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

Summary

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.

Abstract

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.

Introduction

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 metastases^5,7,8. Other experimental metastasis models include injection of tumor cells into the spleen or mesenteric vein which results in the development of liver metastases^9,10. 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 & 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.

Protocol

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.

1. Pre-warm 5 mL of A-Media in a 15 mL conical tube in a 37 ^oC water bath.
2. Melt the 1.2% low melting agarose solution (in sterile water) using the lab microwave.
3. 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.
4. Pre-chill 30 mL of cell-culture grade PBS supplemented with 1X pen/strep in an ice bucket.
5. 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.
6. 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 μL will be used to inject into the tail vein of 1 mouse. Upscale accordingly if more mice are used for the study.
7. 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.
8. 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.
9. 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.
10. Inject 5 x 10⁵ cells in a volume of 100 μL. 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.
11. Place cell suspension in an ice bucket while preparing the mice for injection.

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.

1. Warm a female mouse (age 6-8 weeks) under a heating lamp for 5 min 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.
2. 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.
3. Place the mouse in the restrainer, and swab the tail with an alcohol wipe.
4. Proceed to perform a tail vein injection and inject the 100 μL of the cell suspension. 5 min 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.
5. 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).
6. 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.
7. 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.
8. Cannulate the trachea with a 20 gauge IV catheter. Loosely tie a surgical knot using sterile catgut suture around the cannulated trachea.
9. Attach an IV extension set from the catheterized trachea to the 10 mL syringe of the gravity perfusion apparatus.
10. 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.
11. Fill the lung the agarose/A-media solution until the lung is fully insufflated.
12. 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.
13. 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.
14. 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.
15. 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.

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:

1. 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.
2. 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.
3. 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.
4. For a scale reference, take a digital picture of a micrometer at the same objective.
5. 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.

Image Analysis:
NOTE: The following image processing steps are done with ImageJ 1.51h software package ¹⁷.

1. Open an image file in ImageJ (Figure 3A).
2. Subtract background: Process > Subtract Background > Rolling ball radius (start with 50 pixels), uncheck "Light Background" (Figure 3B).
3. Convert image to 8-bit format: Image > Type > 8-bit (Figure 3C).
4. Set units to pixels: Image > Enter "Pixels" in Unit of Length, enter 1 in "Pixel width", "Pixel Height", "Voxel Depth". Check "Global" box to apply to subsequent images.
5. 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.
6. Threshold image: Image > Adjust > Threshold > highlight "Default" and "B&W" > use the slider to threshold the image such that the majority of tumor cells are accurately highlighted. Pressing "Apply" will result in a black and white image where the lung is all black, and the fluorescent lesions are white (Figure 3D). Pressing "Apply" a second time will invert the image where all fluorescent structures are now black shapes (Figure 3E).
7. Quantification of the number and shape of lesions:
   Analyze > Set Measurements > Check "Area". Uncheck all other boxes.
   Analyze Particles > 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 "Circularity" range as 0-1. "Show" can be set to "Nothing". Alternatively, if "Show" and "Outlines" is selected, a drawing of all outlined shapes is generated. Check "Display results" for a separate window of measurements to pop up. Optionally, having "Add to manager" box checked will save all the shapes quantified to a ROI manager, which can be saved for future reference. After pressing OK, a "Results" window will pop up containing all enumerated shapes and area measurements (Figure 3F).
8. Copy and paste the data from the "Results" 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 (A_A) which is described further by Underwood ¹⁸.
   Lung tumor burden = Sum of metastatic lesion areas/total area of lung slice
9. 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).

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:

1. Immerse lung slices in 10 μμM of DAR4M (in HBSS) for 45 min at 37 °C. DAR4M labels reactive nitrogen species within the cells of the lung.
2. After 45 min of incubation, rinse the lung slices with fresh HBSS.
3. Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.
4. The imaging parameters for the example images shown in Figure 5 are listed in Table 5.
5. 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.

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 tissues^19,20.
Imaging mito-RFP expressing MG63 cells in the PuMA model:

1. Perform the PuMA protocol as outline in Step 1 and 2 with using MG63 cells expressing the mito-RFP construct.
2. Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.
3. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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