Abstract
Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFβ1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFβ1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.
Highlights
Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination
M Bissel’s team elegantly demonstrated the relevance of using 3D systems to investigate cancer mechanisms, by creating a prototypical model of the mammary gland acinus, where transforming growth factor-β1 (TGFβ1)-induced EMT occurred16. 3D models where cells are completely surrounded by a supportive 3D matrix, i.e. hydrogel-based entrapment systems, are the most relevant systems for modulating cell-matrix interactions[17,18,19]
Our 2D model evolved towards a new 3D in vitro model, by combining the inducible epithelial cell line (EpH4)[12,13] and a bioengineered Extracellular matrix (ECM)-like matrix with independently tunable properties, to explore the inter-conversion between E and M states during EMT and its reversion (MET)
Summary
Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Tumor cells may undergo partial EMT with transitory acquisition of mesenchymal characteristics while retaining epithelial features These intermediate states, so-called metastable phenotypes, are characterized by phenotypic heterogeneity and cellular plasticity and likely represent the most aggressive clones in a tumor[6,7,8]. The selected 3D matrix, composed of an optimized soft alginate hydrogel functionalized with cell adhesive RGD peptides[23,24], supported epithelial morphogenesis, promoting the formation of acinar-like structures similar to those present in mammary tissue, and allowed TGFβ1-induced generation of cells with mesenchymal-like and intermediate phenotypes, providing a useful tool to unravel cellular alterations associated with EMT/MET
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