Analysis Of Cell Behavior Research Articles

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform.

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low-throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high-throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet-bioprinting technique for printing cell-laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano-responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.

Comparative proteomic analysis of human mesenchymal stromal cell behavior on calcium phosphate ceramics with different osteoinductive potential.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation in vivo. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive β-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction – one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies.

Spatiotemporal Characterization of Anterior Segment Mesenchyme Heterogeneity During Zebrafish Ocular Anterior Segment Development.

Assembly of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to anterior segment dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and transcriptional identities during early establishment of the zebrafish AS. Two-color fluorescent in situ hybridization suggested that early AS associated POM comprise of a heterogenous population. In vivo and time-course imaging analysis of POM distribution and migratory dynamics analyzed using transgenic zebrafish embryos (Tg[foxc1b:GFP], Tg[foxd3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP]) revealed unique AS distribution and migratory behavior among the reporter lines. Based on fixed timepoint and real-time analysis of POM cell behavior a comprehensive model for colonization of the zebrafish AS was assembled. Furthermore, we generated single cell transcriptomic profiles (scRNA) from our POM reporter lines and characterized unique subpopulation expression patterns. Based on scRNA clustering analysis we observed cluster overlap between neural crest associated (sox10/foxd3), POM (pitx2) and finally AS specified cells (lmx1b, and foxc1b). scRNA clustering also revealed several novel markers potentially associated with AS development and/or function including lum, fmoda, adcyap1b, tgfbi, and hmng2. Taken together, our data indicates that AS-associated POM, or Anterior Segment Mesenchyme (ASM), is not homogeneous but rather comprised of several subpopulations with differing colonization patterns, migration behavior, and transcriptomic profiles.

DCEM: A data cell evolution model for service composition based on bigraph theory

Service composition is an important means of integrating individual web services to create new value-added systems that satisfy complex requirements. Effectively analyzing different types of services and identifying the matching similarity between services is a crucial component of service composition, as it allows failed services to be efficiently substituted in a distributed and dynamic environment. In this paper, we propose a data cell evolution model (DCEM) that supports the dynamic adaptation of service compositions. DCEM combines data service information and biological cell behavior analysis to encapsulate data services into data cells. To optimize the adaptations, we analyze the static and dynamic structure of data cells based on bigraph theory to guarantee the consistency of service evolution. To evaluate the proposed approach, a series of simulation experiments and comparisons are conducted to demonstrate the effectiveness of service composition.

Aging induces aberrant state transition kinetics in murine muscle stem cells.

ABSTRACTMurine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.

Computational Analysis of the Mutual Constraints between Single-Cell Growth and Division Control Models.

Three models of division control are proposed to achieve cell size homeostasis: sizer, timer, and adder. However, few published studies of division control take into account the dynamics of single-cell growth and most assume that single-cell growth is exponential. Here, computational simulations considering exponential, linear, and bilinear growth models are performed. These simulations confirm that a timer division control model alone cannot lead to size homeostasis if the single-cell growth model is exponential. Furthermore, timer and adder division control models cannot be distinguished if the single-cell growth model is linear. Models of division control cannot be easily differentiated by analysis of average cell behavior because the birth sizes of the majority of cells are close to the population average. However, the differences between division control models are amplified in outlier cells whose birth size is far from the average. A method is introduced for vector field analysis of the speed of convergence of outlier lineages toward the steady-state birth size, which can help to distinguish between division control models and single-cell growth models.

Facile Method for Fabricating Microfluidic Chip Integrated with Microwell Arrays for Cell Trapping.

With the development of biomedical technology, personalized diagnosis and treatment at the single-cell level are becoming more important in the medical field. As one of the most powerful tools, microfluidic chips have shown significant potential for various applications related to cell separation, cell proliferation, and cell behavior analysis. However, fabricating microfluidic devices requires complicated procedures and high-cost equipment. In this study, an optofluidic maskless lithography method was proposed for rapid fabrication of microfluidic devices integrated with microwells. Through the use of this approach, microwells can be on-line designed and the exposure patterns can be modulated. Single or multi polystyrene microspheres were successfully trapped by using the designed microwells. The capture of MCF-7 cells and cell arrays indicated that the microfluidic devices fabricated in the present study can be applied for cell research.

Development of Multi-Dimensional Cell Co-Culture via a Novel Microfluidic Chip Fabricated by DMD-Based Optical Projection Lithography.

Establishing a physiological microenvironment in vitro that is suitable for cell and tissue growth is essential for medical research. Microfluidic chips are widely used in the construction of a microenvironment and the analysis of cell behavior in vitro; however, the design and manufacture of microfluidic chips for the long-term culture of a tumor model tends to be highly complex and time-consuming. In this paper, we propose a method for the rapid fabrication of a microfluidic chip for multi-dimensional cell co-culture. A major advantage of this method is that the microfluidic chip can be divided into several sections by micro-pillar arrays to form different functional regions to grow two- and three-dimensional cell culture on the same matrix. At the micro-scale, the surface tension between the gelatin methacryloyl-encapsulated cells and micro-pillars prevents the leakage of the hydrogel, and the hydrogel provides a three-dimensional microenvironment for cell growth. Our results of long-term cell culture and preclinical drug screening showed that cells cultured in a two-dimensional monolayer differ from three-dimensional cultured cells in terms of morphology, area, survival rate, proliferation, and drug resistance. This method shows potential for use in the study of cell behavior, drug screening, and tissue engineering.

Improving Aging Prediction for Electric Vehicle Operation with Combined Electrical, Thermal and Aging Model for Lithium-Ion Battery Packs Using Quantitative Cell Data

Electric vehicles (EV) in the power grid are not necessarily an additional burden, but can also act grid relieving. This is possible by storing energy from renewable sources and shaving the volatility of generation. For EV battery owners it is important to predict the aging effects resulting from secondary battery use. For a future energy system, it is therefore important to address the topic of EV battery aging for the group of stakeholders: EV owners, EV manufacturers, energy providers and grid operators. A physico-chemically motivated, impedance-based lithium-ion battery model has recently been presented in [6] and is available as open source [5]. Additionally, the authors present a new set of quantitative measurement data for parameterization of a battery pack of Daimler’s Smart electric drive. A battery pack was disassembled to gather all information from inside the battery pack. The cells have been aged as part of an extensive aging testing. Therefore, the simulation setup enables in-situ and operando analysis of cell behavior and helps understanding aging effects in electric vehicle operation. For model parameterization, the research contributions for electrical modeling based on electrical impedance spectroscopy from [1], as well as studies on aging behavior of lithium-ion cells with graphite anode from [2] and [3] are considered and used to adapt the method presented in [4]. The model is parameterized with electrical, thermal and aging parameters for an automotive battery pack that consists of 93 high-energy lithium-ion pouch cells with NMC cathode and graphite anode with a capacity of 50 Ah. A 3D-geometrical representation of the battery pack topology including cooling allows a detailed assessment of the simulated cell temperatures. It therefore helps to generate a better understanding of the incremental battery aging effects that are caused by vehicle-to-grid (V2G) operation. Controlled charging and discharging of EVs could help the grid in terms of stability and avoid additional cost for grid expansion. At the same time, V2G services could generate additional revenues to the EV battery owners from service payment. Nevertheless, calculation of EV battery aging is one major purpose for EV battery owners before they agree to the active use of their EV battery by third parties. Sources: [1] Dissertation H. Witzenhausen, https://doi.org/10.18154/RWTH-2017-03437[2] Dissertation A. Warnecke, https://doi.org/10.18154/RWTH-2017-09646[3] Dissertation M. Lewerenz https://doi.org/10.18154/RWTH-2018-228663[4] Schmalstieg et al., https://doi.org/10.1016/j.jpowsour.2014.02.012[6] Dissertation F. Hust, “Physico-Chemically Motivated Parameterization and Modelling of Real-Time Capable Lithium-Ion Battery Models - a Case Study on the Tesla Model S Battery”, January 2019 [5] www.openbat.de

Stochastic single cell migration leads to robust horizontal cell layer formation in the vertebrate retina.

Developmental programs that arrange cells and tissues into patterned organs are remarkably robust. In the developing vertebrate retina, for example, neurons reproducibly assemble into distinct layers giving the mature organ its overall structured appearance. This stereotypic neuronal arrangement, termed lamination, is important for efficient neuronal connectivity. Although retinal lamination is conserved in many vertebrates, including humans, how it emerges from single cell behaviour is not fully understood. To shed light on this issue, we here investigated the formation of the retinal horizontal cell layer. Using in vivo light sheet imaging of the developing zebrafish retina, we generated a comprehensive quantitative analysis of horizontal single cell behaviour from birth to final positioning. Interestingly, we find that all parameters analysed, including cell cycle dynamics, migration paths and kinetics, as well as sister cell dispersal, are very heterogeneous. Thus, horizontal cells show individual non-stereotypic behaviour before final positioning. Yet these initially variable cell dynamics always generate the correct laminar pattern. Consequently, our data show that the extent of single cell stochasticity in the lamination of the vertebrate retina is underexplored.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†.

During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive fraction. To evaluate the migratory capability of the ASGs, we transplanted visualized cells into the peritoneal cavity of nibe croaker (Nibea mitsukurii) larvae. This resulted in incorporated fluorescent cells labeled with antibody No. 152 being detected in the recipient gonads, suggesting that the visualized ASGs possessed migratory and incorporation capabilities. Thus, the donor germ cell visualization method that was developed in this study will facilitate and simplify Pacific bluefin tuna germ cell transplantation.

Behavior of Stem-Like Cells, Precursors for Tissue Regeneration in Urodela, Under Conditions of Microgravity.

We summarize data from our experiments on stem-like cell-dependent regeneration in amphibians in microgravity. Considering its deleterious effect on many tissues, we asked whether microgravity is compatible with reparative processes, specifically activation and proliferation of source cells. Experiments were conducted using tailed amphibians, which combine profound regenerative capabilities with high robustness, allowing an in vivo study of lens, retina, limb, and tail regeneration in challenging settings of spaceflight. Microgravity promoted stem-like cell proliferation to a varying extent (up to 2-fold), and it seemed to speed up source cell dedifferentiation, as well as sequential differentiation in retina, lens, and limb, leading to formation of bigger and more developed regenerates than in 1g controls. It also promoted proliferation and hypertrophy of Müller glial cells, eliciting a response similar to reactive gliosis. A significant increase in stem-like cell proliferation was mostly beneficial for regeneration and only in rare cases caused moderate tissue growth abnormalities. It is important that microgravity yielded a lasting effect even if applied before operations. We hypothesize on the potential mechanisms of gravity-dependent changes in stem-like cell behavior, including fibroblast growth factor 2 signaling pathway and heat shock proteins, which were affected in our experimental settings. Taken together, our data indicate that microgravity does not disturb the natural regenerative potential of newt stem-like cells, and, depending on the system, even stimulates their dedifferentiation, proliferation, and differentiation. We discuss these data along with publications on mammalian stem cell behavior in vitro and invertebrate regeneration in vivo in microgravity. In vivo data are very scarce and require further research using contemporary methods of cell behavior analysis to elucidate mechanisms of stem cell response to altered gravity. They are relevant for both practical applications, such as managing human reparative responses in spaceflight, and fundamental understanding of stem cell biology.

A spatially varying charge model for regulating site-selective protein adsorption and cell behaviors.

Implanted materials that enter the body first interact with proteins in body fluids, and cells then perceive and respond to the foreign implant through this layer of adsorbed proteins. Thus, spatially specific regulation of protein adsorption on an implant surface is pivotal for mediating subsequent cellular behaviors. Unlike the surface modulation strategy for traditional biomaterials, in this research, materials with a nonuniform spatial distribution of surface charges were designed to achieve site-selective protein adsorption and further influence cell behavior by charge regulation. Spatially varying microdomains with different levels of piezoelectricity were generated via a focus laser beam-induced phase transition. In addition, after polarization, the zones with different levels of piezoelectricity showed significant differences in surface charge density. The results of scanning Kelvin probe force microscopy (SKPM) showed that the surface charge on the material exhibits a nonuniform spatial distribution after laser irradiation and polarization. Site-specific charge-mediated selective protein adsorption was demonstrated through a protein adsorption experiment. Cell behavior analysis showed that the increase in charge density was conducive to promoting cell adhesion and the formation of filopodia while the nonuniform spatial distribution of charge promoted an oriented arrangement of cells; both features accelerated cell migration. This study provides a new method for spatially regulating protein adsorption through surface charges to further influence cell behaviors.

Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout

BackgroundThe dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT).ResultsFor the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors.ConclusionsThe transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.

Acquisition and reconstruction of 4D surfaces of axolotl embryos with the flipping stage robotic microscope

We have designed and constructed a Flipping Stage for a light microscope that can view the whole exterior surface of a 2 mm diameter developing axolotl salamander embryo. It works by rapidly inverting the bottom-heavy embryo, imaging it as it rights itself. The images are then montaged to reconstruct the whole 3D surface versus time, for a full 4D record of the surface. Imaging early stage axolotl development will help discover how cell differentiation and movement takes place in the early embryo. For example, the switch from ectodermal to neural plate cells takes place on the top, animal surface portion the egg/embryo and can be observed using the flipping stage microscope. Detailed pictures of the whole surface need to be obtained so that cell tracking and event histories, such as cell divisions and participation in differentiation waves, of individual cells can be recorded. Imaging the whole exterior of the eggs/embryos will allow for the analysis of cell behavior and the forces the cells experience in their natural setting in the intact or manipulated embryo. This will give insights into embryogenesis, development, developmental disruptions, birth defects, cell differentiation and tissue engineering.

Competitive Tuning Among Ca2+/Calmodulin-Dependent Proteins: Analysis of in silico Model Robustness and Parameter Variability.

Calcium/Calmodulin-dependent (Ca2+/CaM-dependent) regulation of protein signaling has long been recognized for its importance in a number of physiological contexts. Found in almost all eukaryotic cells, Ca2+/CaM-dependent signaling participates in muscle development, immune responses, cardiac myocyte function and regulation of neuronal connectivity. In excitatory neurons, dynamic changes in the strength of synaptic connections, known as synaptic plasticity, occur when calcium ions (Ca2+) flux through NMDA receptors and bind the Ca2+-sensor calmodulin (CaM). Ca2+/CaM, in turn, regulates downstream protein signaling in actin polymerization, receptor trafficking, and transcription factor activation.The activation of downstream Ca2+/CaM-dependent binding proteins (CBPs) is a function of the frequency of Ca2+ flux, such that each CBP is preferentially "tuned" to different Ca2+ input signals. We have recently reported that competition among CBPs for CaM binding is alone sufficient to recreate in silico the observed in vivo frequency-dependence of several CBPs. However, CBP activation may strongly depend on the identity and concentration of proteins that constitute the competitive pool; with important implications in the regulation of CBPs in both normal and disease states. Here, we extend our previous deterministic model of competition among CBPs to include phosphodiesterases, AMPAR receptors that are important in synaptic plasticity, and enzymatic function of CBPs: cAMP regulation, kinase activity, and phosphatase activity. After rigorous parameterization and validation by global sensitivity analysis using Latin Hypercube Sampling (LHS) and Partial Rank Correlation Coefficients (PRCC), we explore how perturbing the competitive pool of CBPs influences downstream signaling events. In particular, we hypothesize that although perturbations may decrease activation of one CBP, increased activation of a separate, but enzymatically-related CBP could compensate for this loss, providing a homeostatic effect. First we compare dynamic model output of two models: a two-state model of Ca2+/CaM binding and a four-state model of Ca2+/CaM binding. We find that a four-state model of Ca2+/CaM binding best captures the dynamic nature of the rapid response of CaM and CBPs to Ca2+ flux in the system. Using global sensitivity analysis, we find that model output is robust to parameter variability. Indeed, although variations in the expression of the CaM buffer neurogranin (Ng) may cause a decrease in Ca2+/CaM-dependent kinase II (CaMKII) activation, overall AMPA receptor phosphorylation is preserved; ostensibly by a concomitant increase in adenylyl cyclase 8 (AC8)-mediated activation of protein kinase A (PKA). Indeed phosphorylation of AMPAR receptors by CaMKII and PKA is robust across a wide range of Ng concentrations, though increases in AMPAR phosphorylation is seen at low Ng levels approaching zero. Our results may explain recent counter-intuitive results in neurogranin knockout mice and provide further evidence that competitive tuning is an important mechanism in synaptic plasticity. These results may be readily translated to other Ca2+/CaM-dependent signaling systems in other cell types and can be used to suggest targeted experimental investigation to explain counter-intuitive or unexpected downstream signaling outcomes.Tamara Kinzer-Ursem is an Assistant Professor in the Weldon School of Biomedical Engineering. She received her B.S. in Bioengineering from the University of Toledo and her M.S. and Ph.D. degrees in Chemical Engineering from the University of Michigan, and her post-doctoral training in Molecular Neuroscience at the California Institute of Technology. Prior to joining Purdue she was the Head of R&D in Biochemistry at Maven Biotechnologies and Visiting Associate in Chemical Engineering at the California Institute of Technology.Research in the Kinzer-Ursem lab focuses on developing tools to advance quantitative descriptions of cellular processes and disease within three areas of expertise: 1) Using particle diffusivity measurements to quantify biomolecular processes. Particle diffusometry is being used as a sensitive biosensor to detect the presence of pathogens in environmental and patient samples. 2) Development of novel protein tagging technologies that are used to label proteins in vivo to enable quantitative description of protein function and elucidate disease mechanisms. 3) Computational modeling of signal transduction mechanisms to understand cellular processes. Using computational techniques, we have recently described "competitive tuning" as a mechanism that might be used to regulate information transfer through protein networks, with implications in cell behavior and drug target analysis.

Quantitative phase imaging unravels new insight into dynamics of mesenchymal and amoeboid cancer cell invasion

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells’ activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells’ membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

Osteoblasts infill irregular pores under curvature and porosity controls: a hypothesis-testing analysis of cell behaviours.

The geometric control of bone tissue growth plays a significant role in bone remodelling, age-related bone loss, and tissue engineering. However, how exactly geometry influences the behaviour of bone-forming cells remains elusive. Geometry modulates cell populations collectively through the evolving space available to the cells, but it may also modulate the individual behaviours of cells. To factor out the collective influence of geometry and gain access to the geometric regulation of individual cell behaviours, we develop a mathematical model of the infilling of cortical bone pores and use it with available experimental data on cortical infilling rates. Testing different possible modes of geometric controls of individual cell behaviours consistent with the experimental data, we find that efficient smoothing of irregular pores only occurs when cell secretory rate is controlled by porosity rather than curvature. This porosity control suggests the convergence of a large scale of intercellular signalling to single bone-forming cells, consistent with that provided by the osteocyte network in response to mechanical stimulus. After validating the mathematical model with the histological record of a real cortical pore infilling, we explore the infilling of a population of randomly generated initial pore shapes. We find that amongst all the geometric regulations considered, the collective influence of curvature on cell crowding is a dominant factor for how fast cortical bone pores infill, and we suggest that the irregularity of cement lines thereby explains some of the variability in double labelling data as well as the overall speed of osteon infilling.

Aging and Spaceflight: Catalase targeted to mitochondria alters skeletal structure and responses to musculoskeletal disuse

Accumulation of oxidative damage from excess reactive oxygen species (ROS) contributes to aging and also may mediate adverse tissue responses to the space environment. We aim to explore the importance of cellular redox defense for physiological adaptation and tissue degeneration during spaceflight or prolonged bedrest on Earth. To begin to test this, transgenic mice (male, 16 weeks of age) with human catalase targeted to mitochondria (mCAT), and wildtype (WT) littermates were analysed for responsiveness to a combination of hindlimb unloading (to simulate weightlessness) and a single dose of ionizing radiation (137Cs, 0.83 Gy/min, 2Gy); mCAT mice are known to display improved longevity and reduced age‐related diseases compared to WT mice (Schriner et al. 2005, Dai et al. 2010). After 2 weeks, bones were recovered for analysis of cell behavior (osteoblast and osteoclast differentiation ex vivo from bone marrow cells), oxidative lipid damage (malondialdehyde, 4‐hydroxynonenol) and structure (3D microcomputed tomography). mCAT mice expressed the catalase transgene in skeletal tissues, and in cultured marrow‐derived osteoblasts and pre‐osteoclasts. Treatment with unloading and radiation led to increased lipid oxidative damage markers in bones from WT mice but not mCAT mice, showing that transgene expression effectively mitigated oxidative damage. Ex vivo osteoblast colony growth rate was 95% greater in mCAT mice than WT, and correlated with catalase activity levels (P&lt;0.005, r=0.6679), though terminal differentiation of osteoblasts (mineralization) and osteoclasts (resorption pit formation) remained unaffected. Microarchitectural analysis of high turnover, cancellous tissue revealed diminished size of mCAT bones compared to WT (−16% bone volume/total volume mCAT/WT), and no genotype‐dependent difference in cancellous bone loss caused by treatment. Similarly, slow‐turnover, cortical bones were smaller in control mCAT mice (−7% cortical area), although treatment of mCAT mice showed an unexpected and rapid stimulation of radial expansion (+8% cortical area, +22% moment of inertia, mCAT:Treated/Control), which was not observed in WT mice. Thus, quenching of mitochondrial ROS by over‐expression of catalase appeared to stimulate growth but not differentiation of osteoprogenitors ex vivo; nonetheless, the smaller size of mCAT bones showed that mitochondrial ROS signaling was important for the acquisition of adult bone structure. Further, we conclude that transgene expression led to changes in cortical structure following treatment that are similar to those that occur during aging, indicative of a role for mitochondrial ROS in coordinating skeletal remodeling in response to challenge.Support or Funding InformationGrant funding from NASA Space Biology program (NNH14ZTT001N) and the National Space Biomedical Research Institute through NCC 9–58, Project MA02501. A.S. Schreurs was supported by a NASA Space Biology Postdoctoral Program Fellowship award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gelatin Methacryloyl Hydrogels in the Absence of a Crosslinker as 3D Glioblastoma Multiforme (GBM)-Mimetic Microenvironment.

3D platforms are important for monitoring tumor progression and screening drug candidates to eradicate tumors such as glioblastoma multiforme (GBM), a malignant type of human brain tumor. Here, a new strategy is reported that exploits visible-light-induced crosslinking of gelatin where the reaction is carried out in the absence of an additional crosslinker. Visible light-induced crosslinking promotes the design of cancer microenvironment-mimetic system without compromising the cell viability during the process and absence of crosslinker facilitates the synthesis of the unique construct. Suspension and spheroid-based models of GBM are used to investigate cellular behavior, expression profiles of malignancy, and apoptosis-related genes within this unique network. Furthermore, sensitivity to an anticancer drug, Digitoxigenin, treatment is investigated in detail. The data suggest that U373 cells, in sparse or spheroid form, have significantly decreased expressions of apoptosis-activating genes, Bad, Puma, and Caspase-3, and a high expression of prosurvival Bcl-2 gene within GelMA hydrogels. Matrix-metalloproteinase genes are also upregulated within GelMA, suggesting positive contribution of gels on extracellular remodeling of cancer cells. This unique photocurable gelatin holds great potential for clinical translation of cancer research through the analysis of 3D malignant cancer cell behavior, and hence for more efficient treatment methods for GBM.