Significance: Tumor growth intrinsically generates pressure onto the surrounding tissues, which conversely compress the tumor. These mechanical forces have been suggested to contribute to tumor growth regulation. We developed a microfluidic technique to produce 3D cell-based assays and to interrogate the interplay between tumor growth and mechanics in vitro. Multicellular spheroids are grown in permeable elastic capsules. Capsule deformation provides a direct measure of the exerted pressure. By simultaneously imaging the spheroid by confocal microscopy, we show that confinement induces a drastic cellular reorganization, including increased motility of peripheral cells. We propose that compressive stress has a beneficial impact on slowing down tumor evolution but may have a detrimental effect by triggering cell invasion and metastasis.

Atomic force microscopy (AFM) allows the acquisition of high-resolution images and the measurement of mechanical properties of living cells under physiological conditions. AFM cantilevers with blunted pyramidal tips are commonly used to obtain images of living cells. Measurement of mechanical properties with these tips requires a contact model that takes into account their blunted geometry. The aim of this work was to develop a contact model of a blunted pyramidal tip and to assess the suitability of pyramidal tips for probing mechanical properties of soft gels and living cells. We developed a contact model of a blunted pyramidal tip indenting an elastic half-space. We measured Young’s modulus $\left(E\right)$ and the complex shear modulus $(G^{*}=G^{\prime }+\mathrm{i}{G}^{″})$ of agarose gels and A549 alveolar epithelial cells with pyramidal tips and compared them with those obtained with spherical tips. The gels exhibited an elastic behavior with almost coincident loading and unloading force curves and negligible values of $G^{″}$. $E$ fell sharply with indentation up to $\sim 300\mathrm{nm}$, showing a linear regime for deeper indentations. A similar indentation dependence of $E$ with twofold lower values at the linear regime was obtained with the spherical tip fitted with Hertz’s model. The dependence of $E$ on indentation in cells paralleled that found in gels. Cells exhibited viscoelastic behavior with $G^{″}∕G^{\prime }\sim 1∕4$. Pyramidal tips commonly used for AFM imaging are suitable for probing mechanical properties of soft gels and living cells.

Microrheology of cells suggests that the dynamics of single filaments in the cytoskeleton dominate at high frequencies. This response can be used to detect differences between cell types and states — including benign and malignant cancer cells. Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond1. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce2. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range—up to the frequency characterizing the molecular components—provides a mechanistic understanding of cell mechanics.

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Bridging the Titin Gap: The muscle protein titin is a molecular spring that has been extensively studied by single-molecule unfolding experiments and by molecular simulation. However, experimental and simulated unfolding could not be compared directly because they differ by orders of magnitude in pulling velocity. Rico et al. (p 741 ) developed high-speed force spectroscopy to pull titin molecules at speeds that reach the lower limits of molecular dynamics simulations. Bridging the gap between simulation and experiment clarified the mechanism of conformational changes in titin.