dc electric fields direct breast cancer cell migration, induce egfr polarization, and increase the...
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ORIGINAL PAPER
DC Electric Fields Direct Breast Cancer Cell Migration, InduceEGFR Polarization, and Increase the Intracellular Levelof Calcium Ions
Dan Wu • Xiuli Ma • Francis Lin
� Springer Science+Business Media New York 2013
Abstract Migration of cancer cells leads to invasion of
primary tumors to distant organs (i.e., metastasis). Growing
number of studies have demonstrated the migration of
various cancer cell types directed by applied direct current
electric fields (dcEF), i.e., electrotaxis, and suggested its
potential implications in metastasis. MDA-MB-231 cell, a
human metastatic breast cancer cell line, has been shown to
migrate toward the anode of dcEF. Further characteriza-
tions of MDA-MB-231 cell electrotaxis and investigation
of its underlying signaling mechanisms will lead to a better
understanding of electrically guided cancer cell migration
and metastasis. Therefore, we quantitatively characterized
MDA-MB-231 cell electrotaxis and a few associated sig-
naling events. Using a microfluidic device that can create
well-controlled dcEF, we showed the anode-directing
migration of MDA-MB-231 cells. In addition, surface
staining of epidermal growth factor receptor (EGFR) and
confocal microscopy showed the dcEF-induced anodal
EGFR polarization in MDA-MB-231 cells. Furthermore,
we showed an increase of intracellular calcium ions in
MDA-MB-231 cells upon dcEF stimulation. Altogether,
our study provided quantitative measurements of electro-
tactic migration of MDA-MB-231 cells, and demonstrated
the electric field-mediated EGFR and calcium signaling
events, suggesting their involvement in breast cancer cell
electrotaxis.
Keywords Breast cancer cell � Electrotaxis � EGFR �Calcium � Microfluidic device
Abbreviations
dcEF Direct current electric fields
ECM Extracellular matrix
EGFR Epidermal growth factor receptor
PDMS Polydimethylsiloxane
EI Electrotactic index
SEM Standard error of the mean
MSD Mean square displacement
Introduction
Cell migration plays important roles in mediating a wide
range of biological processes such as wound healing [1, 2],
neuron guidance [3, 4], embryogenesis [5], and cancer
metastasis [6, 7]. Among the diverse cellular guiding
mechanisms, chemotaxis, a process that cells sense and
migrate up a chemical concentration gradient, is well
studied [8, 9]. In addition, physical parameters such as
direct current electric fields (dcEF) can also direct the
migration of various cell types [10–13], a process termed
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12013-013-9615-7) contains supplementarymaterial, which is available to authorized users.
D. Wu � X. Ma � F. Lin (&)
Department of Physics and Astronomy, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada
e-mail: [email protected]
F. Lin
Department of Immunology, University of Manitoba, Winnipeg,
MB R3E 0T5, Canada
F. Lin
Department of Biological Sciences, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada
F. Lin
Department of Biosystems Engineering, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada
123
Cell Biochem Biophys
DOI 10.1007/s12013-013-9615-7
electrotaxis or galvanotaxis. Endogenous dcEF widely
exist in biological systems, and electrotaxis participates in
relevant biological processes such as tissue repair and
embryogenesis [12–15]. Several previous studies demon-
strated electrotaxis of different cancer cells in vitro
including prostate cancer cells [16], lung cancer cells [17],
and breast cancer cells [18], and in most cases the elec-
trotactic migration is correlated with metastatic potential of
the cancer cells, suggesting the possible involvement of
electrotaxis in cancer metastasis. Interestingly, unlike
prostate or lung cancer cells that migrate cathodally, MDA-
MB-231 cells, a human metastatic breast cancer cell line,
move toward the anode of dcEF [18], suggesting that
electrotaxis of different cancer cells can be characteristi-
cally different in specific physiological environments and
disease models. In breast cancers, the most common cancer
in women, the metastatic phase of the disease is the major
cause of death [19, 20]. Therefore, it is important to better
understand the mechanisms of breast cancer cell migration
including the electrical guiding mechanism. Toward this
direction, we in the present study performed quantitative
measurements of breast cancer cell electrotaxis using
MDA-MB-231 cells as a model cell system. Our results
confirmed and quantitatively characterized the anodal
electrotaxis of MDA-MB-231 cells using microfluidic
devices. In addition, we showed that dcEF-induced polar-
ization of epidermal growth factor receptor (EGFR) and an
increase of intracellular calcium ions in MDA-MB-231
cells.
Various environmental factors are involved in mediating
cancer metastasis including extracellular matrix (ECM),
cytokines, growth factors, and their interacting receptors
expressed in cancer cells [21, 22]. Among them, EGFR was
shown to be an essential player in regulating breast cancer
metastasis [23, 24]. Relevantly, electric field can effec-
tively redistribute charged mobile entities in the plane of
cell membrane and EGFR were shown to polarize to the
cathode-facing side of the cell in several cathodally elec-
trotaxing cell types such as bovine corneal epithelial cells
[12, 25, 26], human keratinocytes [27], and lung cancer
cells [17]. Earlier studies also reported similar dcEF-
induced cell surface receptor polarization for concanavalin
A receptor in Xenopus laevis embryonic muscle cells [28]
and murine fibroblastic cells [29]. The re-distribution of
relevant cell surface receptors by dcEF results in more
receptors on the cathode-facing side of the cell, which is
proposed to mediate cell sensing of dcEF and the cathode-
directing electrotactic cell migration [12, 17, 25, 27–29].
Our previous modeling studies support such a receptor
electromigration-based cellular sensing mechanism [30]
and indeed previous studies have shown that EGFR sig-
naling is required for electrotaxis of breast cancer cells
[18]. On the other hand, because EGFR polarizes
cathodally in cathode-directing electrotactic cells while
MDA-MB-231 cells migrate toward the anode of dcEF in
conventional electrotaxis assays, it is interesting to test
whether and how dcEF can polarize EGFR in MDA-MB-
231 cells.
Additionally, both calcium ion release and calcium ion
influx are generally associated with cell migration [31, 32],
and calcium signaling is involved in the migration, inva-
sion, and metastasis of different cancer cells [33]. The
intracellular calcium ion level ([Ca2?]i) in polarized
migrating cells forms a concentration gradient across the
cell body and the elevated [Ca2?]i in the back of the cell
regulates the rear-end retraction [34, 35]. Chemical stim-
ulus such as chemokines can trigger robust calcium flux
through their cell surface G-protein coupled chemokine
receptors [34, 36]. In MDA-MB-231 cells, EGF stimulates
similar [Ca2?]i flux [37]. In electrotaxis, dcEF induces a
transient increase of [Ca2?]i in difference cell types such as
mouse embryo fibroblasts [38] and murine adipose-derived
stromal cells [39], whereas dcEF stimulates a more sus-
tained increase of [Ca2?]i in Dictyostelium cells [40], and
calcium signaling is required for electrotaxis of these cells.
For cancer cells, voltage-gated ion channels such as sodium
channels are shown to mediate electrotactic responses of
prostate cancer cells [16]. Those voltage-gated ion chan-
nels are also expressed in MDA-MB-231 cells, suggesting
the possible involvement of cellular ion signaling in elec-
trotaxis of breast cancer cells.
Taking together, dcEF is a physiologically relevant
guiding mechanism for breast cancer cell migration and
metastasis, and thus there is a great need to better under-
stand the process of electrotactic migration. In the current
study, we performed quantitative measurements of breast
cancer cell (MDA-MB-231 cells) electrotaxis. Further-
more, we focused on EGFR polarization and [Ca2?]i
dynamics to identify these signaling events and their
characteristics in MDA-MB-231 cell electrotaxis. After
initial testing with a simple well plate-based assay, we
further employed microfluidic electrotaxis devices [41] to
test electrotaxis and electrotactic signaling in MDA-MB-
231 cells under better controlled experimental conditions.
Experimental Methods
Cell Culture
MDA-MB-231 cells, a human breast adenocarcinoma cell
line, were purchased from ATCC (Manassas, VA, USA)
with verified lineage and purity. Cells were cultured in
Leibovitz’s L-15 medium supplemented with 10 % FBS
and 1 % penicillin/streptomycin in a 37 �C incubator.
MDA-MB-231 cells were passaged regularly for the use of
Cell Biochem Biophys
123
specific experiments throughout this study. Trypsin–EDTA
was purchased from ATCC (Manassas, VA, USA) as the
cell dissociation buffer.
Electrotaxis Experiments Using Well Plates
A piece of coverslip (VWR micro cover glass No. 1,
thickness is 0.13–0.17 mm) was placed in the well plate
(COSTAR 24 well plate, 15.6 mm well diameter, Corning,
NY, USA). The coverslip was coated with 2 lg/mL of
Collagen Type IV (Sigma-Aldrich, Inc., MO, USA) for
30 min at room temperature followed by 2 % BSA
blocking for another hour at 37 �C before the experiment,
providing a substrate for cell adhesion and migration. The
coating concentration of Collagen Type IV was selected
according to the previous literature [42]. For each experi-
ment, cells were seeded on the coverslip and the well was
filled with 1 mL culture medium. Then a pair of platinum
electrodes connecting to a DC power supply was placed in
the well plate with the separation distance similar to the
diameter of the well to apply the electric field. After cali-
brating the electrical current, the well plate was placed
under a microscope (Nikon Ti-U) and cell migration in the
well was recorded by time-lapse microscopy at 1 frame/
min for 1–2 h using a CCD camera. The image acquisition
was controlled by the NIS Elements software.
Electrotaxis Experiments Using Microfluidic Devices
A previously developed polydimethylsiloxane (PDMS)
microfluidic electrotaxis device was modified for the
present study [41] (Fig. S1). In brief, the microfluidic
device with a simple straight channel was designed in
Freehand 9.0 (Macromedia) and the design was printed to a
transparency mask by a high resolution printer. The mas-
ters were fabricated at The Nano Systems Fabrication
Laboratory (NSFL) at the University of Manitoba. The
design was patterned on a silicon wafer by contact photo-
lithography with SU-8 photoresist (Micro Chem, MA)
through the transparency mask and the SU-8 pattern yields
*100 lm thickness. The PDMS replicas were then fabri-
cated by molding PDMS (Sylgard 184 silicon elastomer,
Dow Corning, MI, USA) against the master. Two 4 mm
diameter medium reservoirs/electrode wells at the two ends
of a 350 lm (W) 9 1 cm (L) channel were punched out
using sharpened needles (Fig. S1). In addition, a 1-mm
diameter hole for the fluidic inlet was punched out in the
PDMS device for infusing medium (Fig. S1). The PDMS
replica was then plasma bonded to a glass slide using a
plasma cleaner (Harrick Plasma, Ithaca, NY, USA). Poly-
ethylene tubing (PE-20, Becton–Dickinson, MD, USA)
was inserted into the fluidic inlet to connect the microflu-
idic device to a syringe pump (KD Scientific, Holliston,
MA, USA) with a 250-lL syringe containing medium for
fluidic infusion. The medium was continuously infused into
the device by the syringe pump through the inlet at the flow
rate of 0.2 lL/min (i.e., linear speed of 5.7 mm/min).
External platinum electrodes (SPPL-010, Omega Engi-
neering, Inc.) that were attached to conducting wires were
inserted into two medium reservoirs and the wires were
connected to a DC power supply to apply electric field to
the microchannel. The microfluidic channel was coated
with Collagen Type IV for 30 min at room temperature
followed by blocking with 2 % BSA for 1 h at 37 �C to
provide a substrate for cell adhesion and migration. A new
microfluidic device was used for each experiment and the
time-lapse imaging of cell migration follows the same
method as in the well plate-based experiments.
The actual potential difference across the well-plate or
microfluidic channel is generally lower than the total
voltage applied from the power supply due to the electrical
potential drop at the electrodes. For simplicity, in this
article, we calculated the dcEF in our experiments using
the total applied voltage from the power supply and pro-
vide a calibration table in the Supplementary Information
(Table S1).
Analysis of Cell Migration Data
Movement of individual cells in the well-plate or micro-
fluidic device was tracked using MetaMorph (Molecular
Devices Inc., Offline Premier Version, v7.7.3). The images
were calibrated to distance and only the cells that migrated
within the field of view were selected for tracking. The
tracking data were exported to Excel and MATLAB for
analysis. Following previously established analysis meth-
ods [43, 44] (Fig. S1), the movement of cells was quanti-
tatively evaluated by (a) the percentage of cells that
migrated toward the anode of the electric field; (b) elec-
trotactic index (EI), which is the ratio of the displacement
of cells toward the anode of the electric field to the total
migration distance, presented as the average value ±
standard error of the mean (SEM) (Fig. S1); (c) the speed
of cells, which is the total migration distance divided by the
migration time, presented as the average value ± SEM
(Fig. S1); (d) statistical analysis of migration angles per-
formed using MATLAB to examine the directionality of
cell movement (Fig. S1). Specifically, migration angles
(calculated from x–y coordinates at the beginning and the
end of the cell tracks) were summarized in a direction plot,
which is a rose diagram showing the distribution of angles
grouped in defined intervals (18�), with the radius of each
wedge indicating the cell number; (e) to quantify the
motility of cells along the direction of electric field, the
mean square displacement (MSD) of cells in the x-direction
(i.e., the dcEF direction) was calculated as a function of
Cell Biochem Biophys
123
time,\x2(t)[. The parameters between different conditions
were compared by the 2-sample t test. 38–142 cells were
analyzed for each experiment. Multiple independent
experiments were performed for each condition with sim-
ilar results and data from representative experiments were
shown in the figures.
Imaging of EGFR Staining
After electrotaxis or control experiments, cells on the
coverslip in either the well-plate or the microfluidic device
were washed quickly with staining buffer (PBS with 0.5 %
BSA), then immediately fixed with 4 % formaldehyde. For
better staining and imaging results of cells in microfluidic
devices, PDMS was reversibly bonded to the coverslip
without plasma treatment. The PDMS top was removed
from the coverslip after cell fixing and then the cell-con-
taining coverslips were used for staining and imaging in
this set of experiment. Alexa 488 tagged monoclonal anti-
human EGFR antibody (Santa Cruz Biotechnology, Inc.,
CA, USA) was diluted in the staining buffer at 1:50 ratio
and was then used to stain cells at 4 �C for 30 min. Cells
were washed three times with the staining buffer and fur-
ther treated by DAPI for nucleus staining followed by
additional washing steps before confocal imaging. The
coverslips were mounted in mountant permafluor (Lab
Vision Corporation, CA, USA) and viewed with a confocal
microscope (Nikon Ti-U microscope equipped with a
C1-Plus confocal system). The image acquisition was
controlled by the EZ-C1 software (ver3.80).
[Ca2?]i Measurement
[Ca2?]i in MDA-MB-231 cells was measured by flow cyto-
metric analysis (FACS analysis) and confocal microscopy.
Cells were loaded with Fluo-4 AM (Molecular Probes, Inc.,
Eugene, Oregon, USA) in IMDM medium (Hyclone Labo-
ratories, Inc., Utah, USA) with 1 % FBS for 30–45 min in a
37 �C incubator in dark. After Fluo-4 loading, the cells were
washed twice with the same medium and recovered in a
37 �C incubator in dark for 30 min before the calcium
experiments. For the FACS experiment, cells were stimu-
lated in the well plate by dcEF of different strength over
defined period of time (as detailed in the ‘‘Results’’ section)
or EGF (recombinant human EGF, Sigma-Aldrich Co. LLC,
MO, USA) of different concentrations (as detailed in the
‘‘Results’’ section; in this case, cells were mixed with EGF
immediately followed by FACS monitoring), and then the
cells were transferred to a FACS tube for FACS analysis
using a flow cytometer (BD FACSCalibur, Becton–Dickin-
son). The Fluo-4 signal was monitored by FACS at 3 s time
resolution. The FACS data was further analyzed using Flow
Jo (Tree Star, Inc., OR). For the confocal experiment, cells
were stimulated in the well-plate or the microfluidic device
by dcEF or EGF and Fluo-4 intensity was monitored in real-
time by time-lapse confocal microscopy at 6 frames/min.
Fig. 1 Electrotaxis of MDA-
MB-231 cells in well plates.
a Angular histogram shows
random cell migration in the
control condition without dcEF.
b Angular histogram shows cell
migration toward the anode of
the applied 1.5 V/cm electric
filed. c Comparison of
electrotactic index (EI),
percentage of electrotaxing
cells, and the speed of cells in
the absence or in the presence of
dcEF. d Comparison of mean
square displacement of cells
along the x-direction as a
function of time \x(t)2[ in the
control experiment or with dcEF
application. The error barsrepresent the standard error of
the mean (SEM). The p values
for each comparison from
2-sample t test are shown.
** p \ 0.01
Cell Biochem Biophys
123
Multiple independent experiments were performed for each
condition with similar results.
Results
Anodal Electrotaxis of MDA-MB-231 Cells
In a previous study, MDA-MB-231 cells were shown to
migrate toward the anode of dcEF using a conventional
dish-based electrotaxis assay [18]. Here, we want to con-
firm this result in our cell migration system. In the first set
of experiment, we applied a 1.5-V/cm of dcEF to MDA-
MB-231 cells seeded in a well plate and measured cell
migration by time-lapse microscopy. As shown by the
migration angle histograms (Fig. 1a, b), cells showed clear
migration toward the anode of the applied dcEF. By con-
trast, cells migrated randomly in the absence of dcEF. This
result was also supported by the higher percentage of
electrotaxing cells to the anode of dcEF and the higher EI
of cells in dcEF compared with it in the absence of dcEF
(Fig. 1c). Furthermore, cell speed (Fig. 1c) and the MSD
(\x2(t)[) analysis showed higher total cell motility on the
2D substrate (i.e., speed) and higher motility along the
dcEF direction (i.e., increase of MSD over time) respec-
tively for cells in dcEF than it in the absence of dcEF
(Fig. 1d). The main limitation of the well-plate assay is the
lack of control of electric field. In particular, the current
set-up of this assay does not allow higher dcEF application,
which produces bubbles and causes significant pH change
due to electrolysis.
Thus, we next performed the electrotaxis experiments
using a microfluidic device. Compared to conventional dish-
based electrotaxis assays, microfluidics devices have
advantages in control of dcEF application and cell migration
environments [45]. This device is in principle similar to our
previously developed microfluidic device for studying
electrotaxis of T cells [41] with the addition of a fluidic inlet
for continuous perfusion of medium into the channel (Fig.
S1). This new flow feature of the device provides continuous
supply of fresh medium and removes waste or other cell
products in the channel. In addition, the medium flow is
configured along the same direction of the dcEF such that the
true anodally electrotaxing cells will need to migrate against
the flow. No bubbles are produced in the channel even at
higher dcEF. It is worth pointing out that there is a significant
change of pH in the two reservoirs, where electrodes were
inserted, before and after applying dcEF. However, the pH in
the channel is not expected to change because fresh medium
from syringe pump is continuously infused into the channel
throughout the experiment (Fig. S1).
Thus, this microfluidic system allowed us to measure
electrotaxis of MDA-MB-231 cells in better controlled dcEF
with higher magnitude comparing to the well plate. In the
Fig. 2 Electrotaxis of MDA-
MB-231 cells in microfluidic
devices. a Angular histogram
shows random cell migration
with a bias along the medium
flow direction in the absence of
dcEF. b Angular histogram
shows cell migration toward the
anode of 5 V/cm dcEF.
c Comparison of electrotactic
index (EI) and speed of cells in
the absence or in the presence of
dcEF. d Comparison of mean
square displacement of cells
along the x-direction as a
function of time \x(t)2[ in the
control experiment or with dcEF
application. The error barsrepresent the standard error of
the mean (SEM). The p values
for each comparison from
2-sample t test are shown.
** p \ 0.01, and * p \ 0.05
Cell Biochem Biophys
123
control experiment without dcEF, cells migrated randomly
with a detectable bias along the medium flow direction as
expected (Fig. 2a, c). By contrast, cells overcame the med-
ium flow and migrated toward the anode of the applied dcEF
(Fig. 2b–d). In this fluidic system, the optimal cell speed is in
the 4 V/cm dcEF (i.e., 33.12 lm/h). This speed is compa-
rable to the previously reported migration speed of MDA-
MB-231 cells in EGF gradients [42]. MSD analysis more
clearly showed the higher motility of cells along the
x-direction in the dcEF experiments comparing to it in the
control experiment (Fig. 2d).
Taking together, the results of electrotaxis experiments
in both the well-plate and microfluidic devices confirmed
the anodal electrotaxis of MDA-MB-231 cells over the
similar range of dcEF as in the previous study [18], pro-
viding the quantitative electrotactic migration characteris-
tics of MDA-MB-231 cells for further electrotactic
signaling-related analysis in this study.
EGFR Polarization in MDA-MB-231 Cells Toward
the Anode of the dcEF
Based on the demonstrated anodal electrotaxis of MDA-
MB-231 cells, we want to next examine the relevant cel-
lular biophysics events in MDA-MB-231 cells upon dcEF
stimulation to identify possible electrotactic signaling. One
candidate event is dcEF-induced polarization of EGFR.
EGFR is expressed in various electrotaxing cells and sev-
eral previous studies have demonstrated polarization of cell
surface EGFR that coincides with the direction of elec-
trotactic cell migration [25–27]. Therefore, in MDA-MB-
231 cells, we expect EGFR will polarize toward the anode-
facing side of the cells in dcEF.
To test this hypothesis, we employed immunofluores-
cence staining against cell surface EGFR to measure EGFR
distribution after electrotaxis experiments either in the
well-plate or microfluidic device. Our results showed that
before dcEF was applied cells typically form round shape
morphology or orient randomly (Fig. 3a). Upon exposure
to 4 V/cm dcEF, many cells formed clear leading edges
and among them most cells oriented toward the anode of
the dcEF (Fig. 3b). Immunofluorescence staining by anti-
EGFR antibody showed that EGFR polarized toward the
anode-facing side of the electrotaxing cells (Fig. 3c), i.e.,
our analysis revealed the detectable more EGFR distribu-
tion on the anode-facing side of more than 50 % of total
cells in confocal images and the remaining cells showed no
clear EGFR polarization at the time of fixation. Quantita-
tive analysis provides complementary indication of EGFR
polarization toward the anode-facing side of the cell
(Supplementary Information; Fig. S2). In the control
experiment without dcEF, EGFR uniformly distributed in
all cells.
dcEF Induce [Ca2?]i Elevation in MDA-MB-231 Cells
Motivated by previous studies showing calcium signaling
in electrotaxing cells [38–40], we in this study analyzed the
dcEF-induced intracellular calcium ion ([Ca2?]i) to test the
possible involvement of calcium signaling in breast cancer
cell electrotaxis.
MDA-MB-231 cells were loaded with Fluo-4 and
stimulated in a well plate by dcEF for 10 min, and Fluo-4
intensity was measured by FACS (before and after 10 min
of dcEF stimulation) or by confocal microscopy (continu-
ous monitoring) to indicate the dynamic changes of
[Ca2?]i. In the FACS analysis, 1.5 V/cm dcEF induced a
very small but more sustained increase of [Ca2?]i (Fig. 4e).
By contrast, the higher magnitude of dcEF (3 V/cm)
induced a clear increase of [Ca2?]i, which decreased over
time and stabilized at a level higher than the baseline
(Fig. 4e). Consistent with the previous research that
showed a transient calcium spike lasting 2 min in MDA-
MB-231 cells in response to EGF stimulation [37], treating
cells with a range of EGF doses in our study caused a
Fig. 3 dcEF-induced morphological change and EGFR polarization
in MDA-MB-231 cells in microfluidic device. a Phase contrast
images of three representative cells before the dcEF exposure.
b Phase contrast images of the same three cells in a after 1.5 h
exposure to a 4-V/cm dcEF. The white arrows point to the leading
edge of the migrating cells. c Confocal images of the same three cells
by anti-EGFR antibody (green) staining and nucleus staining (blue)
showing polarization of EGFR to the anode-facing side of the cell
(Color figure online)
Cell Biochem Biophys
123
transient increase of [Ca2?]i in cells and the duration of
[Ca2?]i spikes also generally lasted *2 min before com-
pletely adapt to the baseline level (Fig. 4f). Unlike the
FACS analysis, which measures [Ca2?]i without concurrent
dcEF stimulation, time-lapse confocal microscopy allows
us to further monitor [Ca2?]i in cells at the population level
(i.e., measuring Fluo-4 intensity of the entire image) in
real-time over the time course of dcEF stimulation. Our
results confirmed the increase of [Ca2?]i by dcEF stimu-
lation (Fig. 4a–d). Consistent with the FACS analysis
results, this increase was only detectable for 3 V/cm but
not 1.5 V/cm dcEF stimulation, and it occurred toward the
end of the 10 min stimulation and lasted even after the
dcEF was turned off. For both the FACS and confocal
measurements, Fluo-4 intensity (F) was normalized to the
initial intensity (F0), and this ratio (F/F0) was used to
indicate the relative [Ca2?]i in cells. Notably, some bubbles
were typically produced around the electrodes in the well
plate in 3 V/cm or higher magnitude of dcEF, which
complicates the interpretation of the observed increase of
[Ca2?]i. Although trypan blue staining showed that 95 %
cells were viable in 3 V/cm of dcEF, we want to further
test the calcium response under better controlled experi-
mental conditions.
To further analyze [Ca2?]i dynamics in a better con-
trolled cellular environment, we used microfluidic devices
as an experimental platform to monitor [Ca2?]i in MDA-
MB-231 cells at the single cell level in dcEF or by EGF
stimulation using time-lapse confocal microscopy. In the
control experiments, Fluo-4 intensity decreased over time
due to photobleaching by confocal scanning and thus set
the baseline curve for comparison. In 3 V/cm dcEF,
[Ca2?]i exhibited a small but long lasting increase that is
more clear a few minutes after the dcEF was applied
(Fig. 5a). In a higher dcEF (4 V/cm), [Ca2?]i significantly
increased (Fig. 5a, b). Similar to the results in well plates,
the increase of [Ca2?]i maintained after dcEF was turned
off, particularly for 4 V/cm. In addition, consistent with the
EGF stimulation results in FACS (Fig. 4f), [Ca2?]i tran-
siently increased upon EGF stimulation in microfluidic
devices (Fig. 5a, c), again suggesting the distinct charac-
teristics of calcium responses to dcEF and EGF stimula-
tions. In addition to analyzing the average [Ca2?]i to
compare the mean difference among conditions, we further
looked at [Ca2?]i in individual cells. dcEF or EGF stimu-
lations-induced [Ca2?]i oscillations in many individual
cells, suggesting similar [Ca2?]i responses as observed in
other electrotaxing cells [40].
Fig. 4 [Ca2?]i elevation in MDA-MB-231 cells in well plates
induced by dcEF or EGF stimulation. a–c Time-lapse confocal
images of cells. 3 V/cm dcEF was applied between the 5th and the
15th min. d Analysis of Fluo-4 intensity of the time-lapse confocal
images. F/F0 is the relative Fluo-4 intensity normalized to the initial
baseline level. e Fluo-4 intensity measured by FACS. Fluo-4 intensity
was measured by FACS before and after 10 min dcEF treatment in
the well plate, i.e., 0–1 min for the baseline before dcEF treatment;
1–5 min after 10 min dcEF treatment in the plot. f Fluo-4 intensity
measured by FACS in cells stimulated by EGF at the indicated
concentrations from the 2nd min
Cell Biochem Biophys
123
It is worth pointing out the different effective dcEF for
triggering significant [Ca2?]i increase in different experi-
mental systems in this study. dcEF below 4 V/cm in
microfluidic device or below 3 V/cm in well plates or
FACS could not elicit significant calcium responses in
cells, likely resulting from the differences of experimental
systems (microfluidic device vs. well plate or FACS) and
analysis method (single cell vs. cell population) that set
different thresholds. This general threshold phenomenon
further suggests the possible regulations of voltage-gated
calcium channels for dcEF-induced calcium responses in
MDA-MB-231 cells. On the other hand, results in all three
experimental systems consistently showed calcium eleva-
tion in MDA-MB-231 cells upon dcEF stimulations, sug-
gesting the possible involvement of calcium signaling in
breast cancer cell electrotaxis.
Conclusion and Discussion
In this study, we quantitatively characterized the migration
of MDA-MB-231 cells in dcEF and tested relevant signal-
ing events with the focus on EGFR polarization and intra-
cellular calcium ion dynamics. Our results confirmed the
anodal electrotaxis of MDA-MB-231 cells. As discussed in
the previous study [18], an estimated dcEF of 6 V/cm
across the epithelial layer exists with lumen side as the
anode resulting from the transepithelial electrical potential
(TEP) difference in breast epithelium. Such physiological
dcEF is comparable to the effective dcEF strength in our
experiments using microfluidic devices. On the other hand,
electrotaxis of MDA-MB-231 cells was observed in the
well-plate assay under much lower dcEF, which is close to
the lower limit of effective dcEF reported in the previous
study using a dish-based electrotaxis assay [18].
Furthermore, our results demonstrated the dcEF-induced
anodal polarization of EGFR and intracellular calcium ion
increase. These results provide further experimental evi-
dence of breast cancer cell electrotaxis with quantitative
details and suggest the possible cellular mechanisms that
involve EGFR and calcium signaling. In particular, previ-
ous study has already shown that EGFR signaling is
required for breast cancer cell electrotaxis [18]. Our results
further suggest the possible involvement of cell surface
EGFR polarization for enabling electric field sensing and
migration of MDA-MB-231 cells. Cathodal EGFR polari-
zation has been reported for several cathodally electro-
taxing cell types such as bovine corneal epithelial cells [12,
25, 26], human keratinocytes [27], and lung cancer cells
[17]. Our results showed that EGFR can also polarize to the
anode-facing side of the cell for anodally electrotaxing
MDA-MB-231 cells, and therefore the same receptor can
be differentially polarized in different cell types depending
on the direction of electrotactic migration.
Despite the different threshold dcEF for triggering cal-
cium response and the detailed [Ca2?]i dynamics in
Fig. 5 [Ca2?]i responses in
MDA-MB-231 cells in
microfluidic devices induced by
electric field or EGF. a [Ca2?]i
over time without stimulation,
or stimulated by dcEF or EGF
(100 ng/mL). The data (F/F0) is
represented by the average
Fluo-4 intensity (F) of all
individual cells analyzed and is
normalized to the initial Fluo-4
intensity (F0). dcEF was applied
to the cells till the 10th min.
EGF-containing medium was
infused to the cells in the
microfluidic channel from the
5th min. b–d The same analysis
was shown for individual cells
for the dcEF (b), EGF (c), and
control experiments (d)
Cell Biochem Biophys
123
different experimental systems used in this study, our
results showed clear elevation of [Ca2?]i in MDA-MB-231
cells upon effective dcEF stimulation. On the other hand,
the results of electrotaxis and calcium elevation derived
from the well-plate assay are not always consistent. We
believe it is due to the limitation of the well-plate assay in
control of dcEF application and cell migration environ-
ments. Although it is a simple assay to test if there is any
response at all to dcEF, the calcium elevation in well-plate
assay may be caused by other factors such as pH change of
the medium or cell death. Therefore, microfluidic devices,
which can better control dcEF application and cell migra-
tion environments, were used to further examine calcium
response of cells to dcEF and EGF stimulations.
Previous studies showed that dcEF induce [Ca2?]i influx
of Dictyostelium cells during electrotaxis [40]. In addition,
embryo fibroblasts stimulated by dcEF showed an increase
of [Ca2?]i. Both studies demonstrated that the cathodal
electrotaxis is calcium-dependent [38, 40]. Our results
showed consistent calcium responses to dcEF for MDA-
MB-231 cells. Importantly, the [Ca2?]i increase induced by
dcEF in MDA-MB-231 cells is more sustained in clear
comparison with the EGF stimulation-induced transient
[Ca2?]i elevation. Previous studies showed that EGF pre-
treatment of cells promotes calcium oscillations in
response to glutamate, ATP, or thimerosal (which directly
activates the inositol-1,4,5-triphosphate receptor) in astro-
cytes [46, 47] and EGF can directly induce transient
[Ca2?]i increase in MDA-MB-468 cells [48]. In the elec-
trotactic Dictyostelium cells, cAMP induces transient cal-
cium flux whereas the [Ca2?]i increase to dcEF is more
sustained [40]. Similar transient calcium response was also
recently reported for MDA-MB-231 cells in direct response
to EGF stimulation [37]. Thus, the characteristics of cal-
cium response to dcEF in electrotaxing cells are different
from it induced by chemoattractants or growth factors.
While some possible factors that may cause the different
calcium response to dcEF and chemical stimulations were
previously discussed such as the delayed calcium spikes
upon dcEF stimulation and passive calcium influx [40], the
underlying mechanisms remain to be determined.
Metastasis is the major cause of death for cancer patients,
which requires the migratory ability of cancer cells. Exis-
tence of physiological electric field between the tumor and
normal tissues during metastasis of breast cancers and the
potential role of such electric field in mediating the invasion
of electrotaxing breast cancer cells into the surrounding
tissues for metastasis have been suggested [16, 18, 49].
Thus, understanding the mechanisms for electrotactic
migration of breast cancer cells will have important impact
on more effectively managing breast cancer metastasis.
Together with other studies [18], our results suggest the
possible involvement of EGFR and calcium signaling in
breast cancer cell electrotaxis. An interesting aspect is the
experimental evidence for EGFR-mediated chemotactic
migration of breast cancer cells to EGF gradients [42] and
the requirement of EGFR signaling for breast cancer cell
electrotaxis [18] as reported in previous studies and the
anodal polarization of EGFR induced by dcEF in MDA-
MB-231 cells as reported in this study that collectively
suggests the comprehensive roles played by EGFR for
mediating breast cancer cell chemotaxis and electrotaxis
during metastasis. However, the EGFR-mediated signaling
mechanisms for chemotaxis and electrotaxis may be char-
acteristically different as evidenced by the different calcium
responses to EGF and dcEF in MDA-MB-231 cells. Further
testing MDA-MB-231 cells in different combinations of
dcEF and EGF fields will provide interesting insight into the
competing or collaborative guidance by chemical and
electrical cues for breast cancer cell migration. In this
regard, our recent study reported a novel microfluidic
device that can better control superimposed chemical and
electric fields and this device was used for studying T cell
migration in co-existing chemokine gradients and dcEF
[50]. Similar approach can be applied to MDA-MB-231 cell
migration and clarify the relative potency of EGF and dcEF
in attracting cells with relevance to developing potential
clinical applications by electrically manipulating cancer
cell trafficking in tissues. More generally, microfluidic
devices have been increasingly developed and applied to
electrotaxis studies [44, 45, 51, 52], providing useful
experimental tools for probing this important cell migration
process. Our study for the first time demonstrated the
effective use of microfluidic device for studying MDA-MB-
231 cell electrotaxis in 2D. However, the process and
mechanism of cell migration on 2D substrates can be lar-
gely different from it in 3D ECM. Although the comparison
of cell migration in 2D and 3D ECM depends on the type of
cells and ECM, cell migration generally replies more on
focal adhesion in 2D than in 3D, and the stiffness of 3D
ECM can significantly affects the morphology and direc-
tional migration of cells [53]. In addition, dcEF is inevitably
more complex when applied to 3D ECM comparing to it in
2D systems. Indeed, a recent study has shown differences of
lung cancer cell electrotaxis between 2D and 3D systems
[54]. In addition, the migratory behaviors of cells are
expected to be different when cells move collectively at
high density [55]. This is clearly the case in epithelial cell
electrotaxis that collective migration of epithelial mono-
layer is more efficient than isolated cells or smaller cell
clusters [56] and thus collective electrotaxis of tumor cells
in 3D tissues can also be sensitive to a lower effective dcEF
as compared with in vitro experiments. Therefore, we are
interested in furthering our studies in the future to test breast
cancer cell electrotaxis in 3D scaffold at the single cell level
or as cell groups.
Cell Biochem Biophys
123
Acknowledgments This Research is supported by Grants from the
Natural Sciences and Engineering Research Council of Canada
(NSERC), the Canada Foundation for Innovation (CFI), the Manitoba
Health Research Council (MHRC), and the University of Manitoba.
We thank The Nano Systems Fabrication Laboratory (NSFL) at the
University of Manitoba, and the Manitoba Centre for Proteomics and
Systems Biology for research support. We thank Saravanan Nan-
dagopal for helping collect chemical reagents, Jing Li and Jiandong
Wu for helping with microfluidic device preparation. D.W. thanks
MHRC for a postdoctoral fellowship.
Conflict of interest The authors declare no conflict of interest.
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