An Active Chemo-mechanical Model to Predict Adhesion and Microenvironmental Regulation of 3D Cell Shapes

AbstractCell shapes are known to regulate cytoskeletal organization, stiffness and the ability of cells to migrate and proliferate. Yet a quantitative understanding of the fundamental biochemical and biophysical mechanisms that determine the cell shapes is currently not available. In this study, we developed a chemo-mechanical feedback model to predict how adhesions and the properties of the 3D microenvironment regulate cell shapes. We find that the cells in 3D collagen matrices remain round or adopt an elongated shape depending on the density of active integrins, the level of contractility regulated by mechanosensitive signaling pathways and the density and mechanics of the matrix. While the formation of actin fibers that run along the cell body mediated by integrins and matrix stiffness drive elongation of cells, the cortical and membrane tension resist elongation. Based on the competition between these mechanisms, we derive phase diagrams for cell shape in the space spanned by the density of active adhesions and the level of biochemical signaling that controls contractility. Our predictions are validated by studying the shapes of HT1080 cells cultured in collagen gels of varying densities and using pharmacological treatments to regulate adhesions and contractility. The predictions of the model are found to be in excellent agreement with our experiments and data reported on a number of cell types in the literature.

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The Effects of Geometry and Static Boundary Conditions on Microvessel Outgrowth in a 3D Model of Angiogenesis

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19587 ◽

2010 ◽

Cited By ~ 1

Author(s):

Clayton J. Underwood ◽

Laxminarayanan Krishnan ◽

Lowell T. Edgar ◽

Steve Maas ◽

James B. Hoying ◽

...

Keyword(s):

Boundary Condition ◽

Boundary Conditions ◽

Direct Relationship ◽

3D Model ◽

Mechanical Strain ◽

Matrix Stiffness ◽

Collagen Gels ◽

3D Collagen ◽

In Vitro Angiogenesis

We reported previously that, in addition to mechanical strain, a constrained boundary condition alone can alter the organization of microvessel outgrowth during in vitro angiogenesis [1]. After 6 days of culture in vitro, microvessels aligned parallel to the long axis of rectangular 3D collagen gels that had constrained edges on the ends. However, unconstrained cultures did not show any alignment of microvessels. The ability to direct microvessel outgrowth during angiogenesis has significant implications for engineering prevascularized grafts and tissues in vitro, therefore an understanding of this process is important. Since there is direct relationship between the ability of endothelial cells to contract 3D gels and matrix stiffness [2], we hypothesize that some constrained boundary conditions will increase the apparent matrix stiffness and in turn will limit gel contraction, prevent microvessel alignment, and reduce microvessel outgrowth. The objective of this study was to compare microvessel growth and alignment under several different static boundary conditions.

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Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices

Gels ◽

10.3390/gels6040033 ◽

2020 ◽

Vol 6 (4) ◽

pp. 33

Author(s):

Jiranuwat Sapudom ◽

Claudia Damaris Müller ◽

Khiet-Tam Nguyen ◽

Steve Martin ◽

Ulf Anderegg ◽

...

Keyword(s):

Extracellular Matrix ◽

Tumor Microenvironment ◽

Average Molecular Weight ◽

Cell Types ◽

Dermal Fibroblasts ◽

Matrix Remodeling ◽

Cancer Associated Fibroblasts ◽

Collagen Matrices ◽

3D Collagen ◽

Tgf Β1

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

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Cell–Matrix Entanglement and Mechanical Anchorage of Fibroblasts in Three-dimensional Collagen Matrices

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0007 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5070-5076 ◽

Cited By ~ 69

Author(s):

Hongmei Jiang ◽

Frederick Grinnell

Keyword(s):

Tight Binding ◽

Three Dimensional ◽

Collagen Matrix ◽

Phagocytic Cells ◽

Collagen Matrices ◽

Cell Matrix ◽

Matrix Interactions ◽

The Matrix ◽

3D Collagen ◽

Cell Matrix Interactions

Fibroblast-3D collagen matrix culture provides a physiologically relevant model to study cell–matrix interactions. In tissues, fibroblasts are phagocytic cells, and in culture, they have been shown to ingest both fibronectin and collagen-coated latex particles. Compared with cells on collagen-coated coverslips, phagocytosis of fibronectin-coated beads by fibroblasts in collagen matrices was found to be reduced. This decrease could not be explained by integrin reorganization, tight binding of fibronectin beads to the collagen matrix, or differences in overall bead binding to the cells. Rather, entanglement of cellular dendritic extensions with collagen fibrils seemed to interfere with the ability of the extensions to interact with the beads. Moreover, once these extensions became entangled in the matrix, cells developed an integrin-independent component of adhesion. We suggest that cell–matrix entanglement represents a novel mechanism of cell anchorage that uniquely depends on the three-dimensional character of the matrix.

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Filamin A–β1 Integrin Complex Tunes Epithelial Cell Response to Matrix Tension

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1186 ◽

2009 ◽

Vol 20 (14) ◽

pp. 3224-3238 ◽

Cited By ~ 79

Author(s):

Scott Gehler ◽

Massimiliano Baldassarre ◽

Yatish Lad ◽

Jennifer L. Leight ◽

Michele A. Wozniak ◽

...

Keyword(s):

Collagen Matrix ◽

Cell Types ◽

Branching Morphogenesis ◽

High Density ◽

Actin Binding ◽

Β1 Integrin ◽

Filamin A ◽

Collagen Gels ◽

Collagen Remodeling ◽

The Matrix

The physical properties of the extracellular matrix (ECM) regulate the behavior of several cell types; yet, mechanisms by which cells recognize and respond to changes in these properties are not clear. For example, breast epithelial cells undergo ductal morphogenesis only when cultured in a compliant collagen matrix, but not when the tension of the matrix is increased by loading collagen gels or by increasing collagen density. We report that the actin-binding protein filamin A (FLNa) is necessary for cells to contract collagen gels, and pull on collagen fibrils, which leads to collagen remodeling and morphogenesis in compliant, low-density gels. In stiffer, high-density gels, cells are not able to contract and remodel the matrix, and morphogenesis does not occur. However, increased FLNa-β1 integrin interactions rescue gel contraction and remodeling in high-density gels, resulting in branching morphogenesis. These results suggest morphogenesis can be “tuned” by the balance between cell-generated contractility and opposing matrix stiffness. Our findings support a role for FLNa-β1 integrin as a mechanosensitive complex that bidirectionally senses the tension of the matrix and, in turn, regulates cellular contractility and response to this matrix tension.

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Magnetic Microtissue Stretching System to Study the Mechanobiology of 3D Fibroblast Populated Collagen Matrix

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80689 ◽

2012 ◽

Author(s):

Ruogang Zhao ◽

Thomas Boudou ◽

Christopher S. Chen ◽

Daniel H. Reich

Keyword(s):

Collagen Matrix ◽

Cell Seeding ◽

Biological Functions ◽

Collagen Gels ◽

Mechanical Environment ◽

Encapsulated Cells ◽

Surrounding Matrix ◽

Property Evaluation ◽

The Matrix ◽

3D Collagen

The biological functions of encapsulated cells in a fibroblast populated collagen matrix (FPCM) are regulated by the mechanical properties of the surrounding matrix. Studies on the mechanobiology of encapsulated fibroblasts have adopted 3D collagen gels either tethered to a 2D substrate or free-floating as the main platforms. However, these approaches have shortcomings in that the cellular mechanical environment can only be estimated based either on the stiffness of the underlying 2D substrate or on that of the matrix before cell seeding. The real time cellular mechanical environment as the cells undergo physiological or pathological transitions is not accessible. Efforts have been made to culture FPCM into centimeter-scale tensile test specimens for mechanical property evaluation, but the bulky size of these specimens has been shown to be not optimal for biochemical intervention.

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Microvascular Oreintation During Angiogenesis: Effects of Mechanical Stretch and Boundary Conditions

ASME 2007 Summer Bioengineering Conference ◽

10.1115/sbc2007-176598 ◽

2007 ◽

Author(s):

Laxminarayanan Krishnan ◽

Clayton J. Underwood ◽

Steve A. Maas ◽

Benjamin Ellis ◽

Tejas C. Kode ◽

...

Keyword(s):

Endothelial Cells ◽

Boundary Conditions ◽

Mechanical Stretch ◽

Cell Types ◽

Mechanical Forces ◽

Collagen Matrices ◽

The Matrix ◽

Tractional Forces ◽

Vitro Model

Mechanical forces affect the behavior of a variety of cell types including fibroblasts, chondrocytes, osteoblasts, smooth muscle cells, and endothelial cells. In vitro cellular orientation is primarily achieved through cell-generated mechanical forces or contact guidance. Endothelial cell (EC) cords align both normal to the stretch direction as well as along pre-tensioned collagen matrices. Confluent ECs orient parallel to grooved surfaces while tractional forces generated by ECs contract the matrix aligning collagen fibers, which in turn are considered a guiding scaffold. The influence of mechanical forces and boundary conditions on the growth and proliferation of microvessels during the process of angiogenesis is unknown. A better understanding of these interactions is essential both from a basic science standpoint as well as in the engineering of artificial vascularized matrices with specific orientation of vessels and endothelial cells. The objective of this study was to examine the role of internal and external mechanical forces in microvessel orientation during angiogenesis using an in vitro model of angiogenesis.

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Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices

Cardiovascular Pathology ◽

10.1016/j.carpath.2010.10.001 ◽

2011 ◽

Vol 20 (6) ◽

pp. 325-333 ◽

Cited By ~ 42

Author(s):

Peter A. Galie ◽

Margaret V. Westfall ◽

Jan P. Stegemann

Keyword(s):

Matrix Stiffness ◽

Collagen Matrices ◽

3D Collagen

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Magnetic probe-based microrheology reveals local softening and stiffening of 3D collagen matrices by fibroblasts

Biomedical Microdevices ◽

10.1007/s10544-021-00547-2 ◽

2021 ◽

Vol 23 (2) ◽

Author(s):

Juho Pokki ◽

Iliana Zisi ◽

Ester Schulman ◽

Dhiraj Indana ◽

Ovijit Chaudhuri

Keyword(s):

Cancer Progression ◽

Cell Cultures ◽

Loss Tangent ◽

Matrix Remodeling ◽

Matrix Stiffness ◽

Biological Matrices ◽

Collagen Matrices ◽

3D Cell Cultures ◽

The Matrix ◽

The Impact

AbstractChanges in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young’s modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell–matrix interactions in cancer progression and other areas.

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