The overall goal of this experimental procedure is to establish a preclinical brain tumor imaging model using fluorescence molecular tomography or FMT. This method can help answer key questions in the fields of brain tumor biology, cancer research, and drug discovery about tumor aggressiveness, heterogeneity, and treatment responsiveness. The main advantage of this technique is that the tumors can be monitored in vivo before, during, or after treatment in a non-invasive, suture free, and quantitative manner.
Another advantage of this technique is the possibility to apply to other types of xenograft models implanted in different organs of the animal. Begin by seeding one times 10 to the sixth glioblastoma cells in five milliliters of medium into a 10 centimeter dish for 24 hour incubation in a cell culture incubator. The next morning, transduce the cells with lentivirus, expressing an appropriate fluorescent protein of interest at a multiplicity of infection of five and return the cells to the incubator.
After 24 hours, replace the supernatant with five milliliters of fresh medium and incubate the culture for another 48 hours. At the end of the incubation, replace the medium with three to five milliliters of tripson for 10 to 15 minutes at 37 degrees Celsius followed by careful pipetting to fully dissociate the detached cells. Collect the cells by centrifugation and re-suspend the pellet in 500 microliters of sorting solution.
Split the cells into the appropriate number of fax tubes and co-stain the cells with DAPI for dead cell exclusion. Then load the tubes onto the flow cytometer and sort the live cells according to their fluorescent construct expression into a 15 milliliter conical tube containing fresh sorting solution. After centrifugation, re-suspend the construct positive, DAPI negative cells in five milliliters of culture medium and seed them onto a new 10 centimeter dish for their culture at 37 degrees Celsius for 48 to 72 hours.
At the end of the incubation, split the cells for seeding into multiple dishes for subsequent in vivo experiments. On the day of the injection, dissociate the fluorescent positive gliomacells into a single cell suspension and re-suspend them at a 0.5 or one times 10 to the sixth cells per two to five microliters of PBS per injection per animal concentration in a 1.5 milliliter microcentrifuge tube on ice. After confirming a lack of response to toe pinch, apply ointment to the eyes of the immuno-deficient Athymic Nude recipient mouse and load a five microliter Hamilton syringe equipped with a blunt tipped needle with the cells for the injection.
Mount the syringe into a probe holder and place the mouse in a small, animal sterotactic frame. Fix the head using the ear bars and the incisor adaptor and disinfect the head with 70%ethanol and batadine solution. Using a small scalpel, make a middle incision and separate the skin and connective tissues.
Using the micromanipulator, place the Hamilton syringe on the bregma point and move the probe holder one millimeter anteroposterior and two millimeters lateral from the bregma point. After marking the position with a pencil, use a micromotor handheld drill to carefully make a hole in the skull, applying a slight downward pressure until the blood vessels become visible. Introduce the needle into the burr hole three millimeters below the pial surface and inject the cells at a one microliter per minute flow right.
When the full experimental cell volume has been injected, gradually remove the needle at a one millimeter per minute ascension rate and clean the injection site with 70%ethanol. Then close the skin wound according to standard protocols and monitor the animal on a warming pad until it has fully recovered. To image the injected cells, place the anesthetized recipient animal in the imaging cassette of a fluorescence molecular tomography imager, head adapter first in the prone position, with the head in the center of the cassette.
With the cassette closed, tighten the adjustment knobs to 17 millimeters. When the animal is secure, insert the cassette into the internal docking station and open the imager and analyzer software. In the experimental tab window, select the appropriate database and study group and open the scan tab window.
Click select subject to select the subject to image and select the laser channel in the laser channel panel. Click capture to acquire an image and click and drag that scan field in the captured image to identify the source locations. Check the add to reconstruction que option and click scan in the scan tab window.
When the scanning is complete, remove the imaging cassette from the docking station and return the animal to its cage with monitoring until full recovery. To analyze the images, in the imager and analyzer software open the analysis tab window and click the plus sign button in the data set selection panel to load the data set and subject for analysis. Then use the ellipsoid icon to select the region of interest and right click the threshold column to adjust the threshold to zero in the statistic data panel.
Glioblastoma cells labeled with near-infrared fluorescent proteins, as just demonstrated, exhibit distinct fluorescent profiles that can be distinguished by fluorescence molecular tomography, even up to five days after injection. Further, the lack of background signal between the constructs facilitates dual in vivo imaging of cancer cell populations of interest. In this experiment, fluorescent construct labeled glioblastoma spheres co-transduced with lentivirus encoding SHRNA targeting death-domain associated protein were injected into recipient immunodeficient Athymic Nude mice, as just demonstrated.
Reverse transcription quantitative preliminaries chain reaction confirmed the efficacy of the SHRNA targeting in both cancer cell lines and fluorescence molecular tomography revealed a decrease in fluorescence signal in the mice engrafted with SHRNA glioblastoma spheres compared to animals implanted with SH control transduced cells. After watching this video, you should have a good understanding of how to generate preclinical data for brain cancer research using a fluorescence molecular tomography system.
