The overall goal of this protocol is to track tumor growth and metastasis in live rodents via imaging. A radionuclidefluorescence fusion reporter allows non invasive in vivo detection by positron emission tomography with the fluorophor aiding ex vivo confirmation of results. This method can help answer key questions whenever cells need to be tracked in live animals over an extended time period.
It can contribute to the understanding of distant metastasis and the impact of treatments on tumor progression. The technique described is cancer cell tracking by highly sensitive and non invasive in vivo imaging with a PET tracer produced by automated synthesis. It offers a significant reduction of animal use as compared to conventional methodology.
Individuals new to this method may feel overwhelmed by its complexity. Particularly the automated synthesis of the PET radiotracer significantly simplifies this process. We believe visual demonstration of this method demonstrates these views of the critical imaging associated steps.
The implications of this technique extend towards preclinical testing of new therapeutics which can also be tracked alongside the cancer cells. This method can provide insight into cancer biology and the development of treatments. Whenever in vivo localization, relocation, expansion, and long-term monitoring of a certain population is of interest.
To begin, use reporter gene plasmids, generate Lentivirus particles, transduce cells and characterize them. Then analyze reporter gene function by radiotracer uptake in the NISFP expressing cell lines. Seed the purified cells and growth medium in six well plates, ensuring all samples are prepared in triplicate.
The next morning wash the cells with serum free growth medium and incubate the plates with 50 kilo becquerels of F18 tetrafluoroborate at 37 degrees Celsius for 30 minutes. Then collect the supernatant and transfer 100 microliters to a pre-labeled collection tube. Discard the remaining supernatant, then wash the cells with one milliliter of ice cold PVS containing calcium and magnesium at physiological levels.
Collect the wash solution and transfer 100 microliters of the wash solution to a pre-labeled collection tube. Then repeat the washing process once more. Add 500 microliters of PVS containing both trypsin and EDTA to lift the cells.
Incubate the samples at 37 degrees Celsius until the cells detach, using a microscope to check for detachment. Then transfer the cell suspension to a labeled collection tube. Next centrifuge the cell samples at 250 G for four minutes at four degrees Celsius.
Use an automated gamma counter to count the samples from each of the four sample types and obtain the percent uptake using equation one. To begin F18 tetrafluoroborate synthesis, set up the automated radio synthesis platform in an appropriate chemical hood and ensure the correct XML file is loaded onto the control computer. While the synthesizer runs, F18 tetrafuoroborate will be produced and then purified on an anionic exchange cartridge.
The anion exchange cartridge will be rinsed with water and then dried with nitrogen gas. The final product is alluded from the anion exchange cartridge with one milliliter of 0.9%sodium chloride and transferred into a glass collection vial via the outlet line. To begin imaging, anesthetize and prepare mice according to the written protocol.
Then use sterile 0.9%saline to dilute the F18 tetrafluoroborate solution to 5 megabecquerels per 50 microliters. Use a syringe with a hypodermic needle to draw 100 microliters of the F18 tetrafluoroborate solution. Measure the radioactivity in the syringe and record the value and the time of measurement.
Administer 50 microliters of the F18 tetrafluoroborate solution into the pre-warmed tail vain intravenously. Then measure the remaining radioactivity in the syringe and record the value and time of measurement. Tail vein injection of the radiotracer is critical.
We perform it under animal anesthesia to minimize risk of mis-injection and radiotracer spillage. The animal remains under anesthesia until the start of PET image acquisition. Precisely 45 minutes after radiotracer injection.
Next, set a timer to count down from 45 minutes and ensure the mouse is positioned on the table in the sternal position. Install the appropriate surgical monitoring instruments and ensure the instruments are functioning properly before moving on. Then, set the perameters for CT imaging and PET image acquisition.
When the countdown timer reads 15 minutes begin the CT image acquisition and when the timer reads zero minutes begin PET image acquisition. First measure the radioactivity of the whole euthanized mouse and record the value and time of measurement. Then dissect the mice and harvest the necessary tissues.
Measure the radioactivity of the remaining carcass with and without the tail. Record these values and note times of measurement. Next, weigh all harvested tissues individually and take photographs of the cancerous organs under daylight and under fluorescence light.
Then, embed the tissues into OCT for downstream histology. Measure the radioactivity of all harvested tissues, record the value and note the time of measurement. Finally, present the data as standard uptake values or SUV as described in equation two.
In this experiment, radionuclide florescence recorder gene imaging was used to track tumor progression in a rodent tumor model. Confocal fluorescence microscopy demonstrated accurate plasma membrane localization of NISFPs. NISFP function and specificity was quantified using NIS afforded radiotracer uptake.
No significant differences between 4T1NISGFP and 4T1NISRFP expressing cell lines with similar NIS expression levels were noted. Importantly, the prechlorate block demonstrated in all cell lines that the obtained radiotracer uptake is due to specific NISFP expression. Whole body PET imaging of tumor bearing animals revealed information on tumor progression and metastatic spread.
The example shown here shows extensive long metastasis with several distinct nodules in the lung. Percent injected dose values and occupied volumes of individual metastasis in the lungs varied. However, volume normalized percent injected dose values were within a similar range.
The fluorescent protein in the dual mode reporter gene enabled cancerous tissue identification under florescence light during animal dissection. Subsequent ex vivo radiotracer bio distribution revealed organs with high radiotracer uptake and thus NIS expressing tissues. While thyroid and salivary glands, as well as the stomach, are indigenously expressing NIS, all other positive signals are indicative of cancerous tissues such as the tumor and metastases.
The radionuclide fluorescence reporter also enables cancer cell identification in downstream tissue sections. This technique paved the way for researchers in the field of cancer to visualize the process of spontaneous metastasis and to test how drugs affect it. While attempting this procedure, it's important to carefully plan all steps ahead.
Cell lines must be established, animal tumor models set up, and all reagents and equipments for imaging must be available on the required day. We recommend small pilot experiments to start with. Once mastered, radiotracer production and in vivo animal imaging can be done with six animals in an eight hour working day if it is performed properly.
After watching this video, you should have a good understanding of how to approach a reporter gene afforded in vivo cell tracking using the radionuclide reporter NIS combined with fluorescent protein. You should also be able to characterize NISFP reporter expressing cell lines to produce the required PET radiotracer for in vivo imaging and to perform in vivo and ex vivo by distribution experiments. Following this procedure, other methods like fluorocytometry can be performed in order to answer additional questions.
For example, how the immune cell profile of a tumor and its metastasis relates to one another or change over time. Do not forget to ensure the cell lines you produce are free of microplasmids as the latter have been reported to affect outcomes. Importantly, do not forget that working with radioisotopes can be extremely hazardous and protection for self and others must always be prioritized while performing this procedure.
