regulatory circuit. The Notch intracellular domain NICD suppresses the endogenous expression of Delta ligands upon activation of Notch receptors by exogenous Delta ligands (top, red color represents the ‘receiver phenotype’ (high notch), green color represents ‘sender phenotype’ (high delta)). Cell patterning of EMT in presence of a strong Notch–Delta signaling (bottom). (B) Coupling of Notch–Jagged signaling with the core EMT regulatory circuit. NICD promotes the endogenous expression of Jagged ligands upon activation of Notch receptors by exogenous Jagged ligands (top, blue color represents a ‘sender/receiver phenotype’ (high Jagged)). Cell patterning of EMT in presence of a strong Notch–Jagged signaling (bottom). Hexagons with different colors depict epithelial (green), hybrid E/M (yellow), and mesenchymal (red) cells. Figures in the right panel are adapted from [53].

Figure 5

The spatial patterning of CSCs with different EMT phenotypes. Tumor-stroma interactions can give rise to a gradient of TGF- β (top, blue scale). In the periphery of the tumor, most cells are mesenchymal (red spheres), while the interior is mostly composed by hybrid E/M and epithelial cells (yellow and green spheres, respectively). CSCs are mostly mesenchymal in the periphery (black-dotted red spheres) and mostly hybrid E/M in the interior (black-dotted yellow spheres).

Figure 6

The relationship between EMT and immune response as shown by various approaches.