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.
