Current research in cancer pathologies, have been showing the role played by mechanical stress in cell malignancy. In this work we hypothesize that this mechanical stimulation can be resembled by the use of three-dimensional polymers-based scaffolds.

To this purpose, MCF7 breast cancer cells were immobilized into alginate-gelatin microcapsules (named as "Artificial Tumors" in this work) having and elasticity of 25 kPa (measured as Young's Moduli). After one week post encapsulation, MCF7 cells were isolated from the bulk of the artificial tumors (i.e. entrapped cells) or recovered from the cell culture media after their release from these matrices (i.e. cells breaking out the confinement), and analyzed by single-cell traction force microscopy and RNA-seq. These results were then compared to the biomechanical and genetic profile shown by cancer cells cultured on flasks, as well as MCF7 cells cultured on flat hydrogels having a similar elasticity to artificial tumours (i.e. 23 kPa, measured as Young’s Moduli).

Our in vitro results show that MCF7 cultured in these artificial tumors enhanced the exertion of cell-matrix adhesion forces, upregulating the expression of proteins involved in the actomyosin machinery, compared to cells cultured on flat surfaces.

In order to validate these results in vivo, MCF7 cells were isolated from 2D or 3D milieus, being then injected into the Zebrafish bloodstream, with the purpose to determine their capability to generate secondary tumours. Interestingly, in vivo results show the same tendency detected in vitro, indicating that confinement and mechanical stress are required to increase the metastatic activity of cancer cells.

Our results are demonstrating that the use of 3D hydrogel-based scaffolds resembles relevant biophysical properties found in primary tumours, enabling us to trigger malignant responses already described in the clinic. Remarkably, these phenotypical characteristics are kept by MCF7 cells, after being transferred from in vitro to in vivo systems, suggesting the existence of a mopho-mechanical memory in cancer cells preconditioned to different 2D or 3D milieus.

Finally, these results are also recommending the use of 3D culture strategies, instead of traditional 2D surfaces, to study the malignant character of cancer cells in vitro.