Using this sophisticated, non-invasive tool, we can real-time monitor tumor growth kinetics and metastatic colonization in mice, which has a great potential in breast cancer therapeutic and disease management. Combining luciferase and fluorescence detection is a helpful strategy to advance the preclinical studies of breast cancer progression and disease management. This can be applied for anticancer drug studies.
This protocol is not restricted to breast cancer and could be applied to other carcinomas, such as lung and pancreatic. Start by growing the MCF-7, MDA-MB-468, and MDA-MB-231 GFP and luciferase-positive cells separately in a 16-centimeter plate to 80%confluency. After harvesting the cells by trypsinization, seed an increasing number of cells in each well into a black 96-well plate.
Then, fill all the wells with 100 microliters of DMEM, and incubate for 16 to 24 hours in 37 degrees Celsius, 5%CO2 incubator. Prepare luciferin solution in PBS at a 1.5-milligrams-per-milliliter concentration, and store the solution at minus 20 degrees Celsius. Post-incubation, wash the cells once with PBS gently before adding 100 microliters of luciferin solution into each well.
Wait for two minutes, and then measure the luciferase activity in all the breast cancer cells using bioluminescence. Before injecting the mouse, transfer five million MCF-7 and MDA-MB-468 cells into 200 microliters of PBS and two million MDA-MB-231 GFP and luciferase-positive cells into 100 microliters PBS. Prepare the anesthetized mouse for injection by placing a cone over the head in a supine position.
Next, wipe the abdominal area of the mouse above the mammary gland with ethanol using a cotton swab, and lift the fourth mammary gland with forceps before inserting a 27-gauge needle under the fat pad to slowly inject 100 microliters of the prepared cell suspension. After the injection, take the mouse out of the hood and transfer it to a new cage under observation until it returns to consciousness. Before the bioluminescence detection, restrain the conscious mouse by holding its neck with the left hand and tilting the hand to the left, resulting in the face of the mouse upward with the lower body in a supine position.
Inject 100 microliters of 30-milligrams-per-milliliter luciferin intraperitoneally into the lower left abdominal quadrant of the mouse using a one-milliliter syringe. Keep the mouse for seven minutes without anesthesia, followed by three minutes within the anesthesia chamber before measuring the tumor kinetics. While the mouse is under incubation, open the software and initialize the imaging system by clicking the Initialize button.
Keep the setup in auto exposure with exposure time in the auto at 60 seconds, binning medium at an aperture of f/stop one, excitation filter blocked, and emission filter open. When initialization ends, select Imaging Wizard and Bioluminescence and then click Next, Open Filter to select mouse in the image subject. In the field view, select stage C at 10 centimeters and subject height at 1.50 centimeters.
Then, click Image Setup Stage to C.Ensure that the mouse is placed on the stage in the proper supine position before closing the door and clicking the Acquire button. Wait for an image to appear on the screen. Repeat the procedure for the other mouse.
To detect lung metastasis, cover the strong signal of the primary tumor using a thick cardboard paper and expose only the ventral side of the lungs towards the camera. Capture the image using the same parameters as described earlier. The breast cancer cell lines were infected with a lentivirus vector expressing fluorescent GFP and subject to FAC sorting.
The GFP-positive cells were sorted two days post-infection, plated and visualized by fluorescence microscope. The in vitro luciferase activity was confirmed with a cell number-dependent increase in the luciferase activity levels. In addition, a linear correlation was found between the luciferase activity and the cell number.
After injecting the breast cancer cell lines, the mice were subjected to bioluminescence reading every two weeks to determine the tumor growth kinetics. The tumor growth kinetics was faster in the MDA-MB-231 cell lines than in the less aggressive cell lines, MCF-7 and MDA-MB-468. The luminescence activity was quantified for three cell lines.
Six weeks post-injection, the harvested tumors were found to maintain the GFP expression. Also, positive bioluminescence readings were obtained from the lung of the whole mouse in real time. The lung was harvested to verify positive metastatic colonies, and the metastatic colonies were observed for GFP and bioluminescence.
It is more important to be careful while performing the injection procedure. An improper injection may lead to deviation in the tumor growth rate or absence of tumor. We can examine the lung metastasis in vivo and ex vivo by detecting GFP expression.
In addition, we can also study the histopathology of lungs by immunohistochemistry-based staining. This technique can be useful to explore the new questions, such as the effect of drug efficacy studies.
