A 3D in vitro model to explore the inter-conversion between epithelial and mesenchymal states during EMT and its reversion

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.

Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes

“…Spheroid cultures are considered to be better suited for studying a wide range of cellular activities, such as tissue development and disease. [9][10][11] MET is an essential initiation step in stem cell reprogramming and thus dedifferentiation of mesenchymal lineage cell types. It has been reported that cancer cell spheroids mimic a more "realistic" cellular response to radioactive [4] and chemical [5] treatments than cancer cells maintained as 2-dimensional (2D) monolayer culture.…”

“…These genes relate to cell differentiation and cell reprogramming, particularly key pluripotency genes Oct4, Sox2, and Nanog. [9][10][11] MET is an essential initiation step in stem cell reprogramming and thus dedifferentiation of mesenchymal lineage cell types. [9] Research has also revealed that spheroid cultures favors mesenchymal-to-epithelial transition (MET), maintenance and even induction of epithelial phenotypes in various cell types.…”

Rebooting the collagen gel: Artificial hydrogels for the study of epithelial mesenchymal transformation

“…With regard to the design of a successor of the aging collagen gel assay, we can focus on biomaterials that will encourage epithelial migration and EMT-stimulated morphological changes and invasion (Gill et al, 2012;Bidarra et al, 2016). As the natural extracellular microenvironment is a highly hydrated network, artificial hydrogels are the material of choice.…”

“…1) (Gill et al, 2012;Weiss et al, 2012;O'Connor et al, 2015;Wei et al, 2015;Bidarra et al, 2016). 1) (Gill et al, 2012;Weiss et al, 2012;O'Connor et al, 2015;Wei et al, 2015;Bidarra et al, 2016).…”

Alginate Matrix of MDA-MB-231 Breast Cancer Cell 3D Culturing Alters CD44 and CD24 mRNA Levels and Induces ALDH1 Expression

“…Among scaffolds culturing the use of alginate, a polymer obtained from brown algae, has been widely applied for 3D cancer cell culturing [21,22] and to cancer stem-cell [23,24]. Additionally, alginate has intrinsic biocompatibility and capacity for forming gel with well-defined features, such as size and density [25] and moreover, it mimics the extracellular matrix or basal membrane to support cell functions and metabolism [26].…”