Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

Abstract

Chemotactic cell migration is a central mechanism during cancer cell invasion and hence metastasis. In order to mimic in vivo conditions, we used a three-dimensional hydrogel matrix made of collagen I and a stable gradient-generating chemotaxis assay system, which is commercially available (μ-Slide Chemotaxis) to characterize epidermal growth factor (EGF)-induced chemotaxis of the human breast cancer cell line MDA-MB-231. Surprisingly, chemotactic effects of EGF on MDA-MB-231 cells could neither be observed in the standard growth medium DMEM/F-12 supplemented with 10% serum nor in starvation medium. In contrast, after adapting the cells to the serum-free growth medium UltraCULTURETM, significant chemotactic effects could be measured with high sensitivity. The extremely time-stable linear gradients, generated in the chemotaxis chamber, led to consistent directional migration of MDA-MB-231 cells. Dose-response experiments showed increased directional and kinetic response of MDA-MB-231 cells towards stable gradients of EGF. While EGF-guided directional migration (chemotaxis) was highly concentration-dependent with the highest response at 1.5 nM/mm EGF, we found that the chemokinetic effect induced by EGF was concentration-independent. Both, blocking the ligand-binding domain of the EGF receptor by an antibody (monoclonal anti-EGFR antibody 225) and inhibition of its kinase domain by a small molecule inhibitor (AG1478) led to a reduction in EGF-induced directed migration. The high sensitivity of the assay even allowed us to observe synergistic effects in EGF-receptor inhibition using a combination of low doses of both inhibitor types. Those results validate the fact that EGF is a potent guidance cue for MDA-MB-231 cell migration and help to understand the mechanism behind chemotaxis-driven cancer metastasis.

Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers

“…The highest level of migration of MDA-MB-231 cells was observed when EGF was present only in the lower chamber, as evidenced in Figure 1 A; demonstrating that EGF elicits a specific increase in directional migration. Our own observations are in accordance with those published in similar studies [ 27 , 28 , 29 , 30 ] and gives evidence to the selection of EGF as a trigger for the processes of migration and invasion in MDA-MB-23 cells.…”

Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers

“…More complex designs that rely on sink and source channels connected with ladder-shaped microgroove arrays have been developed to overcome this problem [31][32][33][34]. In some of these designs, transient gradients can be established [35] in a short period of time; however, fabrication of these multi-depth [36] and multilayer channels [37] can be complex and require extensive usage of cleanroom facilities [38]. However, simpler implementations of these ladder-shaped designs are possible by creating a direct pressure balance between the flow fields in sink and source channels [39][40][41], but these designs are often susceptible to mechanical noise from syringe pumps that introduce pressure imbalance across the gradient chamber [42].…”

Time-lapse Imaging of Mouse Macrophage Chemotaxis

“…During incubation, the slide should be slowly rotated around its long axis so that the cells remain evenly distributed in the X-, Y-and Z-axis directions while the collagen polymerizes into a gel. A related closed chemotaxis slide suitable for 3D chemotaxis assays, with six plugs instead of four plugs, has recently been described 29 . This system allows the collagen-cell mixture to be introduced into the observation area before independently filling each of the flanking reservoirs, because each reservoir has two filling ports, rather than a single port.…”