Name: practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay
Uploaded: 2018-03-12T13:00:00
Description: Watch this Scientific Journal Video about Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay at JoVE.com

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 ...

<ol>
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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 ...

The overall goal of this pulmonary metastasis assay is to directly visualize and study the growth of metastatic osteosarcoma cells in viable lung tissue. This method can help answer really key questions in the osteosarcoma metastasis field. Such as, how metastatic osteosarcoma cells adapt, survive, and proliferate in the lung micro environment.The main advantage of this technique is that it permits a direct visualization of metastatic osteosarcoma cell growth in a relevant three dimensional micro environment. Generally, individuals new to this method will struggle, because of the technically challenging steps of cannulating the trachea and insufflating the lung. The tail vein injection of tumor cells and mouse euthanasia are done according to standard institutional guidelines.After five minutes, place the euthanized mouse in dorsal recumbency on a sterile pad in a laminar flow hood, and use small, sterile scissors to carefully dissect out the sternum, past the thoracic inlet on both sides of the trachea, without puncturing the lungs. Dissecting all of the muscle and connective tissue surrounding the trachea is essential for a successful cannulation. Next, use a 20 gauge IV catheter to carefully cannulate the trachea and use a sterile catgut suture to loosely secure the catheter and the trachea.Attach an IV extension set from the catheter to the 10 milliliter syringe of a gravity profusion apparatus and pour 37 degrees celsius liquid agarose mixed with A medium solution into the syringe. Fill the lungs with the agarose solution until they are fully insufflated. Then remove the cannula and firmly tie off the suture to prevent leakage of the agarose solution.It is important to keep the lung intact during the insufflation. A proper insufflation with agarose and subsequent cooling of the pluck, will maintain the lung in an expanded state. Harvest the pluck, trachea, heart, and lung, taking care not to puncture or damage the surface of the lung.Place the tissues in 30 milliliters of pre-chilled PBS supplemented with antibiotics, and allow the agarose to solidify. Place the pluck on ice for 20 minutes. After 20 minutes, use fine scissors and tweezers to cut the lung into 3 x 1.5 millimeter pieces, and place the slices into individual wells of a 6 well plate containing 2 x 2 centimeter gelatin sponges pre-soaked in B medium.On the day of imaging, carefully transfer the lung slices from the 6 well plate, into a sterile 35 millimeter glass bottom round dish in columns according to their experimental group. Next, on a wide field fluorescence microscope, select the 2.5x objective and set the imaging parameters to provide the best contrast between the fluorescent tumor cells and the non fluorescing background tissue in the control tissue sample group. Image all of the samples using the same parameters as for the control tissues.If the software isn't scale calibrated, image a micrometer using the same objective as the lung tissue in order to obtain a scale reference, and save all of the images as tif files. When all of the images have been obtained, to analyze the images in ImageJ, open the first file. Use the polygon selection tool to outline the entire lung slice to determine the area of the total lung slice.Select process and subtract background. Set the rolling ball radius to a starting value of 50 pixels. This value will be dependent on the degree of background fluorescence and should be optimized for each data set.Make sure to uncheck the light background. Select image and type, 8-bit, then image and properties, enter pixels. Under unit of length, enter 1 for the pixel width, pixel height, and voxel depth.Then, check global to apply these parameters to all of the subsequent images. Next, select image, then adjust, then threshold. Highlight default and black and white, and use the slider to threshold the image so that the majority of the tumor cells are accurately highlighted.Click apply one time to turn the lung black and the fluorescent lesions white. Then, click apply a second time to invert the image so that all of the fluorescent structures turn black. To quantify the number and shape of the lesions, select analyze and set measurements, and check area.Uncheck all of the other boxes. Then, select analyze particles and in the size field use the area of the smallest lesion determined to be a single cell by ImageJ, to set the range of shapes that the program will enumerate. Use an image from day zero vehicle group to choose the smallest area that is considered to be a single tumor cell.When the lower limit has been set, click okay. A results window will pop up containing all of the enumerated shapes and area measurements. Then, copy and paste the data from the results window into a spreadsheet, and use the sum mathematical function to add all of the areas of the metastatic lesions for that particular lung slice.The metastatic propensities for high and low metastatic cell lines are visually apparent over progressive time points. More specifically, these microscopy images show how highly metastatic cells are able to colonize the lung more efficiently than low metastatic cell lines. Image analysis, quantification, and statistical analysis of the image data confirms that highly metastatic tumor cells can efficiently colonize the lung tissue.Whereas low metastatic cells cannot grow at all. High magnification confocal fluorescence microscopy allows visualization of the lung parenchyma cells in great spatial detail compared to EGFP expressing tumor cells. As well as facilitates the analysis of subcellular structures, such as these mitochondria.Depending on the number of mice used, this technique can be completed in four hours if it is performed properly. While attempting this procedure it is important to remember to work with clean, sterile surgical equipment. After it's development, the technique paved the way for researchers in the field of cancer research to explore metastasis progression in the lungs of mice.Following this procedure, other methods like in vitro cell culture assays and western blotting can be performed to answer additional questions about the effects of gene knockdown or drug treatment on cancer cells. After watching this video, you should have a good understanding of how to perform a pulmonary metastasis assay. Don't forget that working with human cancer cells can be extremely hazardous, and that precautions such as working in a bio level safety two laboratory, should always be taken while performing this procedure.

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Research

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Cancer Research

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 ...

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 ...

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 ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...