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

Sarah Altman Author

04/04/2021 Added

30 Plays

Description

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

Searchable Transcript

Search:

[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.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

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

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments