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. 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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><tbody><tr><td>Trypsin</td><td>Corning</td><td>MT25052CV</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Corning</td><td>MT15040CM</td><td></td></tr><tr><td>Phase Contraast Hemacytometer</td><td>Hausser</td><td>02-671-54</td><td></td></tr><tr><td>Micro-Fine IV Insulin Syringes</td><td>BD</td><td>14-829-1D</td><td></td></tr><tr><td>Sterile Alcohol Prep Pads</td><td>Fisherbrand</td><td>22-363-750</td><td></td></tr><tr><td>Mouse Restrainer</td><td>Braintree Scientific</td><td>NC9999969</td><td>Restrainer choice depends on age/size of mice</td></tr><tr><td>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.remotexs.ntu.edu.sg/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 ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Nanyang Technological University

Research

JoVE Journal

Medicine

17.6K 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

Experimental Metastasis Assay

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

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

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

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Cancer Research

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

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

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