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Transendothelial migration of metastatic cancer under the influence of cigarette smoke condensate
h [electronic resource] /
by Daniel Opp.
[Tampa, Fla.] :
b University of South Florida,
ABSTRACT: Cigarette smoke's influence on cancer has primarily been a subject of epidemilogic and tumorigenic studies. There have been no proper investigations with interests focused on how cigarette smoke affects the cellular mechanics of metastasis. Gathering an understanding of how smoke influences metastatic invasion could be vital in regulating or possibly eliminatings cancer's ability to initiate new tumor growth sites. This project focuses on cigarette smoke's influence on cellular mechanics of endothelial cells, and the invasive potential of cancer against a fully active endothelium. It is already known that cigarette smoke has a carcinogenic effect, but it is hypothesized that the cigarette smoke causes the endothelium to exhibit pro-invasive characteristics. Cancer cells are often ignorant to extra-cellular stimuli. It is suspected that there will be a less pronounced degradation of cellular mechanics of cancerous cells than endothelial cells when exposed to similar concentrations of cigarette smoke.
Thesis (M.S.)--University of South Florida, 2007.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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Mode of access: World Wide Web.
Title from PDF of title page.
Document formatted into pages; contains 46 pages.
Advisor: Chun-Min Lo, Ph.D.
Cell adhesion molecules.
t USF Electronic Theses and Dissertations.
Transendothelial Migration of Metastat ic Cancer Under the Influence of Cigarette Smoke Condensate by Daniel Opp A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science Department of Physics College of Arts and Sciences University of South Florida Major Professor: Chun-Min Lo, Ph.D. Garrett Matthews, Ph.D. Dale Johnson, Ph.D. Date of Approval: July 10, 2007 Keywords: ECIS, junctional resistance, cell adhesion molecules, intravasation, extravasation Copyright 2007, Daniel Opp
Table of Contents LIST OF FIGURES ii ABSTRACT v CHAPTER 1. INTRODUCTION AND BACKGROUND 1 CHAPTER 2. MATERIALS AND METHODS 2.1 Electric Cell-substrate Impedance Sensing (ECIS) 5 2.2 Cell Culture Procedures 9 2.3 Cell Attachments and Spreading 10 2.4 Cigarette Smoke Condensate 12 2.5 Cancer Invasion Assay 13 2.6 Reendothelialization/Cell Motility 15 CHAPTER 3. THE ECIS MODEL 3.1 Theoretical Model 18 3.2 Frequency Scan 20 CHAPTER 4. RESULTS AND DISCUSSION 4.1 Reendothelializiation / Tissue Gap Recovery 24 4.2 CSCs effect on confluent HUVEC and ALST layers 26 4.3 CSCS effect on transmigration 34 CHAPTER 5. CONCLUSION AND FUTURE WORK 41 REFERENCES 45 i
List of Figures Figure 2.1 An overview of a single well on the microelectrode array. Here it can be observed how much larger the counter electrode is as compared to the working electrode. 6 Figure 2.2 A diagram of the experimental set up used in the ECIS system. 7 Figure 2.3 Real time data of the resistance measurements of a HUVEC layer as measured by the ECIS system. The resistance increases with time as the HUVEC cells begin to firmly attach to the surface of the electrode. 11 Figure 2.1 The resistance graph of a cancerous invasion of a normal endothelial cell layer can clearly indicate when the monolayer has been compromised. 14 Figure 2.5 A confluent monolayer (A) was scratched with a micropipette tip to (B) create a gap across the HUVEC monolayer (C). 16 Figure 3.1 A logarithmic graph of resistance (A) and capacitance (B) measurements for both confluent cultures on ii
electrodes and naked electrodes as a function of frequency. 22 Figure 3.2 A logarithmic graph of normalized resistance (A) and normalized capacitance (B) measurements as observed through ECIS. The normalized values are determined by dividing the cell-covered and cell-free measurements for the corresponding frequency. 23 Figure 4.1 Images take from an inverted microscope reveal that CSC has the ability to inhibit endothelium reformation. 25 Figure 4.2 Quantification of the recovery of the different cell layers reveal that cancerous cells dont necessarily have a decreased recovery rate as an increasing function of CSC concentration. 27 Figure 4.3 A resistance ratio graph of a HUVEC layer being challenged by various concentrations of CSC. 29 Figure 4.4 An ALST layer being challenged by various concentrations of CSC. 30 Figure 4.5 The junctional resistance (A) and cell-substrate separation (B) of a HUVEC and ALST layer. 32 Figure 4.6 Images of confluent ALST (top) and HUVEC (bottom) layers. The images on the left are not exposed to iii
CSC, while the images on the right are exposed to CSC concentrations of 250 g/mL 33 Figure 4.7 (A) Junctional resistance of a HUVEC layer being exposed simultaneously to CSC concentrations and ALST invasion. (B) Junctional resistance of a HUVEC layer being exposed to CSC 10 hours before ALST invasion. 36 Figure 4.8 (A) Cell-substrate separation of a HUVEC layer being exposed simultaneously to CSC concentrations and ALST invasion. (B) Cell-substrate separation of a HUVEC layer being exposed to CSC 10 hours before ALST invasion. 37 Figure 4.9 The resistance ratio graph of a ten hour delayed ALST invasion of a HUVEC monolayer. 38 Figure 5.1 Images of a HUVEC layer experiencing a fluid flow were taken with an inverted microscope. The times are denoted in the top right corner. 43 iv
Transendothelial Migration of Metastatic Cancer under Cigarette Smoke Condensate Influence Daniel Opp ABSTRACT Cigarette smokes influence on cancer has primarily been a subject of epidemiologic and tumorigenic studies. There have been no proper investigations with interests focused on how cigarette smoke affects the cellular mechanics of metastasis. Gathering an understanding of how smoke influences metastatic invasion could be vital in regulating or possibly eliminating cancers ability to initiate new tumor growth sites. This project focuses on cigarette smokes influence on cellular mechanics of endothelial cells, and the invasive potential of cancer against a fully active endothelium. It is already known that cigarette smoke has a carcinogenic effect, but it is hypothesized that the cigarette smoke causes the endothelium to exhibit pro-invasive characteristics. Cancer cells are often ignorant to extra-cellular stimuli. It is suspected that there will be a less pronounced degradation of cellular mechanics of cancerous cells than endothelial cells when exposed to similar concentrations of cigarette smoke. v
CHAPTER 1 Introduction and Background The endothelium serves as the interface between blood supply and the rest of the tissue within the human body as it lines the entire circulatory system. Through this layer, water and other molecules are allowed to exit the bloodstream and into the surrounding tissue. A secondary purpose of this confluent layer is acting as a protective barrier against vascular diseases. Cancerous cells drifting through blood vessels and lymphatic ducts that have become independent of their original growths attach on to the endothelium in order to initiate new growths. A complete understanding of the cellular mechanics of the endothelial layer is necessary in limiting and possibly nullifying the metastatic potential of cancer through blood vessels. Cellular motility and proliferation is a vital function in reendothelialization. A confluent endothelial cell monolayer demonstrates contact-inhibited latency. When the endothelium is confluent and latent, it allows for an optimized interaction with the blood stream. During the loss of confluency, cellular motility and proliferation increases to recover compromised regions. Motility continues to remain highly active until contact inhibition restricts the 1
monolayer back to latency. The endothelium serves as the initial barrier for metastatic cancer cells through the blood stream searching for new sites to initiate new tumor growths through extravasation. It is also the last barrier invading cancer cells must breach to access the lumen of blood vessels through a process called intravasation. Primary tumors account for about 10% of deaths from cancer while the remaining 90% is a result of transient tumors spawned from primary growth . Metastasis is the ability of cancer cells to detach from primary tumors, travel the body through blood or lymphatic vessels, and initiate new tumor growths in different regions of the body. Current techniques involving cancer penetration utilize polycarbonate membranes containing 8-m pores covered in a thin layer of a protein matrix to simulate an endothelial layer [2, 3], afterwards the penetrated layer is observed through cell staining. The polycarbonate membranes are static and morphologically constant as they do not respond to stimulus in their environment. This utilization of the polycarbonate membrane does not truly observe cancer penetration of the endothelium as it is more of a cellular motility experiment. With ECIS, an active fully responsive endothelium can serve as a barrier against cancer penetration which is quantifiable into electrical impedance measurements. 2
Interactions of cancer cells with the vascular endothelium and basement membrane are crucial in both intravasation and extravasation processes [4, 5], involving the regulation and expression of many genes involved in such diverse activities as endothelial binding , cell signaling resulting in endothelial retraction , the synthesis and secretion of proteolytic enzymes , and cell locomotion . Cigarette smoke-induced disruption of the endothelial integrity may facilitate cancer penetration, but the underlying mechanism is unclear. Cigarette smoke has primarily been utilized as a tumorigenic factor in both in vitro experiments and with live animals. It has been used to induce cancer in samples for investigation of genetic changes and regulations that occur due to the cigarette smoke influence [10, 11]. The interactions between normal cells and cancerous cells within the same smoke exposed environment have not been investigated. This is important because normal and cancer cells respond differently to environmental changes. Endothelial cells and metastatic cancer cells may exhibit different sensitivities and reactive mechanisms to cigarette smoke. Cigarette smoke contains over 4000 different chemicals with 69 of these constituents being individually identified as carcinogens . The possible combined effects of chemicals within the smoke can not 3
be neglected and therefore determination of a total carcinogenic effect is unknown. Even though incidence of cancer is higher with smokers, it has been shown that the unfiltered side stream smoke is in fact more toxic for both smokers and nonsmokers. Cigarette smoke condensate (CSC) has been reported to have adverse effects on endothelial cells, including inhibition of migration , induction of apoptosis , cellular contraction , and transmigration across polycarbonate membranes simulating the endothelium [16, 17]. Cellular contraction  and inhibition of cellular motility  within the endothelium due to cigarette smoke is suspected in contributing towards an aggressive metastasis. 4
CHAPTER2 Materials and Methods 2.1 Electric Cell-substrate Impedance Sensing (ECIS) ECIS observes impedance changes that correlate with changes in junctional resistance and cell substrate separation of tissue cultures. The tissue samples are cultured into gold film microelectrodes wells. As seen in Figure 2.1, the observable electrode is 250 m diameter which sits in the center of the wells, a larger counter electrode sits on the edge of the wells. The significantly larger counter electrode allows for the impedance measurements on the central electrodes to dominate the measurements. The leads from the electrodes connect to an 830 lock-in amplifier from Stanford Research Systems. Impedance analysis was conducted with a 1V 4000 Hz AC signal through a 1M resistor providing a current of 1 A. The ECIS experimental set up with tissue cultures on the microelectrodes is depicted in Figure 2.2. The tissue cultures are maintained in an incubator at 37 C with 5% CO 2 with a coaxial cable connecting the electrodes to the lock-in amplifier. Each electrode well used for the ECIS measurements is well explained by Keese et.al. (10). All eight electrode contains small working electrode (area= 5 -4 cm 2 ) and a 5
Figure 2.1 An overview of a single well on the microelectrode array. Here it can be observed how much larger the counter electrode is as compared to the working electrode. 6
Figure 2.2 A diagram of the experimental set up used in the ECIS system. 7
large counter electrode (area=0.15 cm 2 ). Because of the difference in surface area, the impedance of the small electrode determines the total impedance of the system. This is due to the impedances inverse dependence on the electrode area. The active electrode is delineated by circular openings (diameter = 250 m) in a photoresist overlayer that insulates the rest of the deposited gold film from bulk electrolyte. As cells in the culture attach to the electrode, the cell behavior can be correlated to the impedance measured by this device. The size of the electrode was intentionally selected to be this small as cell related signals are more easily detectable with smaller electrodes. This is because solution resistance in the culture dish is much larger than the electrodes impedance. The impedance of the electrode electrolyte interface (Faradaic impedance) is proportional to the inverse of the electrode area, but constriction resistance (spreading resistance) for the circular disk electrode in a conducting medium of infinite extent varies as /2d, where is the resistivity of the medium and d is the diameter of the electrode. The electrode impedance can be made to always dominate the constriction resistance by using sufficiently small electrodes. When the electrode area is reduced to 10 -4 cm 2 the Faradaic resistance of the electrode-electrolyte interface at 4000 Hz is many times larger than the constriction resistance so the motility of the cells can be easily studied. 8
2.2 Cell Culture Procedures Samuel MOK at Harvard Medical School supplied the OVCA429 (a standard ovarian cancer cell line used in studies) and ALST (another ovarian cancer cell line) cells in frozen ampoules. They were thawed and grown in medium 199 and MCDB 105 (1:1) (Sigma) supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 g/mL streptomycin under 5% CO 2 and 37 0 C, high humidity atmosphere. The Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Clonetics Corp. (San Diego, CA) and were grown in endothelial cell growth medium which is added with following: 10 ng/mL human recombinant Epidermal Growth Factor, 1 g/mL hydrocortisone, 50 g/mL getamicin, 50 ng/mL amphotericinB, 12 g/mL bovine brain extract and 2% fetal bovine serum (amounts indicate final concentration). The HUVECs were cultured at 37C and 5% CO 2 HUVECs were sub-cultured when they were 70% confluent. HUVECs that were passaged less than six times were used in experiments. Confluent HUVEC layers formed in electrode wells using an inoculation density of 8 4 cell/cm 2 For OVCA420 and ALST invasion assays, a cell density of 1 5 cell/cm 2 was added to ensure an observable measurement by the ECIS. The culture medium was changed about 3-4 days after passage to maintain cell integrity for both cancerous and HUVEC cell lines. Cell suspensions were prepared 9
using the standard tissue culture technique with 0.05% or 0.25% of trypsin/EDTA. The cells were then kept in the incubator to acclimate before adding to each wells of the electrode. 2.3 Cell Attachments And Spreading Before HUVEC attachment and spreading, a protein matrix covering the gold electrodes is necessary for proper binding, aiding in monolayer formation upon the electrode surfaces. The electrodes were coated with 200 g/mL gelatin in 0.15 M NaCl and kept in the incubator for 15 minutes. The protein solution created a protein matrix suitable for HUVEC binding and monolayer formation. HUVEC cultures were harvested from culture dishes having confluent layers. The medium was removed, then the cell line washed with Hanks Balanced Salt solution of 5 mL. The dishes were placed in the incubator with 0.05% of trypsin (2.5 mL) working on the cells, causing them to detach from the plastic surface. The trypsin was neutralized with culture medium then centrifuged at 1500 rpm for 5 minutes. After centrifugation, the remaining medium was able to be extracted leaving a pellet of cells to allow for a pure sample. New medium was added, and then 0.4 mL of the sample was added into each of the 16 wells. The electrodes were placed into the incubator where the HUVEC cultures were allowed to reach confluency. Figure 2.3 is a graph of HUVEC cells forming a monolayer as observed by resistance 10
HUVEC Attachment2000500080001100014000048121620Time (hrs)Resistance (ohm) Figure 2.3 Real time data of the resistance measurements of a HUVEC layer as measured by the ECIS system. The resistance increases with time as the HUVEC cells begin to firmly attach to the surface of the electrode. 11
measurements through ECIS. This process was accelerated by adding a high density of cells into the wells. Under ideal conditions, this process takes from 1-2 days depending on HUVEC passage and cell density of sample. Monolayer confluency was verified visually by microscope as well as resistance measurements through ECIS. 2.4 Cigarette Smoke Condensate Cigarette smoking condensate (CSC) was purchased from Murty Pharmaceuticals (Lexington, KY) and was prepared by smoking University of Kentucky's 1R3F Standard Research Cigarettes on a Federal Trade Commission Smoke Machine. The stock solution of CSC was prepared in dimethyl sulfoxide (DMSO) at a concentration of 40 mg/mL and stored in a small aliquot at -70C until used. CSC was diluted with the culture medium to prepare solutions for the experiments. Concentrations of 10 and 50 g/mL of CSC were used which correlated to equivalent CSC concentrations within a light and heavy smokers blood stream respectively. 100 g/mL of CSC was also used to demonstrate an extreme smoke exposure. Out of the total of 16 wells per experimental trial, each concentration of control, 10, 50, and 100 g/mL of CSC were distributed into 4 wells with 0.1 mL of the diluted CSC solutions. Cancer invasion assays were either implemented immediately or 10 hours after the HUVEC monolayers were exposed to the various concentrations of CSC. 12
2.5 Cancer Invasion Assay OVCA429 and ALST cultures that were confluent were used for invasions to ensure proper cell density for observable transmigrations. OVCA429 cultures were trypsinized with 0.25% (2.5 mL) trypsin while 0.05% (2.5 mL) trypsin was used for ALST cell lines. OVCA429 were incubated for 12 minutes while ALST incubated for 7 minutes to allow for trypsin to degrade binding receptors. The trypsin was neutralized with cancer medium then the sample centrifuged at 1500 rpm for 5 minutes. After pellet formation the medium was extracted and replaced with HUVEC medium. Using the hemacytometer, cell density could be calculated then the sample manipulated to ensure a cell density of 10 5 cells/well. 0.1 mL of the sample with the known cancer cell densities were added to the wells. Cancer invasion assays were either implemented immediately or 10 hours after the HUVEC monolayers were exposed to the various concentrations of CSC. Immediate invasions assays were intended to recreate conditions for extravasation while the 10 hour delayed invasion simulated intravasation. Figure 2.4 shows how ECIS is able to observe invasion of cancerous cells upon a normal cell monolayer. The decreasing resistances as compared with control indicate a breaking of junctions of the normal monolayer by the invading cancerous cells. In the case of OVCA429, 13
Cancer Invasion6000900012000150000481216Time (hrs)Resistance (ohm) HOSE15 OVCA429 SKOV3 Control 20 Figure 2.4 The resistance graph of a cancerous invasion of a normal endothelial cell layer can clearly indicate when the monolayer has been compromised. 14
the cells have been able to penetrate to the bottom on the electrode surface and being forming an OVCA429 layer. A continually increasing resistance trace (not shown) indicates OVCA429 beginning to proliferate upon the initial monolayer. 2.6 Reendothelialization/Cell Motility HUVEC, OVCA429, and ALST were cultured to confluency in 35mm x 10mm polystyrene culture dishes. Two lines were drawn on the outside bottom portion of the dish to be used as reference points when acquiring images from a microscope. Gaps were created by gently dragging a 200L pipette tip across the bottom of the cultured dishes. Figure 2.5  demonstrates how the micropipette tip is used to drag cells cleanly away to form a wound in the confluent monolayer. This is a common technique utilized in wound recovery assays. Three gaps perpendicular to the reference lines per dish were created to allow for six observable sections. Images were captured in two hour time increments by a CCD camera attached to a Zeiss Axiovert 200M inverted microscope. Through pixel analysis and time signature imaging the gaps could be measured and recovery rates calculated. When calculating recovery rates, images taken within the first 2 hours were excluded due to misleading accelerated recovery rates of OVCA429 and ALST layers. The accelerated recovery was attributed to overcrowded OVCA429 and ALST populations being able to push cells 15
(A) (B) (C) Figure 2.5 A confluent monolayer (A) was scratched with a micropipette tip to (B) create a gap across the HUVEC monolayer (C). 16
bordering the gap. This occurrence had to be omitted in order to analyze cancerous cells natural motility mechanism. After two hours, it was observed that the recovery rate stabilized and was consistent for the subsequent time and images. For the HUVEC cultures the experiment can be considered a reendothelialization experiment, where as the OVCA429 and ALST cultures are observed as a collective cell motility experiment. Reendothelialization refers to endothelial cells forming a monolayer after being wounded. Upon contact the two opposing groups of cells on either side of the wounds will cease movement and proliferation. With OVCA429 and ALST, cells will continue to proliferate despite forming a monolayer, thus forming cultures with stacked layers. 17
Chapter 3 The ECIS Model 3.1 Theoretical Model Giaever and Keese in 1991  developed a model that describes the various impedance measurements observed by the ECIS and the mathematical analysis needed to determine the junctional resistance and cell-substrate separation. The resistance of the culture medium acts in series with the impedance of both electrodes thereby causing the medium to dominate the impedance measurements. The solution resistance or constriction resistance inherits a dependency on electrode size. The impedance of the electrode-electrolyte interface has to be proportional to the inverse of the area of the circular electrode, 4/d 2 Therefore, the electrode-electrolyte interface can be forced to dominate the constriction resistance by making the electrode sufficiently small. When measured under the proper frequency and properly sized electrode, the real part of the impedance (faradaic resistance) is several times larger than the constrictions resistance. As a result the binding and anchoring behavior of the tissue samples can be measured and calculated. The equations below demonstrate the mathematics of the physical model used to calculate the specific 18
impedance (impedance for a unit area) of a cell covered electrode. A critical assumption is that the cells are approximated to be circular disks of radius r c The model is based on the specific impedance of a cell-free electrode Z n (v) and the cell layer Z m (v) as well as the resistivity of the medium. Both Z n (v) and Z m (v) exhibit frequency v dependence. The assumption has been made that the current flows radially between the ventral surface of the cell and substrate and that the current density in this region is unvarying in the z direction. @ d V d r f f f f f f f f 2 r f f f f f f f f f f I Vc@V Z n2 r d r f f f f f f f f f f f f f f f f dIc V Z m2 r d r f f f f f f f f f f f f f f f f dIi d I d I c@ d I i This equation can be combined to yield dV2 d r 2 f f f f f f f f f f f 1r f f f dVdr f f f f f f f f @2V0 2hffff1Znfffffff1Zmfffffffffg f VchZnfffffffff Where Vc is the potential of the electrode, and h is the height of the 19
space between the ventral surface of the cell and the substratum. The equation can be solved with the sum of modified Bessel functions of first and second kind (18). Using proper boundary conditions, the specific impedance for a cell-covered electrode is obtained as follows: 1Zcffffff1ZnfffffffZnZnZmfffffffffffffffffffffZnZnZmffffffffffffffffffffffffffrc2ffffffffffI0rc`aI1rc`afffffffffffffffffffffffRb1Znfffffffff1ZmfffffffffdeffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffHLLLLJIMMMMK Where I 0 and I 1 are modified Bessel functions of the first kind with order 0 and 1. The solution exhibits a dependency on two variable parameters which are R b (the resistance between the cells for a unit area), and (related to cell substrate separation) which can be given as: rcrchffff1Znfffffff1Zmfffffffffg v uutwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww1Znfffffff1Zmffffffffswwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww With the specific impedances being evaluated as two cell membranes in series, and R b are the only adjustable parameters in the equation stated above. 3.2 Frequency Scan A unique feature of the ECIS system is the application of frequency spectroscopy to the tissue-electrode interface model. In Figure 3.1, the resistance and capacitance are plotted versus frequency on a logarithmic graph. In order to observe impedance attributed to 20
exclusively the cell-layer, impedance measurements of cell-free electrodes as well as cell-covered electrodes must both be taken into account. Notice the two distinct traces in each graph as being labeled without any cells (no cells), and confluent with a HUVEC cell layer (HUVECs). The resistance and capacitance values are normalized by dividing resistance and capacitance values of the cell-free impedance measurements, with the cell-layer impedance measurements for the corresponding frequencies. In Figure 3.2 the normalized values are plotted as a function of logarithmic frequency. The solid lines in Figure 3.2 represent the calculated values of the resistance (Figure 3.2A) and capacitance (Figure 3.2B), as determined from the measured values of impedance. The points represent the calculated values based off the equations stated in previous section developed for the cell-layer model. The trace the points follow are forced to mimic the normalized resistance and capacitance measurements by the adjustment of the parameters and R b within the theoretical model. This method of model fitting is used to determine and R b which directly relate to the cell-substrate separation and binding of the tight junctions between cells. 21
(A) (B) Figure 3.1 A logarithmic graph of resistance (A) and capacitance (B) measurements for both confluent cultures on electrodes and naked electrodes as a function of frequency. 22
(A) (B) Figure 3.2 A logarithmic graph of normalized resistance (A) and normalized capacitance (B) measurements as observed through ECIS. The normalized values are determined by dividing the cell-covered and cell-free measurements for the corresponding frequency. 23
Chapter 4 Results and Discussion 4.1 Reendothelializiation / Tissue Gap Recovery Previous studies indicate that endothelium cells contract and have inhibited migration abilities when exposed to CSC, while certain cancers experience stimulation in cell proliferation and angiogenic factor expression. Wound healing assays with confluent cultures of HUVEC, OVCA429, and ALST were prepared and observed under time signature microscopic imaging. For all cell cultures, the first image was taken two hours after wounding the cultures. This was due to the cancerous cells stacking upon each other where as normal endothelium cell types do not. Within the first 90 minutes the cancerous cultures experience an accelerated recovery due simply to the over crowded cells being pushed into the gap. At 2 hours the cancerous cultures gap recovery reaches a steady rate. Figure 4.1 shows the images for the wounded HUVEC cultures control group in comparison to the 10 g/mL of CSC. Degradation in gap recovery can be observed when comparing the images. The images were analyzed with software having the ability to count the pixels within a designated region. In this case the region was specified as the gap between the 24
Figure 4.1 Images taken from an inverted microscope, revealed that CSC has the ability to inhibit endothelium reformation. 25
encroaching groups of cells. With the known length and area of the gaps in pixels, the average width of the gaps could be calculated and converted to micrometers. Figure 4.2 shows the quantification of the gaps recovered in micrometers per hour as a function of CSC concentration. HUVEC experienced degradation in gap recovery with increasing CSC concentrations as expected. This is attributed to the increased surface expression of cell adhesion molecules (CAM)  that is stimulated by phosphorylation of CAM regulators induced from the CSC. ALST cultures had a degraded gap recovery at 10 g/mL of CSC but then increased almost back to normal at 50 g/mL of CSC. OVCA429 experiences a stimulated wound recovery at 10 g/mL of CSC before degrading back to normal at 50 g/mL of CSC. With CSCs unusual affect of increasing motility of cancerous cells, it was thought that CSC could possibly stimulate cancers intravasation or extravasation abilities thereby making it more aggressive. 4.2 CSCs effect on confluent HUVEC and ALST layers An investigation was conducted to observe how CSC affected the junctional formations and substrate binding capabilities of HUVEC and ALST layers. HUVEC and ALST layers were allowed to reach confluency before being exposed to various concentrations of CSC. Frequency scans were applied before and after 40 hour impedance measurements were taken. Junctional resistance and cell-substrate 26
01020304050600102030405060708090100CSC concentration (g/mL)wound recovery (m/h) ALST OVCA429 HUVEC Figure 4.2 Quantification of the recovery of the different cell layers reveal that cancerous cells dont necessarily have a decreased recovery rate as an increasing function of CSC concentration. 27
separation could be calculated before and after 40 hours of CSC exposure. Within that 40 hour time frame, the overall resistance of the cell layers could be observed to see how the cell layers adjust in terms of tight junction and substrate binding. Figure 4.3 demonstrates the HUVECs response to the CSC. The resistance measurements are normalized to better observe the behavior the cell layers demonstrate. 10 g/mL of CSC has little effect if any at all to the HUVEC layer as the resistance ratio traces act similarly. 50 and 100 g/mL of CSC create an interesting reaction from the HUVEC layer upon exposure. The layers experience an increase in overall resistance of 10% and 30% for 50 and 100 g/mL of CSC respectively approximately 10 hours after CSC exposure. This initial increased resistance correlates with the findings that CSC induces adhesion molecule expression on the surface of cells  creating tighter junctions and substrate binding. These results of subsequent initial increasing resistance traces for HUVEC layers correlates with the data of Figure 4.2. The increased CAM expression increases the resistance of the HUVEC layers initial resistance while decreasing the layers ability to reendothelialize. Figure 4.4 shows the response of an ALST layer to various concentrations of CSC. A slight increase in resistance occurs with 10 g/mL of CSC which can be assumed to be an increase in CAM expression. There is a subtle decrease in the resistance trace 28
HUVEC-CSC 00.20.40.60.8188.8.131.52510152025303540Time (hr)Resistance Ratio control 10 50 100 Figure 4.3 A resistance ratio graph of a HUVEC layer being challenged by various concentrations of CSC. The traces are in units of g/mL of CSC. 29
ALST under CSC influence0.40.50.60.70.80.911.11.2051015202530time (hr)normalized resistance ratio control 10 50 100 Figure 4.4 An ALST layer being challenged by various concentrations of CSC. 30
at 50 g/mL of CSC, suggesting a loss of CAM surface expression. Both these results correspond to the wound healing data that Figure 4.2 is suggesting. At 100 g/mL of CSC the resistance decreases substantially to about 45% below normal. Coupled with the data in Figure 4.2 showing a decrease in motility as well, this suggests that at 100 g/mL of CSC, ALST cells experience an over ruling of the mechanism stimulating CAM expression. The result is degradation in junctional and substrate binding. The graphs in Figure 4.5 show the junctional resistance (Figure 4.5A) and cell-substrate separation (Figure 4.5B) as measured and calculated with the frequency scan before and after a 40 hour CSC exposure. At the time of 40 hours, in comparison to pre-CSC exposure, the HUVEC layer has a similar junctional resistance and cell-substrate separation for the various concentrations of CSC, with the exception of 250 g/mL of CSC. In this situation, the HUVEC cells individually begin to isolate their selves by contraction. Fig. 4.6 shows confluent layers of HUVEC and ALST with the 250 g/mL of CSC in comparison to the layers unexposed to CSC. At this high concentration, the ALST layer is still maintained even though agitated, where as the HUVEC cells are beginning to contract compromising the monolayer formation. With this occurring, the cell layer resistance is virtually zero which is why the junctional resistance and cell-substrate 31
(A) 051015200 hours 40 hours0 hours40 hoursHUVEC ALSTJunctional Resistance (cm2) control 10 50 100 250 (B) 0204060801001201400 hours40 hours0 hours40 hoursHUVEC ALSTcell-substrate seperation (nm) control 10 50 100 250 Figure 4.5 The junctional resistance (A) and cell-substrate separation (B) of a HUVEC and ALST layer. 32
Figure 4.6 Images of confluent ALST (top) and HUVEC (bottom) layers. The images on the left are not exposed to CSC while the images on the right are exposed to CSC concentrations of 250 g/mL 33
separation does not register, and is therefore not represented on the graphs for the HUVEC layers at the time of 40 hours. The ALST layer on the other hand not only maintains a layer at concentrations of 250 g/mL of CSC, but nicely demonstrates a dose dependent increase in cell-substrate separation and decrease in junctional resistance after 40 hours of CSC exposure. This data also serves as preliminary indicators for the cancer invasion assays. The noticeable differences in Figure 4.5 between the junctional resistance (Figure 4.5A) and the cell-substrate separation (Figure 4.5B) of the HUVEC and ALST layers serves as an indication on which tissue layer is present on the electrodes. 4.3 CSCs effect on cancer invasion In observing ALSTs ability to transmigrate across HUVEC layers, two experimental set ups were utilized. The initial experimental set up involved HUVEC cultures simultaneously exposed to ALST invasion and CSC exposure to simulate extravasation. The secondary set up exposed the HUVEC layers to the various concentrations of CSC for approximately 10 hours then adding ALST to allow for invasion. This simulated an intravasation scenario. The justification for 10 hours was that HUVEC cultures experienced higher resistances in 50 and 100 g/mL of CSC at this time after exposure (Figure 4.3). After CSC exposure for 10 hours, the HUVEC layers indicated a slightly higher 34
junctional resistance (Figure 4.7B) and cell-substrate separation (Figure 4.8B) with the greatest increase occurring at 50 g/mL of CSC. 40 hours later, after ALST invasion, the significantly lower cell-substrate separation (the 50 hour group in Figure 4.8B) indicates that there is an ALST layer on the electrodes. The junctional resistance demonstrates dose dependence (the 50 hour group in Figure 4.8B) similar to the ALST behavior suggested in Figure 4.5A. The data in Figures 4.7 and 4.8 suggest that the resistance trace and the degrading nature corresponding to the increased concentrations of CSC of Figure 4.9 can be attributed exclusively to the junctional resistance from the inter-cellular junctions. With simultaneous ALST invasion and CSC exposure there is a significant discrepancy that occurs between 10 and 50 g/mL of CSC. There is an increase of almost 3-fold for the junctional resistance (Figure 4.7A) and an 80% decrease in cell-substrate separation (Figure 4.8A) for the control and 10 g/mL of CSC. This shows ALST invasion for both groups. The subtle decrease in junctional resistance (Figure 4.7A) and increase in cell-substrate separation (Figure 4.8A) indicate a HUVEC layer on the electrode surfaces. Overall this suggests that ALST was not able to invade the HUVEC layers exposed to 50 and 100 g/mL of CSC after 40 hours of simultaneous CSC exposure and ALST invasion. The intravasation experiments allow transmigration of ALST across HUVEC 35
(A) HUVEC-CSC-ALST012345670 hours40 hoursTime (hr)Junctional Resistance(cm2) control 10 50 100 (B) delayed ALST invasion0246810120 hours10 hours50 hoursTime (hr) Control 10 50 100 Figure 4.7 (A) Junctional resistance of a HUVEC layer being exposed simultaneously to CSC concentrations and ALST invasion. (B) Junctional resistance of a HUVEC layer being exposed to CSC 10 hours before ALST invasion. 36
(A) HUVEC-CSC-ALST0204060801001201400 hours40 hoursTime (hr)cell-substrate seperation (nm) control 10 50 100 (B) delayed ALST invasion0204060801001200 hours10 hours50 hoursTime (hr)cell-substrate seperation (nm) Control 10 50 100 Figure 4.8 (A) Cell-substrate separation of a HUVEC layer being exposed simultaneously to CSC concentrations and ALST invasion. (B) Cell-substrate separation of a HUVEC layer being exposed to CSC 10 hours before ALST invasion. 37
delayed ALST invasion00.511.522.50510152025303540Time (hr)resistance ratio control 10 50 100 Figure 4.9 The resistance ratio graph of a ten hour delayed ALST invasion of a HUVEC monolayer. 38
layers for all concentrations of CSC, where as extravasation seems to have inhibited transmigration for higher concentrations of CSC. It is possible that HUVEC layers absorb CSC toxins thereby decreasing CSC concentrations greatly within the well for the intravasation experiments. This situation would simulate the intravasation process providing a better environment for transmigration as compared to the extravasation environment. For the extravasation experiments, the inhibited transmigration is either from the HUVECs increased CAM expression in response to the CSC, or a great decrease in ALST adhesion mechanics due to CSC exposure while the cells are suspended. One lacking quality in the extravasation experiment was that both HUVEC and ALST cells were not pre-exposed to CSC. The fault in this is that ALST and HUVEC were exposed to each other while they physiologically changed in response to the CSC. Despite that, the experiment did yield interesting results as the ALST was inhibited from transmigration. Two factors that need to be resolved in order to attribute the suggested scenario occurring is if the CSC concentrates at the bottom of the wells and whether the limited volume limits the effects of CSC. A non-uniform CSC distribution could affect CSCs effect on the cellular mechanics involved with ALST transmigration of HUVEC layers that wouldnt necessarily occur in a uniformly distributed CSC situation. It 39
is also possible for the HUVEC layers to be absorbing CSC toxins completely from the wells thereby limiting the effect CSC would have on the HUVEC layers. A habitual smoker would have a continual or more consistent CSC concentration within their bloodstream. If these two scenarios are occurring it would lack unrealistic conditions that would occur in vivo. An experimental set up remedying this situation is explained in further detail in the future work section. 40
Chapter 5 Conclusion and Future Work 5.1 Conclusion ALST and OVCA429 experienced different reactive mechanisms towards CSC when compared with normal HUVEC cells. It is reasonable to assume that cancerous cell types are already missing components within the cell signaling pathways that would mediate a normal response towards CSC exposure. The HUVEC cultures responded to CSC with an increase in CAM expression, where as ALST and OVCA429 relieved CAM expression thereby providing an improved motility. ALST cultures demonstrated a nicely reactive unbinding in accordance to increasing concentrations of CSC. HUVEC junctional and substrate binding is smaller in magnitude when compared to ALST, and as a result, no visible CSC concentration dependence can be observed. Transmigration of the HUVEC layer by ALST cells exhibit a possible pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC tissues increased CAM expression in response to CSC during intravasation is possibly responsible for inhibited ALST transmigration at high concentrations of CSC. It needs to determined whether the CAM expression of HUVEC 41
after 10 hours is relieved through decreased CSC, or by HUVEC adapting to the new toxic environment. It is quite possible that CSC has pro-inhibitory effects for transmigrating ALST cells against a HUVEC layer, but experimental set ups reenacting realistic in vivo situations need to be developed to further prove this statement. 5.2 Future Work The investigation into CSCs affect on transmigration of cancer is not complete and future experiments will be performed in order to provide the data necessary to allow for more profound conclusive statements. Fluorescence microscopy will be utilized to verify whether ALST has invaded the HUVEC layers for the various assays. Particular interest will focus on the simultaneous CSC exposure and ALST invasion assays for the 50 and 100 g/mL of CSC. Different cancer cell types will also be investigated since different cancers have different CAM expression as well as different reactive mechanisms. CAM expression should be further investigated through immunofluorescence, western blotting, or immunoprecipitation to observe expression change over time and varying CSC concentrations. Implication of a fluid flow system into the CSC investigations will help in resolving two other issues. A fluid flow system will provide a uniform and consistent CSC concentration which will help to replicate realistic in vivo scenarios. Figure 5.1 shows a HUVEC layer exposed to 42
Figure 5.1 Images of a HUVEC layer experiencing a fluid flow were taken with an inverted microscope. The times are denoted in the top right corner. 43
fluid flow. Images were captured every 5 minutes with the images that were an hour apart displayed in Figure 5.1. Just as under static conditions, the HUVEC cells continually express movement despite forming a monolayer. The movement by a visual inspection seems to be randomized and not dictated in any particular direction. Finally, longer delayed invasions will be explored to simply observe effects caused by longer CSC exposure. This will be better facilitated with a fluid flow system due to the great amount of medium ensuring a more consistent CSC concentration. 44
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