Effect of non-linear strain stiffening in eDAH and unjamming.

In cell clusters, the prominent factors at play encompass contractility-based enhanced tissue surface tension and cell unjamming transition. The former effect pertains to the boundary effect, while the latter constitutes a bulk effect. Both effects share outcomes of inducing significant elongation in cells. This elongation is so substantial that it surpasses the limits of linear elasticity, thereby giving rise to additional effects. To investigate these effects, we employ atomic force microscopy (AFM) to analyze how the mechanical properties of individual cells change under such considerable elongation. Our selection of cell lines includes MCF-10A, chosen for its pronounced demonstration of the extended differential adhesion hypothesis (eDAH), and MDA-MB-436, selected due to its manifestation of cell unjamming behavior. In the AFM analyses, we observe a common trend in both cases: as elongation increases, both cell lines exhibit strain stiffening. Notably, this effect is more prominent in MCF-10A compared to MDA-MB-436. Subsequently, we employ AFM on a dynamic range of 1-200 Hz to probe the mechanical characteristics of cell spheroids, focusing on both surface and bulk mechanics. Our findings align with the results from single cell investigations. Specifically, MCF-10A cells, characterized by strong contractile tissue tension, exhibit the greatest stiffness on their surface. Conversely, MDA-MB-436 cells, which experience significant elongation, showcase their highest stiffness within the bulk region. Consequently, the concept of single cell strain stiffening emerges as a crucial element in understanding the mechanics of multicellular spheroids (MCSs), even in the case of MDA-MB-436 cells, which are comparatively softer in nature.

How tissue fluidity influences brain tumor progression.

Mechanical properties of biological tissues and, above all, their solid or fluid behavior influence the spread of malignant tumors. While it is known that solid tumors tend to have higher mechanical rigidity, allowing them to aggressively invade and spread in solid surrounding healthy tissue, it is unknown how softer tumors can grow within a more rigid environment such as the brain. Here, we use in vivo magnetic resonance elastography (MRE) to elucidate the role of anomalous fluidity for the invasive growth of soft brain tumors, showing that aggressive glioblastomas (GBMs) have higher water content while behaving like solids. Conversely, our data show that benign meningiomas (MENs), which contain less water than brain tissue, are characterized by fluid-like behavior. The fact that the 2 tumor entities do not differ in their soft properties suggests that fluidity plays an important role for a tumor's aggressiveness and infiltrative potential. Using tissue-mimicking phantoms, we show that the anomalous fluidity of neurotumors physically enables GBMs to penetrate surrounding tissue, a phenomenon similar to Saffman-Taylor viscous-fingering instabilities, which occur at moving interfaces between fluids of different viscosity. Thus, targeting tissue fluidity of malignant tumors might open horizons for the diagnosis and treatment of cancer.

Whole tissue and single cell mechanics are correlated in human brain tumors.

Biomechanical changes are critical for cancer progression. However, the relationship between the rheology of single cells measured ex-vivo and the living tumor is not yet understood. Here, we combined single-cell rheology of cells isolated from primary tumors with in vivo bulk tumor rheology in patients with brain tumors. Eight brain tumors (3 glioblastoma, 3 meningioma, 1 astrocytoma, 1 metastasis) were investigated in vivo by magnetic resonance elastography (MRE), and after surgery by the optical stretcher (OS). MRE was performed in a 3-Tesla clinical MRI scanner and magnitude modulus |G*|, loss angle φ, storage modulus G', and loss modulus G'' were derived. OS experiments measured cellular creep deformation in response to laser-induced step stresses. We used a Kelvin-Voigt model to deduce two parameters related to cellular stiffness (µKV) and cellular viscosity (ηKV) from OS measurements in a time regimen that overlaps with that of MRE. We found that single-cell µKV was correlated with |G*| (R = 0.962, p < 0.001) and G'' (R = 0.883, p = 0.004) but not G' of the bulk tissue. These results suggest that single-cell stiffness affects tissue viscosity in brain tumors. The observation that viscosity parameters of individual cells and bulk tissue were not correlated suggests that collective mechanical interactions (i.e. emergent effects or cellular unjamming) of many cancer cells, which depend on cellular stiffness, influence the mechanical dissipation behavior of the bulk tissue. Our results are important to understand the emergent rheology of active multiscale compound materials such as brain tumors and its role in disease progression.

Correction: The role of stickiness in the rheology of semiflexible polymers.

Correction for 'The role of stickiness in the rheology of semiflexible polymers' by Tom Golde et al., Soft Matter, 2019, 15, 4865-4872.

The role of stickiness in the rheology of semiflexible polymers.

Semiflexible polymers form central structures in biological material. Modelling approaches usually neglect influences of polymer-specific molecular features aiming to describe semiflexible polymers universally. Here, we investigate the influence of molecular details on networks assembled from filamentous actin, intermediate filaments, and synthetic DNA nanotubes. In contrast to prevalent theoretical assumptions, we find that bulk properties are affected by various inter-filament interactions. We present evidence that these interactions can be merged into a single parameter in the frame of the glassy wormlike chain model. The interpretation of this parameter as a polymer specific stickiness is consistent with observations from macro-rheological measurements and reptation behaviour. Our findings demonstrate that stickiness should generally not be ignored in semiflexible polymer models.

Glassy dynamics in composite biopolymer networks.

The cytoskeleton is a highly interconnected meshwork of strongly coupled subsystems providing mechanical stability as well as dynamic functions to cells. To elucidate the underlying biophysical principles, it is central to investigate not only one distinct functional subsystem but rather their interplay as composite biopolymeric structures. Two of the key cytoskeletal elements are actin and vimentin filaments. Here, we show that composite networks reconstituted from actin and vimentin can be described by a superposition of two non-interacting scaffolds. Arising effects are demonstrated in a scale-spanning frame connecting single filament dynamics to macro-rheological network properties. The acquired results of the linear and non-linear bulk mechanics can be captured within an inelastic glassy wormlike chain model. In contrast to previous studies, we find no emergent effects in these composite networks. Thus, our study paves the way to predict the mechanics of the cytoskeleton based on the properties of its single structural components.

Jamming transitions in cancer.

The traditional picture of tissues, where they are treated as liquids defined by properties such as surface tension or viscosity has been redefined during the last few decades by the more fundamental question: under which conditions do tissues display liquid-like or solid-like behaviour? As a result, basic concepts arising from the treatment of tissues as solid matter, such as cellular jamming and glassy tissues, have shifted into the current focus of biophysical research. Here, we review recent works examining the phase states of tissue with an emphasis on jamming transitions in cancer. When metastasis occurs, cells gain the ability to leave the primary tumour and infiltrate other parts of the body. Recent studies have shown that a linkage between an unjamming transition and tumour progression indeed exists, which could be of importance when designing surgery and treatment approaches for cancer patients.

Single Actin Bundle Rheology.

Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.

Tuning Synthetic Semiflexible Networks by Bending Stiffness.

The mechanics of complex soft matter often cannot be understood in the classical physical frame of flexible polymers or rigid rods. The underlying constituents are semiflexible polymers, whose finite bending stiffness (κ) leads to nontrivial mechanical responses. A natural model for such polymers is the protein actin. Experimental studies of actin networks, however, are limited since the persistence length (l_{p}∝κ) cannot be tuned. Here, we experimentally characterize this parameter for the first time in entangled networks formed by synthetically produced, structurally tunable DNA nanotubes. This material enabled the validation of characteristics inherent to semiflexible polymers and networks thereof, i.e., persistence length, inextensibility, reptation, and mesh size scaling. While the scaling of the elastic plateau modulus with concentration G_{0}∝c^{7/5} is consistent with previous measurements and established theories, the emerging persistence length scaling G_{0}∝l_{p} opposes predominant theoretical predictions.

Transition from a Linear to a Harmonic Potential in Collective Dynamics of a Multifilament Actin Bundle.

Attractive depletion forces between rodlike particles in highly crowded environments have been shown through recent modeling and experimental approaches to induce different structural and dynamic signatures depending on relative orientation between rods. For example, it has been demonstrated that the axial attraction between two parallel rods yields a linear energy potential corresponding to a constant contractile force of 0.1 pN. Here, we extend pairwise, depletion-induced interactions to a multifilament level with actin bundles, and find contractile forces up to 3 pN. Forces generated due to bundle relaxation were not constant, but displayed a harmonic potential and decayed exponentially with a mean decay time of 3.4 s. Through an analytical model, we explain these different fundamental dynamics as an emergent, collective phenomenon stemming from the additive, pairwise interactions of filaments within a bundle.

Keratins significantly contribute to cell stiffness and impact invasive behavior.

Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cell-cell and cell-matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.

The lensing effect of trapped particles in a dual-beam optical trap.

In dual-beam optical traps, two counterpropagating, divergent laser beams emitted from opposing laser fibers trap and manipulate dielectric particles. We investigate the lensing effect that trapped particles have on the beams. Our approach makes use of the intrinsic coupling of a beam to the opposing fiber after having passed the trapped particle. We present measurements of this coupling signal for PDMS particles, as well as a model for its dependence on size and refractive index of the trapped particle. As a more complex sample, the coupling of inhomogeneous biological cells is measured and discussed. We show that the lensing effect is well captured by the simple ray optics approximation. The measurements reveal intricate details, such as the thermal lens effect of the beam propagation in a dual-beam trap. For a particle of known size, the model further allows to infer its refractive index simply from the coupling signal.

Active contractions in single suspended epithelial cells.

Investigations of active contractions in tissue cells to date have been focused on cells that exert forces via adhesion sites to substrates or to other cells. In this study we show that also suspended epithelial cells exhibit contractility, revealing that contractions can occur independently of focal adhesions. We employ the Optical Stretcher to measure adhesion-independent mechanical properties of an epithelial cell line transfected with a heat-sensitive cation channel. During stretching the heat transferred to the ion channel causes a pronounced Ca(2+) influx through the plasma membrane that can be blocked by adequate drugs. This way the contractile forces in suspended cells are shown to be partially triggered by Ca(2+) signaling. A phenomenological mathematical model is presented, incorporating a term accounting for the active stress exerted by the cell, which is both necessary and sufficient to describe the observed increase in strain when the Ca(2+) influx is blocked. The median and the shape of the strain distributions depend on the activity of the cells. Hence, it is unlikely that they can be described by a simple Gaussian or log normal distribution, but depend on specific cellular properties such as active contractions. Our results underline the importance of considering activity when measuring cellular mechanical properties even in the absence of measurable contractions. Thus, the presented method to quantify active contractions of suspended cells offers new perspectives for a better understanding of cellular force generation with possible implications for medical diagnosis and therapy.

Growth cones as soft and weak force generators.

Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of τ = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone's mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per µm(2). Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.

Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

Biomechanical properties of retinal glial cells: comparative and developmental data.

The biomechanical properties of Müller glial cells may have importance in understanding the retinal tissue alterations after retinal surgery with removal of the inner limiting membrane and during the ontogenetic development, respectively. Here, we compared the viscoelastic properties of Müller cells from man and monkey as well as from different postnatal developmental stages of the rat. We determined the complex Young's modulus E = E' + iE″ in a defined range of deforming frequencies (30, 100, and 200 Hz) using a scanning force microscope, where the real part E' reflects the elastic property (energy storage or elastic stiffness) and the imaginary part E″ reflects the viscous property (energy dissipation) of the cells. The viscoelastic properties were similar in Müller cells from man, monkey, and rat. In general, the elastic behavior dominated over the viscous behavior (E' > E″). The inner process of the Müller cell was the softest region, the soma the stiffest (Einnerprocess(')Eglia(')). These relations were also observed during the postnatal development of the rat. It is concluded that, generally, retinal cells display mechanics of elastic solids. In addition, the data indicate that the rodent retina is a reliable model to investigate retinal mechanics and tissue alterations after retinal surgery. During retinal development, neuronal branching and synaptogenesis might be particularly stimulated by the viscoelastic properties of Müller cell processes in the inner plexiform layer.

Different modes of growth cone collapse in NG 108-15 cells.

In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.

Heavy water induces bundling in entangled actin networks.

Heavy water is known to affect many different biological systems, with the most striking effects observed at the cellular level. Many dynamic processes, such as migration or invasion, but also central processes of cell proliferation are measurably inhibited by the presence of deuterium oxide (D2O). Furthermore, individual cell deformabilities are significantly decreased upon D2O treatment. In order to understand the origin of these effects, we studied entangled filamentous actin networks, a commonly used model system for the cytoskeleton, which is considered a central functional element for dynamic cellular processes. Using bulk shear rheology to extract rheological signatures of reconstituted actin networks at varying concentrations of D2O, we found a non-monotonic behavior, which is explainable by a drastic change in the actin network architecture. Applying light scattering and fluorescence microscopy, we were able to demonstrate that the presence of deuterium oxide induces bundling in reconstituted entangled networks of filamentous actin. This constitutes an entirely novel and previously undescribed actin bundling mechanism.

Re-evaluation of publicly available gene-expression databases using machine-learning yields a maximum prognostic power in breast cancer.

Gene expression signatures refer to patterns of gene activities and are used to classify different types of cancer, determine prognosis, and guide treatment decisions. Advancements in high-throughput technology and machine learning have led to improvements to predict a patient's prognosis for different cancer phenotypes. However, computational methods for analyzing signatures have not been used to evaluate their prognostic power. Contention remains on the utility of gene expression signatures for prognosis. The prevalent approaches include random signatures, expert knowledge, and machine learning to construct an improved signature. We unify these approaches to evaluate their prognostic power. Re-evaluation of publicly available gene-expression data from 8 databases with 9 machine-learning models revealed previously unreported results. Gene-expression signatures are confirmed to be useful in predicting a patient's prognosis. Convergent evidence from [Formula: see text] 10,000 signatures implicates a maximum prognostic power. By calculating the concordance index, which measures how well patients with different prognoses can be discriminated, we show that a signature can correctly discriminate patients' prognoses no more than 80% of the time. Additionally, we show that more than 50% of the potentially available information is still missing at this value. We surmise that an accurate prognosis must incorporate molecular, clinical, histological, and other complementary factors.

Systematic altering of semiflexible DNA-based polymer networks via tunable crosslinking.

In order to understand and predict the mechanical behaviours of complex, soft biomaterials such as cells or stimuli-responsive hydrogels, it is important to connect how the nanoscale properties of their constituent components impact those of the bulk material. Crosslinked networks of semiflexible polymers are particularly ubiquitous, being underlying mechanical components of biological systems such as cells or ECM, as well as many synthetic or biomimetic materials. Cell-derived components such as filamentous biopolymers or protein crosslinkers are readily available and well-studied model systems. However, as evolutionarily derived materials, they are constrained to a fixed set of structural parameters such as the rigidity and size of the filaments, or the valency and strength of binding of crosslinkers forming inter-filament connections. By implementing a synthetic model system based on the self-assembly of DNA oligonucleotides into nanometer-scale tubes and simple crosslinking constructs, we used the thermodynamic programmability of DNA hybridization to explore the impact of binding affinity on bulk mechanical response. Stepwise tuning the crosslinking affinity over a range from transient to thermodynamically stable shows an according change in viscoelastic behaviour from loosely entangled to elastic, consistent with models accounting for generalized inter-filament interactions. While characteristic signatures of concentration-dependent changes in network morphology found in some other natural and synthetic filament-crosslinker systems were not apparent, the presence of a distinct elasticity increase within a narrow range of conditions points towards potential subtle alterations of crosslink-filament architecture. Here, we demonstrate a new synthetic approach for gaining a deeper understanding of both biological as well as engineered hydrogel systems.