controlled-strain-of-3d-hydrogels-under-live-microscopy-imaging

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Tel-Aviv University

Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications.

Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation -resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up (resembling mesenchymal to epithelial transition (MET) phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation-based collective cell motility dynamics. Our quantitative phenotypes may guide future investigation on the molecular and cellular mechanisms of tyrosine kinase-induced coordinate cell motility and morphogenesis in metastasis.

Emergence of HGF/SF-induced coordinated cellular motility.

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.

Propagating waves of directionality and coordination orchestrate collective cell migration.

High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met-Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met-Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy. 

Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy.

The wound healing assay is the common method to study collective cell migration in vitro. Computational analyses of live imaging exploit the rich temporal information and significantly improve understanding of complex phenomena that emerge during this mode of collective motility. Publicly available experimental data can allow application of new analyses to promote new discoveries, and assess algorithms' capabilities to distinguish between different experimental conditions.

Live time-lapse dataset of in vitro wound healing experiments.

Metastasizing tumor cells migrate through the surrounding tissue and extracellular matrix toward the blood vessels, in order to colonize distant organs. They typically move in a dense environment, filled with other cells. In this work we study cooperative effects between neighboring cells of different types, migrating in a maze-like environment with directional cue. Using a computerized model, we measure the percentage of cells that arrive to the defined target, for different mesenchymal/amoeboid ratios. Wall degradation of mesenchymal cells, as well as motility of both types of cells, are coupled to metabolic energy-like resource level. We find that indirect cooperation emerges in mid-level energy, as mesenchymal cells create paths that are used by amoeboids. Therefore, we expect to see a small population of mesenchymals kept in a mostly-amoeboid population. We also study different forms of direct interaction between the cells, and show that energy-dependent interaction strength is optimal for the migration of both mesenchymals and amoeboids. The obtained characteristics of cellular cluster size are in agreement with experimental results. We therefore predict that hybrid states, e.g. epithelial-mesenchymal, should be utilized as a stress-response mechanism. 

Tumor invasion optimization by mesenchymal-amoeboid heterogeneity.

Metastasis is the major cause for cancer patients' death, and despite all the recent advances in cancer research it is still mostly incurable. Understanding the mechanisms that are involved in the migration of the cells in a complex environment is a key step towards successful anti-metastatic treatment. Using experimental data-based modeling, we focus on the fundamentals of metastatic invasion: motility, invasion, proliferation and metabolism, and study how they may be combined to maximize the cancer's ability to metastasize. The modeled cells' performance is measured by the number of cells that succeed in migration in a maze, which mimics the extracellular environment. We show that co-existence of different cell clones in the tumor, as often found in experiments, optimizes the invasive ability in a frequently-changing environment. We study the role of metabolism and stimulation by growth factors, and show that metabolism plays a crucial role in the metastatic process and should therefore be targeted for successful treatment.

The motility-proliferation-metabolism interplay during metastatic invasion.

Biological cells embedded in fibrous matrices have been observed to form intercellular bands of dense and aligned fibers through which they mechanically interact over long distances. Such matrix-mediated cellular interactions have been shown to regulate various biological processes. This study aimed to explore the effects of elastic nonlinearity of the fibers contained in the extracellular matrix (ECM) on the transmission of mechanical loads between contracting cells. Based on our biological experiments, we developed a finite-element model of two contracting cells embedded within a fibrous network. The individual fibers were modeled as showing linear elasticity, compression microbuckling, tension stiffening, or both of the latter two. Fiber compression buckling resulted in smaller loads in the ECM, which were primarily directed toward the neighboring cell. These loads decreased with increasing cell-to-cell distance; when cells were >9 cell diameters apart, no such intercellular interaction was observed. Tension stiffening further contributed to directing the loads toward the neighboring cell, though to a smaller extent. The contraction of two neighboring cells resulted in mutual attraction forces, which were considerably increased by tension stiffening and decayed with increasing cell-to-cell distances. Nonlinear elasticity contributed also to the onset of force polarity on the cell boundaries, manifested by larger contractile forces pointing toward the neighboring cell. The density and alignment of the fibers within the intercellular band were greater when fibers buckled under compression, with tension stiffening further contributing to this structural remodeling. Although previous studies have established the role of the ECM nonlinear mechanical behavior in increasing the range of force transmission, our model demonstrates the contribution of nonlinear elasticity of biological gels to directional and efficient mechanical signal transfer between distant cells, and rehighlights the importance of using fibrous gels in experimental settings for facilitating intercellular communication. VIDEO ABSTRACT.

Nonlinear Elasticity of the ECM Fibers Facilitates Efficient Intercellular Communication.

External forces play an important role in the development and regulation of many tissues. Such effects are often studied using specialized stretchers-standardized commercial and novel laboratory-designed. While designs for 2D stretchers are abundant, the range of available 3D stretcher designs is more limited, especially when live imaging is required. This work presents a novel method and a stretching device that allow straining of 3D hydrogels from their circumference, using a punctured elastic silicone strip as the sample carrier. The system was primarily constructed from 3D-printed parts and low-cost electronics, rendering it simple and cost-efficient to reproduce in other labs. To demonstrate the system functionality, > 100 μm thick soft fibrin gels (< 1 KPa) were stretched, while performing live confocal imaging. The subsequent strains and fiber alignment were analyzed and found to be relatively homogenous throughout the gel's thickness (Z axis). The uniform Z-response enabled by our approach was found to be in contrast to a previously reported approach that utilizes an underlying elastic substrate to convey strain to a 3D thick sample. This work advances the ability to study the role of external forces on biological processes under more physiological 3D conditions, and can contribute to the field of tissue engineering.

Straining 3D Hydrogels with Uniform Z-Axis Strains While Enabling Live Microscopy Imaging.

When seeded in fibrous gels, pairs of cells or cell aggregates can induce bands of deformed gel, extending to surprisingly long distances in the intercellular medium. The formation of bands has been previously shown and studied in collagen systems. In this study, we strive to further our understanding of this fundamental mechanical mechanism in fibrin, a key element in wound healing and angiogenesis processes. We embedded fibroblast cells in 3D fibrin gels, and monitored band formation by real-time confocal microscopy. Quantitative dynamic analysis of band formation revealed a gradual increase in fiber density and alignment between pairs of cells. Such intercellular bands extended into a large-scale network of mechanically connected cells, in which the connected cells exhibited a more spread morphology than the isolated cells. Moreover, computational modeling demonstrated that the direction of cell-induced force triggering band formation can be applied in a wide range of angles relative to a neighboring cell. Our findings indicate that long-range mechanical coupling between cells is an important mechanism in regulating multicellular processes in reconstituted fibrin gels. As such, it should motivate exploration of this mechanism in studies in vivo, in wound healing or angiogenesis, in which fibrin is contracted by fibroblast cells.

Long-range mechanical coupling of cells in 3D fibrin gels.

In vivo, eukaryotic cells are embedded in a matrix environment, where they grow and develop. Generally, this extracellular matrix (ECM) is an anisotropic fibrous structure, through which macromolecules and biochemical signaling molecules at the nanometer scale diffuse. The ECM is continuously remodeled by cells, via mechanical interactions, which lead to a potential link between biomechanical and biochemical cell-cell interactions. Here, it is studied how cell-induced forces applied on the ECM impact the biochemical transport of molecules between distant cells. It is experimentally observed that cells remodel the ECM by increasing fiber alignment and density of the matrix between them over time. Using random walk simulations on a 3D lattice, elongated fixed obstacles are implemented that mimic the fibrous ECM structure. Both diffusion of a tracer molecule and the mean first-passage time a molecule secreted from one cell takes to reach another cell are measured. The model predicts that cell-induced remodeling can lead to a dramatic speedup in the transport of molecules between cells. Fiber alignment and densification cause reduction of the transport dimensionality from a 3D to a much more rapid 1D process. Thus, a novel mechanism of mechano-biochemical feedback in the regulation of long-range cell-cell communication is suggested.

Mechanical Interaction between Cells Facilitates Molecular Transport.

It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.

Article	Year
Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images. PloS one\| PubMed ID: 22096600	2011
Emergence of HGF/SF-induced coordinated cellular motility. PloS one\| PubMed ID: 22970283	2012
Propagating waves of directionality and coordination orchestrate collective cell migration. PLoS computational biology\| PubMed ID: 25058592	2014
Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy. Oncoscience\| PubMed ID: 25593982	2014
Live time-lapse dataset of in vitro wound healing experiments. GigaScience\| PubMed ID: 25722853	2015
Tumor invasion optimization by mesenchymal-amoeboid heterogeneity. Scientific reports\| PubMed ID: 26013062	2015
The motility-proliferation-metabolism interplay during metastatic invasion. Scientific reports\| PubMed ID: 26337223	2015
Nonlinear Elasticity of the ECM Fibers Facilitates Efficient Intercellular Communication. Biophysical journal\| PubMed ID: 30217380	2018
Straining 3D Hydrogels with Uniform Z-Axis Strains While Enabling Live Microscopy Imaging. Annals of biomedical engineering\| PubMed ID: 31802281	2020
Long-range mechanical coupling of cells in 3D fibrin gels. Molecular biology of the cell\| PubMed ID: 32374653	2020
Mechanical Interaction between Cells Facilitates Molecular Transport. Advanced biosystems\| PubMed ID: 32648678	2019
Motion magnification analysis of microscopy videos of biological cells. PloS one\| PubMed ID: 33151976	2020

Sari Natan

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