Abstract
Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.
Highlights
A great deal of experimental evidence has shown that mechanical stimuli from the cell microenvironment play a key role in affecting several types of cell behaviour, both in healthy and in pathological conditions[1,2,3]
The elastic modulus of each gel was measured at the sub-micrometer scale, the same length scale of the actual cell sensing[34], using Atomic Force Microscopy (AFM) nanoindentation technique
A weaker cross-linker content (i.e. 0.2 M CaCl2) did not allow significantly different stiffnesses to be obtained among the gels, despite the increase in alginate percentage
Summary
A great deal of experimental evidence has shown that mechanical stimuli from the cell microenvironment play a key role in affecting several types of cell behaviour, both in healthy and in pathological conditions[1,2,3]. Cells are embedded within a complex three-dimensional gel – the Extracellular Matrix (ECM) – that provides mechanical support while directing cellular behaviour[1,7]. The lack of reliability seems to be associated with the following main aspects: cell source (e.g. phenotype selection), model dimensionality and microenvironment complexity[17]. Another main issue in cancer biology regards the use of animal models. In vivo animal models do not allow direct investigation of specific microenvironmental cues or their influence on cellular evolution, and present well-known ethical and cost-related limits[18]
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