A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

Daniel Koch,Susette C Mueller,Jeffrey Urbach,Robert J Polackwich,Ryan G Mcallister,Kathleen J Jee,John Lazar,Richard Arevalo,Donald Gullberg,Anne M Miermont

doi:10.1371/journal.pone.0058138

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.

Highlights

Tumor cells exertmeasurable forceson their environment,bothfor cell movement and to reshape the surrounding matrix
To study the role of the Src kinase protein on mechanotransduction and motility, we generated stable MDA-MB-231 breast cancer cell clones with similar expression of GFP-tagged wild type Src protein (GFP-wild-type Src (wt-Src), W2E9 clone) and a GFP-tagged mutant of Src protein, c-Src(Y527F), rendering it constitutively active (GFP-constitutively active Src (ca-Src), C1G1 and C2E8 clones) (Fig. 1, a-Src and a-GFP)
GFP-ca-Src was localized mostly at the cell surface and was clustered at sites associated with fine protrusions within 3 hours of the time when cells were cultured within a sandwich of collagen

Summary

Introduction

Tumor cells exertmeasurable forceson their environment,bothfor cell movement and to reshape the surrounding matrix. The introduction of uniform elastic films of controllable stiffness combined with advances in microscopy and analysis approaches have enabled researchers to map traction forces on flat surfaces with high spatial and temporal resolution [2,4,5], and even to map the forces propagating from cells on the 2D surface down into the surface [6]. These approaches all place the cells on a twodimensional environment. Another approach, embedding cells in a three-dimensional (3D) hydrogel with known mechanical properties [7], allows for the measurement of cell force generation in a 3D, non-fibrillar environment

Methods

Results

Conclusion