Mechanical properties of human tumour tissues and their implications for cancer development.

The mechanical properties of cells and tissues help determine their architecture, composition and function. Alterations to these properties are associated with many diseases, including cancer. Tensional, compressive, adhesive, elastic and viscous properties of individual cells and multicellular tissues are mostly regulated by reorganization of the actomyosin and microtubule cytoskeletons and extracellular glycocalyx, which in turn drive many pathophysiological processes, including cancer progression. This Review provides an in-depth collection of quantitative data on diverse mechanical properties of living human cancer cells and tissues. Additionally, the implications of mechanical property changes for cancer development are discussed. An increased knowledge of the mechanical properties of the tumour microenvironment, as collected using biomechanical approaches capable of multi-timescale and multiparametric analyses, will provide a better understanding of the complex mechanical determinants of cancer organization and progression. This information can lead to a further understanding of resistance mechanisms to chemotherapies and immunotherapies and the metastatic cascade.

Myosin-independent stiffness sensing by fibroblasts is regulated by the viscoelasticity of flowing actin.

The stiffness of the extracellular matrix induces differential tension within integrin-based adhesions, triggering differential mechanoresponses. However, it has been unclear if the stiffness-dependent differential tension is induced solely by myosin activity. Here, we report that in the absence of myosin contractility, 3T3 fibroblasts still transmit stiffness-dependent differential levels of traction. This myosin-independent differential traction is regulated by polymerizing actin assisted by actin nucleators Arp2/3 and formin where formin has a stronger contribution than Arp2/3 to both traction and actin flow. Intriguingly, despite only slight changes in F-actin flow speed observed in cells with the combined inhibition of Arp2/3 and myosin compared to cells with sole myosin inhibition, they show a 4-times reduction in traction than cells with myosin-only inhibition. Our analyses indicate that traditional models based on rigid F-actin are inadequate for capturing such dramatic force reduction with similar actin flow. Instead, incorporating the F-actin network's viscoelastic properties is crucial. Our new model including the F-actin viscoelasticity reveals that Arp2/3 and formin enhance stiffness sensitivity by mechanically reinforcing the F-actin network, thereby facilitating more effective transmission of flow-induced forces. This model is validated by cell stiffness measurement with atomic force microscopy and experimental observation of model-predicted stiffness-dependent actin flow fluctuation.

Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.

Atomic force microscopy-mediated mechanobiological profiling of complex human tissues.

Tissue mechanobiology is an emerging field with the overarching goal of understanding the interplay between biophysical and biochemical responses affecting development, physiology, and disease. Changes in mechanical properties including stiffness and viscosity have been shown to describe how cells and tissues respond to mechanical cues and modify critical biological functions. To quantitatively characterize the mechanical properties of tissues at physiologically relevant conditions, atomic force microscopy (AFM) has emerged as a highly versatile biomechanical technology. In this review, we describe the fundamental principles of AFM, typical AFM modalities used for tissue mechanics, and commonly used elastic and viscoelastic contact mechanics models to characterize complex human tissues. Furthermore, we discuss the application of AFM-based mechanobiology to characterize the mechanical responses within complex human tissues to track their developmental, physiological/functional, and diseased states, including oral, hearing, and cancer-related tissues. Finally, we discuss the current outlook and challenges to further advance the field of tissue mechanobiology. Altogether, AFM-based tissue mechanobiology provides a mechanistic understanding of biological processes governing the unique functions of tissues.

Septins Enable T Cell Contact Guidance via Amoeboid-Mesenchymal Switch.

Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes dynamically alternate and combine their amoeboid (non-adhesive) and mesenchymal (adhesive) migration modes. However, which mechanisms guide and balance different migration modes are largely unclear. Here we report that suppression of septins GTPase activity induces an abrupt amoeboid-to-mesenchymal transition of T cell migration mode, characterized by a distinct, highly deformable integrin-dependent immune cell contact guidance. Surprisingly, the T cell actomyosin cortex contractility becomes diminished, dispensable and antagonistic to mesenchymal-like migration mode. Instead, mesenchymal-like T cells rely on microtubule stabilization and their non-canonical dynein motor activity for high fidelity contact guidance. Our results establish septin's GTPase activity as an important on/off switch for integrin-dependent migration of T lymphocytes, enabling their dynein-driven fluid-like mesenchymal propulsion along the complex adhesion cues.

Combined AAV-mediated gene replacement therapy improves auditory function in a mouse model of human DFNB42 deafness.

Hearing loss is a common disorder affecting nearly 20% of the world's population. Recently, studies have shown that inner ear gene therapy can improve auditory function in several mouse models of hereditary hearing loss. In most of these studies, the underlying mutations affect only a small number of cell types of the inner ear (e.g., sensory hair cells). Here, we applied inner ear gene therapy to the Ildr1Gt(D178D03)Wrst (Ildr1w-/-) mouse, a model of human DFNB42, non-syndromic autosomal recessive hereditary hearing loss associated with ILDR1 variants. ILDR1 is an integral protein of the tricellular tight junction complex and is expressed by diverse inner ear cell types in the organ of Corti and the cochlear lateral wall. We simultaneously applied two synthetic adeno-associated viruses (AAVs) with different tropism to deliver Ildr1 cDNA to the Ildr1w-/- mouse inner ear: one targeting the organ of Corti (AAV2.7m8) and the other targeting the cochlear lateral wall (AAV8BP2). We showed that combined AAV2.7m8/AAV8BP2 gene therapy improves cochlear structural integrity and auditory function in Ildr1w-/- mice.

Frequency-dependent nanomechanical profiling for medical diagnosis.

Atomic force microscopy (AFM), developed in the early 1980s, has become a powerful characterization tool in micro- and nanoscale science. In the early 1990s, its relevance within biology and medicine research became evident, although its incorporation into healthcare applications remains relatively limited. Here, we briefly explore the reasons for this low level of technological adoption. We also propose a path forward for the incorporation of frequency-dependent nanomechanical measurements into integrated healthcare strategies that link routine AFM measurements with computer analysis, real-time communication with healthcare providers, and medical databases. This approach would be appropriate for diseases such as cancer, lupus, arteriosclerosis and arthritis, among others, which bring about significant mechanical changes in the affected tissues.

Unbalanced bidirectional radial stiffness gradients within the organ of Corti promoted by TRIOBP.

Hearing depends on intricate morphologies and mechanical properties of diverse inner ear cell types. The individual contributions of various inner ear cell types into mechanical properties of the organ of Corti and the mechanisms of their integration are yet largely unknown. Using sub-100-nm spatial resolution atomic force microscopy (AFM), we mapped the Young's modulus (stiffness) of the apical surface of the different cells of the freshly dissected P5-P6 cochlear epithelium from wild-type and mice lacking either Trio and F-actin binding protein (TRIOBP) isoforms 4 and 5 or isoform 5 only. Variants of TRIOBP are associated with deafness in human and in Triobp mutant mouse models. Remarkably, nanoscale AFM mapping revealed unrecognized bidirectional radial stiffness gradients of different magnitudes and opposite orientations between rows of wild-type supporting cells and sensory hair cells. Moreover, the observed bidirectional radial stiffness gradients are unbalanced, with sensory cells being stiffer overall compared to neighboring supporting cells. Deafness-associated TRIOBP deficiencies significantly disrupted the magnitude and orientation of these bidirectional radial stiffness gradients. In addition, serial sectioning with focused ion beam and backscatter scanning electron microscopy shows that a TRIOBP deficiency results in ultrastructural changes of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, correlating with messenger RNA and protein expression levels and AFM stiffness measurements that exposed a softening of the apical surface of the sensory epithelium in mutant mice. Altogether, this additional complexity in the mechanical properties of the sensory epithelium is hypothesized to be an essential contributor to frequency selectivity and sensitivity of mammalian hearing.

Stiffening of Circumferential F-Actin Bands Correlates With Regenerative Failure and May Act as a Biomechanical Brake in the Mammalian Inner Ear.

The loss of inner ear hair cells causes permanent hearing and balance deficits in humans and other mammals, but non-mammals recover after supporting cells (SCs) divide and replace hair cells. The proliferative capacity of mammalian SCs declines as exceptionally thick circumferential F-actin bands develop at their adherens junctions. We hypothesized that the reinforced junctions were limiting regenerative responses of mammalian SCs by impeding changes in cell shape and epithelial tension. Using micropipette aspiration and atomic force microscopy, we measured mechanical properties of utricles from mice and chickens. Our data show that the epithelial surface of the mouse utricle stiffens significantly during postnatal maturation. This stiffening correlates with and is dependent on the postnatal accumulation of F-actin and the cross-linker Alpha-Actinin-4 at SC-SC junctions. In chicken utricles, where SCs lack junctional reinforcement, the epithelial surface remains compliant. There, SCs undergo oriented cell divisions and their apical surfaces progressively elongate throughout development, consistent with anisotropic intraepithelial tension. In chicken utricles, inhibition of actomyosin contractility led to drastic SC shape change and epithelial buckling, but neither occurred in mouse utricles. These findings suggest that species differences in the capacity for hair cell regeneration may be attributable in part to the differences in the stiffness and contractility of the actin cytoskeletal elements that reinforce adherens junctions and participate in regulation of the cell cycle.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Viscoelastic parameterization of human skin cells characterize material behavior at multiple timescales.

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts-melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression.

Mechanical Counterbalance of Kinesin and Dynein Motors in a Microtubular Network Regulates Cell Mechanics, 3D Architecture, and Mechanosensing.

Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin-MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.

Survey of cancer cell anatomy in nonadhesive confinement reveals a role for filamin-A and fascin-1 in leader bleb-based migration.

Cancer cells migrating in confined microenvironments exhibit plasticity of migration modes. Confinement of contractile cells in a nonadhesive environment drives "leader bleb-based migration" (LBBM), morphologically characterized by a long bleb that points in the direction of movement separated from a cell body by a contractile neck. Although cells undergoing LBBM have been visualized within tumors, the organization of organelles and actin regulatory proteins mediating LBBM is unknown. We analyzed the localization of fluorescent organelle-specific markers and actin-associated proteins in human melanoma and osteosarcoma cells undergoing LBBM. We found that organelles from the endolysosomal, secretory, and metabolic systems as well as the vimentin and microtubule cytoskeletons localized primarily in the cell body, with some endoplasmic reticulum, microtubules, and mitochondria extending into the leader bleb. Overexpression of fluorescently tagged actin regulatory proteins showed that actin assembly factors localized toward the leader bleb tip, contractility regulators and cross-linkers in the cell body cortex and neck, and cross-linkers additionally throughout the leader bleb. Quantitative analysis showed that excess filamin-A and fascin-1 increased migration speed and persistence, while their depletion by small interfering RNA indicates a requirement in promoting cortical tension and pressure to drive LBBM. This indicates a critical role of specific actin crosslinkers in LBBM.

Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion.

Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

Cochlear outer hair cell horizontal top connectors mediate mature stereocilia bundle mechanics.

Outer hair cell (OHC) stereocilia bundle deflection opens mechanoelectrical transduction channels at the tips of the stereocilia from the middle and short rows, while bundle cohesion is maintained owing to the presence of horizontal top connectors. Here, we used a quantitative noncontact atomic force microscopy method to investigate stereocilia bundle stiffness and damping, when stimulated at acoustic frequencies and nanometer distances from the bundle. Stereocilia bundle mechanics were determined in stereocilin-deficient mice lacking top connectors and with detached tectorial membrane (Strc -/-/Tecta -/- double knockout) and heterozygous littermate controls (Strc +/-/Tecta -/-). A substantial decrease in bundle stiffness and damping by ~60 and ~74% on postnatal days P13 to P15 was observed when top connectors were absent. Additionally, we followed bundle mechanics during OHC top connectors development between P9 and P15 and quantified the observed increase in OHC bundle stiffness and damping in Strc +/-/Tecta -/- mice while no significant change was detected in Strc -/-/Tecta -/- animals.

Evaluation of the elastic Young's modulus and cytotoxicity variations in fibroblasts exposed to carbon-based nanomaterials.

BACKGROUND: The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. These assays are strongly dependent on the properties of the nanomaterials, for example; specific surface area (SSA), size, surface defects, and surface charge, and the host response. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. Here, we proposed a structured approach for cytotoxicity assessment complemented with cells' mechanical responses represented as the variations of elastic Young's modulus in conjunction with conventional biochemical tests. Monitoring the mechanical properties responses at various times allowed understanding the effects of NMs to the filamentous actin cytoskeleton. The elastic Young's modulus was estimated from the force volume maps using an atomic force microscope (AFM). RESULTS: Our results show a significant decrease on Young's modulus, ~ 20%, in cells exposed to low concentrations of graphene flakes (GF), ~ 10% decrease for cells exposed to low concentrations of multiwalled carbon nanotubes (MWCNTs) than the control cells. These considerable changes were directly correlated to the disruption of the cytoskeleton actin fibers. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Applying both conventional biochemical approach and cells mechanics, we were able to detect differences in the actin networks induced by MWCNT inside the cells and GF outside the cell's membrane. These results contrast with the conventional live/dead assay where we obtained viabilities greater than 80% after 24 h; while the elasticity dramatically decreased suggesting a fast-metabolic stress generation. CONCLUSIONS: We confirmed the production of radical oxygen species (ROS) on cells exposed to CBNs, which is related to the disruption of the cytoskeleton. Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. We evidenced the importance of not just nanomaterials properties but also the effect of the location to assess the cytotoxic effects of nanomaterials.

Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy.

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.