Effects of morphological patterning on endothelial cell migration

The migration of vascular endothelial cells (ECs) plays an important role in vascular remodeling. Here we studied the effects of cell morphology on the migration of bovine aortic ECs by culturing cells on micropatterned strips of collagen matrix (60-, 30-, and 15-microm wide). The spreading areas of the cells on 15- and 30-microm wide strips were 30% lower than those on 60-microm wide strips and unpatterned collagen. The cells on 15-microm wide strips completely aligned in the direction of the strip, and had significantly lower shape index than those in all other groups. On strips of all widths, ECs tended to migrate in the direction of strips. ECs on 15-microm wide strips had highest speed, particularly in the direction of the strip. Vinculin staining showed that the leading edge of ECs on 15-microm wide strips had focal adhesions that were oriented with their lamellipodial protrusion and the direction of cell migration; this arrangement of the focal adhesions may promote EC migration. The present study provides direct evidence on the role of cell morphology in EC migration, and will help us to understand the mechanisms of EC migration during angiogenesis and wound healing.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction. cytoskeleton; prestress; stress fibers; mechanotransduction; mechanical deformation     

Three-Dimensional Numerical Model of Cell Morphology during Migration in Multi-Signaling Substrates

Cell Migration associated with cell shape changes are of central importance in many biological processes ranging from morphogenesis to metastatic cancer cells. Cell movement is a result of cyclic changes of cell morphology due to effective forces on cell body, leading to periodic fluctuations of the cell length and cell membrane area. It is well-known that the cell can be guided by different effective stimuli such as mechanotaxis, thermotaxis, chemotaxis and/or electrotaxis. Regulation of intracellular mechanics and cell’s physical interaction with its substrate rely on control of cell shape during cell migration. In this notion, it is essential to understand how each natural or external stimulus may affect the cell behavior. Therefore, a three-dimensional (3D) computational model is here developed to analyze a free mode of cell shape changes during migration in a multi-signaling micro-environment. This model is based on previous models that are presented by the same authors to study cell migration with a constant spherical cell shape in a multi-signaling substrates and mechanotaxis effect on cell morphology. Using the finite element discrete methodology, the cell is represented by a group of finite elements. The cell motion is modeled by equilibrium of effective forces on cell body such as traction, protrusion, electrostatic and drag forces, where the cell traction force is a function of the cell internal deformations. To study cell behavior in the presence of different stimuli, the model has been employed in different numerical cases. Our findings, which are qualitatively consistent with well-known related experimental observations, indicate that adding a new stimulus to the cell substrate pushes the cell to migrate more directionally in more elongated form towards the more effective stimuli. For instance, the presence of thermotaxis, chemotaxis and electrotaxis can further move the cell centroid towards the corresponding stimulus, respectively, diminishing the mechanotaxis effect. Besides, the stronger stimulus imposes a greater cell elongation and more cell membrane area. The present model not only provides new insights into cell morphology in a multi-signaling micro-environment but also enables us to investigate in more precise way the cell migration in the presence of different stimuli.     

Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.     

Biophysical regulation of histone acetylation in mesenchymal stem cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.     

Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage

Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (∼ 10,000 μm²) while cells grown on 20 μm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of ∼ 2000 μm². Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.     

Microtopography and flow modulate the direction of endothelial cell migration

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.     

Myosin-II-Mediated Directional Migration of Dictyostelium Cells in Response to Cyclic Stretching of Substratum

Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin.     

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.     

Mechanical effects on endothelial cell morphology: In vitro assessment

Summary Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function. This work was partially supported by grants HL 17437, HL 18072, and HL 23016 from the National Institutes of Health, Bethesda, MD, and grant C-938 from the Robert A. Welch Foundation.     

Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers

Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center toward the leading edge of migrating cells in a Cdc42-dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to 4 days, but not shear stress-induced morphology. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor-free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and induced an increase in the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant-negative Par6, a major downstream effector of Cdc42-induced polarity. Shear stress-induced directed migratory polarity is modulated by exogenous growth factors and dependent on Par6 activity and shear stress direction.     

Endothelial cell response to biomechanical forces under simulated vascular loading conditions

In vivo, endothelial cells (EC) are constantly exposed to the haemodynamic forces (HF) of pressure, wall shear stress and hoop stress. The main aim of this study was to design, create and validate a novel perfusion bioreactor capable of delivering shear stress and intravascular pressure to EC in vitro and to characterise their morphology, orientation and gene expression. Here we report the creation and validation of such a simulator and the dual application of pressure (120/60 mmHg) and low shear stress (5 dyn/cm(2)) to a monolayer of EC established on a non-compliant silicone tube. Under these conditions, EC elongated and realigned obliquely to the direction of applied shear stress in a time-dependent manner. Furthermore, randomly distributed F-actin microfilaments reorganised into long, dense stress fibres crossing the cells in a direction perpendicular to that of flow. Finally, combinatorial biomechanical conditioning of EC induced the expression of the inflammatory-associated E-selectin gene.     

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).     

Trophoblast migration under flow is regulated by endothelial cells

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

Influence of Micropatterning on Human Periodontal Ligament Cells’ Behavior

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells’ peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells’ stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.     

Influence of surface topography on the human epithelial cell response to micropatterned substrates with convex and concave architectures

Background

Understanding the fundamental mechanisms underlying the cellular response to topographical surface features will extend our knowledge regarding the regulation of cell functions. Analyzing the cellular response to different topographical features, over multiple temporal and spatial scales, is central to understanding and guiding several biological functions. We used micropatterned substrates with convex and concave architectures to evaluate the behaviors of human epithelial cells on these substrates.

Results

Pillar and pit substrates caused heterogeneous spatial growth and distribution, with differences in cell density, over 48 h. Regional densities and distribution were significantly increased at pillar sidewalls, and at pit sidewalls and bottoms compared with those on flat unpatterned areas. Time-lapse observations revealed that different mechanisms of cell migration were dependent upon pillar and pit features. Cells on pillar substrate migrated towards the sidewall, whereas cells on pit substrate tended to move towards the sidewalls and bottom. Cytoskeletal staining of F-actin and vinculin showed that this migration can be attributed to difference in spatial reorganization of actin cytoskeleton, and the formation of focal adhesions at various points on the at the convex and concave corners of pillar and pit substrates. Cells cultured on the pillar substrate had stress fibers with extended filopodia and immature focal contacts at the sidewalls and convex corners, similar to those on the flat unpatterned substrate. Cells at the sidewalls and concave corners of pit substrate had more contractile stress fibers and stable focal contacts compared with cells on the pillar substrate. We also found that the substrate structures affect cell-cell contact formation via E-cadherin, and that this was associated with reorganization of the actin cytoskeleton at the sidewall, and at the convex and concave corners of the substrate.

Conclusion

Migration is an important factor affecting spatial growth and distribution. Heterogeneity at various locations was caused by different migratory behaviors at the convex and concave corners of pillar and pit substrates. We propose that this investigation is a valuable method for understanding cell phenotypes and the heterogeneity during spatial growth and distribution of epithelial cells during culture.

     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.