Abstract:The ability of cells to follow gradients of extracellular matrix stiffnessdurotaxis-has been implicated in development, fibrosis and cancer. Durotaxis is established as a single cell phenomenon but whether it can direct the motion of cell collectives is unknown. Here we found that multicellular clusters 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, and required the action of myosin motors and the integrity of cell-cell junctions. By extending traction microscopy to extracellular matrices of arbitrary stiffness profiles we showed that collective durotaxis originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stickslip dynamics of cell-matrix adhesions is 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. One Sentence Summary: Mechanical cooperation between cells enables an emergent mode of collective movement -3- Main Text:The ability of living cells to migrate following environmental gradients underlies a broad range of phenomena in development, homeostasis, and disease (1, 2). The best understood mode of directed cell migration is chemotaxis, the well-established ability of cells to follow gradients of soluble chemical cues (1). Some cell types are also able to follow gradients in the stiffness of their extracellular matrix (ECM), a process known as durotaxis (3-10).Durotaxis has been implicated in development (11), fibrosis (12) and cancer (13), but its underlying mechanisms remain unclear.Most of our understanding of directed cell migration has been obtained in single isolated cells. However, fundamental processes during development, wound healing, tissue regeneration, and some forms of cancer cell invasion are driven by directed migration of cell groups (14-16). Cell-cell interactions within these groups provide cooperative mechanisms of cell guidance that are altogether inaccessible to single cells (14-20). Here we investigated whether cell groups undergo collective durotaxis, and the cooperative nature of underlying mechanisms.Using stencils of magnetic PDMS, we micropatterned rectangular clusters (500 µm width) of human mammary epithelial cells (MCF-10A) on fibronectin-coated polyacrylamide gel substrates exhibiting uniform stiffness or a stiffness gradient (51 ± 17 kPa/mm, Fig. S1) (21). Upon removal of the PDMS stencil, clusters migrating on uniform gels displayed symmetric expansion (Fig. 1A,C,E,G, Fig. S2, Movie S1), whereas clusters migrating on stiffness gradients displayed a robust asymmetry characterized by faster, more persistent expansion towards the stiff edge (Fig. 1B-D-F-H, Fig. S2, Movie S1). This result was also -4-observed in clusters of...

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. 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. 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. 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, 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.

No abstract

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to its specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and micro-damage, accumulated by fatigue or creep, is removed minimizing the risk of fracture. Nevertheless, bone is not always able to repair itself completely. Actually, if bone repairing function is slower than micro-damage accumulation, a type of bone fracture, usually known as "stress fracture", can finally evolve. In this paper, we propose a bone remodelling continuous model able to simulate micro-damage growth and repair in a coupled way and able therefore to predict the occurrence of "stress fractures". The biological bone remodelling process is modelled in terms of equations that describe the activity of basic multicellular units. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation is achieved. In overloading, bone porosity decreases unless the damage rate is so high that causes resorption or "stress fracture".

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions.Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix.In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.

International audienc

No abstract

Cancer cells have the ability to migrate from the primary (original) site to other places in the body. The extracellular matrix affects cancer cell migratory capacity and has been correlated with tissue-specific spreading patterns. However, how the matrix orchestrates these behaviors remains unclear. Here, we investigated how both higher collagen concentrations and TGF-β regulate the formation of H1299 cell (a non-small cell lung cancer cell line) spheroids within 3D collagen-based matrices and promote cancer cell invasive capacity. We show that at low collagen concentrations, tumor cells move individually and have moderate invasive capacity, whereas when the collagen concentration is increased, the formation of cell clusters is promoted. In addition, when the concentration of TGF-β in the microenvironment is lower, most of the clusters are aggregates of cancer cells with a spheroid-like morphology and poor migratory capacity. In contrast, higher concentrations of TGF-β induced the formation of clusters with a notably higher invasive capacity, resulting in clear strand-like collective cell migration. Our results show that the concentration of the extracellular matrix is a key regulator of the formation of tumor clusters that affects their development and growth. In addition, chemical factors create a microenvironment that promotes the transformation of idle tumor clusters into very active, invasive tumor structures. These results collectively demonstrate the relevant regulatory role of the mechano-chemical microenvironment in leading the preferential metastasis of tumor cells to specific tissues with high collagen concentrations and TFG-β activity.