Bioengineering paradigms for cell migration in confined microenvironments

Abstract

Cell migration is a fundamental process underlying diverse (patho)physiological phenomena. The classical understanding of the molecular mechanisms of cell migration has been based on in vitro studies on two-dimensional substrates. More recently, mounting evidence from intravital studies has shown that during metastasis, tumor cells must navigate complex microenvironments in vivo, including narrow, pre-existing microtracks created by anatomical structures. It is becoming apparent that unraveling the mechanisms of confined cell migration in this context requires a multi-disciplinary approach through integration of in vivo and in vitro studies, along with sophisticated bioengineering techniques and mathematical modeling. Here, we highlight such an approach that has led to discovery of a new model for cell migration in confined microenvironments (i.e., the Osmotic Engine Model).

Figure 1

Overview of 2D, 3D, 1D, and microchannel cell migration assays and their limitations. In the wound healing assay, a monolayer of cells is scratched, or a physical barrier is removed, and the cells subsequently migrate towards each other to close the wound. In the durotaxis assay, a gradient of substrate stiffness is created by placing two drops of polymerizing polyacrylamide (PA) of different stiffnesses next to each other, and covering the solutions with a glass coverslip. Cells are then induced to migrate in response to the mechanical gradient of stiffness. In the micropipette assay, cells respond to a chemotactic gradient created by a chemoattractant-filled micropipette. In a 3D matrix, cells must enzymatically degrade the surrounding matrix in order to move, while in fabricated tracks within a 3D matrix, preexisting tracks are created in a collagen gel via microfabrication techniques. In the assay with microprinted 1D lines, cells adhere selectively to 1D protein lines of specific width. In the microchannel assay, cells are induced to migrate into confined or unconfined microchannels in response to a chemoattractant gradient. Some parts of the figure are adapted with permission from [9].

Figure 2

Comparison of major differences between 2D migration and migration through confined spaces (i.e., microchannels). In 2D migration, actin polymerization drives the leading edge forward, and both cortical actin and stress fibers are evident within the cell. Myosin motors are necessary to retract the cell’s trailing edge. Distinct focal adhesions help anchor the cell and traction forces are generated through these focal adhesions. When the actin-disrupting drug latrunculin-A is added to cells in 2D, they lose attachment to the substrate, round up, and cell velocity goes to zero. Blebbistatin, which inhibits myosin II function and decreases cell contractility, decreases cell traction forces in 2D; meanwhile, calyculin A, which inhibits protein phosphatases and increases cell contractility, increases cell traction forces in 2D. During migration in confined microchannels, the cell undergoes dramatic stress fiber and cortical actin remodeling, with both becoming more diffuse throughout the cell. Attenuation of focal adhesion size is also observed in microchannels. In contrast to 2D, the cell can still move in confined microchannels if actin and myosin functions are disrupted. Furthermore, neither blebbistatin nor calyculin A has any effect on the magnitude of cell traction forces in confinement, indicating that cell traction forces play a reduced role during migration through confined spaces. In confined spaces, the net direction of forces is towards the chemoattractant, though appreciable forces are also directed towards the side walls of the microchannels.

Figure 3

Overview of the Osmotic Engine Model of cell migration. (A) Cell migration in confined spaces is driven by water permeation across the cell membrane. Water flows in at the cell’s leading edge, which allows the front of the cell to extend forward, and water flows out at the cell’s trailing edge, which allows the back of the cell to retract. This results in translocation of the cell body forward with little change in cell length (or volume). (B) The Osmotic Engine Model can be tested by applying osmotic shocks to the cell’s leading (or trailing) edge. Here, a hypotonic shock is introduced within the microfluidic device at the cell’s leading edge, causing the cell to reverse direction and migrate away from the chemoattractant gradient. (C) The velocity of cell migration depends on the magnitude of osmolarity of the extracellular medium at the cell’s leading edge. The reversal of cell migration direction in response to a hypotonic shock at the cell’s leading edge is predicted by the theoretical framework of the Osmotic Engine Model. Thus, differences in solute concentration at the leading and trailing ends of the cell can drive cell migration. In the absence of osmotic shocks, the cell’s ion channels and aquaporins must be polarized in order to sustain migration. Figures reproduced with permission from [79].