The European Physical Journal E

The measurement of stresses and forces at the tissue level has proven to be an indispensable tool for the understanding of complex biological phenomena such as cancer invasion, embryo development or wound healing. One of the most versatile tools for force inference at the cell and tissue level are elastic force sensors, whose biocompatibility and tunable material properties make them suitable for many different experimental scenarios. The evaluation of those forces, however, is still a bottleneck due to the numerical methods seen in literature until now, which are usually slow and render low experimental yield. Here we present Bead-Buddy, a ready-to-use platform for the evaluation of deformation and stresses from fluorescently labelled sensors within seconds. The strengths of BeadBuddy lie in the pre-computed analytical solutions of the elastic problem, the abstraction of data into Spherical Harmonics, and a simple user interface that creates a smooth workflow for force inference. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/633835v3_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@80c95corg.highwire.dtl.DTLVardef@123d712org.highwire.dtl.DTLVardef@1fd1ce0org.highwire.dtl.DTLVardef@72c688_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cellular force generation and transmission are fundamental processes driving cell migration, division, tissue morphogenesis, and disease progression. Traction Force Microscopy (TFM) and Monolayer Stress Microscopy (MSM) have emerged as essential techniques for quantifying these mechanical processes, but current software solutions are fragmented across multiple platforms with varying degrees of usability and accessibility. Here, we present napariTFM, a comprehensive open-source plugin for the napari image viewer that integrates state-of-the-art algorithms for both TFM and MSM analysis within an intuitive graphical user interface. The software implements TV-L1 optical flow for displacement analysis, Fourier Transform Traction Cytometry (FTTC) for force reconstruction, and finite element methods for stress calculation, supporting both single-frame and time-series analysis of 2D microscopy data. Systematic validation using synthetic datasets with known ground truth values demonstrated excellent accuracy, with correlation coefficients above 0.9 for most situations. Real-time parameter adjustment and immediate visualization capabilities enable interactive optimization of analysis parameters and quality assessment during processing. Finally, we demonstrate the softwares capabilities through analysis of optogenetic contractility experiments in cell doublets. napariTFM addresses critical gaps in the cellular mechanics software ecosystem by combining algorithmic rigor with practical usability, providing the research community with an accessible platform for quantitative studies of cellular force generation and transmission.

The cell wall is a key component of fungi. It constitutes a stiff shell which counteracts internal cell turgor pressure. Its mechanical properties thus contribute to define cell morphology. Measurements of the elastic moduli of the fungal cell wall have been carried out in many species including Candida albicans, a major human opportunistic pathogen. They mainly relied on atomic force microscopy, and mostly considered the yeast form. We developed a parallelized pressure-actuated microfluidic device to measure the bending stiffness of hyphae. We found that the cell wall stiffness lies in the MPa range. We the used three different ways to disrupt cell wall physiology: inhibition of beta-glucan synthesis, a key component of the inner cell wall; application of an hyperosmotic shock triggering a sudden decrease of the hyphal diameter; deletion of two genes encoding GPI-modified cell wall proteins resulting in reduced cell wall thickness. The bending stiffness values were affected to different extents by these environmental stresses or genetic modifications. Overall, our results support the elastic nature of the cell wall and its ability to remodel at the scale of the entire hypha over minutes.

Computational modeling has emerged as a powerful approach to studying cytoskeletal dynamics. The simulation software Cytosim provides intuitive yet flexible simulations of filament polymerization, cross-linking, and motor activity. Here, we present Cytocalc, a lightweight Python toolkit designed to streamline and standardize the analysis of Cytosim simulation output, supporting studies of biological functionality and physical properties of cytoskeletal systems. After introducing Cytocalc and validating it, we use it to establish a new workflow for quantifying network viscoelasticity from Cytosim simulations. Specifically, we determine the complex shear modulus of cross-linked networks and quantify how the storage modulus increases with cross-linker density. The cross-linker dependence of the networks elasticity exhibits two regimes, a scaling regime consistent with elasticity arising from the suppression of thermal bending fluctuations of filaments as well as a much weaker dependence at high cross-linker concentration.

Spheroids are of great interest in the study of cancer as they can partially mimic the tumour microenvironment, thus allowing to investigate several aspects of cell - microenvironment interactions in healthy and diseased conditions, including those pertaining to mechanobiology. Atomic Force Microscopy (AFM) is a versatile tool for studying biological samples and their mechanobiological properties. In AFM, the tip shape and dimensions determine the contact geometry between the tip and the sample and the length scales at which the mechanical properties are probed. Given the complex multiscale structure of spheroids, the choice of tip geometry and size would allow, in principle, to dissect the mechanical response of the overall system into the contributions of the constituents, from the single cell level to the cellular aggregate. In this work, we studied the mechanical properties of spheroids derived from four cell lines (A549, NHLF, HT-29, CCD-18Co). Our studies revealed that using different contact geometries in the fitting procedure results in significantly different Youngs modulus values, highlighting the multiscale response of these complex cellular systems and the importance of a precise experiment design and choice of the AFM probe for the nano-mechanical measurements. We observed that the location of F-actin filaments is correlated to the rigidity of the spheroids.

Understanding the intertwining of biology and mechanics in tissue architecture is a challenging issue, especially when it comes to the 3D tissue organization. Addressing this challenge requires both a biological model allowing multiscale observations from the cell to the tissue, and theoretical and computational approaches allowing the generation of a synthetic model, relevant to the biological model, and allowing access to the mechanical constraints experienced by the tissue. Here, using human colon epithelium monolayer organoid as biological model, and combining vertex and FEM approaches, we generated a comprehensive elastic finite element model of the human colon organoid and demonstrated its flexibility. This FEM model provides a basis for relating cell shape, tissue deformation, and strain at the cellular level due to imposed stresses. In conclusion, we demonstrated that the combination of vertex and FEM approaches allows for better modeling of the alteration of organoid morphology over time and better assessment of the mechanical cues involved in establishing the architecture of the human colon epithelium.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Theoretical approaches to study electrotactic cell migration until now mainly used reaction-diffusion models, and did not consider the effect of electric field on single-cell motility, or incorporate spatially dependent cell-to-cell interactions. Here, we present a computational model that takes into account cell interactions and describes cell migration in direct current electric field. We compare this model with in vitro experiments in which human primary osteoblasts are exposed to direct current electric field of varying field strength. Our results show that cell-cell interactions and fluctuations in the migration direction together lead to anode-directed collective migration of osteoblasts. Author summaryElectrotactic migration of cells involves directed movement of a large number of single cells under the influence of external electric field. Influencing the migration behaviour of osteoblasts by external direct current electric field offers a promising approach towards building highly effective implants for bone regeneration. We present a computational model for electrotactic migration of osteoblasts subject to external direct current electric field. Our model considers individual cells that interact with each other and the external electric field, and, replicates the experimental observations, based on single-cell analysis, of the response of osteoblasts to electrical stimulation of varying strengths for 7 hours. Our results suggest that tracking trajectories of individual cells provide a way of determining the role of various interactions of a cell in collective migration. Our model provides a framework that links single cell response to the large scale collective dynamics.

3D cell culture models replicate tissue complexity, aiming to study cellular interactions and responses in a more physiologically relevant environment compared to traditional 2D cultures. However, the spherical structure of these models makes it difficult to extract meaningful data, necessitating advanced techniques for proper analysis. In silico simulations enhance research by predicting cellular behaviors and therapeutic responses, providing a powerful tool to complement experimental approaches. Despite their potential, these simulations often require advanced computational skills and significant resources, creating a barrier for many researchers. To address these challenges, we developed an accessible pipeline using open-source software to facilitate virtual tissue simulations. Our approach employs the Cellular Potts Model, a versatile framework for simulating cellular behaviors in tissues. The simulations are constructed from real world 3D image stacks of cancer spheroids, ensuring the virtual models are rooted in experimental data. By introducing a new metric for parameter optimization, we enable the creation of realistic simulations without requiring extensive computational expertise. This pipeline benefits researchers wanting to incorporate computational biology into their methods, even if they do not possess extensive expertise in this area. By reducing the technical barriers associated with advanced computational modeling, our pipeline allows more researchers to utilize these powerful tools. Our approach aims to foster broader use of in silico methods in disease research, contributing to a deeper understanding of disease biology and the refinement of therapeutic interventions.

Chemotaxing Dictyostelium discoideum cells migrate in a step-wise fashion characterized by periodic protrusion, contraction, and rear retraction cycles accompanied by distinct traction force patterns. Traction force microscopy reveals two stationary force spots that exchange identity as the cell advances and generate a convergent stress pattern with both axial and lateral components. To investigate the physical origin of these traction patterns, we developed a continuum, phase-field model that couples cytosolic flow, active stresses, and substrate friction within a cell with a deformable morpholgy. The model incorporates protrusive forces at the front, contractile stresses at the rear and sides, and spatially localized adhesive regions that undergo cyclic activation. While this baseline model reproduces persistent motion, it fails to capture the experimentally observed traction force patterns and cell morphology. Guided by new experiments visualizing myosin dynamics, we extended the model to include a localized contractile myosin patch positioned between the two adhesive regions. This modification yields cell shapes, speeds, and convergent traction patterns consistent with experimental measurements. The results demonstrate that a centrally positioned myosin patch is sufficient to generate the step-wise migration cycle and the characteristic convergent traction pattern of Dictyostelium cells, providing a mechanistic link between intracellular contractility, cytosolic flow, and force transmission during amoeboid motility.

Cancer spheroids offer a valuable experimental model that mimics the complexity and heterogeneity of solid tumors. Characterizing their mechanical response is crucial for understanding tumor development, progression, and drug response. Currently, whole live spheroids are analyzed primarily using image analysis, which is challenging, requires extended incubation times, and has limited imaging depth. Here, we present a new label-free approach for characterizing sub-superficial structures of bladder cancer spheroids and measuring their mechanical response at three distinct stages of cancer progression. We study the microrheological changes induced by aging at the cellular and cluster levels by conducting a multi-physics characterization and modeling approach. We find that spheroids exhibit viscoelastic behavior that can be described by fractional models. We show that spheroids are mechanically heterogeneous, with strong depth and time-dependent variations associated with evolving structural features. Our approach opens new possibilities to study 3D in vitro models, paving the way for the discovery of novel and more precise procedure in cancer diagnosis based on the use of mechanomarkers.

Imposed deformations play an important role in morphogenesis and tissue homeostasis, both in normal and pathological conditions. To perceive mechanical perturbations of different types and magnitudes, tissues need appropriate detectors, with a compliance that matches the perturbation amplitude. By comparing results of selective osmotic compressions of cells within multicellular aggregates with small osmolites and global aggregate compressions with big osmolites, we show that global compressions have a strong impact on the aggregates growth and internal cell motility, while selective compressions of same magnitude have almost no effect. Both compressions alter the volume of individual cells in the same way but, by draining the water out of the extracellular matrix, the global one imposes a residual compressive mechanical stress on the cells while the selective one does not. We conclude that, in aggregates, the extracellular matrix is as a sensor which mechanically regulates cell proliferation and migration in a 3D environment.

External mechanical stresses acting on cellular compartments critically regulate cell behaviour and can induce cell death. Magnetically actuated particles present a promising strategy to apply such forces in a controlled manner, with potential applications in cancer therapy. In this study, we investigate the effects of actuating vortex magnetic microdiscs on a glioblastoma cell line cultured on soft, biomimetic substrates that mimic in vivo stiffness. Using a Halbach array, we applied either static mechanical compression or a combination of compressive and low-frequency vibrational stresses (2-20 Hz). Our results demonstrate that both compressive and vibrational stresses impair important cellular functions associated with glioblastoma persistence in a dose-dependent and stiffness-dependent manner. In particular on soft 2D substrates, sufficiently strong compressive loads limit proliferation, while the addition of vibrations alter cell motility, cell morphology and the acto-myosin machinery. Our findings demonstrate that magnetic particles-mediated mechanical stimulation can disrupt glioblastoma cell aggressiveness in physiologically relevant 2D substrates, supporting its potential as an adjunct to conventional chemo-and radiotherapies by both inducing cell death and limiting resistant populations.

Dendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration both to minimize resistance and to avoid DNA damage. Vimentin-deficiency also led to DNA double strand breaks in the compressed dendritic cells, pointing to a role of vimentin in nuclear positioning. Together, these observations show that vimentin IF-microfilament interactions provide both the specific mechano-dynamics required for dendritic cell migration and the protection the genome needs in compressed spaces.\n\nSummary statementVimentin -- in joint action with actin -- mediates the mechanical stiffness of cells required for amoeboid cell migration through confined spaces and protects the nucleus from DNA damage.

The reciprocal interplay between cancer cells and their local environment, mediated by mechanical forces, necessitates a deeper experimental understanding. This requires precise quantitative measurements of cellular forces within the intricate three-dimensional context of the extracellular matrix. While methods such as traction-force microscopy and micropillar-array technology have effectively reported on cellular forces in two-dimensional cell culture, extending these techniques to three dimensions has proven exceedingly challenging. In the current study, we introduced a novel approach utilizing soft, elastic hydrogel microparticles, resembling the size of cells, to serve as specific and sensitive traction probes in three-dimensional cell culture of collagen-embedded tumoroids. Our methodology relies on high-resolution detection of microparticle deformations. These deformations are translated into spatially resolved traction fields, reaching a spatial resolution down to 1 {micro}m and thereby detecting traction forces as low as 30 Pa. By integrating this high-resolution traction analysis with three-dimensional cell segmentation, we reconstructed the traction fields originating from individual cells. Our methodology enables us to explore the relationships between cellular characteristics, extracellular traction fields, and cellular responses. We observed that cellular stresses ranged from 10 to 100 Pa, integrating to cellular forces from 0.1 to 100 nN, which correlated with the localization of the cells actin skeleton, and the interaction area that cells developed towards the microparticles. Interestingly, the interaction of cells with inert microparticles appeared to be governed by contact mechanics resembling that of two soft spheres. The methodology presented here not only addresses the challenges of extending traditional stress-probe techniques to three dimensions, but also opens a strategy for the study of specific interactions between cells and the local tumoroid environment in a strive to further understand cell-matrix reciprocity in tissue. Here, we present a novel methodology that permits the measurement of quantitative surface stresses on small, inert, elastic, deformable microparticles. Our approach tackles the involved task of mapping local three-dimensional stress fields within tissue. Our methodology was successfully applied to analyze local stresses within a tumor spheroid. We foresee that our research represents a significant advancement toward comprehending the intricate dynamics of cell-matrix reciprocity within tissue.

Musculoskeletal models for multibody dynamic analysis provide unique insights into human and animal movement. Although some biomechanical simulators provide model-building tools, these presuppose substantial preprocessing by the user, and resulting models are generally not cross-platform compatible. Thus, the workflow from anatomical 3D scans to musculoskeletal model is time-consuming, requiring numerous processing and conversions steps between software packages, and the process differs between simulators. Despite the popularity of musculoskeletal modelling within biomechanics, no cross-platform, open source software package exists for constructing musculoskeletal models. Here, I introduce MuSkeMo: A software suite for defining 3D musculoskeletal models entirely within Blender (open-source 3D computer graphics software). MuSkeMo provides a visual interface, enabling users to interactively define all aspects of a musculoskeletal model (including rigid bodies, skeletal geometry, joint centres, muscles, landmarks, and body-fixed reference frames). MuSkeMo can calculate 3D inertial tensors from arbitrary meshes (e.g., from CT scans), and also implements automated convex-hull based mass-estimation approaches from the literature. Joints can be defined using shape-fitting of bony surfaces, and muscles can wrap around primitive shapes. Models can be analyzed within MuSkeMo using popular pose-sampling procedures, or exported to multiple text-based formats for use in biomechanical simulators. A conversion script to OpenSim is included. MuSkeMo is compatible with models created for popular biomechanical simulators (OpenSim and Gaitsym). MuSkeMo can import these models and simulation trajectories, enabling users to create publication-ready stills and animations with Blenders ray tracing. These visualisations include volumetric muscles based on the contractile parameters, which can be more visually intuitive than traditional constant-diameter tube segments. Whether the end goal is a highly-detailed subject-specific human model, or a simplified animal model, MuSkeMo includes features that can aid this process. By consolidating many elements of common model construction workflows into a cohesive package, MuSkeMo substantially simplifies musculoskeletal modelling.

Cell cortex is a thin sheet of actin cytoskeleton spanning cell boundaries with rich out-of-equilibrium dynamics. A theoretical description of the cortex as an active fluid enables to capture cell shapes dynamics in 3D tissues. However models integrated with calibration of parameters and quantitative experiments are lacking so far. Here we report that cells in organoids and in cysts have conserved apico-basal-lateral asymmetric compositions in actin and in myosin, and we quantify their densities and mechanical properties. This allows to calibrate a new model coupling active fluids with a phase field which reproduces the main features of cell shapes. To test our approach, we successfully predict changes in cell shapes by modulating actin polymerisation in experiments and in simulations. Our study shows how active fluid theory integrated with experiments can determine cell shapes in 3D tissues.

Liquid and elastic behavior of tissues drives their morphology and their response to the environment. They appear as the first insight on tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells by developing a fully automated surface tension and Youngs modulus measurement system. Flattening multicellular aggregates under magnetic constraint, we show that rigidity and surface tension act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization and intercellular adhesions, we reveal the role of desmin organization on the macroscopic mechanics of this tissue model.

The development of physics-based 3D models that investigate the behavior of biological tissues requires effective and efficient visualization tools. The open-source software ParaView has such capabilities, but often impose a steep learning curve due to the use of the Visualization Toolkit (VTK) data structures. To overcome this, I show how to setup the components of 3D vertex-like models, i.e., vertices, faces, and polyhedra, into the VTK data format and then output as ParaView unstructured grid files. I present a few relevant tools to visualize and analyze the files in ParaView. All sample codes are available in the Github repository vis3Dvertex.

AO_SCPLOWBSTRACTC_SCPLOWCell migration is involved in key phenomena in biology, ranging from development to cancer. Fibroblasts move between organs in 3D polymeric networks. So far, motile cells were mainly tracked in vitro on Petri dishes or on coverslips, i.e. 2D flat surfaces, which made the extrapolation to 3D physiological environments difficult. We therefore prepared 3D Cell Derived Matrix (CDM) with specific characteristics with the goal of extracting the main readouts required to measure and characterise cell motion: cell specific matrix deformation through the tracking of fluorescent fibronectin within CDM, focal contacts as the cell anchor and acto-myosin cytoskeleton which applies cellular forces. We report our method for generating this assay of physiological-like gel with relevant readouts together with its potential impact in explaining cell motility in vivo.

The mechanical interaction between cells and the extracellular matrix (ECM) is fundamental to coordinate collective cell behavior in multicellular tissues. Relating individual cell-level mechanics to tissue-scale collective behavior is an outstanding challenge which cell-based models such as the cellular Potts model (CPM) are well-positioned to address. These models generally represent the ECM with mean-field approaches, which assume substrate homogeneity. This assumption breaks down with fibrous ECM, which has non-trivial structure and mechanics. Here, we extend the CPM with a bead-spring chain model of ECM fiber networks modelled using molecular dynamics. We model contractile cells pulling with discrete focal adhesion-like sites on the ECM fiber network, and demonstrate agreement with experimental spatiotemporal fiber densification and displacement. We show that contractile cell forces propagate over multiple cell radii scaling with power law exponent of {approx} -0.5 typical of viscoelastic ECM. Further, we use in silico atomic force microscopy to measure local cell-induced network stiffening consistent with experiments. Our model lays the foundation to investigate how local and long-ranged cell-ECM mechanobiology contributes to multicellular morphogenesis.