AFM-based rheology methods enable the investigation of the viscoelastic properties of cancer cells. Such properties are known to be essential for cell functions, especially for malignant cells. Here, the relevance of the force modulation method was investigated to characterize the viscoelasticity of bladder cancer cells of various invasiveness on soft substrates, revealing that the rheology parameters are a signature of malignancy. Furthermore, the collagen microenvironment affects the viscoelastic moduli of cancer cell spheroids; thus, collagen serves as a powerful proxy, leading to an increase of the dynamic moduli vs. frequency, as predicted by a double power law model. Taken together, these results shed new light on how cancer cells and tissues adapt their viscoelastic properties depending on their malignancy and the microenvironment. This method could be an attractive way to control their properties in the future, based on the similarity of spheroids with in vivo tumor models.

Cancer cell migration is a widely studied topic but has been very often limited to two dimensional motion on various substrates. Indeed, less is known about cancer cell migration in 3D fibrous-extracellular matrix (ECM) including variations of the microenvironment. Here we used 3D time lapse imaging on a confocal microscope and a phase correlation method to follow fiber deformations, as well as cell morphology and live actin distribution during the migration of cancer cells. Different collagen concentrations together with three bladder cancer cell lines were used to investigate the role of the metastatic potential on 3D cell migration characteristics. We found that grade-3 cells (T24 and J82) are characterized by a great diversity of shapes in comparison with grade-2 cells (RT112). Moreover, grade-3 cells with the highest metastatic potential (J82) showed the highest values of migration speeds and diffusivities at low collagen concentration and the greatest sensitivity to collagen concentration. Our results also suggested that the small shape fluctuations of J82 cells are the signature of larger migration velocities. Moreover, the displacement fields generated by J82 cells showed significantly higher fiber displacements as compared to T24 and RT112 cells, regardless of collagen concentration. The analysis of cell movements enhanced the fact that bladder cancer cells were able to exhibit different phenotypes (mesenchymal, amoeboid). Furthermore, the analysis of spatio-temporal migration mechanisms showed that cancer cells are able to push or pull on collagen fibers, therefore producing efficient local collagen deformations in the vicinity of cells. Our results also revealed that dense actin regions are correlated with the largest displacement fields, and this correlation is enhanced for the most invasive J82 cancer cells. Therefore this work opens up new routes to understand cancer cell migration in soft biological networks.