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The ability of cells to follow gradients of extracellular matrix stiffness—durotaxis—has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion.

The formation of cellular adhesion complexes is important in normal and pathological cell activity, and is determined by the force imposed by the combined effect of the distribution of extracellular matrix molecules and substrate rigidity. Forcing cellular sticking points Integrin-mediated cell adhesion is a critical parameter in many physiological and pathological processes, and can be harnessed to modulate cellular responses to synthetic biomaterials for various applications. This paper reports that the formation of cellular focal adhesions is regulated by both the distribution of extracellular matrix (ECM) ligands and substrate rigidity. The authors suggest that these parameters dictate how much force is loaded onto each ECM–integrin bond, which they call a 'molecular clutch', from myosin motors within the cell pulling on the bonds via the actin filaments. They propose a model to explain how cells attempt to regulate the force loaded onto each molecular clutch by recruiting more integrins to the adhesion to generate new molecular clutches. If the maximum number of available clutches is generated but the force per clutch is too high, the focal adhesion collapses. Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts 1 , 2 . Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin–ligand bonds are separated by more than a few tens of nanometres 3 , 4 , 5 , 6 . It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly 3 , 7 , 8 , 9 . Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model 10 , 11 , in which individual integrin–ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

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.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli. Lolo et al. show caveolin-1 functions in non-caveolae structures termed dolines. Whereas caveolae respond to high forces over a mechanical threshold, dolines transduce low and medium mechanical forces gradually in a complementary buffering system.

A stretch in the cells How do cells in our body go about their routine mechanical business of stretching, contracting and remodelling? This question has far-reaching implications for understanding airway narrowing in asthma, cell invasion in cancer and vessel constriction in cardiovascular disease. The answer, arrived at in a novel experimental system measuring the 'stretch' of human airway smooth muscle cells, is that the cell has much in common with familiar materials including tomato ketchup, shaving foam and toothpaste. These materials fluidize when deformed, as do granular materials including sugar in a bowl or coffee beans. Such materials act as a glass-like intermediate form of matter, neither solid nor fluid but retaining features of both. How the cell goes about its routine mechanical business of stretching, contracting, and remodelling has implications for understanding excessive airway narrowing in asthma, cell invasion in cancer and vessel constriction in vascular disease. Surprisingly, the cell is an intermediate form of matter, neither solid nor fluid but retaining features of both - that responds to stretch by fluidizing, much as do common pastes, foams, clays, and colloids. With every beat of the heart, inflation of the lung or peristalsis of the gut, cell types of diverse function are subjected to substantial stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodelling and gene expression 1 , 2 . Here, we report that in response to transient stretch the cytoskeleton fluidizes in such a way as to define a universal response class. This finding implicates mechanisms mediated not only by specific signalling intermediates, as is usually assumed, but also by non-specific actions of a slowly evolving network of physical forces. These results support the idea that the cell interior is at once a crowded chemical space 3 and a fragile soft material in which the effects of biochemistry, molecular crowding and physical forces are complex and inseparable, yet conspire nonetheless to yield remarkably simple phenomenological laws. These laws seem to be both universal and primitive, and thus comprise a striking intersection between the worlds of cell biology and soft matter physics.

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus ( G *( ω)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1–100 Hz) and at different loading forces (0.1–0.9 nN) with atomic force microscopy. G *( ω) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G′( ω) increased with frequency following a power law with exponent ∼0.2. The loss modulus G″( ω) was ∼2/3 lower and increased similarly to G′( ω) up to ∼10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G′( ω) and G″( ω). G *( ω) conformed to the power-law model with a structural damping coefficient of ∼0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.

An adverse role for obstructive sleep apnea (OSA) in cancer epidemiology and outcomes has recently emerged from clinical and animal studies. In animals, intermittent hypoxia (IH) mimicking OSA promotes tumor malignancy both directly and via host immune alterations. We hypothesized that IH could potentiate cancer aggressiveness through activation of the cyclooxygenase-2 (COX-2) pathway and the concomitant increases in prostaglandin E2 (PGE 2 ). The contribution of the COX-2 in IH-induced enhanced tumor malignancy was assessed using celecoxib as a COX-2 specific inhibitor in a murine model of OSA bearing Lewis lung carcinoma (LLC1) tumors. Exposures to IH accelerated tumor progression with a tumor associated macrophages (TAMs) shift towards a pro-tumoral M2 phenotype. Treatment with celecoxib prevented IH-induced adverse tumor outcomes by inhibiting IH-induced M2 polarization of TAMs. Furthermore, TAMs isolated from IH-exposed mice treated with celecoxib reduced the proliferation of LLC1 naïve cells, while the opposite occurred with placebo-treated IH-exposed mice. Finally, in vitro IH exposures of murine macrophages and LLC1 cells showed that both cell types increased PGE 2 release in response to IH. These results suggest a crucial role for the COX-2 signaling pathway in the IH-exacerbated malignant processes, and designate macrophages and lung adenocarcinoma cells, as potential sources of PGE 2 .

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.