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A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

ABSTRACT: New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

SUBMITTER: Polackwich RJ

PROVIDER: S-EPMC3594227 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

Polackwich Robert J RJ Koch Daniel D Arevalo Richard R Miermont Anne M AM Jee Kathleen J KJ Lazar John J Urbach Jeffrey J Mueller Susette C SC McAllister Ryan G RG

PloS one 20130311 3

New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction ...[more]

PMID: 23536784

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Project description:Biomechanical changes to the collagen fibrillar architecture in articular cartilage are believed to play a crucial role in enabling normal joint function. However, experimentally there is little quantitative knowledge about the structural response of the Type II collagen fibrils in cartilage to cyclic loading in situ, and the mechanisms that drive the ability of cartilage to withstand long-term repetitive loading. Here we utilize synchrotron small-angle X-ray scattering (SAXS) combined with in-situ cyclic loading of bovine articular cartilage explants to measure the fibrillar response in deep zone articular cartilage, in terms of orientation, fibrillar strain and inter-fibrillar variability in healthy cartilage and cartilage degraded by exposure to the pro-inflammatory cytokine IL-1β. We demonstrate that under repeated cyclic loading the fibrils reversibly change the width of the fibrillar orientation distribution whilst maintaining a largely consistent average direction of orientation. Specifically, the effect on the fibrillar network is a 3-dimensional conical orientation broadening around the normal to the joint surface, inferred by 3D reconstruction of X-ray scattering peak intensity distributions from the 2D pattern. Further, at the intrafibrillar level, this effect is coupled with reversible reduction in fibrillar pre-strain under compression, alongside increase in the variability of fibrillar pre-strain. In IL-1β degraded cartilage, the collagen rearrangement under cyclic loading is disrupted and associated with reduced tissue stiffness. These finding have implications as to how changes in local collagen nanomechanics might drive disease progression or vice versa in conditions such as osteoarthritis and provides a pathway to a mechanistic understanding of such diseases. STATEMENT OF SIGNIFICANCE: Structural deterioration in biomechanically loaded musculoskeletal organs, e.g., joint osteoarthritis and back pain, are linked to breakdown and changes in their collagen-rich cartilaginous tissue matrix. A critical component enabling cartilage biomechanics is the ultrastructural collagen fibrillar network in cartilage. However, experimental probes of the dynamic structural response of cartilage collagen to biomechanical loads are limited. Here, we use X-ray scattering during cyclic loading (as during walking) on joint tissue to show that cartilage fibrils resist loading by a reversible, three-dimensional orientation broadening and disordering mechanism at the molecular level, and that inflammation reduces this functionality. Our results will help understand how changes to small-scale tissue mechanisms are linked to ageing and osteoarthritic progression, and development of biomaterials for joint replacements.

Dataset Information

A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

Publications

A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

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