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

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.

Quantitative studies of neuronal chemotaxis in 3D.

During development a variety of cell types are guided by molecular concentration gradients to form tissues and organ systems. In the nervous system, the migration and neuronal pathfinding that occurs during development is organized and driven by "guidance cues." Some of these cues are substrate bound or nondiffusible, while many are diffusible and form gradients within the developing embryo to guide neurons and neurites to their appropriate destination. There have been many approaches used to discover and characterize the multitude of guidance cues, their cognate receptors, and how these cues and receptors are regulated to achieve the highly detailed connections found in the nervous system. Here we present a method for creating precisely controlled gradients of molecular factors within a three-dimensional culture environment. The method is based on a non contact mediated delivery of biomolecules to the surface of a collagen gel. The factors are printed in a pattern on the top of a gel containing the tissue or cell type of interest embedded in the gel. The formation of the gradient is dependent upon the diffusion of the printed molecule in the gel. The concentration of the factor within the gel becomes independent of depth rapidly, and the gradient becomes smooth on a similar time scale. The gradients formed can remain relatively stable for a day or more. Moreover, the steepness and molar concentration of tropic or trophic factors within the gradient can be controlled.

Optical neuronal guidance in three-dimensional matrices.

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

Design and optimization of a high-speed, high-sensitivity, spinning disk confocal microscopy system.

We describe the principles, design, and systems integration of a flexible, high-speed, high-sensitivity, high-resolution confocal spinning disk microscopy (SDCM) system. We present several artifacts unique to high-speed SDCM along with techniques to minimize them. We show example experimental results from a specific implementation capable of generating 3-D image stacks containing 30 2-D slices at 30 stacks per second. This implementation also includes optics for differential interference contrast (DIC), phase, and bright-field imaging, as well as an optical trap with sensitive force and position measurement.

A new chemotaxis assay shows the extreme sensitivity of axons to molecular gradients.

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.