RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.

The influence of ions on the lubricative abilities of mucin and the role of sialic acids.

Mucus reduces friction between epithelial surfaces by providing lubrication in the boundary and mixed regime. Mucins, the main macromolecule, are heavily glycosylated proteins that polymerise and retain water molecules, resulting in a hydrated biogel. It is assumed that positively charged ions can influence mucin film structure by screening the electrostatic repulsions between the negatively charged glycans on mucin moieties and draw in water molecules via hydration shells. The ionic concentration can vary significantly in different mucus systems and here we show that increasing the ionic concentration in mucin films leads to an increase in lubrication between two polydimethylsiloxane surfaces at sliding contact in a compliant oral mimic. Mucins were found to bind sodium ions in a concentration-dependent manner and increased ionic concentration appears to cause mucin films to swell when assessed by Quartz Crystal hiMicrobalance with Dissipation (QCM-D) analysis. Furthermore, we determined that the removal of negatively charged sialic acid moieties by sialidase digestion resulted in reduced adsorption to hydrophilic surfaces but did not affect the swelling of mucin films with increasing ionic concentrations. Moreover, the coefficient of friction was increased with sialic acid removal, but lubrication was still increased with increasing ionic concentrations. Taken together this suggests that sialic acids are important for lubrication and may exert this through the sacrificial layer mechanism. Ionic concentration appears to influence mucin films and their lubrication, and sialic acids, at least partly, may be important for ion binding.

Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program.

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.

Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic.

Neutrophils were previously shown to digest oxidized carbon nanotubes through a myeloperoxidase (MPO)-dependent mechanism, and graphene oxide (GO) was found to undergo degradation when incubated with purified MPO, but there are no studies to date showing degradation of GO by neutrophils. Here we produced endotoxin-free GO by a modified Hummers' method and asked whether primary human neutrophils stimulated to produce neutrophil extracellular traps or activated to undergo degranulation are capable of digesting GO. Biodegradation was assessed using a range of techniques including Raman spectroscopy, transmission electron microscopy, atomic force microscopy, and mass spectrometry. GO sheets of differing lateral dimensions were effectively degraded by neutrophils. As the degradation products could have toxicological implications, we also evaluated the impact of degraded GO on the bronchial epithelial cell line BEAS-2B. MPO-degraded GO was found to be non-cytotoxic and did not elicit any DNA damage. Taken together, these studies have shown that neutrophils can digest GO and that the biodegraded GO is non-toxic for human lung cells.

Lubrication synergy: Mixture of hyaluronan and dipalmitoylphosphatidylcholine (DPPC) vesicles.

Phospholipids and hyaluronan have been implied to fulfil important roles in synovial joint lubrication. Since both components are present in synovial fluid, self-assembly structures formed by them should also be present. We demonstrate by small angle X-ray scattering that hyaluronan associates with the outer shell of dipalmitoylphophatidylcholine (DPPC) vesicles in bulk solution. Further, we follow adsorption to silica from mixed hyaluronan/DPPC vesicle solution by Quartz Crystal Microbalance with Dissipation measurements. Atomic Force Microscope imaging visualises the adsorbed layer structure consisting of non-homogeneous phospholipid bilayer with hyaluronan/DPPC aggregates on top. The presence of these aggregates generates a long-range repulsive surface force as two such surfaces are brought together. However, the aggregates are easily deformed, partly rearranged into multilayer structures and partly removed from between the surfaces under high loads. These layers offer very low friction coefficient (<0.01), high load bearing capacity (≈23MPa), and self-healing ability. Surface bound DPPC/hyaluronan aggregates provide a means for accumulation of lubricating DPPC molecules on sliding surfaces.

Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.

In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively.

Wetting hysteresis induced by temperature changes: Supercooled water on hydrophobic surfaces.

The state and stability of supercooled water on (super)hydrophobic surfaces is crucial for low temperature applications and it will affect anti-icing and de-icing properties. Surface characteristics such as topography and chemistry are expected to affect wetting hysteresis during temperature cycling experiments, and also the freezing delay of supercooled water. We utilized stochastically rough wood surfaces that were further modified to render them hydrophobic or superhydrophobic. Liquid flame spraying (LFS) was utilized to create a multi-scale roughness by depositing titanium dioxide nanoparticles. The coating was subsequently made non-polar by applying a thin plasma polymer layer. As flat reference samples modified silica surfaces with similar chemistries were utilized. With these substrates we test the hypothesis that superhydrophobic surfaces also should retard ice formation. Wetting hysteresis was evaluated using contact angle measurements during a freeze-thaw cycle from room temperature to freezing occurrence at -7°C, and then back to room temperature. Further, the delay in freezing of supercooled water droplets was studied at temperatures of -4°C and -7°C. The hysteresis in contact angle observed during a cooling-heating cycle is found to be small on flat hydrophobic surfaces. However, significant changes in contact angles during a cooling-heating cycle are observed on the rough surfaces, with a higher contact angle observed on cooling compared to during the subsequent heating. Condensation and subsequent frost formation at sub-zero temperatures induce the hysteresis. The freezing delay data show that the flat surface is more efficient in enhancing the freezing delay than the rougher surfaces, which can be rationalized considering heterogeneous nucleation theory. Thus, our data suggests that molecular flat surfaces, rather than rough superhydrophobic surfaces, are beneficial for retarding ice formation under conditions that allow condensation and frost formation to occur.

Gemini surfactants: new synthetic vectors for gene transfection.

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.