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We have
used the ability of ECIS to detect changes in cell morphology to design
whole-cell assays related to the behaviour of the cancer cell including
metastatic potential.
The measurement is based upon observations of transformed cells interacting
with established normal cell layers. Several years ago Kramer and Nicolson
presented scanning electron micrographs demonstrating, in cell culture, the
metastatic-like behaviour of cancer cells. In this work they first
established a confluent layer of endothelial cells in culture. This cell
layer was then challenged with highly metastatic cells (in this case
melanoma B16 cells). Electron micrographs taken at different time intervals
showed the melanoma cell attaching to the endothelial cell layer, with
subsequent
retraction of the endothelial cells, and finally penetration of the melanoma
to the tissue culture substratum.
Since the
ECIS system should easily detect this alteration in the endothelial cell
layer, we have explored an assay to monitor these changes to look for
correlations of impedance changes with the metastatic potential of cancer
cells.
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Our
studies were done in collaboration with researchers in Dr. Donald Coffey’s
group at Johns Hopkins Brady Urological Institute in Baltimore, MD. Workers
there had previously established a series of cancer cell sublines. These all
originated from a culture established from a single rat prostatic tumour,
that, upon extensive tissue culture passages and mutagenesis, resulted in
several sublines that exhibited a wide range of metastatic potential. When
injected subcutaneously into rats, some of these sublines formed tumours
only at the site of injection (G subline). Others would form distant
metastases approximately 10 to 20% of the time (AT1, ST3 sublines); whereas,
others would formed distant metastases at several locations over 90% of the
time (AT3, MLL sublines). The basic plan of our experiments was to first
establish a confluent layer of endothelial cells and then follow impedance
changes as the layer was exposed to different rat prostatic sublines. |
Establishment of the endothelial cell layer
These ECIS data show the establishment of the endothelial cells layer
for the assay. The ECIS wells were first coated with an adsorbed layer of
gelatin (1.0 mg/ml gelatin in 0.15M NaCl applied for 5 minutes and then
washed out of the well with sterile medium). At time zero, a HUVEC (human
umbilical vein endothelial cell) suspension was added to several wells and
their attachment and spreading monitored with standard ECIS measurements.
The resistive portion of the impedance at 4000 Hz is shown as a function of
time. 100,000 cells were added to each well in complete medium (DMEM with
10% FBS). Spreading is completed in the first 2 hours after inoculation and
the subsequent fluctuations are characteristic of this cell and are related
to cellular motion. |
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Challenge of the HUVEC layer |
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The
drawing shows the type of activities expected to occur during the challenge
of the normal endothelial cell layer with the metastatic cell lines. |
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The
data shows a control culture receiving only additional medium. The other
traces are of duplicated cultures challenged with the weakly metastatic G
subline or the metastatic AT3 subline. All additions were at the two
vertical marks on the x-axis. Note that the addition of both cell sublines
tends to reduce the resistance of the system and result in reduction of the
fluctuations. The activity of the AT3 subline is considerably larger than
that of its weakly metastatic counterpart (G). The lost of the resistance is
due to the lost of integrity of the endothelial cell layer in response to
the activities of the cancer cells. |
Challenge with active and heat-killed cells
These curves show similar activity where the highly metastatic MLL cell line
rapidly breaks down the resistance of the established endothelial cell
layer, but the same cells when first heat killed (15 minutes at 56 degrees
C) have no effect verifying that the assay is indeed seeing biological
activities. In these data all additions are at time zero. |
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