The work was funded by the Fund for Medical Research and Education, Wayne State University, Detroit, MI.

1. Cell Culture
<ol>
	<li>Seed 3x105 HUVEC cells in 10 ml of complete growth medium10 in a 75 cm flask.</li>
	<li>Incubate cells at 37 &#176;C in 5% CO2 to 70-80% confluence.</li>
</ol>
2. Tubule Formation Assay
<ol>
	<li>Thaw either the growth factor-reduced (GFR) extracellular matrix or normal extracellular matrix overnight on ice at 4 &#176;C. 
	Note: For the sake of brevity &#39;extracellular matrix&#39; will henceforth be used to reference both GFR and normal extracellular matrix. The process to prepare them is identical.</li>
	<li>Keep 96-well culture plates on ice, and add 50 &#181;l of chilled extracellular matrix per well using pre-cooled tips. Prepare triplicate of 5 wells containing only the normal extracellular matrix. Prepare another triplicate of 5 wells containing only GFR extracellular matrix.</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 30 min.</li>
	<li>Wash the HUVECs once with phosphate-buffered solution without calcium and magnesium (PBS), and add 1 ml 0.05% trypsin-EDTA. Incubate the dish at 37 &#176;C for 1-5 min, checking the cells every minute until most cells round up. Dislodge the cells by tapping the flask once.
	<ol>
		<li>Add 5 ml basal medium (i.e., endothelial cell growth medium without anything (e.g., supplements) added) with 1% Fetal Bovine Serum (FBS). Collect the cells in the flask by pipetting and transfer to a 15 ml tube. Centrifuge the cells at 200 x g for 5 min and discard the supernatant.</li>
		<li>Re-suspend with 2 ml basal medium, count the cells using a hemocytometer, and adjust the cell concentration to 2-3 x 105 cells/ml.</li>
	</ol>
	</li>
	<li>Prepare 100 &#181;l of the following 10x concentrations of CXCR2 inhibitor SB225002 in basal medium: 11 &#181;M, 5.6 &#181;M, 1.1 &#181;M, 0.56 &#181;M and 0 &#181;M (control).</li>
	<li>Pipette 300&#160;&#181;l of the HUVEC suspension into each 5 microcentrifuge tubes. Add 33&#160;&#181;l of the inhibitor concentrations (11 &#181;M, 5.6 &#181;M, 1.1 &#181;M, 0.56 &#181;M and 0 &#181;M) into one tube each containing the HUVECs, and vortex.</li>
	<li>Pipette 50 &#181;l of each of the cell suspensions (1-1.5 x 104 cells) to individual wells of extracellular matrix. Plate each suspension in triplicate and incubate for 4-16 hr at 37 &#176;C, 5% CO2.</li>
	<li>After 4-16 hr, take 4 pictures of the tubes formed per well using an inverted microscope camera11 (original magnification x 100).</li>
</ol>
3. Tubule Disruption Assay
<ol>
	<li>Prepare the extracellular matrix and plates according to the same specifications as the Tube Formation Assay (steps 2.1 to 2.3).</li>
	<li>Prepare HUVEC suspension as described in Steps 2.4- 2.4.2 of the Tube Formation Assay and aliquot into a 96-well plate containing the extracellular matrix at 50 &#181;l per well. 
	Note: Plate enough wells so that there are 3 wells per drug treatment.</li>
	<li>Grow cells on extracellular matrix for 4 hr&#160;at 37 &#176;C, 5% CO2. Take images before the initiation of drug treatment.</li>
	<li>Prepare 2x concentrations of the CXCR2 inhibitor SB225002 as described in Step 2.5 of the Tube Formation assay, and add 50 &#181;l of each concentration per well of the 96 well plate containing the HUVECs. Plate each concentration in triplicate.</li>
	<li>Incubate the plate for another 2-12 hr at 37 &#176;C, 5% CO2. Take 4 images of the tubes formed in each well using an inverted microscope camera (original magnification &#215; 100) 11.</li>
</ol>
4. Quantifying Data
<ol>
	<li>Evaluate tube formation captured in the images by measuring the total tube length of tubes in four random microscopic fields using the microscope camera software.</li>
	<li>Open the image in the microscope camera software, and click &#39;Annotations and Measurements&#39;. Under the &#39;length&#39; section, select the &#39;simple line&#39; tool. Click on the image, then draw a line along the length of the tube, then right click. 
	Note: The software will automatically calculate the length of the line in pixels and put the data into a file.</li>
	<li>Repeat the procedure for all the tube lines in the image by selecting the &#39;simple line&#39; tool. Click on the image, then draw a line along the length of the tube, then right click. 
	Note: The software will record the length of each line and display the data on the screen.</li>
	<li>After measuring every tube in the picture, click &#39;Export&#39;, then chose &#39;Length to spreadsheet&#39;. 
	Note: The length data will be exported to a spreadsheet.</li>
	<li>Add all of the tube lengths from the spreadsheet to get the total tube length. Calculate and record the average length of the tubes. 
	Note: Four replicates are recommended with mean and standard deviation determined.</li>
</ol>
5. Recovering Cells from the Extracellular Matrix Culture
<ol>
	<li>Culture and perform a tube formation assay (see section 2) on a larger scale as a 96 well plate will not yield sufficient cell count. For 12 well plates, use 500 &#181;l of extracellular matrix and 500 &#181;l of the HUVEC cell suspension (1.5x105 cells).</li>
	<li>Incubate the cells for 4-16 hr&#160;in order to form tubes. Then, aspirate the cell culture medium. Rinse the cells with 1 ml of cold 1x PBS without calcium and magnesium.</li>
	<li>Add 1&#160;ml of ice cold PBS-2.5 mM EDTA buffer onto the culture and keep on ice for 10 min.</li>
	<li>Dislodge the cells and extracellular matrix mixture from the dish using a 1,000 &#181;l pipette tip with tip cut off, and then transfer to a cold 15 ml tube, wash the well with 4 ml of ice cold PBS-2.5 mM EDTA and put into the tube.</li>
	<li>Put the tubes on ice for 1-4 hr and invert the tube a few times until all of the extracellular matrix is dissolved.</li>
	<li>Centrifuge for 10 min at 1,620 x g at 0 &#176;C</li>
	<li>Re-suspend the cell pellets with 1 ml cold PBS to a 1.ml tube on ice.</li>
	<li>Centrifuge again for 5 min at 3,000 x g at 0 &#176;C, then dispose of the supernatant.</li>
	<li>Store the cells in -80 &#176;C.</li>
</ol>

<ol><li>Sharma, B., Nawandar, D. M., Nannuru, K. C., Varney, M. L., Singh, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+CXCR2+enhances+chemotherapeutic+response%2C+inhibits+mammary+tumor+growth%2C+angiogenesis%2C+and+lung+metastasis%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis.</a> Mol Cancer Ther. 12 (5), 799-808 (2013).</li>
<li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type-2+pericytes+participate+in+normal+and+tumoral+angiogenesis%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Type-2 pericytes participate in normal and tumoral angiogenesis.</a> Am J Physiol Cell Physiol. 307 (1), 25-38 (2014).</li>
<li>Ferrara, N., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenesis+as+a+therapeutic+target%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Angiogenesis as a therapeutic target.</a> Nature. 438 (7070), 967-974 (2005).</li>
<li>Speyer, C. L., Hachem, A. H., Assi, A. A., Johnson, J. S., DeVries, J. A., Gorski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabotropic+glutamate+receptor-1+as+a+novel+target+for+the+antiangiogenic+treatment+of+breast+cancer%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Metabotropic glutamate receptor-1 as a novel target for the antiangiogenic treatment of breast cancer.</a> PLoS One. 9 (3), 88830(2014).</li>
<li>Gu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Apurinic%2FApyrimidinic+Endonuclease+siRNA+Inhibits+the+Angiogenesis+Induced+by+X-Ray+Irradiation+in+Lung+Cancer+Cells%5BTitle%5D)+AND+%22International+Journal+of+Medical+Sciences%22%5BJournal%5D)">Human Apurinic/Apyrimidinic Endonuclease siRNA Inhibits the Angiogenesis Induced by X-Ray Irradiation in Lung Cancer Cells.</a> International Journal of Medical Sciences. 10 (7), 870-882 (2013).</li>
<li>Manjunathan, R., Ragunathan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chicken+chorioallantoic+membrane+as+a+reliable+model+to+evaluate+osteosarcoma-an+experimental+approach+using+SaOS2+cell+line%5BTitle%5D)+AND+%22Biol+Proced+Online%22%5BJournal%5D)">Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma-an experimental approach using SaOS2 cell line.</a> Biol Proced Online. 17, 10(2015).</li>
<li>Nidhyanandan, S., Boreddy, T. S., Chandrasekhar, K. B., Reddy, N. D., Kulkarni, N. M., Narayanan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphodiesterase+inhibitor%2C+pentoxifylline+enhances+anticancer+activity+of+histone+deacetylase+inhibitor%2C+MS-275+in+human+breast+cancer+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Eur+J+Pharmacol%22%5BJournal%5D)">Phosphodiesterase inhibitor, pentoxifylline enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human breast cancer in vitro and in vivo.</a> Eur J Pharmacol. 764, 508-519 (2015).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper+improves+the+anti-angiogenic+activity+of+disulfiram+through+the+EGFR%2Fsrc%2FVEGF+pathway+in+gliomas%5BTitle%5D)+AND+%22Cancer+Lett.+Aug%22%5BJournal%5D)">Copper improves the anti-angiogenic activity of disulfiram through the EGFR/src/VEGF pathway in gliomas.</a> Cancer Lett. Aug. , (2015).</li>
<li>Park, H. J., Zhang, Y., Georgescu, S. P., Johnson, K. L., Kong, D., Galper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+umbilical+vein+endothelial+cells+and+human+dermal+microvascular+endothelial+cells+offer+new+insights+into+the+relationship+between+lipid+metabolism+and+angiogenesis%5BTitle%5D)+AND+%22Stem+Cell+Rev%22%5BJournal%5D)">Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis.</a> Stem Cell Rev. 2 (2), 93-102 (2006).</li>
<li>Clonetics Endothelial Cell System Technical Information & Instructions. , Lonza Walkersville, Inc. Available from: 
 <a target="_blank" href="http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_Instructions__Technical_Info_-_Endothelial_Cell_Systems.pdf">http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_Instructions__Technical_Info_-_Endothelial_Cell_Systems.pdf</a> (2015).</li>
<li>The Essence of Cutting-edge Microscopy Research. , Nikon Corporation. Available from: 
 <a target="_blank" href="http://www.nikoninstruments.com/images/stories/PDFs/Eclipse_Ti_Brochure.pdf">http://www.nikoninstruments.com/images/stories/PDFs/Eclipse_Ti_Brochure.pdf</a> (2008).</li>
<li>de Wit, C., Fautz, C., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+quantitative+PCR+for+retrovirus-like+particle+quantification+in+CHO+cell+culture%5BTitle%5D)+AND+%22Biologicals%22%5BJournal%5D)">Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.</a> Biologicals. 28 (3), 137-148 (2000).</li>
<li>50% of what? How exactly are IC50 and EC50 defined. , Graphpad Software. Available from: 
 <a target="_blank" href="http://www.graphpad.com/support/faqid/1356/">http://www.graphpad.com/support/faqid/1356/</a> (2015).</li>
<li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+of+human+endothelial+cells+for+derived+from+umbililcal+veins.+Identification+by+morphologic+and+immunologic+criteria%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Culture of human endothelial cells for derived from umbililcal veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li>
</ol>

The authors declare that they have no competing financial interests.

When conducting this assay, it is essential to keep the extracellular matrix on ice or at 4 &#176;C at all times unless otherwise specified. If the extracellular matrix is allowed to warm above 4 &#176;C, it will polymerize, and the assay will be ruined. It is also important to ensure that anything that comes into contact (e.g., pipet tips, the plate) with the extracellular matrix is pre-cooled for the aforementioned reason. The number of cells seeded in each well is critical, too few cells will not give the expected web in the control sample, too many cells will form large cell clusters or a monolayer and the assay will not be valid. Another important factor in the success of this assay is the cell passage number of the HUVECs. The passage number should always be lower than ten, otherwise robust tube formation may not occur. In addition, one should ensure that the cell culture medium being used has not expired, or the cells will not be viable. Alternatively, one can aliquot the medium and its components and store them at -20 &#176;C to preserve for a period of time after the expiration date. Of course, it is still preferable to use the medium prior to the expiration date
Like all procedures, there are some disadvantages to conducting the endothelial cell tube formation assay. One major problem is that, because there are different types of endothelial cells and support matrices, the results of the assay can vary greatly depending on which type of cell and matrix is used. The endothelial cells used (HUVECs, HAECs or HMVECs) are primary cells, they are costly to get and have variability compared to immortalized cells. The primary cells have limited passages for use, and are therefore not suitable for long term angiogenesis experiments14. For reliable data, one should always use the same types for the assay. As with other in vitro assays, the results of a tube formation assay should be confirmed in vivo, because results from the controlled and artificial conditions of two-dimensional tissue culture may not always be reflected in the complex biosystem of a living organism. It is also important to keep in mind that this type of assay can only be used to demonstrate endothelial cell tube formation, and should not be used to test other, non-endothelial, tube forming cells.
This assay is a rather simple and quick method to quantify angiogenic potential of a compound. It also forms a platform for further experiments, as alone, it does not yield information regarding the specific mechanism by which the compound actually affects the vessel forming process. However, the use of varied extracellular matrices is an example of how to further create a framework for research questions which is not commonly used. There are approaches to study the specific bio-chemical mechanisms of the compound including specific inhibitors that target components of the angiogenesis pathway. Another approach may be to extract RNA from the endothelial cells and use RT-PCR (Real Time PCR)12 to analyze alterations of gene expression which may cause the growth behavior observed in the assay. Future applications of this method include transfected HUVECs to create knock-down assays for specific steps of the angiogenesis pathway. There is potential to apply this approach to study the lymphangiogenesis pathway. By altering the extracellular matrix components, the interaction of matrix and cellular process supporting angiogenesis in cancer can be further elucidated. The extraction of RNA and proteins from endothelial cells under these specific conditions provide additional quantifiable data corresponding to imaging data.

In order to obtain the nutrients necessary for survival, malignant tumors require access to the patient&#39;s blood stream. To get that access, tumor cells release chemical signals that stimulate the growth of new blood vessels to the tumor, thus hijacking the normal physiologic process known as angiogenesis2. It is also through the blood vessels created via angiogenesis that metastasis (i.e., the spread of cancer to other organs) can occur. Because the process of angiogenesis is so vital to the progression of such a wide variety of cancers, it is an attractive target in anticancer therapy research3.
One method used to quantify angiogenesis is to measure the endothelial progenitor cell&#39;s ability to form tubes when placed on an extracellular matrix. Because formation of these tubes is a critical early step in angiogenesis, testing environmental conditions (e.g., presence or lack of a given compound) that can stimulate or inhibit tube formation provides insight into specific steps that can be targeted to inhibit angiogenesis. The endothelial cell tube formation assay is one of the most widely used4 in vitro methods that measures the cells&#39; ability to form tubes. It is a simple assay, requiring relatively few components and a short culture period5. Perhaps most importantly, however, is that the data gained from this type of assay is quantifiable.
An example for the use of the tube formation assay involves comparing the development of tubes from vascular endothelial cells grown on extracellular matrix vs. growth factor reduced (GFR) extracellular matrix. The extracellular matrix is a basement membrane-like material isolated from sarcoma cells and its main components are laminin, type IV collagen, growth factors and proteoglycans. Some compounds have different effects on the cell&#39;s ability to form tubes when in an environment with reduced growth factors and reduced heparan sulfate proteoglycans (HSPGs). HSPGs are a component of the extracellular matrix which is significantly reduced in GFR extracellular matrix. As an example, if the hypothesis is that angiogenesis inhibitors, such as SB225002, which blocks the CXCR2 receptor, have considerably weaker ability to interrupt tube formation when HSPGs are abundant, then a comparison between tube formation activity in extracellular matrix and GFR extracellular matrix is important in exploring the possibility of angiogenesis inhibition via the control of HSPGs.
Other assays that are commonly used to determine the effects of compounds on angiogenesis are in vivo methods. Notable examples of this are the chick chorioallantoic membrane (CAM) assay6 using chicken eggs, and the in vivo Matrigel plug angiogenesis assay7 using mice. While in vivo methods measure angiogenesis in three dimensions and are more representative of the human body compared to the in vitro tube formation assay, they suffer the flaw of requiring significantly more time and are considerably more difficult to perform. Both the CAM assay and the extracellular plug assay take at least a week8 to do, while in comparison, the tube formation assay can be done in a single day and it does not require animal usage.
It is important to note that the endothelial cells used in this report are human umbilical vein endothelial cells (HUVEC). These cells play a key role in vascular sprout and growth of blood vessels, and are sufficiently analogous to endothelial cells in cancers to be used to evaluate anti-angiogenesis activity in both in vitro and in vivo experiments9. Other cells such as primary microvascular endothelial cells may also be used.
It is also important to note that besides having lower levels of HSPGs, the GFR extracellular matrix also has reduced levels of many components when compared to normal extracellular matrix. This includes, but is not limited to: EGF, IGF-1, PDGF, and TGF-beta. Those performing experiments studying the significance of these compounds in relation to angiogenesis may be interested in using GFR extracellular matrix.

<table><tbody><tr><td>EGM-2 complete growth medium (EGM-2 bullet kit CC-3162 )</td><td>Fisher</td><td>NC9525043</td><td>HUVEC complete growth medium: EGM-2 base medium mixed with BBE (Bovine Brain Extract), hEGF (Human Epidermal Growth Factor), Hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), 2% FBS (Fetal Bovine Serum), VEGF (Vascular Endothelial Growth Factor), hFGF-&amp;beta; (Human Fibroblast Growth Factor Beta), R3-IGF-1 (Long R Insulin-like Growth Factor One), Ascorbic Acid, and Heparin. Aliquot the medium and its components and store them at -20&amp;nbsp;&amp;deg;C, warm in 37&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>MATRIGEL MATRIX 10ML Growth Factor Reduced (GFR); 10&amp;nbsp;ml; LDEV-Free</td><td>Fisher</td><td>CB-40230</td><td>Growth Factor Reduced (GFR) extracellular matrix, keep in -20&amp;nbsp;&amp;deg;C, warm in 4&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>MATRIGEL MATRIX 10ML; 10&amp;nbsp;ml; LDEV-Free</td><td>Fisher</td><td>CB-40234</td><td>extracellular matrix, keep in -20&amp;nbsp;&amp;deg;C, warm in 4&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>SB225002</td><td>Fisher</td><td>559405</td><td>CXCR2 inhibitor, keep in -20&amp;nbsp;&amp;deg;C, warm in room temperature before use.</td></tr><tr><td>Primary human umbilical vein endothelial cells (HUVEC)&amp;nbsp;</td><td>ScienCell Research Laboratories&amp;nbsp;</td><td>8000</td><td>culture it according reference&amp;nbsp; 10.</td></tr><tr><td>Trypsin 0.05%-EDTA phenol red</td><td>Invitrogen</td><td>25300-054</td><td>keep in 4&amp;nbsp;&amp;deg;C, warm in 37&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>Nikon ECLIPSE Ti</td><td>Nikon</td><td></td><td>Inverted Research Microscope.&amp;nbsp;</td></tr><tr><td>NIS-Elements AR 4.20.02 64 bit</td><td>Nikon</td><td></td><td>Inverted Research Microscope software</td></tr><tr><td>Graph-pad Prism 5</td><td>GraphPad Software, Inc.</td><td></td><td>scientific 2D graphing and statistics software, to calculate IC&lt;sub&gt;50&lt;/sub&gt;.</td></tr></tbody></table>

strategic endothelial cell tube formation assay: comparing extracellular matrix and growth factor reduced extracellular matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

Considerable, healthy endothelial tube formation can easily be contrasted to inhibited tube formation in the microscopy images. Healthy tube formation appears as an organized web of the capillary-like structures (Figure 1). In comparison, inhibited tube formation manifests itself as scattered cells (Figure 2). Tube formation assay data is quantified by measuring the total tube length of capillary tubes (Table 1). Besides total tube length, the average tube length, total number of tubes, or total number of branch points can be measured. Structured tube formation yields greater net tube length than scattered inhibited tube growth. In Figure 3, a dose response curve permits calculation of IC50 under the experimental conditions of HUVECs on GFR extracellular matrix and a CXCR2 inhibitor SB225002.
<img alt="Figure 1" src="/files/ftp_upload/54074/54074fig1.jpg" /> 
Figure 1. Endothelial tube formation on extracellular matrix and growth factor-reduced (GFR) extracellular matrix. Two images show successful tube formation on extracellular matrix (A) and GFR extracellular matrix (B). The interconnected network of tubes clearly shows that the growth of tubes is healthy in these HUVECs. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54074/54074fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54074/54074fig2.jpg" /> 
Figure 2. Inhibition of tube formation on extracellular and growth factor-reduced (GFR) extracellular matrix by a CXCR2 inhibitor SB225002 (5.6 &#181;M). Two images show tube formation that has been inhibited by a CXCR2 inhibitor SB225002 (5.6 &#181;M) on extracellular matrix (A) and GFR extracellular matrix (B). The isolated clumps of HUVEC cells seen in this image show that the cells were not able to form the tubes necessary to connect to each other. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54074/54074fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54074/54074fig3.jpg" /> 
Figure 3. Dose response curve on growth factor-reduced (GFR) extracellular matrix. The X-axis shows tube length and the Y-axis shows the concentration of a CXCR2 inhibitor. The dose response curve shows that the HUVEC response to the CXCR2 inhibitor on GFR extracellular matrix is dose dependent. The IC50 (half maximal inhibitory concentration) was calculated by using software13 and can be compared with a dose response curve for extracellular matrix, for example. IC50 is the concentration of the inhibitor that decreases the response to 50%. Error bars represent standard deviation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Growth Factor Reduced (GFR) extracellular matrix&#160; and inhibitor (4 replicates each)</td>
			<td>Total Tube Length Average (pixels) (1 pixel=0.34 &#181;m)</td>
			<td>Standard Deviation</td>
			<td>&#160;P Value</td>
		</tr>
		<tr>
			<td>GFR extracellular matrix control</td>
			<td>2447</td>
			<td>168.174</td>
			<td></td>
		</tr>
		<tr>
			<td>GFR extracellular matrix + 0.56 &#181;M SB225002</td>
			<td>1422.5</td>
			<td>185.4218</td>
			<td>0.000395</td>
		</tr>
		<tr>
			<td>GFR extracellular matrix + 1.1 &#181;M SB225002</td>
			<td>1004</td>
			<td>126.1784</td>
			<td>2.14 x 10-5</td>
		</tr>
		<tr>
			<td>GFR extracellular matrix + 5.6 &#181;M SB225002</td>
			<td>519.25</td>
			<td>129.6368</td>
			<td>4.19 x 10-6</td>
		</tr>
		<tr>
			<td>GFR extracellular matrix + 11 &#181;M SB225002</td>
			<td>393.75</td>
			<td>212.6857</td>
			<td>1.21 x 10-5</td>
		</tr>
	</tbody>
</table>
Table 1. Quantifying total tube lengths on Growth factor-reduced (GFR) extracellular matrix under varying conditions. A portion of a table using total tube lengths recorded in samples grown on GFR extracellular matrix with varying concentrations of CXCR2 inhibitor. The data indicate that the CXCR2 inhibitor does inhibit tube formation. Included are associated values such as average of four replicates, standard deviation, and P value. Measured in pixels to show the data that are exported. IC50 can be calculated by using statistical software13. (1 pixel = 0.34 &#181;m)

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Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

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13.4K Views.  Wayne State University School of Medicine. This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Video: Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Biology

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

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