This method uses D-peptide DPA, labeled with Gallium-68. The radio tracer Gallium-68 to label the DPA enables real-time visualization of PDL-1 expression in the whole body in a noninvasive manner. The use of D-peptide confers the radio tracer with hyper-resistant to proteolytic degradation and remarkably prolong their metabolic half lives.
In particular, the technique can be applied to evaluate different disease, subjected to the discovery of specific targets and the development of radio tracers with good phone call kinetics. The technique can also be applied to the treatment of PDL-1 positive tumors while labeling DPA with other radio nucleotides, such as Lutetium-177 and Actinium-225. Demonstrating the procedure will be Wu Jiang, a researcher from my laboratory.
For radiolabeling the peptide d-dodecapeptide antagonist, or DPA, pipette five microliters of the prepared stock buffer containing DPA into a 1.5 milliliter polypropylene container with a screw cap and add 400 microliters of 68 gallium trichloride to the container. Vortex the mixture for five seconds. Measure the pH of the mixture using pH test strips and adjust the pH to 4 to 4.5 with 0.1 molar sodium hydroxide.
Incubate the solution at room temperature for five to 10 minutes. Then following the conditions mentioned in the text, subject the reaction mixture to radio HPLC for analyzing the radiolabeling yield. To test the tracer stability in PBS, add 10 microliters of Gallium-68-labeled DPA in sodium acetate solution to 990 microliters of PBS.
Incubate the mixture at 37 degrees Celsius for one, two, and four hours, with slight agitation. Collect 200 microliters of the solution at each said time point and analyze it by radio HPLC. To test the tracer stability in mouse serum, add 10 microliters of Gallium-68-labeled DPA in sodium acetate solution to 90 microliters of the freshly prepared mouse serum and incubate the mixture at 37 degrees Celsius for one, two, and four hours with slight agitation.
Then collect 20 microliters of the solution at each said time point and add 100 microliters of aceto nitrile water mixture into the collected aliquot. Centrifuge the mixture at 5, 000 G for 10 minutes at 25 degrees Celsius. Analyze the supernatant using radio HPLC.
Culture the U-87 MG cells in 12-well plates until 80%confluency is reached. Then remove the medium and wash the cells with 0.5 milliliters of PBS. Dilute the Gallium-68-labeled DPA in fresh medium to a concentration of 74 kilobecquerel per milliliter.
Then add 0.5 milliliters of the diluted Gallium-68-labeled DPA buffer to each well. Incubate the cells with Gallium-68-labeled DPA at 37 degrees Celsius for the durations of 10, 30, 40, and 120 minutes. Post incubation, aspirate the medium using a pipette and wash the cells with 0.5 milliliters of PBS three times.
Next, add 300 microliters of 0.5 molar sodium hydroxide solution per well to lyse the cells. After 30 seconds, collect the viscous cell lysates in 1.5 milliliter tubes. Wash the plate by adding 0.4 milliliters of PBS per well twice, and collect the washing solution in the mentioned 1.5 milliliter tubes.
Start the built-in computer of the automatic gamma counter and put the tubes into the built-in shelf. After loading all the samples onto the conveyor, press the start button. Results are calculated in the internal software, while the readout records the decay correlated counts per minute, or CPM, of each tube.
Collect the U-87 MG cells following the protocol mentioned in the text and aspirate the cells into a 0.5 milliliter syringe. Next, take BALB/c Nude mice and inject the cells subcutaneously, maintaining a count of two tumors per mouse. Monitor tumor growth after injection until the tumor volume is 100 to 300 cubic millimeters.
To perform PET imaging, inject the tracers intravenously through a pre-installed tail vein catheter. Create a scanning workflow in a host computer using the reference software, followed by creating a study folder and setting the acquisition protocol per the manufacturer's guidelines. Perform dynamic scans on each mouse for 60 minutes in 3D list mode.
Next, define a histogram protocol and a reconstruction protocol, following the manufacturer's instruction. Use Hanning's filter with a Nyquist cutoff of 0.5 cycles per pixel to reconstruct PET dynamic images for 25 to 30 minutes, and 55 to 60 minutes through filtered back projection. Generate maximum intensity projection, or MIP images, for all mice.
Administer Gallium-68-labeled DPA to the U-87 MG bearing BALB/c Nude mice through tail vein injection. After euthanasia, dissect the chest wall of the animal and open the heart. Using a one-milliliter syringe, draw blood and squeeze the blood from the syringe into a radio immunoassay tube, or RIA, measuring 13 millimeters in diameter for the gamma counter.
Excise the major organs and tumors and place them in similar RIA tubes for the gamma counter. Weigh all the major organs mentioned in the text. Measure the radioactivity within the collected organs using an auto gamma counter and decay correct the values.
Calculate the percentage of injected dose per gram of wet tissue. The Gallium-68-labeled DPA showed high radio chemical yield impurity. It was highly stable in both PBS and mouse serum, showing no Gallium-68 decomposition or peptide hydrolysis after the four hour incubation.
Approximately 60%of the U-87 MG cells were found to be programmed to death ligand one, or PDL-1 positive by flow cytometry, and immunofluorescent staining confirmed strong expression of PDL-1 in the U-87 MG cells. U-87 MG cells showed a time-dependent uptake of Gallium-68-labeled DPA, which reduced significantly upon using a PDL-1 inhibitor, BMS-202, as a blocking agent. The estimated binding affinity for BMS-202 was 43.8 nanomoles per liter when Gallium-68-labeled DPA was used as a competitor.
Whole body PET images revealed high Gallium-68-labeled DPA accumulation in the tumor after 30 and 60 minutes of injection. A parallel experiment demonstrated little Gallium-68-labeled DPA accumulation in the negative control Bon-1 tumors at 60 minutes post-injection. Further, immunohistochemical staining revealed that the U-87 MG cells presented considerable PDL-1 expression, but not the Bon-1 tumor.
Hematoxylin and eosin staining showed a similar cell morphology between the two tumor tissues. The ex-vivo bio-distribution study showed rapid clearance of radioactivity and blood in most analyzed organs, including the heart, liver, lung, and muscle. The kidney accumulated the highest amount of radioactivity, while the tumor exhibited the second highest tracer uptake at all time points.
The structure of D-peptide being different using an efficient method for labeling DPA with Gallium-68 is the most important step. This technique paved the way to exploit the PET test as effective radio pharmaceuticals to manage different cancers, both as PET tracers and radio diagnostics agents.
