Name: Experimental Metastasis Assay (Video) | JoVE Journal
Uploaded: 2010-08-24T17:10:00.000+00:00
Duration: 8 min 28 s
Description: Learn experimental metastasis assay for oncology research using tail vein injections in immunodeficient mice. This method evaluates metastatic potential of human cancer cell lines through lung metastasis quantification.

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5 minutes

This protocol was tested and optimized initially in the laboratory of Dr. Richard Hynes (MIT). Funding is provided by the NYSTEM IDEA AWARD (to L.X.) and Ruth L. Kirschstein National Research Service Award (to L.X.).

1. Sample Preparation
<ol>
 <li> Grow cells to ~70% confluent in their specific media with growth factors or FBS (e.g., DMEM with 10% FBS for MC-1 cells). Aspirate media from plate and gently wash several times with 1 x PBS (8 g/L of NaCl, 0.2 g/L of KCl, 1.15 g/L of Na2HPO4.7H2O, 0.2 g/L of KH2PO4, pH 7.3).</li>
 <li> Aspirate PBS and add 2 mL of 0.05% Trypsin in versene (0.014 g/L of phenol red and 0.2 g/L of EDTA-Na in 1 x PBS, pH 7.2). Gently rock plate to facilitate cell detachment from plate. Observe the cells under a microscope. It usually takes 2-5 minutes for the cells to detach.</li>
 <li> Add an ample volume of media containing FBS (or soybean trypsin inhibitor) to quench the trypsin activity and collect cells in a 50mL falcon tube. Count cells using a hemacytometer. Load 10μL of cell suspension onto a clean hemacytometer. The number of cells per mL is equal to the average # of cells in each of the five squares multiplied by 104. </li>
 <li> Centrifuge the cells at 1000 RPM (or ~200 xg) in a benchtop centrifuge for 3 minutes.</li>
 <li> Carefully remove the media without disturbing the cell pellet. Resuspend cells in an appropriate volume of Hanks Balanced Buffer Solution (HBSS) to reach a final concentration of ~5 x 106 cells/mL. </li>
 <li> Filter cells through a Falcon 70 μm cell strainer to exclude large cell aggregates. Place the tip of the pipette directly onto the filter over a labeled 5mL Falcon culture tube. Quickly eject the suspension through the filter into the culture tube.</li>
 <li> Count cells again using a hemacytometer and dilute them to a final concentration of 2.5 x 106/mL. Keep cells on ice.</li>
 <li> Determine the viability of the cells. Mix some cells with trypan blue and measure the percentage of dead (blue) cells over the total cells using a hemacytometer. The viability of cells should be ≥ 90% prior to injection.</li>
</ol>
2. Intravenous Injection
<ol>
 <li>Change gloves. Gently grab the tail of an immunodeficient mouse (nude, NOD-SCID, or NSG, Jackson's Laboratory) and pull it into the mouse restrainer, with its back against the slit and its tail sticking out of the small opening in the back of the restrainer. </li>
 <li> Slowly slide the ring inward along the slit and lock it in place once the ring catches the mouth of the mouse. The mouse should not be able to move freely, but should have normal rate of breathing. </li>
 <li> Find the major tail veins. Four major blood vessels are present in a mouse tail. Blood vessels on the dorsal and ventral sides of the tail are arteries. Veins are on the lateral sides of the tail. </li>
 <li> Draw more than 200 μL of the prepared cells into a 1mL syringe. Attach the 30 G1/2 inch needle and push out any air bubbles that may exist. The final volume of cells in the syringe should be 200 μL (i.e., 5 x 105 cells in total).</li>
 <li> Inject cells into the tail vein.
 <ol class="lalpha">
 <li> Start from the distal end of the tail, so if the first trial fails, a more proximal region of the tail could be used for a second try.</li>
 <li> Wipe the tail with 70% ethanol. Pull the tail straight. Hold the tip of tail with thumb and support the point for injection with the index finger.</li>
 <li> Insert the needle to the vein and inject cells. Make sure the needle and syringe are parallel to the vein during injection, otherwise the needle will poke through the vessel wall and inject the cells into the adjacent tail tissues.</li>
 <li> Withdraw the needle after injection. Blood should profuse from the injection site if the injection went successfully. Press a clean piece of paper towel or cotton swab on the injection site to facilitate clotting, and palpate the tail upwards to push any residual sample in the vein into circulation.</li>
 <li> Release the mouse from the restrainer and return it to the cage. Record the injection process (e.g., how many trials it took to inject the cells and how much cells were injected) in a lab notebook.</li>
 <li> Determine the viability of cells after injection, as described in the Section of Sample Preparation, Step 9. This step provides reassurance that cells stay alive throughout the injection process. At the end of an injection experiment, the viability of cells will decline, but should be above 80%.</li>
 <li> (optional) Spin down the leftover cells and rinse the pellets with PBS once. Freeze the pellets at -80 oC for future analyses (e.g., western blots to confirm gene expression or knockdowns).</li>
 </ol>
 </li>
 <li> Typically after one or two months, the mice will be dissected and the locations of metastases are grossly determined. Lung is the primary site for metastasis, since it contains the first capillary bed that the cells encounter after they enter the circulation. </li>
 <li> After rinsing with PBS, each lobe of the lung (or other tissues containing metastases) is observed under a dissecting microscope (Figure 1). The number of detectable metastases on both sides of the lung is counted and the numbers of lung metastases on all the four lobes are added together as the total number of lung metastases.1 Lung metastases are more easily detected if the lungs are fixed in formalin overnight, since the metastases will apppear as whitish spots in contrast to the adjacent dark brown lung tissues.</li>
</ol>
3. Culturing Cells from Lung Metastases
<ol>
 <li> Lung metastases are isolated from injected mice. Each should be cultured separately.</li>
 <li> Each metastasis is minced by the end of a needle cap (sterile) on a 70 μm cell strainer.</li>
 <li> Rinse the cell strainer with several mLs of medium and collected cells that pass through the filter in a culture dish. </li>
 <li> Incubate the cells at 37 °C for at least four days without disturbance.</li>
 <li> Wash away blood or tissue debris on the dish with PBS. </li>
 <li> Add fresh medium. At the beginning, both cancer cells and fibroblast cells grow on the plates, but gradually, the fibroblast cells will die out and be replaced by cancer cells. If the cancer cells carry any drug resistant genes, select the cells with corresponding antibiotics.</li>
 <li> The purity of the derived cells is assessed by immunostaining using antibodies against human-specific proteins. We use an anti-human vimentin antibody (NCL-VIM-V9, Novocastra). The cells we derived contain typically over 99% of human cells, even in the absence of drug selection. The newly derived cells could be injected again into immunodeficient mice to test their metastatic abilities, as described above.</li>
</ol>
4. Representative Results
<ol>
 <li> At the end of this assay, a highly metastatic cancer cell line typically gives rise to many lung metastases (Figure 1),1 while very few lung metastases will come from a poorly metastatic cancer cell line.</li>
 <li> The cells derived using this method usually give rise to more metastases than the parental line, when they are tested again using this assay.1-3</li>
</ol>
 <img src="/files/ftp_upload/1942/1942fig1.jpg" alt="Tissue sample under microscope, cell morphology study in histology experiment, biological analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. Representative images of mouse lungs after tail vein injections of cancer cells. 5 x 105 of the metastatic human melanoma cell line, SM cells,4 were injected into the tail vein of immunodeficient mice. Two months later, the lungs were isolated and metastases were found dispersed among the normal lung tissue.

<ol><li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases%5BTitle%5D)+AND+%22Mol+Cancer+Res%22%5BJournal%5D)">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li>
<li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li>
<li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GPR56%2C+an+atypical+G+protein-coupled+receptor%2C+binds+tissue+transglutaminase%2C+TG2%2C+and+inhibits+melanoma+tumor+growth+and+metastasis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li>
<li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice%5BTitle%5D)+AND+%22J+Natl+Cancer+Inst%22%5BJournal%5D)">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li>
<li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multigenic+program+mediating+breast+cancer+metastasis+to+bone%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li>
</ol>

Metastasis is the leading cause of death in cancer patients. It involves four major steps: detachment of cancer cells from their primary loci, their entry into circulation (intravasation), their exit from circulation (extravasation), and survival and growth in a distant organ. Metastasis in human is considered a slow process and often manifested after years of latency. To study its progression in a timely manner, the above relatively quick experimental assay was established in immunodeficient mice.4 Since its...

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>10313-021</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30088.03</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14175-095</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>KCl</td><td>Mallinckrodt Baker Inc.</td><td>6838-04</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt; HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mallinckrodt Baker Inc.</td><td>7896-04</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt Baker Inc.</td><td>7100-12</td><td></td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P3532</td><td></td></tr><tr><td>EDTA-Na</td><td>EMD Millipore</td><td>4010</td><td></td></tr><tr><td>Trypan Blue</td><td>Invitrogen</td><td>15250-061</td><td></td></tr><tr><td>Trypsin 2.5%</td><td>Invitrogen</td><td>15090-046</td><td></td></tr><tr><td>Ethanol</td><td>Ultrapure</td><td>200-CSGP</td><td></td></tr><tr><td>50 ml Falcon Tube</td><td>VWR international</td><td>89039-656</td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Axygen Scientific</td><td>MCT-175-C</td><td></td></tr><tr><td>Falcon 70&amp;mu;m Cell Strainer</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>5ml Falcon Culture Tube</td><td>VWR international</td><td>60818-576</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>15170-208</td><td></td></tr><tr><td>1 ml syringe</td><td>BD Biosciences</td><td>329650</td><td></td></tr><tr><td>30 &amp;frac12; gauge needle</td><td>BD Biosciences</td><td>305106</td><td></td></tr><tr><td>Mouse restrainer</td><td>Plas Labs Inc.</td><td>551-BSRR</td><td></td></tr><tr><td>Surgical scissors</td><td>Fisher Scientific</td><td>08-940</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>08-902</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Nikon Instruments</td><td>SMZ1500</td><td></td></tr><tr><td>Benchtop Centrifuge</td><td>Thermo Fisher Scientific, Inc.</td><td>75003491</td><td></td></tr></tbody></table>

experimental metastasis assay

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

The overall goal of the following experiment is to test the metastatic abilities of human cancer cells by tail vein injections. In immunodeficient mice, the cells are prepared for injection. The cells are then injected into immunodeficient mice, which will generate lung metastases after one to two months.The mice are dissected and lung metastases are quantified and cultured. Results of this type of assay indicate the extent of metastases of a particular type of cancer cell. Hi, I am Sonali Mohanty from Dr.LE's Lab in the Department of Biomedical Genetics at the University of Rochester.Today we will show you a procedure to perform experimental metastasis assay. We use this procedure in our lab to study cancer metastasis. So let's get started.Begin with cells grown to about 70%Co fluency aspirate media from the plate and gently wash once with one XPBS. Next, add two milliliters of 0.05%Trypsin inversing. Gently rock the plate to facilitate cell detachment.Observe the cells under a microscope as they detach. Add an ample volume of media containing FBS to quench the trips in activity. Then transfer to a 50 milliliter tube.Count the cells using a hemo cytometer. Once the concentration has been determined, centrifuge at about 200 times gravity for three minutes without disturbing the cell pellet. Carefully remove the media Resus suspending an appropriate volume of Hank's balance buffer solution or HBSS to reach a final concentration of about five times 10 to the six cells per milliliter.Next, filter through a Falcon's 70 micrometer cell strainer. To exclude large cell aggregates, place the tip of the pipette directly onto the filter, placed over a five milliliter culture tube. Quickly eject the suspension through the filter into the tube.Count the cells again using trian blue to determine viability. The viability should be greater than 90%prior to injection. Then dilute to a final concentration of 2.5 times 10 to the six cells per milliliter.Keep on ice. Gently grab the tail of an immunodeficient mouse and pull it into the mouse restrainer with its back against the slit and its tail sticking outta the small opening in the back. Slowly slide the ring inward along the slit.Once the ring catches the mouth of the mouse, lock it into place. The mouse should not be able to move freely, but should have a normal rate of breathing. Find the major tail veins.Four major blood vessels are present in a mouse tail. The arteries are located on the dorsal and ventral sides of the tail. The veins are located on the lateral sides.Draw at least 200 microliters of the prepared cells into a one milliliter syringe. Attach a 30 gauge half needle and push out any air bubbles. The final volume in the syringe should be 200 microliters.Prepare to inject into the distal end of the tail. So if the first trial fails, a more proximal region of the tail could be used for a second try. After wiping the injection site with 70%ethanol, pull the tail straight.Hold the tip with the thumb and support the point for injection with the index finger. Insert the needle into the vein and inject the cells. Make sure the needle and syringe are parallel to the vein during injection.Otherwise, the needle will poke through the vessel wall and cells will be injected into the adjacent tail tissues. Withdraw the needle if the procedure went successfully. Blood should perfuse from the injection site.Press a clean cotton ball to facilitate flossing and palpate the tail upwards to push any residual sample into circulation. Release the mouse from the restrainer and return it to the cage. Record the number of injection trials and the number of cells injected in a laboratory notebook.Determine the viability of cells after injection. As before this step provides reassurance that cells stay alive throughout the injection process. At the end of an injection experiment, the viability of cells will decline, but should remain above 80%As an optional step, spin down the leftover cells and rinse the pellets with PBS once.Then freeze the pellets at minus 80 degrees Celsius for future analyses such as western blotting. To confirm gene expression on knockdowns After one to two months, dissect the mice to determine the locations of metastases. Begin by placing the euthanized mouse on a dissection station.Spray the mouse generously with ethanol. Cut open the rib cage to expose the lungs and heart. Then carefully excise them and place them in PBS.The lung is the primary site for metastasis since it contains the first capillary bed that cells encounter. After they enter the circulation, remove the heart and rinse the lungs in PBS then observe each lobe of the lung under a dissecting microscope. Count the number of detectable metastases on both sides of the lung, then at the numbers of lung metastases on all four lobes together to determine the total number of lung metastases.Next, isolate individual lung metastases by excision Using scissors. Transfer each metastasis to a 70 micrometer cell strainer and mince it using the end of a sterile needle cap. Rinse the cell strainer with several milliliters of medium and collect the cells that pass through the filter.In a culture dish, each dish contain the cells from one metastasis. Incubate the cells at 37 degrees Celsius for at least four days without disturbance. Initially, both cancer cells and fibroblast cells will grow on the plates, but gradually the fibroblast cells will die out and be replaced by cancer cells.If the cancer cells carry any drug resistant genes, select the cells with corresponding antibiotics After three days, wash away any blood or tissue debris, debris on the dish with PBS and add fresh medium. Assess the purity of the derived cells by immuno staining with antibodies against human specific proteins here and anti-human menting antibody is used even in the absence of drug selection. Greater than 99%of the cells should be human.The newly derived cells can now be injected again into immunodeficient mice to test their metastatic abilities at the end of this assay. A highly metastatic cancer cell line typically gives rise to many lung metastases, while very few lung metastases will come from a poorly metastatic cancer cell line. These images show lung metastases two months after injection of the human melanoma cell line.A 3 7 5 sm. The cells derived using this method usually give rise to more metastasis than the parental line when they're tested. Again using this assay, We have just shown you the procedure to perform experimental metastasis assay.When doing this procedure, it is very important to remember to ensure that the cells are injected into the tail vein, but not the its and tail tissue. So that's it. Thank you for watching and good luck with your experiments.

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Learn experimental metastasis assay for oncology research using tail vein injections in immunodeficient mice. This method evaluates metastatic potential of human cancer cell lines through lung metastasis quantification.

Experimental Metastasis Assay

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