Angiogenesis play important role in tumor metastasis and progression. In this protocol, we will use dual bioluminescence imaging method to monitor the dynamic tumor progression and angiogenesis in a mouse model of breast cancer. In this model, we can track the tumor growth and angiogenesis simultaneously in the single mouse through Firefly luciferase and Renilla luciferase imaging, respectively.
This model may be widely applied in antitumor drug screening and oncology research. This dual bioluminescence model can be used to track tumor progression and regression and to detect tumor-related molecular processes in response to therapeutic strategies. Angiogenesis is a crucial process of tumor progression.
Therefore, monitoring tumor progression and angiogenesis in a visual and sensitive manner is necessary for developing effective tumor treatments. The procedures will be demonstrated by Kaiyue Zhang, Chen Wang, and Shang Chen, students from our lab. For lentivirus packaging and production seed one times 10 to the sixth of 293T cells in two milliliters of DMEM supplemented with 10%fetal bovine serum per well into a six-well plate for overnight culture in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.
The next morning mix 7.5 microliters of liposome with 250 microliters of Minimal Essential Medium in two separate 1.5-milliliter tubes for a five minute incubation at room temperature. To prepare the DNA solutions add the plasmid lentivirus RR vector and helper plasmids to 250 microliters of Minimal Essential Medium in individual 1.5-milliliter tubes as outlined in the table. At the end of the incubation gently add the DNA RR solutions to the liposome suspensions in a drop-wise manner and allow the DNA to bond to the lipid membranes for 20 minutes at room temperature.
While the mixture is incubating, replace the supernatant of the 293T cell culture with one milliliter of fresh culture medium, and gently add 0.5 milliliters of the appropriate liposome DNA mixture to each well. Return the cells to the cell culture incubator for 12 to 16 hours before replacing the supernatant in each well with one milliliter of fresh culture medium supplemented with antibiotics. After an additional 36 hours of incubation, pool the supernatants into one conical tube per lentivirus strain to sediment the 293T cells by centrifugation.
Then transfer the lentivirus RR-containing supernatants into individual 1.5-milliliter sterile polypropylene storage tubes for storage at minus 80 degrees Celsius. For lentiviral transduction for gene expression in 4T1 mammary tumor cells, replace the supernatant in each well of a six-well 4T1 cell culture plate with one milliliter of fresh RPMI-based cell culture medium supplemented with fetal bovine serum and one milliliter of lentiviral stock. Then add eight micrograms per milliliter of Polybrene to each well, and gently pipette a few times to mix.
Centrifuge the plate to help increase the transduction efficiency, and return the cells to the cell culture incubator for four to 12 hours. At the end of the incubation, replace the supernatant in each well with fresh culture medium supplemented with serum and antibiotics to remove any lentiviral particles and Polybrene. To select for the lentivirus-transduced cells, passage the transduced 4T1 cell cultures at a one-to-three to one-to-four ratio with selection medium, changing the medium every two to three days.
After seven days of selection medium culture, view the transduced cell cultures under a fluorescence inverted phase contrast microscope and count the number of red fluorescent protein-positive or RFP-4T1 cells and all of the 4T1 cells in three fields of vision to estimate the RFP-positive ratio. To set up a tumor-bearing mouse model, wash 80%confluent transduced cell cultures with PBS before detaching the cells with two milliliters of trypsin-EDTA per 60-millimeter culture plate. When the cells have lifted from the plate bottoms stop the reaction with five to 10 milliliters of serum supplemented medium per plate.
And transfer the cultures into individual 15-milliliter centrifuge tubes for counting. Dilute the cells to a one times 10 to the sixth cells per 100 microliters of culture medium without serum concentration. And subcutaneously inject 100 microliters of the 4T1-RR cell line into the left shoulder of an anesthetized tumor-bearing model mouse, and 100 microliters of the 4T1-RRT cell line into the right shoulder of the same mouse.
After the injections, place each mouse in an appropriate recovery area with a thermal support until fully recovered. Palpate the tumor masses every day for seven days to confirm that the mice are tumor-bearing. At day seven post-implantation, intraperitoneally inject 50 micrograms per kilogram of Ganciclovir into each tumor-bearing mouse two times per day until the end of the experiment.
For tumor growth, open the Living Imaging System. Initialize the Living Imaging software and initialize the system. While the system is initializing, use an insulin syringe to inject each mouse to be imaged with the appropriate volume of coelenterazine into the retrobulbar.
Place the injected mice into the camera chamber and immediately obtain several pictures of the mouse dorsally to acquire the Renilla luciferase signals from 4T1 cells until the bioluminescent signals fade away. 10 minutes after the Renilla luciferase imaging, use an insulin syringe to intraperitoneally inject the appropriate volume of D-luciferin into each animal. 10 minutes after the D-luciferin injection, place the mice into the camera chamber and obtain several images of the mouse dorsal to acquire the Firefly luciferase signals from angiogenesis.
After lentivirus transduction, more than 99%of the cells are RFP-positive with no differences in the cell culture morphology observed between the two transduced cell lines. Subsequently, bioluminescence imaging of the transduced cells reveals that both cell lines emit strong bioluminescent signals of a similar strength. After subcutaneous injection of the transduced cell lines into a transgenic tumor-bearing mouse model, angiogenesis-induced tumor growth can be evaluated by Firefly luciferase signals in the presence of D-luciferin in the same animal.
At seven day post-implantation, Ganciclovir administration induces death in the 4T1-RRT cells resulting in a dramatic reduction in the Renilla luciferase signal in the tumors established by these cells. Firefly luciferase signals also increase in conjunction with an increase in Renilla luciferase expression and decrease following luciferase decline. Taken together, these results suggest that there is a direct correlation between tumor angiogenesis and tumor growth.
Indeed, Ganciclovir-induced tumor cell death may lead to an inhibition of tumor angiogenesis. Further, immunostaining for anti-vascular endothelial growth factor receptor II, as a marker of angiogenesis, reveals a more significantly established microvascular structure in 4T1-RR tumor tissue sections than in sections from 4T1-RRT tumors, consistent with the decline in Firefly luciferase signal observed after Ganciclovir administration. The most critical step of the protocol is using two kinds of luciferase, such as FLuc and RLuc for simultaneous tracking of the cells and the angiogenesis.
This technology could be used to treat cancer cells within different locations, and it can be used widely in virus cancer models. This mouse model allowing the tumor growth and the anti-tumor effects of the HSV-ttk/GCV tracking system to be visualized dynamically in a living animal. A biosafety level II facility is required to prevent a lentivirus transfection, as the media contains lentivirus particles that could be harmful to humans.
