We would like to thank Dr. Arnulfo Mendoza who provided training in the PuMA technique. Additionally, we would like to acknowledge Drs. Chand Khanna, Susan Garfield (NCI/NIH), and Sam Aparicio (BC Cancer Agency) for providing use of their microscopes during the course of this study. This research was supported (in part) by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, Pediatric Oncology Branch. M.M.L. was supported by the National Institutes of Health Intramural Visiting Fellow Program (award 15335), and is currently supported by a Joan Parker Fellowship in Metastasis Research. P.H.S. is supported by British Columbia Cancer Foundation.

All animal protocols from which imaging data were obtained were performed with approval of the Animal Care and Use Committee of the National Cancer Institute, National Institutes of Health. All animal protocols discussed and portrayed in the article video have been approved by the University of British Columbia Animal Care Committee.
1. Preparation of tumor cells for injection and materials for the PuMA model
NOTE: The amount of solutions and cells will be enough for 1 mouse. Scale up as necessary if more mice are used in the study. For media recipes, refer to Table 1 and Table 2.
<ol>
	<li>Pre-warm 5 mL of A-Media in a 15 mL conical tube in a 37 oC water bath.</li>
	<li>Melt the 1.2% low melting agarose solution (in sterile water) using the lab microwave.</li>
	<li>Transfer 5 mL of the molten agarose into a 15 mL conical tube, and keep warm in a 37 oC water bath. Ensure the melted agarose is at 37 oC and liquid prior to the lung insufflation step.</li>
	<li>Pre-chill 30 mL of cell-culture grade PBS supplemented with 1X pen/strep in an ice bucket.</li>
	<li>In one well of a 6-well plate, pre-soak a gelatin sponge in 1.5 mL of B-media. The sponge will be the support anchor for the lung slices.</li>
	<li>Ensure the OS cells are about 70-90% confluent on the day of the procedure. Do not use cells that are over-confluent or if the media is orange to yellow since the cells are usually stressed and have diminished viability at this point. For the osteosarcoma cell lines, MNNG and HOS, 5 x 105 in a volume of 100 &#956;L will be used to inject into the tail vein of 1 mouse. Upscale accordingly if more mice are used for the study.</li>
	<li>Harvest the tumor cells using 0.25% trypsin-EDTA (3 mL for a T75 flask or 2 mL in a 10 cm culture dish). When cells are beginning to lift off the plate, neutralize the trypsin-EDTA with complete media. Spin down cells and rinse once with cell-culture grade PBS. Resuspend pellet in 5 mL of PBS.</li>
	<li>Perform a cell count by standard methods. It is best to make more cell suspension than what is actually needed since loss of cell suspension often occurs during needle draw-up and failed injection attempts.</li>
	<li>Perform a Trypan-Blue Exclusion Assay on a sample of the cell suspension to assess the viability of the cells. Proceed only if the cell show 90% viability or higher.</li>
	<li>Inject 5 x 105 cells in a volume of 100 &#956;L. To prepare an excess of 2X of this amount, 1 x 106 cells should be spun down and resuspended in 0.2 mL of HBSS.</li>
	<li>Place cell suspension in an ice bucket while preparing the mice for injection.</li>
</ol>
2. Tail Vein Injection and Lung Insufflation
NOTE: The amount of solutions and cells in this section will be enough for 1 mouse. Scale up as necessary if more mice are used in the study. For a list of the equipment, materials and surgical instruments used in the following steps, refer to Table 3 and Table 4.
<ol>
	<li>Warm a female mouse (age 6-8 weeks) under a heating lamp for 5 min&#160;in order to make their tail vein more visibly apparent. For murine K7M2 or K12 cells, use Balb/c mice; for human MG63.3, MG63, MNNG, and HOS cells, use severe combined immunodeficiency mice.</li>
	<li>Make sure the cell suspension is uniform by gently shaking the tube. Carefully draw up the cell suspension into the 1 mL syringe without the needle. Cap the syringe with a 27 gauge needle. Make sure the needle bevel is on the same side as the volume markings on the needle.</li>
	<li>Place the mouse in the restrainer, and swab the tail with an alcohol wipe.</li>
	<li>Proceed to perform a tail vein injection and inject the 100 &#956;L of the cell suspension. 5 min&#160;after the injection, place the mouse in a CO2 chamber and begin the euthanasia standard operating procedure (as outline by your institutional animal care committee). Cervical dislocation should not be used as a means of euthanasia since the procedure will damage the trachea.</li>
	<li>Once the mouse is euthanized, begin preparation of the laminar flow hood for insufflation of the mouse lung with the agarose/A-media solution. Within the laminar flow hood, set up a work area with your sterile pad. Onto this pad you will place your sterilized instruments, IV catheter, IV extension set, and gravity perfusion apparatus (see Supplemental Figure 1).</li>
	<li>Place the mouse in dorsal recumbancy. With sterile small scissors, carefully dissect out the sternum to expose the chest cavity. Take care to not puncture the lung since an agarose/A-media solution will be used to insufflate the lung.</li>
	<li>When dissecting out the sternum, dissect past the thoracic inlet on both sides the trachea. Expose the trachea by dissecting away the surrounding soft tissue.</li>
	<li>Cannulate the trachea with a 20 gauge IV catheter. Loosely tie a surgical knot using sterile catgut suture around the cannulated trachea.</li>
	<li>Attach an IV extension set from the catheterized trachea to the 10 mL syringe of the gravity perfusion apparatus.</li>
	<li>Combine the pre-warmed 37 oC agarose (5 mL) and A-media (5 mL) into a 1:1 mixture. Pour the liquid agarose/A-media solution into the 10 mL syringe of the gravity perfusion device. Prior to insufflation of lungs with agarose/A-media solution, ensure that the entire length of the extension set has been primed with agarose/A-media, thereby negating inadvertent insufflation of lung samples with air.</li>
	<li>Fill the lung the agarose/A-media solution until the lung is fully insufflated.</li>
	<li>Once the lung is fully insufflated, remove the cannula and firmly tie off the surgical knot to prevent leakage of the agarase/A-media solution through the trachea.</li>
	<li>Proceed to dissect out the pluck (trachea, heart, and lung) from the chest cavity. Take care to not puncture or damage the surface of the lung.</li>
	<li>Place the pluck (in no particular orientation) in the pre-chilled 30 mL of PBS supplemented with 1X pen/strep, and allow the agarose/A-media to solidify for 20 min.</li>
	<li>Using fine scissors and tweezers, cut small pieces of the lung (3 mm x 1.5 mm) as shown in Figure 1A. Smaller lung slices can be easily imaged using a 2.5X objective. Multiple slices can be cut per experimental condition, typically 4-10 slices per group. 
	NOTE: For each experimental group, place the lung slices into a separate well (6-well plate) containing a 2 x 2 cm gelatin sponge pre-soaked in B-media. The amount of media per well should be 1.5 mL. Change the media every 2-3 days. For drug studies, the frequency of changing the media/drug is user determined.</li>
</ol>
3. Widefield Fluorescence Imaging of Lung Slices and Analysis
NOTE: For widefield fluorescence imaging, smaller slices are cut (3 mm x 1.5 mm x 1 mm) in order to fit the lung section into 1 image using a 2.5X objective.
Image acquisition on a widefield fluorescence microscope:
<ol>
	<li>Lung slices are typically imaged at 0, 3, 7, and 14 days post-injection. In the sterile environment of the biological cabinet, carefully transfer the lung slices from the gelatin sponges to a sterile 35 mm glass-bottom round dish. Take care to dab off the excess fluid from the lung slice as the excess fluid will act as a mirror and reflect fluorescent light coming from the tumor cells.</li>
	<li>Arrange the lung slices in a similar manner depicted in Figure 1A. Each column would represent a different experimental condition. Take care to not cross-contaminate the lungs with the tweezers. Rinse with 70% ethanol and dry before handling lung slices from another experimental group.</li>
	<li>Optimize the imaging parameters (ie. gain, offset, exposure time, binning) that provides the best contrast between the fluorescent tumor cells and background lung tissue in the control (vehicle untreated) group. Use the same parameters from the control group to image the experimental groups. Save as tiff image format.</li>
	<li>For a scale reference, take a digital picture of a micrometer at the same objective.</li>
	<li>Since lung auto-fluorescence changes over time, the imaging parameters must be adjusted to the control lungs each imaging session. The imaging parameters for one session may not necessarily be optimal for subsequent imaging sessions.</li>
</ol>
Image Analysis: 
NOTE: The following image processing steps are done with ImageJ 1.51h software package 17.
<ol>
	<li>Open an image file in ImageJ (Figure 3A).</li>
	<li>Subtract background: Process &#62; Subtract Background &#62; Rolling ball radius (start with 50 pixels), uncheck &#34;Light Background&#34; (Figure 3B).</li>
	<li>Convert image to 8-bit format: Image &#62; Type &#62; 8-bit (Figure 3C).</li>
	<li>Set units to pixels: Image &#62; Enter &#34;Pixels&#34; in Unit of Length, enter 1 in &#34;Pixel width&#34;, &#34;Pixel Height&#34;, &#34;Voxel Depth&#34;. Check &#34;Global&#34; box to apply to subsequent images.</li>
	<li>Using the Polygon selection tool, outline the shape of the entire lung slice and determine the area (pixel2) of the total lung slice. This value will be used to calculate the percent tumor burden of the lung.</li>
	<li>Threshold image: Image &#62; Adjust &#62; Threshold &#62; highlight &#34;Default&#34; and &#34;B&#38;W&#34; &#62; use the slider to threshold the image such that the majority of tumor cells are accurately highlighted. Pressing &#34;Apply&#34; will result in a black and white image where the lung is all black, and the fluorescent lesions are white (Figure 3D). Pressing &#34;Apply&#34; a second time will invert the image where all fluorescent structures are now black shapes (Figure 3E).</li>
	<li>Quantification of the number and shape of lesions: 
	Analyze &#62; Set Measurements &#62; Check &#34;Area&#34;. Uncheck all other boxes. 
	Analyze Particles &#62; Size (pixel2): 0-infinity represents the range of shapes that ImageJ will enumerate. Empirically determine the smallest lesion that is deemed to be a single tumor cell in Day 0 vehicle images. Use the area of this single tumor cell as the lower limit for the remaining images in the data set. For the work presented in this paper, 11 pixel2 is set as the lower limit. For the work presented in the video example, 24 pixel2 is set as the lower limit. Leave &#34;Circularity&#34; range as 0-1. &#34;Show&#34; can be set to &#34;Nothing&#34;. Alternatively, if &#34;Show&#34; and &#34;Outlines&#34; is selected, a drawing of all outlined shapes is generated. Check &#34;Display results&#34; for a separate window of measurements to pop up. Optionally, having &#34;Add to manager&#34; box checked will save all the shapes quantified to a ROI manager, which can be saved for future reference. After pressing OK, a &#34;Results&#34; window will pop up containing all enumerated shapes and area measurements (Figure 3F).</li>
	<li>Copy and paste the data from the &#34;Results&#34; window into a spreadsheet. Use the SUM mathematical function in Excel to sum all the areas of the metastatic lesions for that particular lung slice. To assess the percent lung tumor burden of lung slice, divide the sum of the areas of metastatic lesions by the total area of the lung slice. This parameter is also known as Area fraction (AA) which is described further by Underwood 18. 
	Lung tumor burden = Sum of metastatic lesion areas/total area of lung slice</li>
	<li>Calculate the lung tumor burden for the rest of the lung sections in control group and remaining groups. Plot the average lung tumor burden per group over 0, 3, 7 and 14 days. Representative widefield fluorescent pictures of high and low metastatic human OS cells growing in the PuMA model at progressive time points are shown (Figure 4A). A line graph showing the fold-change (normalized to Day 0) in percent metastatic tumor burden over time is shown (Figure 4B).</li>
</ol>
4. Confocal Fluorescence Imaging of the PuMA Model
NOTE: For confocal imaging, tissue processing is similar to that in the previous section except that larger lung slices are cut (complete transverse sections, 1-2 mm thick) to allow for more ROIs to be imaged.
Labelling of lung parenchyma with DAR4M:
<ol>
	<li>Immerse lung slices in 10 &#956;&#956;M of DAR4M (in HBSS) for 45 min&#160;at 37 &#176;C. DAR4M labels reactive nitrogen species within the cells of the lung.</li>
	<li>After 45 min&#160;of incubation, rinse the lung slices with fresh HBSS.</li>
	<li>Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.</li>
	<li>The imaging parameters for the example images shown in Figure 5 are listed in Table 5.</li>
	<li>Acquire a Z-stack for a region of interest. Use the suggested Z slice thickness for Nyquist sampling. A representative movie of a 3D stack showing green fluorescent OS cells in the DAR4M-labeled lung tissue is provided in Movie 1 and Supplemental Movie 1.</li>
</ol>
For the example confocal images shown in Figure 5, the imaging session was terminal. If longitudinal imaging is required, the use of a 2-photon or multi-photon equipped confocal LSM microscope for imaging is advised since there is less photodamage to living tissues19,20. 
Imaging mito-RFP expressing MG63 cells in the PuMA model:
<ol>
	<li>Perform the PuMA protocol as outline in Step 1 and 2 with using MG63 cells expressing the mito-RFP construct.</li>
	<li>Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.</li>
	<li>The imaging parameters for the example images shown in Figure 6 are listed in Table 6. A representative movie of a 3D stack showing green fluorescent OS cells with red fluorescent mitochondria is provided in Movie 2 and Supplemental Movie 2.</li>
</ol>

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The authors have nothing to disclose.

The following technical article describes some practical aspects of the PuMA model in studying lung colonization in OS. Some critical steps in the protocol where researchers should take extra care include the following:
a) Cannulation of the trachea. The trachea can be easily damaged while dissecting the surrounding muscle and connective tissue. In addition, the needle of the catheter can easily be pushed through the trachea. Pay close attention to how the bevel of the needle enters the trache...

Improved outcomes for pediatric patients with metastatic osteosarcoma (OS) still remains a critical unmet clinical need 1. This underscores the importance of developing new molecularly-targeted therapies. Conventional chemotherapeutics that target tumor cell proliferation have not proven to be effective in treating metastatic disease, and thus novel strategies must target the metastatic process itself 2. The current article discusses the practical aspects of a relatively new type of ex vivo lung metastasis model, the pulmonary metastasis assay (PuMA) developed by Mendoza and colleagues3, which provides a useful tool in discovering new molecular drivers in lung metastasis progression in OS 4,5. Before proceeding, however, it would be prudent to briefly touch upon several current models of metastasis, and how the PuMA model offers several advantages over conventional in vitro assays.
Most experimental models used to study metastasis comprise of in vitro and in vivo systems that recapitulate either a specific step or several steps of the metastatic cascade. These steps include: 1) tumor cells migrating away from the primary tumor, 2) intravasation into nearby vessels (blood or lymphatic) and transit within circulation, 3) arrest at the secondary site, 4) extravasation and survival at the secondary site, 5) formation of micrometastases, and 6) growth into vascularized metastases (Figure 1). In vitro models of metastasis can include 2-dimensional (2D) migration and 3-dimensional (3D) Matrigel invasion assays which are reviewed in detail elsewhere 6. For in vivo models, the two commonly used model systems include: 1) the spontaneous metastasis model is where a tumor cells are orthotopically injected into a specific tissue type to form a local tumor which spontaneously sheds metastatic cells to distant sites; 2) the experimental metastasis model is where tumor cells are injected into the blood vessel upstream of the target organ. For example, a tail vein injection of tumor cells results in the development lung metastases5,7,8. Other experimental metastasis models include injection of tumor cells into the spleen or mesenteric vein which results in the development of liver metastases9,10. Practical considerations of these in vivo models are discussed in detail by Welch 11. Another in vivo model used to study metastasis in pediatric sarcomas is the renal kidney subcapsular tumor implantation model which results in local tumor formation and spontaneous metastasis to the lungs 12,13. A more technically demanding technique such as intravital videomicroscopy can directly visualize, in real-time, interactions between metastatic cancer cells and the microvasculature of a metastatic site (ie. lung or liver) as described by MacDonald14 and Entenberg15, or cancer cell extravasation in the chorioallantoic membrane as described by Kim 16.
The PuMA model is an ex vivo, lung tissue explant, closed culture system where the growth of fluorescent tumor cells can be longitudinally observed via fluorescence microscopy over a period of a month (see Figure 2A). This model recapitulates the initial stages of lung colonization (steps 3 to 5) in the metastatic cascade. Some major advantages of the PuMA model over conventional in vitro models are: 1) it provides an opportunity to longitudinally measure metastatic cancer cell growth in a 3D microenvironment that retains many features of the lung microenvironment in vivo 3; 2) PuMA allows the researcher to assess whether the knockdown of a candidate gene or drug treatment has anti-metastatic activity in the context of a 3D lung microenvironment; 3) the PuMA model is flexible with many types of fluorescence microscopy platforms (Figure 2B) such as widefield fluorescence microscopy or laser-scanning confocal microscopy, examples of each are shown in Figure 2C &#38; D, respectively. This article will discuss how to use the PuMA model to obtain longitudinal imaging data on the metastatic growth of enhanced green fluorescent protein (eGFP)-expressing, human high and low metastatic osteosarcoma cells (MNNG and HOS cells, respectively) using low-magnification widefield fluorescence. Examples of imaging a fluorescent dye which labels the lung parenchyma, and a red-fluorescent protein genetic reporter which labels mitochondria in OS cells in the PuMA model using confocal laser-scanning microscopy are also discussed.

<table>
	<tbody>
		<tr>
			<td>Table 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture reagents for A-media, B-media, and complete media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MNNG-HOS</td>
			<td>ATCC</td>
			<td>CRL-1547</td>
			<td>highly metastatic OS cell line</td>
		</tr>
		<tr>
			<td>HOS</td>
			<td>ATCC</td>
			<td>CRL-1543</td>
			<td>poorly metastatic OS cell line</td>
		</tr>
		<tr>
			<td>MG63.3</td>
			<td>Amy LeBlanc Laboratory (NCI)</td>
			<td>N/A</td>
			<td>highly metastatic OS cell line</td>
		</tr>
		<tr>
			<td>MG63</td>
			<td>ATCC</td>
			<td>CRL-1427</td>
			<td>poorly metastatic OS cell line</td>
		</tr>
		<tr>
			<td>10X M199 media</td>
			<td>Thermofisher</td>
			<td>11825015</td>
			<td>Base media for A-media and B-media</td>
		</tr>
		<tr>
			<td>Distilled Water (sterilized)</td>
			<td>Thermofisher</td>
			<td>15230-147</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>7.5% sodium bicarbonate solution</td>
			<td>Thermofisher</td>
			<td>25080094</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>Hydrocortizone</td>
			<td>Sigma-Alrich</td>
			<td>H6909</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>Retinol acetate-water soluable</td>
			<td>Sigma-Alrich</td>
			<td>R0635-5MG</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10X concentrated (10000 U/ml) solution</td>
			<td>Thermofisher</td>
			<td>15140122</td>
			<td>Component of A-media &#38; B-media, complete media.</td>
		</tr>
		<tr>
			<td>Bovine insulin solution (10mg/ml)</td>
			<td>Sigma-Alrich</td>
			<td>I0516-5ML</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Thermofisher</td>
			<td>11965092</td>
			<td>Base media of Complete Media</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermofisher</td>
			<td>25030081</td>
			<td>Component of Complete Media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermofisher</td>
			<td>16000044</td>
			<td>Component of Complete Media</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Thermofisher</td>
			<td>14190144</td>
			<td>Used in cell culture.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Buffered Salts Solution, no calcium, no magnesium, no phenol red</td>
			<td>Thermofisher</td>
			<td>14175095</td>
			<td>Used to resuspend cell pellet prior to injection</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermofisher</td>
			<td>25200114</td>
			<td>Used in cell culture.</td>
		</tr>
		<tr>
			<td>DAR4M</td>
			<td>Enzo</td>
			<td>ALX-620-069-M001</td>
			<td>Used to label lung parenchyma.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for PuMA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 710 Confocal LSM</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Upright LSM confocal microscope</td>
		</tr>
		<tr>
			<td>Zeiss 780 Confocal LSM</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Inverted LSM confocal microscope</td>
		</tr>
		<tr>
			<td>SCID mice</td>
			<td>Charles River</td>
			<td>N/A</td>
			<td>NOD.CB17-Prkdcscid/NcrCrl, female, age 6-8 weeks</td>
		</tr>
		<tr>
			<td>GelFoam</td>
			<td>Harvard Apparatus</td>
			<td>59-9863</td>
			<td>Used as a support for lung tissue sections.</td>
		</tr>
		<tr>
			<td>SeaPlaque Agarose</td>
			<td>Lonza</td>
			<td>50100</td>
			<td>Used during insufflation of the lung.</td>
		</tr>
		<tr>
			<td>1 ml syringe with 27 gauge needle</td>
			<td>Fisherscientific</td>
			<td>14-826-87</td>
			<td>Used for tail vein injection.</td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td>Used for insufflation of the lung.</td>
		</tr>
		<tr>
			<td>20 gauge catheter</td>
			<td>Terumo</td>
			<td>SR-OX2032CA</td>
			<td>Used during insufflation of the lung.</td>
		</tr>
		<tr>
			<td>Abbott IV extension set (30&#34;, Sterile)</td>
			<td>Medisca</td>
			<td>8342</td>
			<td>Used during insufflation of the lung.</td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>BD</td>
			<td>326895</td>
			<td>For wiping tail vein before injection</td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>Fisherscientific</td>
			<td>Varies with size</td>
			<td>Asceptic handing of mouse lungs</td>
		</tr>
		<tr>
			<td>30 cm ruler</td>
			<td>Staples</td>
			<td></td>
			<td>Used for insufflation of the lung.</td>
		</tr>
		<tr>
			<td>Support stand for ruler</td>
			<td>Pipette.com</td>
			<td>HS29022A</td>
			<td>Used for insufflation of the lung.</td>
		</tr>
		<tr>
			<td>35 mm glass-bottomed culture dish</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td>Used during imaging of lung slices</td>
		</tr>
		<tr>
			<td>Absorbent Underpads with Waterproof Moisture Barrier</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>Used to line the sterile work area in the biological hood.</td>
		</tr>
		<tr>
			<td>Catgut Plain Absorbable Suture</td>
			<td>Braun</td>
			<td>N/A</td>
			<td>Used to tie off cannulated trachea.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments for PuMA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors 3.5&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>For cutting lung sections</td>
		</tr>
		<tr>
			<td>4&#8221; (10 cm) Long Serrated Straight Extra Delicate 0.5mm Tip</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td>For manipulating/holding lung sections.</td>
		</tr>
		<tr>
			<td>4&#8221; (10 cm) Long Serrated Slight Curve 0.8mm Tip</td>
			<td>Roboz</td>
			<td>RS5135</td>
			<td>For manipulating/holding lung sections.</td>
		</tr>
		<tr>
			<td>Thumb Dressing Forceps; Serrated; Delicate; 4.5&#34; Length; 1.3 mm Tip Width</td>
			<td>Roboz</td>
			<td>RS-8120</td>
			<td>For general dissection.</td>
		</tr>
		<tr>
			<td>Thumb Dressing Forceps 4.5&#34; Serrated 2.2 mm Tip Width</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td>For general dissection.</td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors 3.5&#34; Straight Sharp/Sharp, 20mm blade</td>
			<td>Roboz</td>
			<td>RS-5880</td>
			<td>For general dissection.</td>
		</tr>
		<tr>
			<td>Knapp Scissors; Straight; Sharp-Blunt; 27mm Blade Length; 4&#34; Overall Length</td>
			<td>Roboz</td>
			<td>RS-5960</td>
			<td>For general dissection.</td>
		</tr>
	</tbody>
</table>

practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

Low-magnification widefield fluorescence microscopy
For widefield fluorescence microscopy of PuMA lung slices, representative images and quantification data are shown in Figure 2C, and Figure 4A and B. The metastatic propensities for high and low metastatic cell lines are visually apparent over progressive time points. MNNG cel...

Watch this Scientific Journal Video about Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay at JoVE.com

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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10.0K Views.  National Institutes of Health. The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope. 

Video: Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Perfusion and Inflation of the Mouse Lung for Tumor Histology

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

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 ...

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 ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...