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, ....

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.<....

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.