The overall goal of this procedure is to use OV luminescence imaging to monitor cancer therapy in tumor bearing mice. This is accomplished by first growing subcutaneous tumor xenografts for both treatment and control groups. The second step is to perform small animal PET studies for validation purposes.
Next, the optical imaging studies are performed immediately after PET studies. The final step involves quantifications of PET and clea images and subsequent cross validations of the two imaging modalities. Ultimately, results are obtained to show that CLEA is a viable alternative to pet in monitoring preclinical cancer drug therapy.
The main advantage of chew called Luce Imaging or clay over existing nuclear imaging modalities like PET is that clay takes advantage of the lower cost and the widely available optical imaging instruments to image radionuclides, which could only be imaged by nuclear imaging modalities in the past. The implications of this technique extend toward preclinical applications such as cancer drug screening, thanks to please short acquisition time, high sensitivity, high throughput, low cost wide availability, and a flat learning curve. For this protocol, the choice of cell lines, culture, mediums, and other factors should be tailored to the goals of a particular research study.
To begin the project design presented here, first culture H four 60 cells in RPMI 1640, medium supplemented with 10%fetal bovine serum and 1%penicillin streptomycin. Next, maintain the sound lines in a humidified atmosphere of 5%carbon dioxide at 37 degrees Celsius. Be sure to change to fresh medium every other day.
Then once a 75%confluent monolayer of cells has formed, detach the monolayer with trypsin and dissociate the cells into a single cell suspension in PBS for inoculation. Suspend approximately one times 10 to the sixth H four 60 cells in phosphate buffered saline. Then implant the cell suspension subcutaneously in both left and right shoulders.
A four to six week old female athymic nude mice. The location of inoculation and number of xenografts per mouse should be tailored to the specific study goals. Allow tumors to grow to 150 to 200 cubic millimeters using standard caliper measurement to track tumor sizes.
Note that it will take approximately two weeks for H four 60 tumor xenografts to grow to this size. Once tumors reach the ideal size, the tumor bearing mice are ready for treatment. Followed by in vivo imaging with both PET and chiho luminescence imaging or clea.
Perform PET studies according to the schedule seen here, or adjust according to a specific project. The CLEA studies should follow the same schedule as the PET studies with clea performed immediately after the corresponding pet. The main purpose of the PET studies is for validation of the CLEA results.
When using PET for validation, the PET and clea instruments must be located within close proximity for the validation to be successful. To begin this phase of the experiment, first, divide the tumor bearing mice into treatment and control groups. To begin this phase of the experiment, first, divide the tumor bearing mice into treatment and control groups.
One day pre injection of bevacizumab, perform a pre-scan with both PET and clea. Next for the treatment group, administer the first injection of 20 milligrams per kilogram of bevacizumab. Define this as day zero, the day of first injection.
Also administer the same dosage again on day two, the day after each injection being day one and day three of the experiment perform pet, followed by clea first anesthetize all mice with 2%isof, fluorine, and assure the animals are fully anesthetized. Then using the pet probe diluted in saline, inject via the tail vein. Be sure to follow all radiation safety protocols at your institution and use radiation protective barriers and personal badge dosimeters then after one hour, fully anesthetize the mice again and place prone and near the center of field of view of the small animal pet scanner.
Now obtain three minute static scans and reconstruct the images using a two dimensional ordered subsets expectation. Maximum algorithm background correction is not necessary. Next, draw regions of interest with five pixels for coronal and trans AAL slices over the tumors on the decay corrected.
Whole body coronal images obtain the maximum counts per pixel per minute from the regions of interest and convert to counts per milliliter per minute by use of a calibration constant. Here clea is performed with an IVUS spectrum system, and acquisition and analysis of images are carried out using living image 3.0 software. As a reminder for each mouse, perform clea immediately after pet.
To minimize the amount of radioactive decay, begin by placing animals in a light tight chamber. Under Isof fluorine anesthesia, multiple mice can be placed simultaneously to increase throughput. A special lead shielding is used for protection from radioactivity, acquire images using a three minute exposure time.
Use the same illumination settings for lamp voltage filters. F-stop fields of view and bending to acquire all images. Next, use the dorsal skin area to calculate the signal intensity of background tissue and normalize fluorescence emission to photons per second per centimeter squared per Sterrad.
Here we see that both clea and PET revealed significantly decreased signals from H four 60 xenografts in treated mice from pre-treatment to day three, suggesting significant therapeutic effect. Visual inspection shows good consistency between tumor contrast visualized from clea and PET resolution is sufficient to show central necrosis of the tumor secondary to the anti-cancer treatment regimen. Here we can see the quantifications of clea and PET images and a simple fitting via linear regression showed that the two modalities are highly correlated.
Clea can be performed along with pet validation as we did in this video. However, we can certainly perform standalone clea as a good correlation with PET has already been established in the literature while attempting this procedure. Along with PET validation, it's important to remember to minimize the downtime between the two procedures, particularly if a radioisotope with short half-life is used after its development.
This technique paved a way for researchers in the field of cancer, drug development, cancer biology, and the clinical oncology to effectively monitor cancer drug therapy without having to use nuclear imaging modalities and this potentially benefiting development of preclinical drug screening, clinical therapy monitoring, and a patient stratification among other applications.
