Excessive endometrial PlGF- Rac1 signalling underlies endometrial cell stiffness linked to pre-eclampsia.

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).

Spatial patterning of nanofibrous collagen scaffolds modulates fibroblast morphology.

Current knowledge about cell-biomaterial interactions is often based on two-dimensional (2D) cell culture systems like protein-coated glass slides. However, such smooth surfaces cannot mimic the nanofibrous environment of the native extracellular matrix (ECM). It is therefore a major challenge to transfer the results from 2D surfaces to 3D protein scaffolds with biomimetic nanofiber architecture. To understand the influence of different protein topographies on the cell response we introduce a new process to fabricate binary collagen scaffolds of variable thickness with spatially controlled regions of nanofibrous and smooth topography. We used pH-induced self-assembly to prepare collagen nanofibers with diameters between 130 and 150 nm on glass surfaces, which were partly covered with a polymer mask. After cross-linking with glutaraldehyde, smooth collagen films were prepared on the remaining glass regions. Atomic force microscopy revealed a much lower surface roughness of smooth collagen compared to nanofibers. Subsequently, we studied the viability, morphology and migration of 3T3 fibroblasts on both collagen topographies. We found small, elongated fibroblasts with few, long filopodia on collagen nanofibers whereas large, flat fibroblasts with many short filopodia were observed on smooth collagen. Actin stress fibers on collagen nanofibers were substantially reduced in comparison to smooth collagen. Live cell tracking revealed that fibroblasts on thin nanofibrous collagen migrated faster than on smooth collagen. In summary, binary collagen scaffolds enabled us for the first time to study cell responses to topographical cues on a single protein scaffold. In future, it will be intriguing to transfer our patterning process to other proteins to study fundamental principles of topography-dependent cell recognition processes.

Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study.

Lung surfactants are used for reducing alveolar surface tension in preterm infants to ease breathing. Phospholipid films with surfactant proteins regulate the activity of alveolar macrophages and reduce inflammation. Aberrant skin wound healing is characterized by persistent inflammation. The aim of the study was to investigate if lung surfactant can promote wound healing. Preclinical wound models, e.g. cell scratch assays and full-thickness excisional wounds in mice, and a randomized, phase I clinical trial in healthy human volunteers using a suction blister model were used to study the effect of the commercially available bovine lung surfactant on skin wound repair. Lung surfactant increased migration of keratinocytes in a concentration-dependent manner with no effect on fibroblasts. Significantly reduced expression levels were found for pro-inflammatory and pro-fibrotic genes in murine wounds. Because of these beneficial effects in preclinical experiments, a clinical phase I study was initiated to monitor safety and tolerability of surfactant when applied topically onto human wounds and normal skin. No adverse effects were observed. Subepidermal wounds healed significantly faster with surfactant compared to control. Our study provides lung surfactant as a strong candidate for innovative treatment of chronic skin wounds and as additive for treatment of burn wounds to reduce inflammation and prevent excessive scarring.

Direct evidence that tumor cells soften when navigating confined spaces.

The mechanical properties of cells strongly regulate many physiological and pathological processes. For example, in cancer, invasive and metastatic tumor cells have often been reported to be softer than nontumor cells, raising speculation that cancer cells might adaptively soften to facilitate migration through narrow tissue spaces. Despite growing interest in targeting cell softening to impede invasion and metastasis, it remains to be directly demonstrated that tumor cells soften as they migrate through confined spaces. Here, we address this open question by combining topographically patterned substrates with atomic force microscopy (AFM). Using a polydimethylsiloxane open-roof microdevice featuring tapered, fibronectin-coated channels, we followed the migration of U2OS cells through various stages of confinement while simultaneously performing AFM indentation. As cells progress from unconfined migration to fully confined migration, cells soften and exclude Yes-associated protein from the nucleus. Superresolution imaging reveals that confinement induces remodeling of actomyosin stress fiber architecture. Companion studies with flat one-dimensional microlines indicate that the changes in cytoarchitecture and mechanics are intrinsically driven by topographical confinement rather than changes in cellular aspect ratio. Our studies represent among the most direct evidence to date that tumor cells soften during confined migration and support cell softening as a mechanoadaptive mechanism during invasion.

Nano-mechanical mapping of interdependent cell and ECM mechanics by AFM force spectroscopy.

Extracellular matrix (ECM), as a dynamic component of the tissue, influences cell behavior and plays an important role in cell mechanics and tissue homeostasis. Reciprocally, this three-dimensional scaffold is dynamically, structurally and mechanically modified by cells. In the field of biophysics, the independent role of cell and ECM mechanics has been largely investigated; however, there is a lack of experimental data reporting the interdependent interplay between cell and ECM mechanics, measured simultaneously. Here, using Atomic Force Microscopy (AFM) we have characterized five different decellularized matrices diverse in their topography, ECM composition and stiffness and cultured them with normal and pathological fibroblasts (scar and Dupuytren's). We investigated the change in topography and elasticity of these matrices due to cell seeding, by using AFM peak force imaging and mechanical mapping, respectively. We found normal fibroblasts soften these matrices more than pathological fibroblasts, suggesting that pathological fibroblasts are profoundly influencing tissue stiffening in fibrosis. We detected different ECM composition of decellularized matrices used here influences fibroblast stiffness, thus highlighting that cell mechanics not only depends on ECM stiffness but also on their composition. We used confocal microscopy to assess fibroblasts invasion and found pathological fibroblasts were invading the matrices deeper than normal fibroblasts.

Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome.

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts.

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-ß1 (TGF-ß1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-ß1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-ß1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

The role of the microenvironment in the biophysics of cancer.

During the last decades, cell mechanics has been recognized as a quantitative measure to discriminate between many physiological and pathological states of single cells. In the field of biophysics of cancer, a large body of research has been focused on the comparison between normal and cancer mechanics and slowly the hypothesis that cancer cells are softer than their normal counterparts has been accepted, even though in situ tumor tissue is usually stiffer than the surrounding normal tissue. This corroborates the idea that the extra-cellular matrix (ECM) has a critical role in regulating tumor cell properties and behavior. Rearrangements in ECM can lead to changes in cancer cell mechanics and in specific conditions the general assumption about cancer cell softening could be confuted. Here, we highlight the contribution of ECM in cancer cell mechanics and argue that the statement that cancer cells are softer than normal cells should be firmly related to the properties of cell environment and the specific stage of cancer cell progression. In particular, we will discuss that when employing cell mechanics in cancer diagnosis and discrimination, the chemical, the topographical and - last but not least - the mechanical properties of the microenvironment are very important.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

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.

Comparison of viscoelastic properties of cancer and normal thyroid cells on different stiffness substrates.

We used atomic force microscopy (AFM) technique to measure the viscoelastic response of cancer and normal thyroid cells on different stiffness polyacrylamide gels. After applying a step in contact we recorded the stress relaxation of cells in order to measure their viscous and elastic properties. With the help of an extended version of the Hertz model, we could quantify for the first time by AFM the elastic modulus and the dynamic viscosity of cells on substrates with different stiffnesses. We have cultured anaplastic carcinoma and normal thyroid cells on three different substrates: polyacrylamide gels with elastic modulus in a range of 3-5 and 30-40 kPa and "infinitely" stiff Petri dishes. Whereas normal thyroid cells adapted their mechanical properties to different stiffness substrates, cancer cells were less affected by the surrounding stiffness. Normal cells changed the elastic modulus from 1.2 to 1.6 and to 2.6 kPa with increasing substrate stiffness; the dynamic viscosity values varied from 230 to 515 and to 470 Pa·s, accordingly. By contrast, the values for cancer cells were rather constant regardless of substrate stiffness (in average the elastic modulus was 1.3 kPa and the dynamic viscosity was 300 Pa·s). This difference in sensing and reacting to the mechanical properties of the substrate shows that normal and cancer cells interact differently with the neighboring tissue, which may be related to the ability of cancer cells to form metastases.

Measuring the viscoelastic creep of soft samples by step response AFM.

We have measured the creep response of soft gels and cells after applying a step in loading force with atomic force microscopy (AFM). By analysing the creep response data using the standard linear solid model, we can quantify the viscous and elastic properties of these soft samples independently. Cells, in comparison with gels of similar softness, are much more viscous, as has been qualitatively observed in conventional force curve data before. Here, we quantify the spring constant and the viscous damping coefficient from the creep response data. We propose two different modes for applying a force step: (1) indirectly by increasing the sample height or (2) directly by employing magnetic cantilevers. Both lead to similar results, whereas the latter seems to be better defined since it resembles closely a constant strain mode. The former is easier to implement in most instruments, and thus may be preferable from a practical point of view. Creep analysis by step response is much more appropriate to analyse the viscoelastic response of soft samples like cells than the usually used force curve analysis.

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Reversible Holographic Patterns on Azopolymers for Guiding Cell Adhesion and Orientation.

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nanopatterning techniques have been employed to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated the response of NIH-3T3 cells to reversible topographic signals encoded on light-responsive azopolymer films. Switchable patterns were fabricated by means of a well-established holographic setup. Surface relief gratings were realized with Lloyd's mirror system and erased with circularly polarized or incoherent light. Cell cytoskeleton organization and focal adhesion assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as a trigger. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way toward an in situ and real-time investigation of the material-cytoskeleton crosstalk caused by the intrinsic properties of azopolymers.

Topographic cell instructive patterns to control cell adhesion, polarization and migration.

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features' dimensions and surface chemistry.