Our protocol can be used to quickly and easily test the role of any candidate gene in metastatic colonization and growth. The protocol also allows realtime monitoring of metastasis, formation, and growth, as well as quantification of metastasis number and size in the same animals without the need for resectioning or histological staining. Plate four T1 cells at 150, 000 cells per well in a 12-well plate in complete growth media.
Incubate at 37 degrees Celsius 5%carbon dioxide overnight. The next day, aspirate the growth media from the cells. Add 500 microliters of luciferase viral supernatant and 500 microliters of fluorescent protein viral supernatant to simultaneously infect the cells with both the luciferase and fluorescent protein viral supernatants.
Then, add one microliter of eight milligram per milliliter hexadimethrine bromide and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 to 48 hours. Trypsinize the four T1 cells with 500 microliters of trypsin. After two to five minutes, transfer all the cells to a six centimeter dish in four milliliters of selection media, thereby quenching the trypsin.
After selection is complete, confirm that the infected four T1 cells are expressing ZsGreen fluorescent protein using an inverted fluorescent microscope at 50 to 100 times magnification. Also confirm that the infected four T1 cells are expressing luciferase using a commercially available luciferase activity kit. Proceed to perform optimization of the in vivo experimental design as described in the text protocol.
Aspirate the media and rinse the cell plates with 1X PBS. Trypsinize the cells with five milliliters of trypsin per 15 centimeter plate for two to five minutes and transfer all of the cells to a conical tube. Wash remaining cells from the tissue culture dish with enough complete growth media to quench the trypsin.
Add the wash to the same conical tube. Count the cells using an automated cell counter to determine the total cell number. Next, centrifuge the cells at 122 times G for three minutes, and aspirate the supernatant.
Re-suspend the cells in 1X PBS at the desired concentration. Here, 25, 000 cells are injected into each mouse and 100 microliters of PBS. So the re-suspended cells are at 250, 000 cells per milliliter.
Keep the cell suspensions on ice until injection. Working in a hood at the animal facility, gently but thoroughly mix the cells by inverting the tube or using a one milliliter syringe to ensure that they are uniformly re-suspended. Now, load a one milliliter Luer Lock syringe with the cell suspension and expel excess air bubbles.
Place a half inch 30 gauge needle on the syringe with the bevel up and expel air bubbles. Gently place the mouse in a rodent restrainer. The lateral tail vein should be visible and dilated.
If not, gently pinch the base of the tail and dip the tail in warm tap water to dilate the veins. Use an alcohol wipe to clean the tail, then insert the needle into the tail vein, bevel side up, and inject 100 microliters of cell suspension. If the needle is inserted properly into the vein, it should easily slide slightly forward and back, and there should not be resistance when the plunger is pushed.
Successful injections should also result in a flush in which the blue color of the vein turns white for a few seconds following the injection. Slowly remove the needle, and using a sterile gauze, apply pressure to the injection site to stop any bleeding. Return the mouse to its cage and monitor for 15 minutes to ensure full recovery.
Turn on the in vivo live animal imaging device, open the image software, and log in. Click the initialize button and wait for the machine to initialize. Change the field of view to D.For first time use, edit exposure settings by clicking edit, preferences, acquisition, and auto exposure.
Change the maximum exposure time from the default 60 seconds to 300 seconds and click okay. Load a one milliliter syringe with D-luciferin, then add a half inch 30 gauge needle to the syringe and expel air bubbles. Measure and record the mass of an anesthetized mouse.
Restrain the mouse by pinching the scruff of the neck using the thumb and pointer fingers and grasping the tail between the pinky finger and the base of the hand. Invert the mouse at a 45 degree angle with its head pointing downward. Insert the needle, bevel side up, into the mouse's left side IP space.
Confirm entry into the IP space by drawing back a small volume. There should not be color at the base of the needle when drawing back in the IP space. Inject the appropriate volume of D-luciferin for a dose of 150 milligrams per kilogram.
Immediately after D-luciferin administration, start a timer. Place the mouse flat on its back in the imaging device with its nose in the nose cone, and ensure that one half to two and a half percent isoflurane is being administered. Click the luminescent and photograph boxes.
Change the exposure time to auto, and then click acquire in the acquisition control panel. After imaging, return the mouse to its cage and monitor for 15 minutes to ensure full recovery. Following euthanasia, as described in the text protocol, isolate and remove the lungs from each mouse and rinse in 1X PBS to remove excess blood.
Acquire images of ZsGreen metastases in the lobes at 10X in Brightfield and fluorescence using a fluorescent stereoscope with a GFP wide band filter. Use image analysis software to quantify the size and number of metastases from the images. To process and analyze the data from the images acquired with the in vivo live animal imaging device, open all image files for each mouse in the image software.
Ensure that the units are in radiance for bioluminescent data and efficiency for fluorescent data by clicking the arrow at the top left of the image window and changing it to the appropriate unit. Use the image from the last time point to create a region of interest, or ROI, by first clicking ROI tools in the tool palette window. Then, insert one ROI by clicking the arrow and selecting one.
Click the border of the ROI and move it over to the chest of the mouse. Adjust the size of the ROI so it covers the chest of the mouse and does not exclude signal. Then, click measure ROIs and copy or type the raw number into an Excel sheet.
Plot and analyze the data as described in the text protocol. Right click in the image file to copy the ROI, then paste it into image files from the other time points and click measure ROIs. Retroviral vector expressing tandem miR-30-based shRNAs targeting both YAP and TAZ reduces YAP and tax protein expression and transcriptional activity in four T1 cells.
Four T1 luciferase cells were stably transduced with a ZsGreen expressing version of this tandem YAP/TAZ shRNA vector or a controlled miR-30-based shRNA. Bioluminescent images show that the rate at which metastatic burden increased was significantly faster in the mice injected with control cells compared to the mice injected with YAP/TAZ knockdown cells. A plot of the log10 transformed luciferase signal measured for each mouse over the course of the experiment also shows that the rate was faster in the mice injected with the control cells compared to the mice injected with YAP/TAZ knockdown cells.
Significantly fewer metastases formed in the mice injected with the YAP/TAZ knockdown cells compared to mice with control cells when counted manually. The same trend was observed when using the image analysis software. Not only did many more metastases form in the control mice, but they were generally larger.
Consistently, the metastatic burden in the lungs was also drastically reduced by YAP/TAZ knockdown. Successful injection of equal numbers of healthy cells into each mouse is critical to ensure high quality data. Remember to use caution and follow safety guidelines when working with infectious retroviruses and lentiviruses, particularly if the viruses are human infectious.
This technique has allowed us to illustrate the roles of candidate genes and metastatic colonization and growth, providing a way to identify and test novel therapeutic targets for the treatment of metastatic disease. Following this procedure, metastases containing lungs can be further analyzed by immunofluorescent or histological staining to investigate how the altered gene is influencing metastases and identify other proteins that may be involved.
