Soluble Gradients Research Articles

Gene Expression Profiles in 3D Tumor Analogs Indicate Compressive Strain Differentially Enhances Metastatic Potential

Non-physiological mechanobiological stimuli typically occur in tumors and are considered to promote cancer spreading. Non-fluid related pressure (solid stress), which arises as tumors grow against adjacent tissues, is among the least studied endogenous stimuli due to challenges in replicating the in vivo environment. To this end, the novel devices well-pressor and the videomicroscopy-compatible optic-pressor were developed to exert precise compressive strain on cells in 3D gels in absence of other mechanical stimuli and soluble gradients. Glioblastoma (U87, HGL21) and breast cancer (MDA-MB-231) cells in 1% agarose hydrogels were exposed to 50% compressive strain for 3h (0.25-0.05kPa). Live imaging showed that cells elongate and deflect vertically to the load. This stimulation is shown for the first time to differentially regulate metastasis-associated genes. Furthermore, a group of differentially expressed genes was identified in all cell types, both by microarrays and confirmed by RT-PCR for select genes (caveolin-1, integrin-β1, Rac1), indicating shared response mechanisms. These genes are functionally linked and involved in decreasing cell-cell contact, increasing ECM degradation, and ultimately promoting invasion. Caveolin could orchestrate these responses while the uPA and PI3K/Akt systems could play major roles. Future work will focus on specific molecular partnerships under compression and their impact on cancer progression.

Immobilized Chemokine Fields and Soluble Chemokine Gradients Cooperatively Shape Migration Patterns of Dendritic Cells

Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

A process-oriented and stochastic simulation model for asparagus spear growth and yield

To obtain a better understanding of the factors affecting asparagus spear growth and yield, a process-oriented and stochastic model for asparagus ( Asparagus officinalis L.) grown under soil ridges was developed and tested. This model describes a population of asparagus plants with a binomial distributed bud cluster number per plant. Each bud cluster starts to grow after exposure to an exponential distributed temperature sum. The length growth response of spears, which is mainly dependent on soil temperature and spear length, is affected by the soluble carbohydrate ( CHO) concentration in the storage root system and the temperature gradient between the spear tip and the rhizome. The spear growth rate is assumed to be normally distributed, while a stochastic dependency to the required temperature sum for bud break is assumed. For each bud cluster, a broadly varied apical dominance relation between successive spears is described by an exponential distributed temperature sum for spear length growth induction. After removal of the dominating spear, the required temperature sum decreases exponentially. The time progression of spear diameter is a function of spear generation number within a bud cluster and the mean temperature during spear growth induction and can be used to derive the spear fresh weight. The change of soluble CHO concentrations in the storage root system is derived from maintenance and growth respiration requirements. Some of the model parameters are derived from independent measurements of spear growth. Regarding the tremendous variability of daily yields, the model performed well in the prediction of daily spear number, diameter and yield over the whole harvest season across two production sites with various soil temperatures induced by different types of ridge covering. The effects of soluble CHO concentration and soil temperature gradient on spear growth rate are regarded as preliminary and should be tested in further experiments. Due to the apical dominance relation between spears, asparagus is subject to endogenous rhythmic growth, which invalidates simple statistical yield forecast approaches. In its current state, our yield forecast model is well suited to test the impact of various plant traits on asparagus yield, and thus to guide early selection in crop improvement projects. Finally, the current model could be used to derive easier to handle yet physiological sound yield models to be useful for field-scale applications.

Self-organization of intracellular gradients during mitosis

Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

Adaptive force transmission in amoeboid cell migration

The leading front of a cell can either protrude as an actin-free membrane bleb that is inflated by actomyosin-driven contractile forces, or as an actin-rich pseudopodium, a site where polymerizing actin filaments push out the membrane. Pushing filaments can only cause the membrane to protrude if the expanding actin network experiences a retrograde counter-force, which is usually provided by transmembrane receptors of the integrin family. Here we show that chemotactic dendritic cells mechanically adapt to the adhesive properties of their substrate by switching between integrin-mediated and integrin-independent locomotion. We found that on engaging the integrin-actin clutch, actin polymerization was entirely turned into protrusion, whereas on disengagement actin underwent slippage and retrograde flow. Remarkably, accelerated retrograde flow was balanced by an increased actin polymerization rate; therefore, cell shape and protrusion velocity remained constant on alternating substrates. Due to this adaptive response in polymerization dynamics, tracks of adhesive substrate did not dictate the path of the cells. Instead, directional guidance was exclusively provided by a soluble gradient of chemoattractant, which endowed these 'amoeboid' cells with extraordinary flexibility, enabling them to traverse almost every type of tissue.

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen–receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.

Cell Guidance by 3D-Gradients in Hydrogel Matrices: Importance for Biomedical Applications

Concentration gradients of soluble and matrix-bound guidance cues in the extracellular matrix direct cell growth in native tissues and are of great interest for design of biomedical scaffolds and on implant surfaces. The focus of this review is to demonstrate the importance of gradient guidance for cells as it would be desirable to direct cell growth onto/into biomedical devices. Many studies have been described that illustrate the production and characterization of surface gradients, but three dimensional (3D)-gradients that direct cellular behavior are not well investigated. Hydrogels are considered as synthetic replacements for native extracellular matrices as they share key functions such as 2D- or 3D-solid support, fibrous structure, gas- and nutrition permeability and allow storage and release of biologically active molecules. Therefore this review focuses on current studies that try to implement soluble or covalently-attached gradients of growth factors, cytokines or adhesion sequences into 3D-hydrogel matrices in order to control cell growth, orientation and migration towards a target. Such gradient architectures are especially desirable for wound healing purposes, where defined cell populations need to be recruited from the blood stream and out of the adjacent tissue, in critical bone defects, for vascular implants or neuronal guidance structures where defined cell populations should be guided by appropriate signals to reach their proper positions or target tissues in order to accomplish functional repair.

The induction of cell alignment by covalently immobilized gradients of the 6th Ig-like domain of cell adhesion molecule L1 in 3D-fibrin matrices

Concentration gradients of matrix-bound guidance cues in the extracellular matrix direct cell growth in native tissues and are of great interest for the design of biomedical scaffolds and on implant surfaces.This study describes effects of covalently immobilized gradients of the 6th Ig-like domain of cell adhesion molecule L1 (TG-L1Ig6) within 3D-fibrin matrices on cell alignment. Linear gradients of TG-L1Ig6 were established and shown to be stable for at least 24h whereas soluble gradients disappeared completely. Fibroblast alignment along the gradients was observed when cultured on top and within TG-L1Ig6-gradient matrices. Fibroblasts responded to an increase of 0.2μg TG-L1Ig6/ml per mm matrix, corresponding to a concentration change of <1% per cell. Significant differences were observed when fibroblasts were cultured within the TG-L1Ig6-gradient matrices as the number of aligned cells decreased by 20–30% in the middle of the gradient when compared to cells cultivated on top of the gradient. This finding might be explained by ∼13% reduction in the average cell length of fibroblasts within compared to fibroblasts cultured on top of the gradient matrix. In contrast to fibroblasts endothelial cells did not show any alignment with TG-L1Ig6-gradient matrices. The study indicates that cells exposed to gradients of matrix-bound TG-L1Ig6 are able to respond differentially to 2D- or 3D-environments suggesting the use of gradients for cell guidance within 3D-scaffolds and on implant surfaces to improve their biomedical functions.

Controllable Soluble Protein Concentration Gradients in Hydrogel Networks

Here we report controlled formation of sustained, soluble protein concentration gradients within hydrated polymer networks. The approach involves spatially localizing proteins or biodegradable, protein-loaded microspheres within hydrogels to form a protein-releasing "depot". Soluble protein concentration gradients are then formed as the released protein diffuses away from the localized source. Control over key gradient parameters, including maximum concentration, gradient magnitude, slope, and time dynamics, is achieved by controlling the release of protein from the depot and subsequent transport through the hydrogel. Results demonstrate a direct relationship between the amount of protein released from the depot and the source concentration, gradient magnitude, and slope of the concentration gradient. In addition, an inverse relationship exists between the diffusion coefficient of protein within the hydrogel and the slope of the concentration gradient. The time dynamics of the concentration gradient profile can be directly correlated to protein release from the localized source, providing a mechanism for temporally controlling gradient characteristics. Therefore, each key, biologically relevant parameter associated with the protein concentration gradient can be controlled by defining protein release and diffusion. We anticipate that the resulting materials may be useful in three-dimensional cell culture systems, and in emerging tissue engineering approaches that aim to regenerate complex, functional tissues.

Spatially patterned gene expression for guided neurite extension

Axon pathfinding by localized expression of guidance molecules is critical for the proper development of the nervous system. In this report, we present a well-defined spatially patterned gene expression system to investigate neurite guidance in vitro. Nonviral gene delivery was patterned by combining substrate-mediated gene delivery with soft lithography techniques, and the amount of protein produced at the region of localized expression was varied by altering the vector concentration and the width of the pattern, highlighting the flexibility of the system. A neuronal coculture model was used to investigate responses to spatial patterns of nerve growth factor (NGF) expression. The soluble NGF gradient elicited a guidance cue, and the degree of guidance was governed by the distance a neuron was cultured from the pattern and the time between accessory cell and neuron seedings. A portion of the diffusible NGF bound to the culture surface in the extracellular space, and the surface-associated NGF supported neuron survival and neurite outgrowth. However, the surface-bound NGF gradient alone did not elicit a guidance signal, and in fact masked the guidance cue by soluble NGF gradients. Mathematical modeling of NGF diffusion was used to predict the concentration gradients, and both the absolute and fractional gradients capable of guiding neurites produced by patterned gene expression differed substantially from the values obtained with existing engineered protein gradients. Spatially patterned gene expression provides a versatile tool to investigate the factors that may promote neurite guidance.

A Gradient-generating Microfluidic Device for Cell Biology

The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described. A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients. Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles. Here, we developed simple gradient-generating microfluidic devices with three separate inlets. Three microchannels combined into one microchannel to generate concentration gradients. The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF). Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations. The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis. Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

Spatiotemporal Micropatterning of Cells on Arbitrary Substrates

This paper presents a readily accessible patterning platform--based upon geometric constraints, discrete cell suspension droplets, and controlled cell settling--that provides both temporal and spatial patterning capabilities. As a demonstration, single-cell (and bead) suspensions as well as multicellular human embryonic stem cell colonies are spatiotemporally patterned onto arbitrary substrates. These substrates include tissue culture surfaces, cell monolayers, protein-coated surfaces, and 3D gel matrices. The generation of soluble factor gradients is also demonstrated. This method is completely passive and does not require external power sources. Spatiotemporal patterning provides a foundation for future biological studies that explore the time-dependent relationships between cell-cell signaling and cellular responses.

EphB2 and EphB4 receptors forward signaling promotes SDF-1–induced endothelial cell chemotaxis and branching remodeling

AbstractThe complex molecular mechanisms that drive endothelial cell movement and the formation of new vessels are poorly understood and require further investigation. Eph receptor tyrosine kinases and their membrane-anchored ephrin ligands regulate cell movements mostly by cell–cell contact, whereas the G-protein–coupled receptor CXCR4 and its unique SDF-1 chemokine ligand regulate cell movement mostly through soluble gradients. By using biochemical and functional approaches, we investigated how ephrinB and SDF-1 orchestrate endothelial cell movement and morphogenesis into capillary-like structures. We describe how endogenous EphB2 and EphB4 signaling are required for the formation of extracellular matrix–dependent capillary-like structures in primary human endothelial cells. We further demonstrate that EphB2 and EphB4 activation enhance SDF-1–induced signaling and chemotaxis that are also required for extracellular matrix–dependent endothelial cell clustering. These results support a model in which SDF-1 gradients first promote endothelial cell clustering and then EphB2 and EphB4 critically contribute to subsequent cell movement and alignment into cord-like structures. This study reveals a requirement for endogenous Eph signaling in endothelial cell morphogenic processes, uncovers a novel link between EphB forward signaling and SDF-1–induced signaling, and demonstrates a mechanism for cooperative regulation of endothelial cell movement.

Microfluidic “jets” for generating steady-state gradients of soluble molecules on open surfaces

The ability to control the spatiotemporal microenvironment of cultured cells, such as cells in soluble gradients of signaling molecules, is critical for understanding the quantitative relationship between physicochemical signals and the induced biological responses. Existing methods for generating biomolecule gradients either offer little control, result in poor reproducibility, and/or place cells in potentially confounding or adverse conditions. Here the authors report the generation of soluble gradients by a system of microfluidic “jets” that inject picoliter amounts of fluid into an open pool with negligible exposure of the surface to flow. A stable, reproducible gradient can be formed in minutes.

Immobilized concentration gradients of nerve growth factor guide neurite outgrowth.

Axons are guided to their targets by a combination of haptotactic and chemotactic cues. We previously demonstrated that soluble neurotrophic factor concentration gradients guide axons in a model system. In an attempt to translate this model system to a device for implantation, our goal was to immobilize a stable neurotrophic concentration gradient for axonal (or neurite) guidance. Nerve growth factor (NGF) was immobilized within poly(2-hydroxyethylmethacrylate) [p(HEMA)] microporous gels using a gradient maker. The NGF was stably immobilized, with only approximately 0.05% of the amount originally incorporated into the gel released over an 8-day period. Immobilized NGF was bioactive: the percent of PC12 cells extending neurites on NGF-immobilized p(HEMA) gels was 16 +/- 2%, which was statistically the same as those exposed to soluble NGF (22 +/- 6%). We were able to predict and reproducibly create stable NGF concentration gradients in the gel. At an NGF concentration gradient of 357 ng/mL/mm, PC12 cell neurites were guided up the gradient. The facile, flexible, and reproducible nature of this method allowed us to translate soluble growth factor gradient models to stable growth factor gradient devices that may ultimately enhance axonal guidance and regeneration in vivo.

Selective expression of stromal-derived factor-1 in the capillary vascular endothelium plays a role in Kaposi sarcoma pathogenesis

AbstractKaposi sarcoma (KS), the most common neoplasm in patients with AIDS, typically presents with multiple skin lesions characterized by “spindle cells,” the vast majority of which are infected with KSHV (Kaposi sarcoma herpes virus, also named HHV-8). In patients with AIDS, the presence of cell-associated KSHV DNA in blood is predictive of subsequent KS development, but the mechanisms by which circulating KSHV-infected cells contribute to AIDS-KS pathogenesis are unclear. Here, we show that the chemokine stromal-derived factor-1 (SDF-1), which is constitutively expressed by skin capillary endothelium and displayed on the endothelial cell surface in association with heparan sulfate, can trigger specific arrest of KSHV-infected cells under physiologic shear flow conditions. Moreover, in the presence of soluble SDF-1 gradients, SDF-1 expressed on the endothelial barrier can promote transendothelial migration of KSHV-infected cells. By triggering specific adhesion of circulating KSHV-infected cells and favoring their entry into the extravascular cutaneous space, endothelial cell–associated SDF-1 in cutaneous capillaries may dictate the preferential occurrence of KS in the skin.

Effect of dissolved oxygen concentration on sludge settleability.

This laboratory study presents a detailed evaluation of the effects of dissolved oxygen concentration and accumulation of storage polymers on sludge settleability in activated sludge systems with an aerobic selector. The oxygen and substrate availability regime were simulated in laboratory sequencing batch reactor systems. The experiments showed that low dissolved oxygen concentration (< or =1.1 mg O2 l(-1)) had a strong negative effect on sludge settleability, leading to the proliferation of filamentous bacteria (Thiothrix spp., Type 021N and Type 1851). This negative effect was stronger at high chemical oxygen demand loading rate. This indicates that a compartmentalised (plug flow) aerobic contact tank, designed at short hydraulic residence time to guarantee a strong substrate gradient, with low dissolved oxygen concentration, might be worse for sludge settleability than an "overdesigned" completely mixed contact tank. Contrary to the general hypothesis, the maximum specific acetate uptake rate, poly-beta-hydroxybutyrate production rate, and resistance to short starvation periods are similar in both poor- and well-settling sludge. The results of this study support our previous hypothesis on the importance of substrate gradients for the development of filamentous structures in biological flocs, from soluble organic substrate gradients to dissolved oxygen gradients in sludge flocs.

Chemically-bound nerve growth factor for neural tissue engineering applications

In order to promote regeneration after spinal cord injury, growth factors have been applied in vivo to rescue ailing neurons and provide a path finding signal for regenerating neurites. We previously demonstrated that soluble growth factor concentration gradients can guide axons over long distances, but this model is inherently limited to in vitro applications. To translate the use of growth factor gradients to an implantible device for in vivo studies, we developed a photochemical method to bind nerve growth factor (NGF) to microporous poly(2-hydroxyethylmethacrylate) (PHEMA) gels and tested bioactivity in vitro. A cell adhesive photoreactive poly(allylamine) (PAA) was synthesized and characterized. This photoreactive PAA was applied to the surface of the PHEMA gels to provide both a cell adhesive layer and a photoreactive handle for further NGF immobilization. Using a direct ELISA technique, the amount of NGF immobilized on the surface of PHEMA after UV exposure was determined to be 5.65 ± 0.82 ng/cm2 or 3.4% of the originally applied NGF. A cell-based assay was performed to determine the bioactivity of the immobilized NGF. Using pheochromocytoma (PC-12) cells, 30 ± 7% of the cell population responded to bound NGF, a response statistically similar to that of cells cultured on collagen in the presence of 40 ng/ml soluble NGF of 39 ± 12%. These results demonstrate that PHEMA with photochemically bound NGF is bioactive. This photochemical technique may be useful to spatially control the amount of NGF bound to PHEMA using light and thus build a stable concentration gradient.

Chemokines have diverse abilities to form solid phase gradients.

Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC > TARC > RANTES > MIP1 beta > MCP1 > MIP1 alpha > FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.

Presentation of chemokine SDF-1α by fibronectin mediates directed migration of T cells

The role of chemokine–matrix interactions in integrin-dependent T-cell migration was examined to address the critical question of how chemokines provide directional information. The chemokine SDF-1α binds fibronectin (Fn) with a low nanomolar Kd(equilibrium dissociation constant). SDF-1α presented by Fn induced directed migration. Spatial concentration gradients of chemokine were not required to maintain directed migration. Fn-presented chemokine induced the polarization of cells, including the redistribution of the SDF-1α receptor, to the basal surface and leading edge of the cell. A new model for directed migration is proposed in which the co-presentation of an adhesive matrix and chemokine provides the necessary positional information independent of a soluble spatial gradient.