Supported in part by a National Cancer Institute grant 1R03CA172923.

Ethics Statement: The use of mice in research is only acceptable in instances where it might advance human understanding of fundamental biology or towards the eventual treatment of disease.
1. Ordering
<ol>
	<li>Purchase female C57BL/6 mice (age of 6 - 8 weeks), 5 mice per group. Allow several days for the mice to adjust.</li>
</ol>
2. Preparation of B16-BL6 Cells
<ol>
	<li>Remove cell culture plates from 37-degree incubator and place in a sterile hood. Plates should be approximately 70% confluent with B16-BL6 murine melanoma cells. A greater confluence may alter the cells metastatic potential.</li>
	<li>Add 1 ml of 0.05% Trypsin-EDTA per plate, let sit for 1 min, and then aspirate the trypsin-media solution.</li>
	<li>Add 1 ml of trypsin per plate and return plates to the incubator for 10 min.</li>
	<li>After moving the plates from the incubator to the sterile hood, add 4 ml of serum-free Rosewell Park Memorial Institute Medium (RPMI) to each plate.</li>
	<li>Collect the cells with a sterile, motorized pipette. Eject cells against the bottom of the plate, with the tip pressed at a very slight angle, to break up the cells. Repeat several times in order to ensure adequate separation of cell clumps, which might otherwise clog the ejection needle. Recollect and transfer to a 50 ml conical tube.</li>
	<li>Use a hemocytometer to determine the cell density. Keeping the total number of B16-BL6 cells constant, determine the volume of serum-free RPMI needed to reach final concentrations of 0, 0.065, 0.015, 0.25, and 0.5 million cells/ml.</li>
	<li>Equally aliquot the media in the 50 ml tube to 15 ml conical tubes. Centrifuge the conical tubes at 2,250 x g for 5 min. Decant the media. Add an appropriate volume of serum-free RPMI and confirm the desired cell density via hemacytometer. Adjust densities- by adding additional RPMI or spinning and resuspending in a smaller volume- accordingly.</li>
	<li>Aliquot 500 &#181;l of each cell suspension into labeled 1.8 ml tubes on ice.</li>
</ol>
3. Tail Vein Injection
<ol>
	<li>Take a mouse, held by the tail, and guide it rear-first into the mouse restrainer. With the torso in the main chamber of the restrainer, with the tail outside it, pull the mouse towards the end of the restrainer. Insert the restrainer-plunger, continuing to push until the mouse is secure.</li>
	<li>Rotate the mouse&#160;90 degrees such that one of the lateral tail veins is facing upwards. Using an alcohol pad, vigorously clean the side of the mouse&#39;s tail, particularly where the tail vein is most visible. 
	Note: Good lighting is essential for effective tail vein visualization on B57BL/6J mice. Heat lamps or heated surgery pads, while not necessary, will also help by increasing the volume of the tail vein.</li>
	<li>In a sterile culture hood, collect 300 &#181;l of cell culture media from the 2 million cells/ml tube. Invert the needle, flicking the side and pushing the plunger, to eject bubbles from the syringe. Eject culture-RPMI to result in a final volume of 250 &#181;l.</li>
	<li>Extend the mouse-tail with non-dominant hand. Hold the tail with the proximal end of the tail elevated with the forefinger of the non-dominant hand, and the distal portion of the tail depressed with the thumb. 
	Note: In this way, the tail vein is held in a lateral position, parallel to the restrainer surface, with a congruent entry possible in the more distal part of the tail.</li>
	<li>Insert the needle into the tail vein, towards the distal end, at a minute downward angle to begin with, and then adjust the needle to match the angle of the tail vein upon further insertion (lowering plunger end of the syringe relative to the needle).</li>
	<li>Insert the needle approximately 1 cm into the tail vein. After insertion, begin to push the plunger, holding the needle steady. 
	Note: If the needle is in the tail vein and the end of the needle is not obstructed the media should flow unimpeded into the vein.&#160;Effective injection is characterized by blood clearance from the vein. If there is resistance, or a bubble begins to form at the site in injection, remove the needle and try again at a more proximal location along the tail vein.&#160;</li>
	<li>Remove the needle from the vein and discard it. Hold gauze against the entry site to stop any bleeding.</li>
	<li>Remove the plunger from the restrainer and place the mouse in the recipient cage.</li>
	<li>Repeat for all other concentrations. 
	Note: Lung foci establishment takes approximately 2 - 3 weeks, with variability depending on cell line and the desired size of the foci.9&#160;In the interim, mice should be monitored for symptoms that could warrant early euthanasia, like excessive panting.&#160;</li>
</ol>
4. Counting the Lung Foci
<ol>
	<li>Euthanize the mouse by placing in a CO2 chamber and exposing to 100% CO2 introduced at 10-30% of chamber volume per minute for 5 min. Follow with a cervical dislocation.&#160;</li>
	<li>Splay the mouse on Styrofoam. Using surgical scissors, create a surgical incision along the sternum.</li>
	<li>Make two additional incisions, both from the top of the sternum incision and branching towards the shoulders of the mouse, exposing the rib cage.</li>
	<li>Make two cuts, each along the lateral sides of sternum, to remove the rib cage from the torso, exposing the lungs and heart.</li>
	<li>Holding the heart with surgical tweezers, cut the esophagus and windpipe, freeing the heart and lungs from the mouse. Place beneath a dissecting microscope, at 10X magnification, for better visualization.</li>
	<li>Dissect the heart from the lungs and remove the thymus, leaving only the two lungs.</li>
	<li>With the lungs beneath a dissecting scope, begin to count the lung foci. Each foci should appear as a circular, brown obtrusion on the surface of the lungs.
	<ol>
		<li>For consistency between mice, count the number of foci on the surface of a lobe, and then invert that lobe. Doing each of the four lobes individually allows for easier counting.</li>
	</ol>
	</li>
	<li>Save and fix the lungs if performing IHC, otherwise, discard the remains.</li>
</ol>

<ol>
	<li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J Clin Invest. 119 (6), 1420-1428 (2009).</li><li>Stetler-Stevenson, W. G., Aznavoorian, S., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+interactions+with+the+extracellular+matrix+during+invasion+and+metastasis.">Tumor cell interactions with the extracellular matrix during invasion and metastasis.</a> Annu Rev Cell Biol. 9, 541-573 (1993).</li><li>Allard, W. J., Matera, J., Miller, M. C., 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=Tumor+cells+circulate+in+the+peripheral+blood+of+all+major+carcinomas+but+not+in+healthy+subjects+or+patients+with+nonmalignant+disease.">Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant disease.</a> Clin Cancer Res. 10, 6897-6904 (2004).</li><li>Dunn, G. P., Bruce, A. T., Ikeda, H., 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=Cancer+immunoediting:+from+iimmunosurveillance+to+tumor+escape.">Cancer immunoediting: from iimmunosurveillance to tumor escape.</a> Nat Immunol. 3, 991-998 (2002).</li><li>Hugo, H., Ackland, M. L., Blick, T., 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=Epithelial-mesenchymal+and+mesenchymal-epithelial+transition+in+carcinoma+progression.">Epithelial-mesenchymal and mesenchymal-epithelial transition in carcinoma progression.</a> J Cell Physiol. 213, 374-383 (2007).</li><li>Kang, Y., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+Twist+in+development+and+metastasis.">Epithelial-mesenchymal transitions: Twist in development and metastasis.</a> Cell. 118, 277-279 (2004).</li><li>Hodi, S. F., 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=Improved+survival+with+Ipilimumab+in+patients+with+metastatic+melanoma.">Improved survival with Ipilimumab in patients with metastatic melanoma.</a> NEJM. 363 (13), 711-723 (2002).</li><li>Ekwueme, D. U., Guy, G. P., Li, C., Rim, S. H., Parelkar, P., Chen, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+burden+and+economic+costs+of+cutaneous+melanoma+motality+by+race/ethnicity-United+States.">The health burden and economic costs of cutaneous melanoma motality by race/ethnicity-United States.</a> J. Am. Acad. Dermatol. 65, 133-143 (2011).</li><li>Alizadeh, A. M., Shiri, S., Farsinejad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+review:+from+bench+to+bedside.">Metastasis review: from bench to bedside.</a> Tumor. Bio.Volume. 35, 8483-8523 (2014).</li><li>Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+metastasis+predetermined.">Is metastasis predetermined.</a> Mol. Oncol. 1 (3), 263-264 (2007).</li><li>Ishiguro, T., Nakajima, M., Naito, M., Muto, T., Tsuruo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+differentially+expressed+in+B16+murine+melanoma+sublines+with+different+metastatic+potentials.">Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials.</a> Cancer Res. 56 (4), 875-879 (1996).</li><li>Clark, E. A., Todd, R. G., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406, 532-535 (2000).</li><li>Terranova, V. P., Hic, S., Diflorio, R. M., Lyall, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+metastasis.">Tumor cell metastasis.</a> Crit. Rev. Oncol. Hematol. 5 (2), 87-114 (1986).</li><li>Elkin, M., Vlodavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tail+vein+assay+of+cancer+metastasis.">Tail vein assay of cancer metastasis.</a> Curr Protoc in Cell Biol. , (2001).</li><li>Hagedorn, H. G., Bachmeier, B. E., Nerlich, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+degradation+of+basement+membranes+and+extracellular+matrix+and+their+regulation+by+TGF-beta+in+invasive+carcinomas+(Review).">Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-beta in invasive carcinomas (Review).</a> Int. J. Oncol. 18 (4), 669-681 (2001).</li><li>Hart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+selection+and+characterization+of+an+invasive+variant+of+the+B16+melanoma.">The selection and characterization of an invasive variant of the B16 melanoma.</a> Am J Pathol. 97 (3), 587-600 (1979).</li><li>Overwijk, W. W., Restifo, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N.P.+B16+as+a+Mouse+Model+for+Human+Melanoma.">N.P. B16 as a Mouse Model for Human Melanoma.</a> Curr Protoc Immunol. 20 (1), (2001).</li></ol>

The authors declare that they have no competing financial interests.

Circulating tumor cells from a tail vein injection represent the metastases that evade sites of primary growth and migrate through routes like the bloodstream or lymphatic system, occasionally establishing themselves in the capillary beds of distant locations.14 The protocol described above serves as a model for secondary metastases. With an optimal number of B16-BL6 cells injected, experimenters can determine the relative effect of an administered drug or immune cell population, post-neutralization, on cancer cell metastasis.
This protocol has limitations. The circulating tumor cells have been selected for their ability to metastasize and are therefore non-immunogenic, having lower levels of major histocompatibility complex class 1 molecules.12 Furthermore, the sudden and clustered introduction of large number of metastases is unlike the typical scenario in patients where small numbers of metastatic tumor cells dissipate over a prolonged period of time from the primary tumor location. Additionally, the intravenously introduced tumor cells are from tissue culture and not of a primary tumor origin. Therefore, these cells are unlike the secondary metastases that result from alterations in cell adhesion, migration, immune recognition and recipient organ establishment.15,16
An alternative protocol would be the subcutaneous injection of B16/BL6 for the generation of primary tumors and the analysis of the resulting &#34;spontaneous&#34; pulmonary metastases. These &#34;spontaneous&#34; metastases have been found to contain more heterogeneity than their &#34;experimental&#34; counterparts, more closely matching those of human patients. The protocol is limiting in its ability to determine the influence of an experimental compound on metastasis. With a lateral tail vein injection, the number of melanoma cells injected into the bloodstream can be approximated via hemocytometer. With &#34;spontaneous&#34; metastasis, however, there is greater heterogeneity between tumors, adding an additional variable to the number of resulting lung foci.16
In conclusion, the tail vein B16-BL6 murine melanoma model represents a simple model for determining the influence of a treatment or immunotherapy on metastasis. By intravenously injecting circulating tumor cells, researchers can replicate, to some extent, patients post-metastasis. Considering the destructive effects of metastatic tumor cells in the cancer patients, this model is a powerful tool for understanding the multistep process of metastasis.

Metastasis is a major cause of death among patients with malignancies. The process of metastatic dissemination of cancer cells is poorly understood but appears to involve several steps, including invasion of adjacent tissues, intravasation into the lymphatics and vasculature, survival and translocation within the circulatory system, extravasation from the vasculature at the site of metastasis, adaptation to the new microenvironment of the new site, and colonization at the new site by proliferation and formation of secondary tumors.1 Each of these biological events involve the transformation, dissemination and survival of metastatic tumor cells. For example, the transformation of the epithelial phenotype of the tumor cell into a mesenchymal phenotype, coupled with the successful interaction with the extracellular matrix, enable them to invade adjacent tissues and metastasize to other parts of the body.2,3 Furthermore, these metastatic cells must survive within the circulating bloodstream and evade immune surveillance from the host.4,5 Finally, when implanted at a distant site within the body, the tumor cells must adapt to their microenvironment in order to proliferate and form secondary tumors.6,7 Therefore, although metastasis is a common phenomenon among cancer patients, metastatic tumor cells have multiple areas of vulnerability that are amenable to therapeutic intervention.
Given the immunogenic nature of melanoma, and the recent interest in immunotherapies, models for melanoma are increasingly useful.8 In the United States alone, melanoma is the cause of an estimated 9,000 deaths per year.9 Common locations of melanoma metastasis are bones, brain, liver, and lungs. The majority of metastases to distant sites occur through the bloodstream. Circulating tumor cells in the blood must evade immune clearance, reach a capillary bed of a distal organ and invade through the endothelial cells of the blood vessel in order to successfully establish themselves.4-7,10, 11
To mimic the common and virulent phenomena of metastasis, the murine B16-BL6 cell line was created. A decedent of the parental C57BL/6 melanoma cell line B16-F0, B16-BL6 is the end product of 10 successive selections for lung metastasis from intravenous injection (resulting in B16-F10), followed by 6 successive selections for bladder membrane penetration.12 As such, it is a reliable melanoma cell line for the establishment of metastatic foci in the mouse, particularly when injected intravenously.13
After injection of a sufficient quantity of B16-BL6 cells into the tail vein of a B57BL/6 mouse, followed by two or more weeks to allow implantation and growth of the B16/BL6 cells, metastatic foci will form in the lungs. Upon euthanasia and inspection by dissecting microscopy, the number of individual foci present can be quantified. This, in turn, can be used to establish a dose-metastasis effect, as the number of injected B16-BL6 cells correlates with the number of foci formed on the surface of the lungs. This model is known as &#34;experimental metastasis,&#34; where known-metastatic cells are introduced directly into the bloodstream, facilitating a rapid and predictable spread and establishment in the lungs, liver, or other organ of investigation. This is in contrast to &#34;spontaneous metastasis,&#34; where tumor cells are implanted, often subcutaneously, and metastases originate from organically shed cancer cells.14,15
It is important to inject an appropriate quantity of B16/BL6 cells into the tail veins of the mice. Too many, and the lungs will be covered with metastases and neighboring foci will be indistinguishable from one another. Too few, and the influence of a therapeutic agent will be indiscernible due to inherent variations between injections. In order to obtain an optimal number of foci on the lungs of the C57BL/6 mice, it is necessary to correlate the number of injected cells with the quantity of established metastatic foci on the surface of the lungs while taking into account the distinguishability of individual foci. This protocol will demonstrate a metastatic murine melanoma model for C57BL/6 mice.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Trypsin</td>
			<td style="width:275px;">Corning</td>
			<td style="width:288px;">MT25052CV</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT15040CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase Contraast Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine IV Insulin Syringes</td>
			<td>BD</td>
			<td>14-829-1D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Restrainer</td>
			<td>Braintree Scientific</td>
			<td>NC9999969</td>
			<td>Restrainer choice depends on age/size of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating Pad</td>
			<td>Harry Schein</td>
			<td>NC0012697</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

injection of syngeneic murine melanoma cells to determine their metastatic potential in the lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Upon visual inspection, the variation in surface foci is evident. Counting the number of tumors under a dissecting microscope should yield a linear relationship between the number of injected tumor cells and number of resulting and countable foci. Using an optimal number of injectable cells, the goal is one where the lungs are not completely covered, as they are at 500,000 cells/ml in Figure 1A, and not too sparsely covered, as they are at 62,500 cells/ml and 125,000 cells/ml. This is also quantifiable by linear correlation (Figure 1B).
<img alt="Figure 1" src="/files/ftp_upload/54039/54039fig1.jpg" /> 
Figure 1. Lung Foci Resulting from Tail Vein Injected B16/BL6 Cells. (A) Lung foci can be visualized on the surface of lungs from C57BL/6 mice as brown circular masses (representative foci under arrows). As the density of the injected cell culture (above each image) increases, so does the corresponding number of resulting lung foci (below each image). The lungs from the mouse with the highest density of injected cells has the greatest visual coverage of lung foci. Scale bars represent 20 mm. (B) Enumeration of the lung foci relative to the cell culture density. There is an approximately linear relationship above 125,000 cells/ml, as determined by hemacytometer. <a href="https://www.jove.com/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

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17.5K Views.  Beth Israel Deaconess Medical Center & Harvard Medical School. Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Video: Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

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An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

The Measurement and Treatment of Suppression in Amblyopia

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Myocardial Infarction and Functional Outcome Assessment in Pigs

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Measurement of the Pressure-volume Curve in Mouse Lungs

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery