Name: the use of mouse mammary tumor cells in an in vitro invasion assay as a measure of oncogenic cell behavior
Uploaded: 2019-06-12T14:30:00
Description: Watch this Scientific Journal Video about The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior at JoVE.com

This work was supported by grant R15CA169978 from the National Institutes of Health. Additional funding came from Villanova University.

All experiments and methods were performed as authorized by Villanova University Institutional Animal Care and Use Committee (IACUC).
1. Gene Expression in Cultured Mouse Mammary Tumor Cells
<ol>
	<li>First, prepare the cell lines to be tested.</li>
	<li>Use a breeding colony of BALB/cV mice. These mice carry the BALB/cV strain of mouse mammary tumor virus, transmitted to pups in milk6,7. Fifty percent of breeding females dev...

<ol>
	<li>He, X., Lee, B., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-ECM+Interactions+in+Tumor+Invasion.">Cell-ECM Interactions in Tumor Invasion.</a> Advances in Experimental Medicine and Biology. 936, 73-91 (2016).</li><li>Albini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+in+vitro+assay+for+quantitating+the+invasive+potential+of+tumor+cells.">A rapid in vitro assay for quantitating the invasive potential of tumor cells.</a> Cancer Research. 47 (12), 3239-3245 (1987).</li><li>Simon, N., Noel, A., Foidart, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+in+vitro+reconstituted+basement+membrane+assay+to+assess+the+invasiveness+of+tumor+cells.">Evaluation of in vitro reconstituted basement membrane assay to assess the invasiveness of tumor cells.</a> Invasion and Metastasis. 12 (3-4), 156-167 (1992).</li><li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research.">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Advanced Drug Delivery Reviews. 79-80, 3-18 (2014).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+basement+membrane+matrix+with+biological+activity.">Matrigel: basement membrane matrix with biological activity.</a> Seminars in Cancer Biology. 15 (5), 378-386 (2005).</li><li>Kang, J. J., Schwegel, T., Knepper, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+similarity+between+the+long+terminal+repeat+coding+regions+of+mammary-tumorigenic+BALB/cV+and+renal-tumorigenic+C3H-K+strains+of+mouse+mammary+tumor+virus.">Sequence similarity between the long terminal repeat coding regions of mammary-tumorigenic BALB/cV and renal-tumorigenic C3H-K strains of mouse mammary tumor virus.</a> Virology. 196 (1), 303-308 (1993).</li><li>Slagle, B. L., Lanford, R. E., Medina, D., Butel, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mammary+tumor+virus+proteins+in+preneoplastic+outgrowth+lines+and+mammary+tumors+of+BALB/cV+mice.">Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice.</a> Cancer Research. 44 (5), 2155-2162 (1984).</li><li>Schmidt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+oncogenic+phenotype+by+the+nuclear+body+protein+ZC3H8.">Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8.</a> BMC Cancer. 18 (1), 759 (2018).</li><li>Neophytou, C., Boutsikos, P., Papageorgis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+and+Emerging+Therapeutic+Targets+of+Triple-Negative+Breast+Cancer+Metastasis.">Molecular Mechanisms and Emerging Therapeutic Targets of Triple-Negative Breast Cancer Metastasis.</a> Frontiers in Oncology. 8, 31 (2018).</li><li>Al-Alwan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin+is+a+key+regulator+of+breast+cancer+invasion+that+acts+via+the+modification+of+metastasis-associated+molecules.">Fascin is a key regulator of breast cancer invasion that acts via the modification of metastasis-associated molecules.</a> PloS One. 6 (11), e27339 (2011).</li><li>Wang, M. J., Zhang, H., Li, J., Zhao, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-98+inhibits+the+proliferation,+invasion,+migration+and+promotes+apoptosis+of+breast+cancer+cells+by+binding+to+HMGA2.">microRNA-98 inhibits the proliferation, invasion, migration and promotes apoptosis of breast cancer cells by binding to HMGA2.</a> Bioscience Reports. 38 (5), (2018).</li><li>Zheng, Y. F., Luo, J., Gan, G. L., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+microRNA-98+inhibits+cell+proliferation+and+promotes+cell+apoptosis+via+claudin-1+in+human+colorectal+carcinoma.">Overexpression of microRNA-98 inhibits cell proliferation and promotes cell apoptosis via claudin-1 in human colorectal carcinoma.</a> Journal of Cellular Biochemistry. 120 (4), 6090-6105 (2019).</li><li>Yan, W., Cao, Q. J., Arenas, R. B., Bentley, B., Shao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA3+inhibits+breast+cancer+metastasis+through+the+reversal+of+epithelial-mesenchymal+transition.">GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition.</a> Journal of Biological Chemistry. 285 (18), 14042-14051 (2010).</li></ol>

The in vitro invasion assay is an inexpensive, rapid, quantitative, and straightforward method for studying the factors promoting cancer cell invasion. Breast cancer is the most commonly diagnosed cancer among women. Of the three major subtypes of breast cancer, triple negative, (or ER-, PR-, HER2/neu-), is the most aggressive, most likely to metastasize, and most deadly9. Therefore, understanding the genes and expression that result in metastasis can help find new therapeutic targets and...

The in vitro invasion assay can be a useful tool when measuring a cell&#39;s ability to migrate through a protein-coated membrane, analogous to the first steps in metastasis. A key feature of malignant cancer cells is their ability to migrate through and invade nearby tissues. Cancer that has spread or metastasized poses more treatment challenges and has lower rates of long-term survival, while localized tumors are easier to treat and have higher rates of long-term survival. In order to metastasize, cancer cells must leave the primary tumor and migrate into the circulatory or lymphatic system, a process which requires passing through the extracellular matrix and basement membrane1. In the process called the epithelial mesenchymal transition (EMT), the tumor cells must break cell-cell contacts, migrate directionally, and invade nearby blood or lymph vessels. The initial steps of this metastasis cascade are of great interest since these steps are what can make cancer deadlier. The genetic and epigenetic factors involved in the early steps of metastasis are the focus of a great amount of research, but accurate and reliable experimental tools are needed to test these early steps both in vivo and in vitro.
Tools to measure changes in cell migration such as wound healing (scratch) assays or growth in 3D environments such as soft agar assays can partially address the need for experimental methods of measuring early steps of metastasis, but an assay to measure invasion is more challenging since the process occurs in the body within a complex tumor microenvironment. For the purposes of screening drugs or gene alterations to determine important factors in invasion and metastasis, a system that can be used in vitro with cultured cells and mimic the challenges faced by metastatic cells in vivo is the invasion assay2,3. Breast cancer is the most commonly diagnosed type of cancer in women and the second leading cause of cancer death in women, so understanding the genes responsible for breast cancer cell invasion and metastasis is critically important for public health. Moreover, mouse cells are a useful model system for studying breast cancer and its progression.
The in vitro invasion assay is based on the Boyden Chamber assembly where two chambers of growth media are separated by a porous membrane3. To mimic the tumor microenvironment, a protein-rich gel is also included to separate cells in one chamber from a chemoattractant in the other and act as a basement membrane barrier. In order to migrate towards the chemoattractant, cells must first pass through the protein-rich barrier then pass through the porous membrane - a process analogous to how metastatic cells migrate through stroma. The protein-rich gel can be altered based on the needs of the experiment, but usually consists of collagen, or basement membrane extract (e.g., Matrigel)4. It is a complex mixture of proteins, proteoglycans and growth factors, but mostly consists of laminins and collagen IV 4,5. Cells must then pass through a porous membrane typically made of polycarbonate, polyester, or polytetrafluoroethylene (PTFE). Membranes may be purchased commercially with or without a protein gel (typically collagens), or the gel may be purchased separately and added. The pore size can be adjusted based on the cell size. While pore sizes are available from 0.4 - 8.0 &#181;m, only pores from 3.0 - 8.0 &#181;m are large enough for cell migration. The invasion assay has been used to determine the effectiveness of inhibitors on the ability of cells to migrate and invade. While lacking the exact tumor microenvironment that is present in vivo, the in vitro invasion assay is beneficial at screening many conditions in a short time while minimizing the need for animal models. The goal of these experiments is to compare gene expression of suspected oncogenes and determine the effects on cancer cell behavior and disease aggressiveness using the in vitro invasion assay and other tests. Overall, the invasion assay provides consistent, quantitative, and rapid results for determining metastatic potential while also being a relatively inexpensive, straightforward, and adaptable method.

<table>
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>ThermoFisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Treated Flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical cenrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Puritan</td>
			<td>25-806</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma Aldrich</td>
			<td>C0775</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>10566-016</td>
			<td>high glucose, GlutaMAX</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcepts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slide</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025076</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Invasion Chambers (24-well)</td>
			<td>Corning</td>
			<td>354480</td>
			<td>Cat. #354481 for 6-well</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Transfection Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel, disposable</td>
			<td></td>
			<td></td>
			<td>#11</td>
		</tr>
		<tr>
			<td>shRNA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Transfer pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse mammary tumor cells in an in vitro invasion assay as a measure of oncogenic cell behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

This method of in vitro invasion through a protein matrix was used to assess the aggressive phenotypes and oncogenic cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein ZC3H88. In conjunction with other approaches that also examine cell migration and growth in 3D environments, it was found that higher levels of expression of Zc3h8 in tumor cell lines, or by promoter-mediated expression from a plasmid, resulted in rapid rat...

Watch this Scientific Journal Video about The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior at JoVE.com

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

This protocol, the in vitro invasion assay, is a useful tool for quantitatively measuring a cells lines ability to migrate through a protein rich matrix and pass through a porous membrane, in a process similar to the early stages of cancer cell invasion. These cell lines can be genetically altered in order to over express a gene or to have reduced expression of a gene in order to determine if that gene plays a role in cell migration or cell invasion. Many aggressive cancers involve dramatic changes in cell behavior, including increased migration and invasion.So this in vitro invasion assay can allow us to better understand the genes and other factors involved in this process. To begin this procedure, grow adherent mouse mammary tumor cells in a T25 flask at 37 degrees Celsius with 5%carbon dioxide and 100%humidity until they are 70%to 90%confluent for day one of the experiment. Retrieve the Boyden chamber inserts from storage and transfer them to a cell culture hood to warm to room temperature for 20 minutes.Use three inserts to generate statistically valid data for each cell line for each experiment. And then add 500 microliters of the pre-warmed serum-free DMEM media to the inserts so that the porous membrane and gel are hydrated on both sides. Incubate at 37 degrees Celsius with 5%carbon dioxide and 100%humidity for at least two hours.After this, prepare the cells by removing the growth media and rinsing the cells with five milliliters of HBSS. Remove the HBSS and add one milliliter of 0.25%trypsin EDTA solution. Incubate at 37 degrees Celsius for one to five minutes or until cells appear rounded or show signs of detachment.Then gently tap on the flask to detach all the cells. Re-suspend the cells in five milliliters of DMEM with 10%FBS. And transfer the cell suspension to a sterile 15 milliliter centrifuge tube.Centrifuge the cells and 1000 times g for five minutes to gently pellet the cells. Remove the media and re-suspend the pellet in five milliliters of serum-free DMEM. Repeat the centrifugation and re-suspending process two additional times for a total of three washes.After this, thoroughly re-suspend the cells in the final five milliliters of serum-free DMEM and ensure there are no clumps of cells. Use a hemocytometer to determine the cell concentration making sure to only count viable cells. And dilute the cell suspension to 50, 000 cells per milliliter in five milliliters of serum-free DMEM.Next, remove the dish with the inserts from the incubator, and gently aspirate the media from an insert. Lift the insert and aspirate the media from the well. Working quickly, add the chemoattractant to the lower chamber.Place the insert into the well, and for a 24 well dish, add 500 microliters of the cell suspension to the insert. Ensure that there are no air bubbles present on either side of the membrane. Return the dish to the cell culture incubator for 22 hours.After 22 hours, prepare a solution of 1%paraformaldehyde and one X PBS for fixation. In a clean 24 well cell culture dish, add one milliliter of the fixative to individual wells so there's one well for each insert. Then, prepare the staining solution of 0.1%crystal violet in a solution of PBS with 10%ethanol.Add one milliliter of this staining solution to a clean well for each insert. Using forceps, remove each insert one at a time. Place a sterile cotton swab inside each insert and swab the upper side of the membrane to remove any unmigrated cells.Repeat this process with a second cotton swab. Next, remove any remaining media from the inside of the insert and add 750 microliters of PBS to wash away any detached cells. Remove the PBS and repeat the wash with fresh PBS.Then place the insert into a well containing fixative to fix the migrated cells on the underside of the membrane. Repeat this process for all inserts and let the inserts fix for 15 minutes at room temperature. After this, remove each insert one at a time from its well and wash it with 750 microliters of PBS.Place the insert into a well containing staining solution and leave it for 15 minutes to stain all migrated and fixed cells. Then remove the inserts and dip them into a beaker containing distilled water until the water running off the insert is clear. Remove any excess water droplets and place the insert sideways onto a piece of filter paper.Let the inserts air dry. Prepare the membrane for imaging by labeling a clean glass microscope slide for each insert, and placing a small drop of microscope immersion oil into the center of each slide. Using a scalpel, cut around the perimeter of the membrane on the inside of the plastic insert to detach the membrane.Use forceps to remove the membrane and place it on top of the oil drop on the labeled slide. Using a compound microscope, view cells at five times, 10 times, or 20 times magnification. For quantification, take multiple non-overlapping images at 10 times magnification.Determine the total number of invading cells or cells per area for all samples. For each experiment, perform each condition in the assay with three replicate inserts and repeat multiple times for statistically useful results. In this study, in vitro invasion through a protein matrix is used to assess the aggressive phenotypes in oncogenics cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein, Zc3h8.Higher levels of Zc3h8 expression is seen in tumor cell lines or by promoter mediated expression from a plasmid, resulting in rapid rates of cell proliferation, fast migration, growth in 3D environments, and increased encroachment within the in vitro invasion assay. Conversely, decreased expression by shRNA constructs results in less aggressive proliferation, migration and invasion. While the cell invasion decreases upon shRNA knockdown of Zc3h8 expression, that invasion is rescued when the expression is rescued.The in vitro invasion assay technique is extremely useful as it is a rapid, inexpensive, flexible, and relatively easy approach to studying cell behavior. Cells grown in culture are easy to manipulate genetically. They can even be tested for specific mutations.The in vitro invasion system also allows for the analysis of small molecule inhibitors as well biological agents like micro RNAs for their effects on cell migration and invasion. This assay also includes a great amount of flexibility, allowing for the alteration of the protein content of the matrix, pore size and chemoattractant.

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