The overall goal of the following experiment is to non-invasively assess tumor hypoxia dynamics in orthotopic breast cancer brain metastases in a mouse model. This is achieved by stably transducing breast cancer MDA MB 2 31 cells with the hypoxia reporter construct. Then in vitro luciferase assays are used to ensure successful transfection and expression of the hypoxia reporter construct.
Next in vivo bioluminescence imaging is performed to monitor longitudinal changes in tumor hypoxia of the intracranial metastases. Results show the initiation and evolution of tumor hypoxia of breast cancer brain metastases based on bioluminescence imaging of the expression pattern of the hypoxia reporter gene. The main advantage of this technique over existing methods such as EOL oxygen electrode, is that this matter is totally noninvasive and allows inva monitoring of dynamic change longitudinally.
The implications of this technique extend toward therapy or diagnosis of brain tumors because tumor hypoxia reduces the effectiveness of conventional therapy such as radiation. Therefore, detecting the extent of tumor hypoxia will be helpful in designing therapeutic interventions. First culture, the MDA MB 2 3 1 5 H-R-E-O-D-D luciferase cells, which are a human metastatic breast cancer cell line stably transfected with a novel HIF one alpha dependent reporto gene in supplemented DMEM medium for the in vitro HIF one alpha bioluminescence assay plate three times 10 to the five MDA MB 2 3 1 5 H-R-E-O-D-D luciferase cells in each well of two six well plates incubate the cells overnight in a tissue culture incubator set at 37 degrees Celsius and 5%carbon dioxide to allow the cells to attach to the dish.
The next day. Place one of the six well plates into the hypoxia chamber, along with a dish containing 10 to 20 milliliters of sterile water to prevent EVA aberration of the cultures. The second plate remains in the tissue culture incubator at 21%oxygen and serves as a normoxia control.
Connect the inlet port tubing to a gas cylinder containing 0.1%oxygen, 5%carbon dioxide, and 94.9%nitrogen. Open both the inlet and outlook port clamps and gas the hypoxia chamber to achieve a 0.1%oxygen concentration. Disconnect the gas source and seal the chamber by closing the plastic clamps.
Then place the chamber into the tissue culture incubator for 24 hours. After 24 hours, perform the bioluminescence assay first aspirate the media from both six well plates and quickly wash the cells twice with ice cold PBS. Then add one milliliter, revised cold PBS containing 100 microliters of Lucifer in acquired by luminescence images, using the caliper Enogen spectrum with exposure times of 1 30, 60 and 180 seconds.
And measure the intensity of bioluminescence in each well using living imaging software on the day of intracranial injection, harvest the MDA MB 2 3 1 5 H-R-E-O-D-D luciferase cells at 80%confluence by trypsin and centrifugation. Re suspend the pellet in fresh, medium and perform a cell count. Dilute the cell suspension to a final concentration of 10 to the five cells in a four microliter volume and place the resuspended cells on ice after anesthetizing.
Four to six week old female nude mice with 3%ice of fluorine mixed with oxygen. In an induction chamber, transfer the animal to a nose cone administering 2%isof fluorine to maintain anesthesia, sterilize the scalp by wiping with Betadine solution and then 70%alcohol. Next, using a sharp scalpel, perform a midline incision and expose the right hemisphere of the skull.
Identify the point that is one millimeter anterior to the coronal suture and two millimeters lateral to the sagittal suture. Use a high speed drill to bur a one millimeter hole. At this point, draw the four microliters of cell suspension into a 10 microliter Hamilton syringe.
Fitted with a 32 gauge Hamilton needle. Position the Hamilton syringe over the hole in the skull. Carefully insert the tip of the needle one millimeter below the URA mater.
Once this depth is reached, carefully depress the plunger of the syringe to inject the cells directly into the right corridor. D kelon. Once the cells have been injected, wait around 30 seconds, then carefully withdraw the needle.
Fill the bur hole with bone, close the scalp with absorbable sutures and sterilize the region with 70%alcohol. Administer the first buprenorphine injection for analgesia. Buprenorphine analgesia is administered every 12 hours for two days.
Ensure that the animal has fully recovered from the anesthetic. Then return to the home cage. Two weeks after intracranial implantation of cells, initiate longitudinal bioluminescence using the ivus spectrum system.
After simultaneously anesthetizing three mice with 3%isof, fluorine, and oxygen. Administer a solution of 120 milligrams per kilogram Lucifer in a total volume of 80 microliters in the nape of the neck. Immediately after the administration of the injection, set a timer to five minutes.
Place the mice into the imaging chamber and maintain anesthesia with 1%isof, fluorine, and oxygen at a flow rate of one cubic decimeter per minute. After five minutes have elapsed from the time of Luciferian injection. Acquire bioluminescence images with exposure times of 1 30 60 and 180 seconds.
After capturing the final image, remove the animals from the chamber and place them back in the home cage to allow them to recover from anesthesia. Analyze the data using the living imaging software manually draw a region of interest to outline the bioluminescence signal and then use absolute photon counts per second to quantify the intensity of the signal. Repeat bioluminescence imaging once a week for eight to 10 weeks.
Then immediately after the last session of bioluminescence imaging administer piol the hypoxia marker and sacrifice the mice. One hour later after removing the whole brain's embed in optimal cutting temperature medium and freezing the minus 80 degrees Celsius freezer. Subsequent histological crestal violet staining and immunohistochemical staining against luciferase hypoxia.
Marker OLE and H one alpha is performed to validate imaging observations. This image shows representative results from the in vitro HIF one alpha bioluminescence assay. The scale bar on the right shows from bottom to top.
The pseudo color changes associated with increasing bioluminescence. The upper two wells of MDAM 2 3 1 5 HEDD Lucifer cells were maintained under hypoxic conditions. According to the procedure described in this video article, the green color indicates increased transcription of the luciferase reporter from the HIF one alpha promoter in response to hypoxic conditions.
The lower two wells are control wells maintained under normoxia and only emitted very low levels of bioluminescence. This image shows the results of longitudinal in vivo bioluminescence imaging of tumor hypoxia dynamics. The same animal was imaged once a week from five to 10 weeks after intracranial administration of M-D-A-M-B 2 31 5 H-R-E-O-D-D luciferase cells.
A weak light signal from the right side of the mouse brain was first visualized. Five weeks after intracranial implantation of the breast cancer cells increased optical signal was observed over additional six weeks indicating increased tumor hypoxia. This plot shows the time course curve of quantitative photon counts of light signal over the six weeks of imaging.
This image shows a 10 micron section of a frozen mouse brain bearing a metastasis of breast cancer immunostain with the hypoxic marker pi Monosol al intratumoral heterogeneity of hypoxia is apparent here, the same section as immunostain with anti luciferase antibody. This overlay image shows that intratumoral heterogeneity of hypoxia correlates spatially with lucifer's expression. After watching this video, you should have good understanding of how to apply bio bioluminescence imaging to monitor intracranial tumor hypoxia dynamics based on their hypoxia reported gene HRE look.
