Evaluation of EGFR and RTK Signaling in the Electrotaxis of Lung Adenocarcinoma Cells under Direct-Current Electric Field Stimulation

Abstract

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells’ response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment

“…The stiffness of the ECM and stromal cells can also regulate the invasiveness of cancer cells [183,184]. Finally, transepithelial potential differences in tissues can generate physiological electric field and guide the migration of cancer cells through electrotaxis [185][186][187]. In short, many metastatic niche studies verified that invasive cancer cells could interact with and modulate the biophysical and biochemical properties of the stroma, as well as all the cellular components in the complex microenvironment.…”

Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

“…300 V m −1 dcEFs were established in section I and V (FIG. 5.c) through two home-made silver/silver chloride (Ag/AgCl) wire electrodes 51,139 inserted in the PBS reservoirs on component B by a source measure unit (2410, Keithley, USA). Time lapse phase contrast images of each condition were taken on an automated phase contrast microscope (Ti-E, Nikon, Japan).…”

The use of electric, magnetic, and electromagnetic field for directed cell migration and adhesion in regenerative medicine

“…This asymmetrical activated signaling pathway appears to promote both conformational changes and reorganization in the cell's cytoskeletal structure that are involved in cell motility . Cell receptors have been reported to move from the leading edge of the cell to the trailing edge under the influence of exogenously applied EF with intensities between 1 and 5 V cm −1 . In the case of applied MF or EMF action on cell motility, most studies suggest a frequency dependency, which points to a nonlinear, discrete (or quantized) mechanism of energy transfer .…”

“…156,157 Cell receptors have been reported to move from the leading edge of the cell to the trailing edge under the influence of exogenously applied EF with intensities between 1 and 5 V cm 21 . 77,158 In the case of applied MF or EMF action on cell motility, most studies suggest a frequency dependency, which points to a nonlinear, discrete (or quantized) mechanism of energy transfer. 159 Multiple experiments on isolated cell systems suggest that the Ca 21 signaling pathway is the target of MF and EMF in the first step of coupling.…”