«Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt ...»
20: Collagenase digestion of control and glutaraldehyde-treated gels. (A) Integrated reflected light intensity of image stacks versus time (mean ± se of 3 different gels).
At t=0, collagenase was added to the gels. Every 40 s, an image stack over a volume of 185 µm × 185 µm × 50 µm was recorded. After collagenase treatment, collagen fibers disappeared faster in control gels (black line) compared to glutaraldehyde-treated gels (red line). (B-G) Confocal reflected microscopy images of control gels (top row, B-D) and glutaraldehyde-treated gels (bottom row, E-G) at different time points after collagenase addition. Scale bar represents 20 µm.
III RESULTS AND DISCUSSION 49
Influence of matrix metalloproteinases on cell invasion
Cells have the ability to secrete MMPs that allow them to remodel and digest the surrounding matrix. It is suspected that the MMPs secretion support cell migration through a dense extra cellular matrix. To investigate if cells rely on matrix degradation through secretion of metalloproteinases for invasion [90-92], we repeated the invasion assays in the presence of the broad-band MMP inhibitor GM 6001. Cell invasiveness in glutaraldehyde-stabilized gels was not altered by MMP-inhibition (Fig 3.1.18, orange bars). This finding was expected as the glutaraldehyde-treated gels remain stable in the presence of MMPs (Fig 3.1.20, B). Interestingly, MMP inhibition also had little or no effect in untreated gels (Fig 3.1.18, B, grey bars), demonstrating that cell migration in these cells did not depend critically on MMP secretion and matrix degradation. We found only at the highest collagen concentration (2.4 mg/ml) a reduced invasion in the presence of the MMP inhibitor.
Inhibition of matrix metalloproteinase had only minor effects on the migration behavior of the breast carcinoma cells studied here. Our data shows that the combination of small pore sizes with high gel stiffness can severely impair cell invasion.
Here we analyzed the behavior of a breast carcinoma cell line in collagen gels of different stiffnesses, pore size and collagen concentrations. MDA invasiveness followed a biphasic response with a maximum invasiveness in gels at intermediate collagen concentrations, stiffness and pore size. To evaluate influences of stiffness changes without introducing changes in collagen concentration and pore size, we stiffened the gels by crosslinking with glutaraldehyde. Interestingly, gel stiffening lead to an increase of cell invasiveness in diluted gels with large pores but to a decrease of cell invasiveness in dense gels with small pores. This resulted in a similar biphasic response as seen in the untreated gels but with a shift of the maximum invasiveness to larger pores. This demonstrates the critical role of matrix stiffness for cell migration and invasion in a porous 3D biopolymer network such as a collagen gel. Cells fail to invade very soft matrices, whereas high matrix stiffness promotes 3D cell invasion as long as the pore size remains above a critical value.
50 III RESULTS AND DISCUSSION
3.2 Effects of transfection with fluorescently tagged proteins on cell migration in 3D 3.2.1 Introduction – why do we use transfection?
As cell migration is a fundamental process involved in organ development, wound healing and in the building of metastasis [2, 3] it is crucial to understand the process of cell migration in detail. The processing from simple light microscopy to fluorescence microscopy and the development of fluorescent cell staining enabled scientists to observe not only cellular movement but also intracellular processes. However, the observation of single proteins and their interactions inside a living was difficult to monitor until the technique of transfection was developed in the early 1970s. There are several techniques of transfection, for example electroporation , DEAE-dextran based transfection  or lipofection . All of them combine the task to introduce nucleic acid sequences of fluorescent proteins into cells such that the cells express the fluorescently tagged proteins. Those “cell-expressed” fluorescent proteins are expressed constantly during normal cell cycles and allow therefore imaging over hours without major photobleaching difficulties. Cells can either be transfected stably or transiently. In our study we used lipofection for transient transfection because it is highly effective and, once the protocol is established for a cell line, it is highly reproducible. With lipofection a cationic lipid forms an aggregate with the anionic nucleic acid that should be transferred into the cell . As a transfection reagent, we used lipofectamine, which is a cationic liposome that forms complexes with negatively charged DNA plasmids and facilitates DNA transfer into cells. The lipofectamine-DNA complex is then able to fuse with the cell membrane such that the foreign DNA is transported into the cytoplasm of the cell and eventually transported to the nucleus where the DNA-sequence can be transcripted and subsequently translated .
Together with the improvement of transfection methods, the development of fluorescent proteins as molecular tags has revolutionized modern research allowing complex biochemical processes to be correlated with the function of proteins in living cells. Fluorescent proteins like the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its variants can be fused to nearly any protein to visually analyze protein movement and protein interaction in living cells.
52 III RESULTS AND DISCUSSIONNumerous studies have been made in which cells were transfected with fluorescencetagged proteins of the cytoskeleton (actin, myosin), the focal adhesion complexes (vinculin, talin, FAK) or intracellular signaling cascades (Rho, ROCK) to understand the process of cell migration on a molecular level [97-102]. By using cells transfected with fluorescence-tagged proteins it was possible to reveal that cell migration on a flat, stiff 2D substrate follows a four step process: 1. Polymerization of actin filaments at the leading edge is translated into protrusive force. 2. Membrane protrusion facilitates the binding of trans-membrane cell surface receptors to the substratum. Nascent adhesions are rapidly linked to the network of actin filaments. 3. The combined activity of retrograde actin movement and contractile forces produced by stress fibers generates tension to pull the cell body forward. 4. The forces produced by the contractile network combined with actin filament and FA disassembly help to retract the trailing cell edge .
Although it is known that transfection is potentially toxic for cells, transfected cells are in general considered healthy if their morphological appearance does not change after transfection. Sometimes cells are tested for a change in spreading area or cell shape index after a transfection . In some cases, even 2D migration, in terms of cell speed, is monitored to evaluate if cellular behavior changed.
However, in a physiological 3D environment cells face a completely different situation than in 2D because they have to overcome the steric hindrance imposed by the extracellular matrix. This ECM environment is suspected to influence also intracellular signaling through its properties and compositions . To elucidate intracellular processes while migrating through a 3D environment, again transfection with fluorescence-tagged proteins is used.
In this section we reveal that cellular migration in 3D can be impaired after transfection, even if results from 2D measurements show no difference between transfected and non-transfected cells. Therefore we measured 2D migration, 2D cell spreading area, 3D invasion, cell stiffness and binding strength. In our measurements we used a GFP-actin plasmid, where G-actin was coupled with a GFP-sequence (established in our lab). Furthermore we used GFP-LifeAct, which is a small protein that binds on F-actin that is already present in the cell . Moreover we used a single EGFP-plasmid, without any further protein, so that EGFP was expressed in the cytoplasm.
III RESULTS AND DISCUSSION 533.2.2 Transfection with fluorescent proteins can impair cell behavior Transfection with fluorescently tagged proteins has no influence in 2D cell shape and migration In general it is assumed that the transfection procedure with fluorescence-tagged proteins did not change cellular behavior when cells appear morphologically normal, demonstrate no membrane “blebbing” and do not round up after transfection. Mostly studies confirmed with 2D cell shape measurements  that their transfection procedure was not harmful to cells.
To test whether our transfection protocol resulted in unaltered cellular behavior, we evaluated the 2D cell spreading area. Therefore we transfected the cell line MDA MB 231 with 3 different fluorescence-tagged protein plasmids: GFP-actin, GFP-lifeAct and EGFP (Fig 3.2.1). Furthermore we performed all measurements with non-transfected control cells, as well as lipofectamine only treated cells, to test whether the reagent alone was harmful for the cells. Our measurements confirmed that there was no difference in the spreading area of non-transfected cells, lipofectamine treated cells and transfected cells, regardless of which protein sequence was transfected (Fig. 3.2.2, A).
Another method to test whether a transfection influences cellular behavior is to monitor 2D cell migration. In this study we tested the 2D migration of MDA MB 231 cells that were expressing fluorescence-tagged proteins. We tracked the cellular movement from transfected cells over 24 hours. Subsequently we analyzed the cell trajectory and the mean squared displacement of the migrating cells, which combines information about migration speed and directionality as well as the general migration behavior. With a MSD slope of about 1.1 MDA MB 231 cells showed a weakly superdiffusive migration behavior. Again, we confirm that the transfection with any fluorescently tagged protein, as well as the lipofectamine treatment alone, did not change the 2D migratory behavior in terms of their MSD function (Fig. 3.2.2, B).
54 III RESULTS AND DISCUSSIONFigure 3.2.
1: MDA cells transfected with pEGFP. MDA MB 231 cell were transfected with pEGFP constructs and imaged in order to analyze their spreading area. (A) Brightfield image of MDA MB 231 cells. (B) Fluorescent image of the same cells as in (A). (C) Same brightfield image as in (A) with illustrated spreading area detected from the edge detection algorithm implanted in Matlab (white line). Images were obtained with a confocal microscope (512 x 512 pixels, 279 nm x 279 nm). Bar represents 20 µm.
2: 2D Cell spreading and migration after transfection. To test whether the transfection with fluorescently labeled proteins alter cell behavior, we evaluated the cell spreading area of cells seeded in cell culture dishes. (A) 15,000 transfected cells were seeded into cell culture dishes. 2D spreading area was detected via an edge detection algorithm implemented in matlab. No difference in 2D spreading area between transfected and nontransfected cells could be detected (mean ± SE). (B) 30,000 cells were seeded in a cell culture dish and tracked over 20 hrs in phase contrast mode. The mean squared displacement shows no difference in 2D MSD slopes detected between transfected and non-transfected cells (inset).
III RESULTS AND DISCUSSION 55Transfection with fluorescently tagged proteins influences 3D migration Although the measurements above show that transfection had no influence on cell behavior in 2D, transfected cells appeared less motile when seeded into 3D collagen gels and monitored over several hours (Fig. 3.2.3, SI Movie_2).
3: Transfected cells in a 3D collagen gel built protrusions but did not migrate.
Maximum projection of a confocal image stack (512 x 512 pixels, 423 nm x 423 nm x 448 nm pixel size, total thickness 22.4 µm). MDA MB 231 cells were transfected with an EGFP construct and seeded into 1.2 mg/ml gels. Although the cells were considered to be vital, during a 12 h period of time, cell movement simply consisted of the building of several protrusions (white arrows; SI Movie_2). In contrast to their non -transfected counterparts, they did not migrate more than 1 µm in 70 minutes. Scale bar represents 20 µm.
transfected cells did migrate through the collagen gels (red arrows indicate the building of protrusions and the direction of cell movement; arrow length indicates speed; SI Movie_3 and SI Movie_4). Scale bar represents 20 µm.