Cancer Cell Chemotaxis in
Microfluidic Chambers
Dallas Reilly, Chemistry (ACS)
Carthage College
Mentor- Noo Li Jeon, Biomedical Engineering
University of California Irvine
Graduate Student- Carlos Huang, Biomedical Engineering
University of California Irvine
Abstract In recent years microfluidic devices have been used to obtain valuable information
about cells, namely cancer cells. Firstly it is important to study the true value of these devices in
their usefulness in many things including cancer research. Secondly, the main chambers of
microfluidic devices can provide us with an area to obtain important information on the various
migrations of cancer cells towards or away from specific gradients composed of various
chemicals (chemo-attractant or chemo-repellant gradients). With proper sterilization techniques
to culture MDA-MB 231 breast cancer cells and by following the proper protocols to create the
devices, we can use a 10x microscope (with mercury lamp), fluorescence/growth factor
(epidermal growth factor) solution, and various computer programs we can monitor the cells and
accurately quantify their migrations. Different variations of conditions can also be used to study
these cells in even further depth (i.e. different concentrations of growth factor, starving the cells,
and eventually using chemo-repellants to study cancer fighting drugs). In this project I
discovered that metastatic cancer cells do migrate towards gradients containing natural amounts
of growth factor, and I concluded that other conditions affect these cells as well. These results
are important because they show promise in the study of cancer cell migrations due to different
positively or negatively stimulating chemicals.
Key Terms
Apoptosis
Cancer
Epidermal Growth Factor (EGF)
Metastasis
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Introduction Cancer is a wide range of diseases distinguished by anomalous, unusual cell
growth and the spread of those abnormal cells to other parts of the body. Normal cells will grow,
divide, and eventually die through a process called apoptosis (programmed cell death). Cancer
cells however, will continue to grow and divide until eventually they develop into what is called
a tumor. The uncontrolled growth and migration of those cells, called metastasis, can cause
death in cancer victims. When cells reach the stage of metastasis, they travel through the
bloodstream or the lymphatic system.
Environmental factors are the main cause of cancer, but it is important to keep in mind that
the abnormal cell growth that is cancer is a naturally occurring process. What is meant by
environmental factors? These can include just about anything, but the most obvious factors
include: tobacco use, alcohol abuse, diet, radiation and sunlight exposure, occupation in rare
cases, sometimes race, and in recent years it has been shown that viral infections greatly
contribute to risk of cancer. Risk factor also increases with age.
Breast cancer affects about one in every ten women, and kills about 40,000 women per year
in the United States alone (American Cancer Society, 2005). Although most breast cancers are
benign and are not deadly, there are some strains that are metastatic and easily spread to other
parts of the body. It is extremely important to understand why and how metastatic cancers
spread to other parts of the body. If we can understand why cancers migrate to other tissues, we
can begin to create drugs that slow or completely stop metastasis. Cancer is classified in many
ways: historically neoplasia or neoplasm(s), tumors, or simply cancer. However, tumors are
lumps of cancer cells, and there are two types: benign and malignant. Although benign tumors
can invade other tissues, they generally grow slowly and do not invade other tissues of the body.
Malignant tumors on the other hand grow fast and have a high risk of moving to other tissues and
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organs of the body. Once a tumor reaches a certain point, cancer cells begin to “fall off” of the
main tumor. Most of these cells die, but others sometimes find their way into the lymphatic
system or the bloodstream. The term for this is metastasis- a stage when cancer is at its
deadliest. Although it is rare for a cancer cell to survive the many opposing forces in other parts
of the body (and few do survive), when a cell does metastasize successfully it can reach much
more important organs and tissues of the body, making it so dangerous.
Figure 1 Normal cells compared to cancer cells forming a tumor (CancerBackup).
Discovering how and why cancer cells metastasize could prove extremely useful in the fight
against cancer. Cancer cells that do break away from the original tumor attach to proteins (such
as epidermal growth factor), and somehow escape into other tissues of the body. In this research
project I studied how epidermal growth factor influences the migration of cancer cells.
Epidermal growth factor (EGF) is a small protein produced by cells in the body. Other cells,
including cancer cells have EGF receptors. These receptors bind to EGF and send signals to the
cell. These signals cause the cell to become healthier in several ways (increase in certain
chemicals and actions). These actions play a large role in cell growth, division, and
differentiation (the ability to become a different type of cell). Cancer cells have an overexpression of the EGF receptor, which gives them a much higher sensitivity to the growth factor,
as well as a greater chance of binding with it. The question to study here is: what role does EGF
have (if any) on the migration of cancer cells?
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In this research project I studied how exactly epidermal growth factor can influence metastatic
breast cancer cells with the use of a microfluidic device. Microfluidic devices are devices
(usually made of polydimethyl-siloxane) that control fluids in channels in micro-sized channels.
Fluids in such small channels experience “laminar flow,” smooth flow of different fluids in
parallel without any turbulence or diffusion of fluids. These devices have a huge range of
applications including chemical synthesis, public health, defense, biological analysis,
information technology, biotechnology, life sciences, pharmaceuticals, and there are many
promising fields and studies in each application. In research involving cells, often times the
fluids that are used are expensive. Microfluidics allow researchers to use extremely small
amounts (picoliters to microliters) making it much cheaper to operate with expensive chemicals.
They also allow researchers to view and analyze small numbers of cells and easily track their
movement and health. Finally, and most importantly, these devices can be manipulated to
produce many types of gradients containing chemo-repellant or chemo-attractant chemicals (such
as EGF).
Materials
Sylgard 184 Silicone Elastomer base (Polydimethyl-siloxane) and curing agent
3 inch silicon wafer master mold
Nalgene vacuum chamber
Cimarec hot plate
Harrick Scientific plasma cleaner/sterilizer
MDA-MB 231 metastatic breast cancer cells (ATCC.org)
Fetal Bovine Serum (FBS)
Leibovit’s L15 media with 10% FBS (none for starving)
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Shel Lab incubator (no carbon dioxide)
Falcon 60x15 tissue culture dishes
Falcon T-75 tissue culture flasks
.01M phosphate buffer saline (PBS)
Gibco .25% trypsin-EDTA
Sterile hood
Intramedic polyethylene tubing (.38mm)
Disposable 3mL syringes with short needles
Harvard PicoPlus syringe pump (70-2213)
3-way valves
Epidermal growth factor (Combrex CC-43317A)
FTIC-Dextran fluorescent marker
Enclosed Hoffman 10X microscope (with mercury lamp and Q-Capture imaging system and
heater)
Metamorph Offline cell tracking software
Oriana circular orientation software
Microsoft Excel tracking templates
Experimental Procedures and Protocols
Cell Culture:
MDA-MB 231 cells were cultured in T-75 flasks with L15 media (containing 10% FBS) placed
in an incubator containing no carbon dioxide. The cells were taken out by removing the media,
washing with phosphate buffer saline solution, and adding about 2 mL trypsin-EDTA. This was
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left to sit for five minutes (placing the flask in an incubator speeds the process) until the cells
were no longer attached to the bottom of the flask. Then this was washed with approximately 8
mL L15 media (to have a total of 10 mL in the flask). This solution containing the cells was
placed in a 15 mL test tube and centrifuged for five minutes. By this time the cells would form a
clump at the bottom of the test tube, so that the media/trypsin mixture could be removed without
removing the cells. Then, more media was added to break the cells up to distribute them evenly
in the media. The cells could then be placed into smaller dishes for experimentation or other
flasks for further culturing.
Device fabrication:
An amount of PDMS elastomer base was mixed with one tenth that amount of curing agent.
This was poured evenly over a silicon master. The bubbles were brought to the top of the PDMS
with a vacuum and were popped with an air gun. The silicon master dish was placed on a hot
plate (80C ) for one to two hours. These device molds (four per silicon master) were cut out and
reservoirs and tubing holes were punched.
Experimental procedure:
The heat pumps in the microscope chamber were turned on (30C ) first. The EGF/FTIC/L15
solution was then prepared and the device was plasma treated with the Harrick plasma cleaner
apparatus. The pump was prepared and the vacuum syringe, tweezers, scissors, and dish lid were
sprayed with ethanol and placed in the sterile hood. The pump and vacuum syringe tubes were
cut at the ends (to avoid contamination), fed through the hole in the lid, and attached to the
tubing holes on the device. The device was carefully placed upon the dish of cells by holding the
tubing lines and pressing down with the tweezers simultaneously. Suction was applied with the
vacuum syringe so that the device was attached to the dish. The EGF/FTIC/L15 solution was
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applied to the desired reservoir and the lid was carefully placed upon the dish. The pump,
vacuum syringe, and dish were then carried to the microscope chamber where the permanent
vacuum could be attached (using the 3-way valves), the device secured in the microscope
chamber, and the pump plugged in and turned on.
Analysis:
Metamorph Offline was used to track the cell’s movement over a three hour period (one picture
every two minutes). Once the data of the cell’s tracked movement was fully collected, Excel
spreadsheets were used to collect and calculate various data from the cell’s movement such as:
total distance traveled, average velocity, distance towards or away from the gradient, velocity
towards or away from the gradient, absolute angle, and the chemotactic index or CI (total
distance towards gradient divided by the total distance) and standard deviation of that
measurement. Oriana software was used to measure the mean vector angle ( 90 for the right
high gradient and 270 for the left high gradient is ideal). It also calculates Rayleigh’s test or “pvalue.” Rayleigh’s test is basically a number describing the randomness of a group of circular
angles. A higher number such as .75 relates that the results from the case in question are 75%
random. In this study a lower number means that our results mean something (95% randomness
is a good goal)
Results Throughout this project I aimed to use three experiments for each different set of
conditions that I planned on studying. In each dish I cultured approximately 450,000 cells for an
experiment, and in every experiment I placed the EGF/FTIC/ media solution in the middle
channel, creating what I known as a “bell-curve” gradient. In the first triplet of experiments I
studied the MDA-MB 231 cells’ migration with 50 ng/mL EGF concentration without starving
the cells. Refer to figures 2 and 3 for the results.
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Angles
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Figure 2 Average angles for the cells left of the gradient (known as a right-high gradient) (left). Average angles for
the left-high gradient (right).
Figure 3 Examples of the cell movement taken from the second experiment from the series with a picture of the
gradient in the middle for a reference. The illumination is created by the FTIC in the solution with a mercury lamp.
For the next set of three experiments, a much higher concentration of EGF (500 ng/mL, 10
times the amount originally used) was used, again without starving the cells. Refer to figure 4
and 5 for the results.
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Angles
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Figure 4 Average angles for the right-high gradient (left) and average angles for the left-high gradient (right)
Figure 5 Examples from experiment 3 of the series of three experiments of the cells’ tracked movement (assuming
the same bell-curve gradient as the first triplet).
For the third group of three experiments I reverted to 50 ng/mL concentrations of EGF and
starved the cells. I had assumed the length of time of starving the cells would impact my results
less than it did. Refer to figures 6 and 7 for the results from the first two experiments, and
figures 8 and 9 for the last experiment.
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Angles
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Figure 6 Angles for the first two starved-cell experiments: right-high gradient (left) and left-high gradient (right)
Figure 7 Examples from experiment 2 of the series of two experiments with about 12 hours of cell starvation
(assuming the same bell-curve gradient as the other triplets).
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Figure 8 Angles from the experiment with five hours of cell starvation: right-high gradient (left) and left-high
gradient (right).
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Figure 9 Cells’ tracked movement from the experiment with five hours of cell starvation: right-high gradient (left)
and left-high gradient (right).
Based upon my findings from the last three experiments I did three experiments with 50 ng/mL
EGF concentration, but this time I starved the cells 1-3 hours. Refer to figures 10 and 11 below
for the results.
Angles
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Figure 10 Angles from the experiments with 1-3 hours of cell starvation: right-high gradient (left) and left-high
gradient (right).
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Figure 11 Cells’ tracked movement from the experiment with 1-3 hours of cell starvation: right-high gradient (left)
and left-high gradient (right).
Table 1 Numerical results from all experiments and conditions (refer to the discussion section for explanations).
Discussion Cancer includes many different types if irregular cell growths, including many types
that can invade important tissues of the body. Breast cancer affects many people around the
world (mostly women) and encompasses several types of metastatic cancer cells. This metastasis
of cancer can have extremely detrimental affects to its victims because it often times spreads to
vital organs and tissues of the body. Studying metastasis with the use of microfluidic devices
that create gradients offers many valuable avenues to study metastatic cancer cells. In this study
I used time lapsed experiments to study how epidermal growth factor influences the chemotaxis
of MDA-MB 231 metastatic breast cancer cells in a microfluidic chamber. I placed the EGF in
the middle channel (of three channels) to create a bell-curved gradient.
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In the first few weeks of the study I simply cultured cells, learned the proper sterilization
techniques, constructed devices, and perfected the experimental procedure and my own
techniques used in that procedure. Once I was ready to collect consistent data, I started running
experiments. Previous studies (Saadi, Lin, Jeon, Wang 109-117) have shown that 50 ng/mL
EGF concentration creates positive migration in MDA-MB 231 cells, so I decided to start with
that concentration without starving the cells. Figures 2 and 3 and table 1 show the results from
those three experiments. Although the right-high gradient produced more random results than
the left high gradient, overall the cells did move towards the EGF gradient. The cell tracking
figures shown in figure 3 are similar for the other two experiments as well, which also indicate
positive migration. Overall 69% of the cells moved towards the gradient. The average angle for
the cells were 97 / 262 . This is a good results because in a perfect experiment the angles
would be 90 / 270 . The P-value is quite low, indicating a randomness of less than 2%. The
chemotactic index is relatively high, although the standard deviation for the right-high gradient is
quite high. These results prove that healthy cancer cells do show a migration due to the presence
of epidermal growth factor.
For the next three experiments I decided to increase the concentration of EGF by a factor of
10 (500 ng/mL). This unnaturally high concentration of EGF produced extremely random
results. Less than 50% of the cells moved towards the gradient, the average angles were random,
and the P-value showed a randomness of over 60%. The CI value for the right-high gradient is
very high, but the standard deviation is as well. The CI value for the left-high gradient is very
low, with a very high deviation. These results show that healthy cells show no obvious
migration to a very high concentration of epidermal growth factor. I concluded that the cells are
probably “confused” by such an abnormal amount of EGF.
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Based upon the negative results I obtained from the high concentration of EGF, I reverted
back to 50 ng/ml EGF concentration. This time however, I planned on starving the cells. For the
first two experiments of starving the cells I starved for about 12 hours. Only 47% of the cells
moved towards the gradient, and the results were even more random than when using the
extremely high EGF concentration. I had assumed that the starvation time of the cells would not
make a large difference in their migration, and that the overall migration would increase because
the cells would be desperate for food and move even more. For the third experiment I only
starved the cells for about 5 hours, which gave me much better results. 75% of the cells moved
towards the gradient, although I was only able to analyze 16 cells, which is probably the reason
my P-value showed such high randomness (24%). The angles: 90 / 279 were almost perfect.
The CI value and deviation for the right-high gradient were the same, indicating a bad result, but
the values for the left-high gradient (.14/.07) looked fairly good. Based upon these results I
concluded that although starving can have a positive affect on the cells’ migration, too much
starving drives them to such an unhealthy point that instead of migrating, the cells’ main goal is
to simply survive.
For my last set of three experiments I wanted to starve the cells again, but this time I would
starve for only 1-3 hours (again using 50 ng/mL EGF concentration). I was able to analyze more
cells and the results were quite good. 66% of the cells moved towards the gradient, the angles of
106 / 260 were close to ideal, and the P-value indicated a lot randomness of about 98%. Not
only that but the CI values were both somewhat high, with deviations lower than the CI values
themselves. Based upon this I concluded that starving does not have an extremely significant
affect whether it is negative or positive on the cancer cells’ migrations.
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This research experience has taught me a tremendous amount. I experienced difficulties
learning the sterilization, cell culture techniques, and consistently preparing adequate
experiments, but I feel confident in all of those areas now. I believe that I accurately concluded
that epidermal growth factor has a significant role in the migration of MDA-MB 231 breast
cancer cells. Healthy cancer cells undergoing a gradient containing natural amounts of EGF will
indeed migrate towards that gradient. In the future (and in other studies currently being
undertaken) researchers will do similar studies involving chemo-repellants, other chemoattractants, possibly combining both in the same device, other types of metastatic cancer cell
lines, and possibly most importantly, extracellular matrices and their relation to the migration of
cancer cells.
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Acknowledgements
•
I’d like to thank my mentor, Noo Li Jeon, my graduate student, Carlos Huang, and the
rest of the great people in my lab for teaching me such an incredible amount in such a
short time and for taking their time to assist my research
•
I’d also like to thank the UROP program, with which this research opportunity would
have never been possible, their time and effort
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References and works cited:
George M. Whitesides. The Origins and the Future of Microfluidics. Nature Publishing Group,
July 2006.
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Ingber. Soft Lithography in Biology and Biochemistry. Annual Review of Biomedical
Engineering, 2001 (335-573).
Noo Li Jeon, Harihara Baskaran, Stephan K.W. Dertinger, George M. Whitesides, Livingston
Van De Water, and Mehmet Toner. Neutrophil Chemotaxis in Linear and Complex Gradients of
Interleukin-8 Formed in a Microfabricated Device. Nature Publishing Group, 2002.
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Differential Effects of EGF Gradient Profiles oN MDA-MB-231 Breast Cancer Cell Chemotaxis.
Elsevier, INC, 2004.
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Wajeeh Saadi · Shur-Jen Wang · Francis Lin · Noo Li Jeon. A Parallel-Gradient Microfluidic
Chamber for Quantitative Analysis of Breast Cancer Cell Chemotaxis. Biomedical Devices (109118), 2006.
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