Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

All procedures performed with mice were approved by the Animal Research Committee of Tokyo Women&#39;s Medical University.
1.Tail Vein Injection of Lewis Lung Carcinoma (LLC) Cells
<ol>
	<li>Maintain LLC cells in DMEM supplemented with 10% FCS, in a humidified 5% CO2 incubator at 37 &#176;C. Cells should contain a fluorescent protein expression system (e.g., GFP).</li>
	<li>Remove cells from culture dish by using a non-enzymatic cell dissociation reagent. Follow manufactu...

<ol>
	<li>Valastyan, S., 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=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, S., 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. 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=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, Y., 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, 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+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. 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+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. J., 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=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, S., 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, K., 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=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

Pre-treatment of mice with antibodies, drugs, a high-fat diet or tumor-conditioned medium prior to tumor cell injection is possible. The most difficult step in this assay is the tail vein injection. Incomplete injection results in inconclusive data. The tail vein in C57BL/6 is particularly difficult to identify, resulting in failed injections. Placing the mice on a heating pad (37 &#176;C) helps dilate the tail vein so that injection becomes easier.
This assay uses a large number of (1 x 10<su...

Tumor metastasis accounts for high mortality in cancer-related diseases. From the viewpoint of investigations at the molecular level, metastasis can be divided into multiple steps: tumor initiation at the primary tumor site, primary tumor growth and invasion into surrounding tissues, intravasation, circulation through blood vessels, extravasation at distant organs, and tumor re-growth. Each step involves different sets of molecules/signals1.
Studies to date have been concerned with events occurring in distant organs before the tumor cells start circulating in the blood, which is also known as the pre-metastatic phase2,3,4,5,6. Our mouse model studies revealed that the pre-metastatic phase facilitates lung metastasis by creating long-lasting and low-level inflammatory responses in the lungs. The pre-metastatic phase is characterized by hyperpermeability of themicrovasculature, recruitment of bone marrow derived cells (BMDC), and upregulation of cytokine/chemokine-like molecules including S100A8 and SAA33,4. It has been reported that lysyl oxidase modifies the extracellular matrix in the lungs to recruit BMDCs7. Another report has revealed the importance of Angpt2, MMP3, and MMP10 in the pre-metastatic phase8. In other words, the intricate interactions between primary tumors, bone marrow, and remote organs need to be understood and properly modulated (for instance, by using drugs that target proteins involved in these interactions) to prevent future metastasis9. To regulate the pre-metastatic phase, it should first be visualized and evaluated for diagnostic or therapeutic interventions. Here, we report a lung tumor cell recruitment assay that enables the study of the pre-metastatic phase in the lungs.
Tumor cells directly injected into the blood vessel of the mouse are similar to circulating tumor cells in cancer patients, although there may be differences between cultured cells and circulating tumor cells. Circulating tumor cells themselves undergo some characteristic changes when they enter circulation from the primary tumor. Nevertheless, cultured cells can form tumors in immunodeficient mice when they are injected in the tail vein. Additionally, in the mouse model system, fluorescence labeled tumor cells can be used that allow tracking of these cells in the body. The behavior of circulating tumor cells is critical as they determine the ultimate consequences in cancer patients10. Blood is not a good place for survival of tumor cells2. They need to escape from attack by the host immune system and need anti-apoptotic signals to prevent apoptosis due to a lack of physical support. Tumor cells with higher abilities of tumor initiation at metastatic sites generate more tumor nodules. If the tumor cells show specific organ tropism, metastasis should be observed in those particular organs. In this assay, the initial steps of the metastatic cascade (primary tumor growth and invasion) are not included, and so the focus is on the interaction between circulating tumor cells and the stromal cells (endothelial cells, epithelial cells, and blood cells). In conventional tumor cell injection assays, researchers have to wait until tumor nodules grow to a tangible size to detect the metastasis. In this case, the exact number of tumor cells in the blood vessel cannot be quantified. Additionally, this process includes a tumor regrowth phase, indicating that other factors in the tumor cells may affect the outcome.
Counting labeled tumor cells under a fluorescence stereomicroscope is a simple process. The stereomicroscope provides a wide field of view so that the whole lung can be scanned. Flow cytometry is also a useful tool that instantly gives an accurate number of tumor cells in the tissue. Counting fluorescent cells in the tissue under a confocal microscope is also possible and displays the most accurate pictures of metastatic tumor cells, but it is time-consuming and may give biased results if only a selected area is counted. The ultimate goal of this assay is to observe how easily tumor cells accumulate in the lungs. As reported previously3,4, if the lungs are in pre-metastatic phase due to inflammatory signaling from the primary tumor, the injected tumor cells are prone to accumulate in the lungs, thus implying higher chances of lung metastasis. Other conditions (rheumatoid arthritis, asthma, etc.), and their treatment with anti-inflammatory agents (such as anti-TNF&#945;) may attenuate lung metastasis. Thus, we conclude that this assay is a powerful tool to assess the possibility of lung metastasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

The lungs present many GFP-LLC cells 2 h after injection (Figure 1A). It should be noted that the fluorescent spots also detected in the red filter should be excluded from the cell number count. The vast majority of LLC cells disappear from the lungs 24 h after injection (Figure 1C).
To confirm the number of the GFP-LLC in the lungs, one of the lobes can be used for flo...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

This method can help answer key questions in the tumor biology field about the molecular mechanisms of metastasis. The main advantage of this technique is that the recruitment of tumor cells within a remote organ can be quantitate. Begin by using a non-enzymatic cell dissociation reagent to remove fluorescent protein-expressing Lewis lung carcinoma, or LLC, cells from their culture container according to standard protocols.Transfer the resulting cell solution into a 15-milliliter conical tube before centrifugation, and centrifuge wash the pellets two times in five milliliters of PBS per wash. After the second wash, filter the cells through a 40-micrometer pore cell strainer, and dilute the suspension to a one times 10 to the six cells per milliliter concentration. Then load the cells into a one-milliliter syringe equipped with a 29-gauge needle, and inject 100 microliters of cells into the tail vein of each eight-to-10-week-old, C57-Black-6, male mouse.At the appropriate experimental endpoint, use scissors to remove the skin from the ventral surface of each mouse, and cut the ribs and the diaphragm to expose the thoracic cavity. Use a 25-gauge winged infusion needle and tubing to flush 15 milliliters of PBS through the right ventricle, and remove the heart and the thymus. Gripping the trachea with forceps, pull up, dissecting the connective tissues around the trachea, to harvest the lungs.Then wash the lungs in PBS, and detach one lobe for visualization of the fluorescent cells in situ under a fluorescence microscope. To digest the lung tissue, first dice the caudal lobe with scissors, and place the lung pieces in a 10-milliliter syringe. Close the syringe with the plunger, and aspirate five milliliters of digestion solution into the syringe, moving the plunger in and out of the syringe shaft until the lung tissue is disseminated throughout the solution.Dispense the lung slurry into a 50-milliliter tube, and digest the tissue on a reciprocal shaker for 15 minutes at 37 degrees Celsius and 150 rpm. At the end of the incubation, triturate the remaining tissue until the lung cells are completely dispersed, and return the tube to the shaker for an additional 30 minutes. Then filter the cells through a 40-micrometer pore strainer, and collect the cells by centrifugation.To analyze the isolated lung cells by flow cytometry, resuspend the pellet in two milliliters of red blood cell lysis buffer for one minute at room temperature, and collect the cells by centrifugation. Then centrifuge the pellet two times in five milliliters of fresh PBS supplemented with 1%bovine serum albumin per wash. After the second centrifugation, resuspend the pellet in one milliliter of fresh PBS plus BSA, and filter the cells through a new 40-micrometer pore strainer for quantification of the tumor cells by flow cytometry.The lungs present many GFP-LLC cells two hours after injection, with the vast majority of cells disappearing from the lungs within 24 hours. Note that any fluorescent spots that are detected within the red filter should be excluded from the cell count. To confirm the number of GFP-LLC in the lungs, one of the lobes can be used for flow cytometric analysis as demonstrated.While attempting this procedure, it's important to remember that a frozen section procedure is a more accurate method for observing the exact location of tumor cells.

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