Utilizing Galvanotactic Assays for the Mechanical Separation of Metastatic Breast Cancer Cell Lines

Sarah Altman Author

04/04/2021 Added

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Galvanotaxis can be used to isolate highly metastatic breast cancer cells for treatment.

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[00:00:01.590]Hello.
[00:00:02.520]My name is Sarah Altman and I am a UCare undergraduate majoring in biological
[00:00:06.510]systems engineering. Today I'll be discussing my research project,
[00:00:10.650]which is utilizing galvanotactic assays for the mechanical separation of
[00:00:14.400]metastatic breast cancer cell lines.
[00:00:18.390]During metastasis cancer cells break away from the original or primary tumor and
[00:00:22.890]travels to the blood and lymph system to form new tumors and other tissue
[00:00:26.280]systems of the body. Breast cancer.
[00:00:28.560]Fatality is heavily linked to metastasis with nearly 97% of breast cancer deaths
[00:00:33.420]resulting from cell migration,
[00:00:35.400]metastasis or migration is influenced by a variety of mechanical and chemical
[00:00:39.690]stressors at the cellular level,
[00:00:42.180]specifically the application of mechanical force on ECM proteins,
[00:00:46.170]such as integrin and focal adhesion
[00:00:47.880]kinase or FAK results in protein conformational changes,
[00:00:51.030]which alter cell signaling cascades.
[00:00:53.550]This in turn alters the regulatory processes of the cell and can lead to
[00:00:57.300]increased motility and adhesion, factors which advance metastasis.
[00:01:01.920]Thus FAK is a key regulatory protein and modulating the metastatic potential of
[00:01:06.540]breast cancer cell lines.
[00:01:08.280]The expression of FAK can be altered by the application of a small electric
[00:01:12.180]field across breast cancer cells seen in monolayer and in tumor environments.
[00:01:16.530]The application of an electric field increases the phosphorylation of FAK and
[00:01:20.190]contributes to increased migratory speed and directness in metastatic breast
[00:01:23.970]cancer cell lines. The effects of Galvanotaxis,
[00:01:27.780]which is the directed movement of a cell under the action of an EF is unique
[00:01:31.590]and pronounced and can be used to separate cells based upon a gradient of
[00:01:35.250]metastatic potential.
[00:01:38.430]Figure One displays the effects of a small physiological strength electric field upon
[00:01:42.570]trophoblast cells exposed to 150 millivolts per millimeter for an hour.
[00:01:47.010]It shows that exposure to an electric field increase the expression of
[00:01:50.820]Phospho-FAK, or P-FAK and FAK in trophoblasts, underscoring
[00:01:54.570]The effects of galvanotaxis on the expression of the key regulatory protein
[00:01:59.310]Figure 2 B shows that cell directness in human breast cancer cell line,
[00:02:04.230]MDA-MB-231 increases significantly under the application of a small
[00:02:09.240]physiological electric field in the range of a hundred to 200 volts per
[00:02:12.420]centimeter,
[00:02:13.650]Figure 2 C displays that migratory speed of human breast cancer cell lines
[00:02:17.640]increases upon the application of a small physiological electric field.
[00:02:22.500]This data serves to indicate that the migration of cells under an electric field
[00:02:26.370]is voltage dependent is more direct and occurs at faster rates upon application
[00:02:30.630]of an electric field.
[00:02:31.890]It also implicates that highly metastatic breast cancer cells migrate to the
[00:02:35.670]anode.
[00:02:36.240]And with up to three times the directness and 2.5 times the migration speed of
[00:02:40.110]weekly metastatic breast cancer cell lines, which migrate towards the cathode.
[00:02:45.960]The purpose of this research is to design develop and test galvanotactic assays
[00:02:50.760]which can be used to mechanically separate breast cancer cell lines based upon a
[00:02:54.030]gradient of metastatic potential.
[00:02:56.310]The main hypothesis of this research is that upon application of an electric
[00:03:00.190]field breast cancer cell cultures will naturally separate themselves based upon
[00:03:04.180]a gradient of metastatic potential due to altering levels of P-FAK
[00:03:07.430]expression, which is heavily linked to metastasis.
[00:03:11.200]This research centers around cultivating a method by which highly metastatic
[00:03:14.560]cells will migrate under an electric field to delineated regions of the
[00:03:18.560]galvanotactic assay for de-seeding and future use. the goal is to develop a
[00:03:23.320]general method,
[00:03:24.430]which can be used by researchers in order to isolate highly metastatic breast
[00:03:27.490]cancer cells for treatment.
[00:03:32.260]The methods and materials are as such: MDA-MB-231
[00:03:36.340]which are cells of higher metastatic potential and
[00:03:38.950]MDA-MB-468, of lower metastatic potential breast cancer cell lines will be
[00:03:43.120]cultured using Dulbecco's modified Eagle,
[00:03:45.370]medium supplemented with 10% fetal bovine serum,
[00:03:48.640]and 1% penicillin streptomycin as previously used in the Lim lab.
[00:03:53.080]These cell lines will then be cultured in a suspension of extracellular matrix
[00:03:56.800]proteins in order to mimic InVivo growth.
[00:03:59.530]The chamber for electric field application will be constructed from three glass
[00:04:03.130]cover strips, forming a flow channel for cells seated in a glass culture dish.
[00:04:08.050]The width of the flow chamber is to be approximately 10 millimeters in width
[00:04:12.460]cells in the chamber will be coated with an excess of culture.
[00:04:14.980]Medium containing 10% FBS.
[00:04:18.940]The electric field will be generated from a DC power source using two silver,
[00:04:22.630]silver chloride electrodes placed in beakers of Steinberg solution.
[00:04:26.260]And these speakers will be connected to the flow chamber via Agarose salt bridges.
[00:04:29.860]Approximately 15 centimeters in length. Cells will be exposed to an electric field
[00:04:34.480]of physiological strength of approximately 150 millivolts per millimeter for an
[00:04:37.870]hour. The flow apparatus will be placed on an inverted Leica DMI,
[00:04:41.650]4,000 microscope and contrast images will be recorded every minute.
[00:04:45.610]The open-source software time-lapse analyzer will be used for data processing of
[00:04:49.000]live cell imaging.
[00:04:50.080]We will then use the Sobel operator edge detection method to detect the
[00:04:53.500]boundaries of individual cells.
[00:04:55.420]These cells will be tracked and existing MATLAB scripts developed in the Lim
[00:04:59.080]lab will be used to track and quantify migration speed.
[00:05:01.660]And directedness. Western blot analysis of FAK
[00:05:04.840]and P-FAK will be performed strategically in order to identify regions of the glass
[00:05:08.890]cell culture dish which contain cells expressing high levels of P-FAK.
[00:05:12.730]We will integrate the results of the Western blot analysis,
[00:05:15.880]which will reveal areas of the cell dish containing cells expressing high
[00:05:19.150]levels of the metastatic marker P-FAK with time-lapse imagery and MATLAB speed
[00:05:23.230]and directness data to identify regions of the dish where you are likely to find
[00:05:27.280]cells with high metastatic potential. Upon repeating the Galvanotaxis experiment multiple times,
[00:05:32.530]we hope to delineate regions with a consistently high probability of containing
[00:05:36.010]highly metastatic breast cancer cells.
[00:05:38.260]The cells can then be carefully deseeded from these regions and preserved for
[00:05:41.620]future treatment.
[00:05:45.570]This is the galvanotactic assay that is used most commonly in applying electric
[00:05:48.840]fields of physiological strength across cell cultures. Silver,
[00:05:52.170]silver chloride electrodes are used because they minimize sediment deposit and
[00:05:55.590]are able to maintain a rather constant temperature during electric field application and
[00:05:59.450]Salt bridges reduce sediment deposit into the cell culture and are able to
[00:06:03.260]apply a constant voltage across the medium.
[00:06:05.180]So they are also very important for the design.
[00:06:10.060]In terms of the results,
[00:06:10.780]And data we have begun to make Agarose bridges using a 0.3 millimeter inner diameter
[00:06:15.790]polyethylene tubing prepared and stored in phosphate buffered saline or PBS.
[00:06:20.410]The agarose salt bridges are prepared at a concentration of 20 milligrams of Agarose
[00:06:24.340]per one milliliter of PBS
[00:06:26.920]Ideally the resistance would be in the range of one to 10 kilohms. However,
[00:06:31.060]through our research,
[00:06:31.660]we have found that both three and seven centimeter Salt bridges,
[00:06:35.080]whether they're fresh or they've been intubated for 14 days have nominal
[00:06:38.470]resistance values that are 10 to a hundred times greater than what we are
[00:06:41.650]looking for. So currently we are looking at using possibly glass tubing or 0.86
[00:06:46.210]millimeter polyethylene tubing as well as longer
[00:06:49.540]salt bridges of length 15 centimeters in order to reduce this resistance.
[00:06:55.630]There's still a lot of work to be done. At present,
[00:06:58.090]We are working on determining the optimal saltbridge configuration by testing
[00:07:01.090]different variables of length, tubing material, inner diameter,
[00:07:04.330]and solvent composition against nominal resistance values in order to pick the
[00:07:07.780]best salt bridge that is able to have a low resistance and also reduce the sediment
[00:07:12.100]deposit. Once an optimal salt bridge configuration is determined,
[00:07:15.910]We will proceed with the construction of the chamber and we will proceed with
[00:07:18.970]running experiments,
[00:07:20.170]which expose cells to electric fields of small physiological strength.
[00:07:23.710]We can then proceed with Western blot and MATLAB analysis to identify delineated
[00:07:27.250]regions of the flow apparatus,
[00:07:28.840]which are likely to contain cells of high metastatic potential.
[00:07:34.630]I would like to thank my research advisor, my faculty research advisor,
[00:07:37.780]Dr. Jung Yul Lim of the department of mechanical and materials engineering and my
[00:07:41.410]personal advisor,
[00:07:42.490]Brandon Riehl of the Lim Biomaterials and Mechanotransduction laboratory.
[00:07:46.000]I would also like to thank UCARE for their funding and support throughout this project.
[00:07:51.430]These are the references for my presentation,
[00:07:53.200]and I thank you for watching my presentation today.

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Utilizing Galvanotactic Assays for the Mechanical Separation of Metastatic Breast Cancer Cell Lines

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