With our migration chip, channel structures of sub-cellular dimensions have been applied to study cell deformation and invasiveness as well as migration dynamics and coordination of cellular movement. In addition, our system is well suited for fluorescence live-cell imaging. Due to its precise channel architecture, it allows for a quantitative analysis of mechanical deformation of cell migration through confined environments. In the last few years, similar channel architectures for studying leukocyte migration inside a confined 3D environment have been used 
. Very recently Irimia et al
examined the migrational persistence of cancer cells inside micro-sized channels, demonstrating the usefulness of such single-cell based approaches.
Using the functionality of our migration chip, we address the question whether the known effect of SPC on Panc-1 cells (decreasing the cells' stiffness) also enables the cell-driven invasion into confined spaces. Beil et al
. measured a drop in the Panc-1 cells' elastic modulus from about 28 to 16 mN m−1
upon SPC-treatment using a micro-plate based single-cell stretcher 
. Using a Boyden chamber assay they correlated this decrease in stiffness to an increased ability of the cells to squeeze through the membranous pores. We characterized and quantified the behavior of cells initiating contact with channels of a cross-section of 7×11µm, a size at which the Panc-1cells were hardly able to squeeze through. With these channel architectures we were not only able to confirm the result of Beil et al
. as we observed a five-fold increase in the number of cells permeating the channels upon SPC-treatment but could additionally observe and quantify the migration dynamics inside the channels.
The enhanced invasive behavior upon treatment with SPC may be explained by using a simple two-component model. First, there is the motor unit with the driving actin-polymerization (lamellipod) in the front and the contractive acto-myosin assembly at the rear of the cell. Secondly, there is the passive and voluminous cell body being pulled upon migration. For an invasion
into the channel, the cell needs to deform the cell body requiring compression of the keratin envelope and the nuclear region. Inside the channels, nucleus shape and keratin network structure deviate from the normal spatial distribution in the cell as shown in . Assuming that Panc-1 cells are able to generate only a finite force, we speculate that the motor unit of a non-SPC-treated Panc-1 cell is not able to pull the cell body into a channel with a width of 7 µm. This speculation is supported by observations on neutrophil leukocytes migrating inside small capillaries. These cells are able to counterbalance a maximal hydrostatic counter-pressure of 1.5 kPa by generating a traction force of 38 nN inside the capillaries 
. Additionally, it was shown for leukocytes that the spatial organization of intermediate filaments plays a major role for the cells' migratory behavior 
Beyond the effects of size exclusion on the invasive behavior of cells, our migration chip permitted the quantification of drastic differences in 2D and 3D migration speeds. This property is of particular importance since recent studies attributed increases in migration speed to the dimensionality of the cell environment 
. Our observed increase in 2D speed after SPC-treatment might be attributed either to the keratin reorganization and softening of the cell or to the known enhancement of filamentous actin formation by SPC 
. As the speed inside the channels is not affected by SPC-treatment we speculate that the SPC-effect on actin may not be the major factor, but that the different keratin morphology might cause the increased migration speed on flat surfaces. Assuming that the nuclear region and keratin envelope of a cell inside a channel is in a compacted state, its steric hindrance should have only a minor influence on the migration, independent of SPC-treatment. If SPC-enhanced actin dynamics were the predominant reason for the observed increase in migration speed on 2D surfaces, this should also lead to an increased speed inside the channels which we did not observe. Thus, SPC facilitates the initial invasion into the channel but seems not to affect the further migration speed inside the 3D environment.
Recent research has identified different migration phenotypes in 2D and 3D 
. In particular, an adhesion independent migration mechanism has been reported for leukocytes in a 3D matrix 
. Based on a theoretical model such a migration mechanism may be attributed only to partial pressure differences between the leading and rear edge without the necessity of specific cell-surface adhesion 
. With our migration chip we did not test this particular migration mechanism but we could reveal two distinct migration phenotypes that are proposed to occur in either a 2D or 3D environment. Doyle et al
. observed a coordinated migration in fibroblasts moving on thin (1D) lines with a width of up to 5 µm 
. They argue that this behavior depends exclusively on the width of the adhesive lines and resembles the behavior in 3D. Similarly, we found in our experiments that the majority of the cells showed a 2D-typical push-and-pull
-like movement when migrating on 7 µm wide lines. However, inside the channels with the same width the majority of the cells showed a sliding
-like movement. Therefore, we propose that the transition from a 2D to a 3D or 1D migration mechanism depends not only on the line width but is also determined by the contact area with the environment, in our case the channel walls.
In summary, we report a detailed investigation of migration dynamics of human pancreatic cancer cells inside micro-channels with a particular focus on the effects of keratin reorganization induced by the bioactive phospholipid SPC. Beyond previous knowledge, we demonstrate that the SPC treatment of Panc-1 cells increased their ability to invade and permeate narrow channels. Hence, our study may contribute to a more detailed understanding of how cancer cells invade the surrounding tissue and also escape from a primary tumor (permeate through the stroma), the first step in tumor spreading. Furthermore, we demonstrated the existence of two different migration phenotypes depending on the dimensionality of the cell environments. Thus, our migration chip provides an easy to use experimental to promote current research on 3D migration behavior on a single-cell level.