Extracellular Matrix Fiber Network Research Articles

Hybrid cellular Potts and bead-spring modeling of cells in fibrous extracellular matrix

The mechanical interaction between cells and the extracellular matrix (ECM) is fundamental to coordinate collective cell behavior in tissues. Relating individual cell-level mechanics to tissue-scale collective behavior is a challenge that 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 nontrivial structure and mechanics. Here, we extend the CPM with a bead-spring model of ECM fiber networks modeled using molecular dynamics. We model a contractile cell pulling with discrete focal adhesion-like sites on the fiber network and demonstrate agreement with experimental spatiotemporal fiber densification and displacement. We show that at high network cross-linking, contractile cell forces propagate over at least eight cell diameters, decaying with distance with power law exponent n= 0.35 – 0.65 typical of viscoelastic ECMs. Further, we use in silico atomic force microscopy to measure local cell-induced network stiffening consistent with experiments. Our model lays the foundation for investigating how local and long-ranged cell-ECM mechanobiology contributes to multicellular morphogenesis.

A computational modeling of invadopodia protrusion into an extracellular matrix fiber network

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

Three-dimensional pore network characterization of reconstructed extracellular matrix.

The extracellular matrix (ECM) has a fiber network that provides physical scaffolds to cells and plays important roles by regulating cellular functions. Some previous works characterized the mechanical and geometrical properties of the ECM fiber network using reconstituted collagen-I. However, the characterization of the porous structure of reconstituted collagen-I has been limited to the pore diameter measurement, and pore network extraction has not been applied to reconstituted collagen-I despite the importance of pore interconnectivity. Here, we aim to show the importance of characterizing the pore network of reconstituted collagen-I by comparing the pore networks of structures that have different fiber alignments. We show that the fiber alignment significantly changes the pore throat area but not the pore diameter. Also, we demonstrate that larger pore throats are directed in the direction of the fiber alignment, which may help in understanding the enhanced cell migration when fibers are aligned.

Predicting Confined 1D Cell Migration from Parameters Calibrated to a 2D Motor-Clutch Model

Biological tissues contain micrometer-scale gaps and pores, including those found within extracellular matrix fiber networks, between tightly packed cells, and between blood vessels or nerve bundles and their associated basement membranes. These spaces restrict cell motion to a single-spatial dimension (1D), a feature that is not captured in traditional in vitro cell migration assays performed on flat, unconfined two-dimensional (2D) substrates. Mechanical confinement can variably influence cell migration behaviors, and it is presently unclear whether the mechanisms used for migration in 2D unconfined environments are relevant in 1D confined environments. Here, we assessed whether a cell migration simulator and associated parameters previously measured for cells on 2D unconfined compliant hydrogels could predict 1D confined cell migration in microfluidic channels. We manufactured microfluidic devices with narrow channels (60-μm2 rectangular cross-sectional area) and tracked human glioma cells that spontaneously migrated within channels. Cell velocities (vexp = 0.51 ± 0.02 μm min−1) were comparable to brain tumor expansion rates measured in the clinic. Using motor-clutch model parameters estimated from cells on unconfined 2D planar hydrogel substrates, simulations predicted similar migration velocities (vsim = 0.37 ± 0.04 μm min−1) and also predicted the effects of drugs targeting the motor-clutch system or cytoskeletal assembly. These results are consistent with glioma cells utilizing a motor-clutch system to migrate in confined environments.

Impact of crosslink heterogeneity on extracellular matrix mechanics and remodeling

Mechanical interactions between cells and the extracellular matrix (ECM) lead to the formation of biophysical cues, notably in the form of cell-generated tension, stiffness, and concentration profiles in the ECM. Fibrillar ECMs have nonlinear stiffnesses, linked to the reorientation of fibers under stress and strain, and nonelastic properties, resulting from the force-induced unbinding of transient bonds (crosslinks) that interconnect fibers. Mechanical forces generated by cells can lead to local ECM stiffening and densification. Cell tension is also propagated through the ECM network. The underlying factors that regulate the relative emergence of these signals are not well understood. Here, through computational simulations of 3D ECM fiber networks, we show that the composition of ECM crosslinks is a key determinant of the degree of densification and stiffening that can be achieved by cell-generated forces. This also regulates the sustainability of tensions propagated through the ECM. In particular, highly transient force-sensitive crosslinks promote nonelastic densification and rapid tension relaxation, whereas permanent crosslinks promote nonlinear stiffening and stable tension profiles. A heterogeneous population of crosslinks with different unbinding kinetics enables ECMs to exhibit accumulation, tension propagation, and stiffening simultaneously in response to mechanical interactions with cells.

Multi-cell ECM compaction is predictable via superposition of nonlinear cell dynamics linearized in augmented state space

Cells interacting through an extracellular matrix (ECM) exhibit emergent behaviors resulting from collective intercellular interaction. In wound healing and tissue development, characteristic compaction of ECM gel is induced by multiple cells that generate tensions in the ECM fibers and coordinate their actions with other cells. Computational prediction of collective cell-ECM interaction based on first principles is highly complex especially as the number of cells increase. Here, we introduce a computationally-efficient method for predicting nonlinear behaviors of multiple cells interacting mechanically through a 3-D ECM fiber network. The key enabling technique is superposition of single cell computational models to predict multicellular behaviors. While cell-ECM interactions are highly nonlinear, they can be linearized accurately with a unique method, termed Dual-Faceted Linearization. This method recasts the original nonlinear dynamics in an augmented space where the system behaves more linearly. The independent state variables are augmented by combining auxiliary variables that inform nonlinear elements involved in the system. This computational method involves a) expressing the original nonlinear state equations with two sets of linear dynamic equations b) reducing the order of the augmented linear system via principal component analysis and c) superposing individual single cell-ECM dynamics to predict collective behaviors of multiple cells. The method is computationally efficient compared to original nonlinear dynamic simulation and accurate compared to traditional Taylor expansion linearization. Furthermore, we reproduce reported experimental results of multi-cell induced ECM compaction.

Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability.MethodsPANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs.ResultsCo-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling.ConclusionsHere, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs.

Local alignment vectors reveal cancer cell-induced ECM fiber remodeling dynamics

Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics.

Cell Invasion Dynamics into a Three Dimensional Extracellular Matrix Fibre Network.

The dynamics of filopodia interacting with the surrounding extracellular matrix (ECM) play a key role in various cell-ECM interactions, but their mechanisms of interaction with the ECM in 3D environment remain poorly understood. Based on first principles, here we construct an individual-based, force-based computational model integrating four modules of 1) filopodia penetration dynamics; 2) intracellular mechanics of cellular and nuclear membranes, contractile actin stress fibers, and focal adhesion dynamics; 3) structural mechanics of ECM fiber networks; and 4) reaction-diffusion mass transfers of seven biochemical concentrations in related with chemotaxis, proteolysis, haptotaxis, and degradation in ECM to predict dynamic behaviors of filopodia that penetrate into a 3D ECM fiber network. The tip of each filopodium crawls along ECM fibers, tugs the surrounding fibers, and contracts or retracts depending on the strength of the binding and the ECM stiffness and pore size. This filopodium-ECM interaction is modeled as a stochastic process based on binding kinetics between integrins along the filopodial shaft and the ligands on the surrounding ECM fibers. This filopodia stochastic model is integrated into migratory dynamics of a whole cell in order to predict the cell invasion into 3D ECM in response to chemotaxis, haptotaxis, and durotaxis cues. Predicted average filopodia speed and that of the cell membrane advance agreed with experiments of 3D HUVEC migration at r2 > 0.95 for diverse ECMs with different pore sizes and stiffness.

Midfacial rejuvenation by hyaluronic acid fillers and subcutaneous adipose tissue – A new concept

In midface rejuvenation, hyaluronic acid (HA) fillers are commonly used as a versatile tool to improve appearance and to correct V-deformities and loss of volume. The induction of collagen as a major constituent of extracellular matrix (ECM) has been considered to be a basic effect of the rejuvenation procedure. Although commonly described as “dermal” soft fillers, histologic studies localized HA filler in the subcutaneous adipose tissue.Deep injection whenever possible lead to prolonged efficacy. Since volumizing HA filler induce mechanical stress not only to fibroblasts but adipocytes and deep injection itself causes minor trauma in the subcutaneous adipose tissue we suggest that the activation of adipose tissue-derived mesenchymal stem cells (ADMSC) is responsible for the observed clinical effects. We present a concept of filler action that discusses interactions of HA with adipocytes, ECM fiber network and ADMSC. Such a concept can explain the prolonged efficacy of deep midfacial filler placement and offers a new understanding to tailor HA fillers in the future.