This study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-04) and the CAMS Innovation Fund for Medical Sciences (nos. 2021-I2M-1-026, 2022-I2M-1-026-1, 02120101, 02130101, and 2022-I2M-2-002).

The animal experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University or the National Institutes of Quantum Science and Technology. Mice experiments were strictly performed in compliance with the institutional guidelines of the Committee for the Care and Use of Laboratory Animals.
1. Peptide synthesis
<ol>
	<li>Swell 100 mg of 4-methylbenzhydrylamine (MBHA) resin (loading capacity of 0.37 mmol/g) in 1 mL of&#160;N-methyl-2-pyrrolidone (NMP) for 30 min under mild N2 bubbling.</li>
	<li>Prepare a fresh stock buffer consisting of Fmoc-protected amino acids (5.0 equiv), HCTU (4.9 equiv), and DIPEA (10.0 equiv) in 1 mL of NMP, and add to the resin (100 mg). Allow the coupling reaction to proceed for 1.5-2 h.</li>
	<li>Prepare deprotection buffer consisting of 50% (vol/vol) morpholine in NMP. Wash the resin (100 mg) 2 x 30 min with 1 mL of the deprotection buffer in each wash to remove the Fmoc group on the amine group. Wash the resin with dichloromethane (DCM; 1 mL) for 1 min, remove the DCM, and rewash the resin with NMP (1 mL) for another 1 min. Next, remove the NMP and rewash the resin with DCM for 1 min. 
	NOTE: All the washing procedures are conducted under mild N2 bubbling. The above procedures are repeated until the last amino acid.</li>
	<li>For adding 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the peptide, pre-activate the DOTA with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC&#183;HCl) and N-hydroxysuccinimide (NHS). Incubate the stock buffer of DOTA/EDC&#183;HCl/NHS at a molar ratio of 1:1:1 in dimethyl sulfoxide (DMSO) for 3 h, with a final DOTA concentration of 1 M. Next, add the stock buffer (1 mL) to the resin (100 mg), and allow the reaction to proceed for 2 h with gentle N2 bubbling. 
	NOTE: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) can also be utilized as a chelator for 68Ga complexation. The same procedures can be used for NOTA coupling.</li>
	<li>Rinse the resin thoroughly from the top using DCM and apply a vacuum to remove residual reaction solution simultaneously. Rinse the resin 3x using DCM (1.5 mL), and then rinse with MeOH (1.5 mL) to shrink the resin. Put the resin under nitrogen overnight, or under high vacuum for at least 4 h, until the resin becomes dry.</li>
	<li>Place the dried DPA peptide-containing resin in a 2 mL polypropylene container with a screw cap. Add the appropriate cleavage cocktail (95/2.5/2.5 TFA/TIS/H2O in volume; 1 mL/100 mg resin) and seal the container tightly using a screw cap. Gently agitate the reaction on an orbital shaker in a fume hood at 20-25 &#176;C for 2 h. 
	CAUTION: Prepare trifluoroacetic acid (TFA) in a fume hood wearing protective clothing, due to its highly corrosive nature.</li>
	<li>Remove most of the TFA by evaporation under nitrogen in the fume hood. Precipitate the peptides by adding diethyl ether (~1.5 mL/100 mg of resin).</li>
	<li>Vortex the mixture to triturate the peptides and centrifuge at room temperature (10,000 &#215; g, 5 min). Carefully pour the solvent out of the container. Repeat steps 1.7-1.8.</li>
	<li>Air-dry the residue in the open container for 10 min. Add 50% (vol/vol) aqueous acetonitrile, and vortex for 1-2 s to dissolve the products (1 mL/100 mg of resin).</li>
	<li>Remove the resin by filtering the mixture, and then wash the resin 2x using 0.2 mL of 50% (vol/vol) aqueous acetonitrile. Mix the filtrates for high-performance liquid chromatography (HPLC) purification. Use the following HPLC conditions. Column: YMC-Triat-C18 (4.6 mm internal diameter [i.d.], 150 mm, 5 mm); solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% TFA); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate: 1 mL/min.</li>
	<li>Freeze the collected HPLC eluents overnight at -80 &#176;C and lyophilize (-50 &#176;C and &#60;1 pa). For radiolabeling, dissolve the solid peptide in sodium acetate buffer (100 mM, pH 5.0) at a final concentration of 1 mg/mL as a stock buffer.</li>
</ol>
2. 68Ga radiolabeling
NOTE 68Ga was generated in-house at Nanjing First Hospital (Nanjing, China) using a 68Ge/68Ga generator.
<ol>
	<li>Pipette 5 &#181;L of stock buffer into a 1.5 mL polypropylene container with a screw cap. Add 200 MBq [68Ga]GaCl3 (400 &#181;L) to the container.</li>
	<li>Vortex the mixture for 5 s. Measure the pH using pH test strips. Adjust the pH to 4-4.5 with NaOH (0.1 M). 
	NOTE: An appropriate pH is critical for complexation between 68Ga and DOTA.</li>
	<li>Incubate the solution at room temperature for 5-10 min. Subject the reaction mixture to radio-HPLC for radiolabeling yield analysis under the following conditions: column: YMC-Triat-C18 (4.6 mm i.d., 150 mm, 5 mm); solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% TFA); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate: 1 mL/min. 
	&#8203;NOTE: Radio thin-layer chromatography (TLC) can be used as an alternative approach to examine radiolabeling yield. The suggested TLC elution buffer is 0.1 M Na3C6H5O7, pH 4.</li>
</ol>
3. Tracer stability test
<ol>
	<li>Tracer stability test in PBS
	<ol>
		<li>Add [68Ga]DPA (10 &#181;L, 3.7 MBq, in NaOAc) to the PBS (990 &#181;L). Incubate at 37 &#176;C for 1, 2, and 4 h with slight agitation.</li>
		<li>Collect 200 &#181;L of the solution at each time point. Inject it into the radio-HPLC for analysis.</li>
	</ol>
	</li>
	<li>Tracer stability in mouse serum
	<ol>
		<li>Add [68Ga]DPA (10 &#181;L, ~3.7 MBq, in NaOAc) to the mouse serum (90 &#181;L, freshly prepared). Incubate at 37 &#176;C for 1, 2, and 4 h with slight agitation.</li>
		<li>Collect 20 &#181;L of the solution at each time point. Add MeCN and water (100 &#181;L, 1:1, v/v).</li>
		<li>Centrifuge the mixture for 10 min (5,000 &#215; g, 25 &#176;C). Analyze the supernatant using radio-HPLC.</li>
	</ol>
	</li>
</ol>
4. Analysis of PD-L1 expression by flow cytometry
<ol>
	<li>Prepare culture medium by supplementing RPMI-1640 medium with 10% (vol/vol) fetal bovine serum and 1% penicillin-streptomycin (vol/vol). Resuspend the U87MG cells in culture medium, and seed in 12-well plates at a density of 105 cells/well. Place the cells in an incubator (5% CO2, 37 &#176;C), and culture for at least 24 h without disturbing.</li>
	<li>Wash the cells with 0.5 mL of PBS and add 250 &#956;L of trypsin-EDTA (0.25%). Place the cells back in the incubator (5% CO2, 37 &#176;C) for 2 min.</li>
	<li>Add 1 mL of culture medium to stop the cell dissociation. Add medium to the cells to detach them from the dishes by pipetting up and down and collect them in 1.5 mL tubes. Centrifuge the cells for 5 min (100 &#215; g).</li>
	<li>Remove the supernatant and resuspend the cells in 1 mL of PBS. Centrifuge the cells for 5 min (100 &#215; g). Repeat this step one more time.</li>
	<li>Dilute the fluorescein isothiocyanate (FITC)-conjugated PD-L1 antibody with 3% bovine serum albumin (BSA) to 20 nmol/L. Add to the cells and incubate at 4 &#176;C for 1 h.</li>
	<li>Centrifuge the cells for 5 min (100 &#215; g), followed by washing with cold PBS twice. Analyze the PD-L1-positive cells using a flow cytometer and analysis software.</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Seed the U87MG cells in glass bottom cell culture dishes (35 mm) at a density of 2.5 &#215; 105 cells per well. When 60% confluency is reached, aspirate the culture medium and add 1 mL of PBS. Gently shake several times and aspirate. Perform the washing step 3x.</li>
	<li>Add 500 &#181;L of 4% paraformaldehyde to the dishes. Place the cells at room temperature and fix for 30 min.</li>
	<li>Wash the cells 3x with PBS. Add 1 mL of 3% BSA (wt/vol, in PBS) and block the fixed cells for 2 h at room temperature.</li>
	<li>Remove the 3% BSA and directly incubate the cells with primary anti-PD-L1 antibody (mAb,1:100, diluted in 3% BSA) overnight at 4 &#176;C. 
	NOTE: After blocking the cells with BSA, do not wash with PBS.</li>
	<li>Wash the cells 3x with 3% BSA buffer. Incubate the cells with FITC-conjugated anti-human IgG Fc secondary antibody (1:500, diluted in PBS) for 1 h.</li>
	<li>Wash the cells 3x with PBS. Add 0.5 mL of DAPI (1 &#181;g/mL) to the cells and incubate at room temperature for 1 h. Wash the stained cells 3x with PBS, and observe using confocal fluorescence microscope.</li>
</ol>
6. Cellular uptake and inhibition experiment
<ol>
	<li>Cellular uptake experiment
	<ol>
		<li>Culture the U87MG cells in 12-well plates until 80% confluency is reached. Remove the medium and wash the cells with 0.5 mL of PBS.</li>
		<li>Dilute the [68Ga]DPA in fresh medium to a concentration of 74 KBq/mL. Add 0.5 mL of the diluted [68Ga]DPA buffer to each well.</li>
		<li>Incubate the cells with [68Ga]DPA at 37 &#176;C for different durations (10, 30, 40, and 120 min). Aspirate the medium using a pipette. Wash the cells with PBS (0.5 mL) three times.</li>
		<li>Add NaOH solution (0.5 M, 300 &#181;L per well) to lyse the cells. After 30 s, collect the viscous cell lysates in 1.5 mL tubes.</li>
		<li>Wash the plate with 0.4 mL of PBS twice. Collect the washing solution in the above 1.5 mL tube.</li>
		<li>Start the built-in computer of the automatic gamma counter; 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. The read-out records the decay-correlated counts per minute (CPM) of each tube.</li>
	</ol>
	</li>
	<li>Competitive binding assay
	<ol>
		<li>Culture the U87MG cells in 12-well plates until 80% confluency is reached. Remove the medium and wash the cells with 0.5 mL of PBS.</li>
		<li>Dilute [68Ga]DPA in fresh medium to a concentration of 74 KBq/mL. Dissolve an appropriate amount of BMS202 compounds in 10% DMSO to yield a 10 mM concentration (400 &#181;L).</li>
		<li>Dilute 4 &#181;L of 10 mM BMS202 in 396 &#181;L of PBS to obtain a concentration of 100 &#181;M. Repeat this step to yield various concentrations of BMS202 (1 &#181;M, 10 nM, 100 pM, and 1 pM).</li>
		<li>Add 0.5 mL of the diluted [68Ga]DPA buffer to each well (0.37 MBq per well). Add 5 &#181;L of the BMS202 solutions to each well (three wells for each concentration). Incubate the cells in a cell incubator at 37 &#176;C for 120 min.</li>
		<li>Aspirate the medium using a pipette. Wash the cells with 3 x 0.5 mL of PBS.</li>
		<li>Add the NaOH solution (0.5 M, 300 &#181;L per well) to lyse the cells. After 30 s, collect the viscous cell lysates into 1.5 mL tubes. Wash the plate with 2 x 0.4 mL of PBS.</li>
		<li>Collect the washing solution in the above 1.5 mL tube. Put the tubes into the built-in shelf of the automatic gamma counter.</li>
		<li>Follow the same procedures as step 6.1.6.</li>
	</ol>
	</li>
</ol>
7. PET imaging
NOTE: Perform small animal PET imaging, using a micro PET scanner that provides 159 transverse axial sections spaced 0.796 mm apart (center-to-center), with a horizontal field of view of 10 cm and an axial field of view of 12.7 cm. All data collected in the list mode are organized into three-dimensional sinograms. The Fourier is then reassembled into two-dimensional sinograms (frame &#215; min: 4 &#215; 1, 8 &#215; 2, 8 &#215; 5).
<ol>
	<li>Use male, 5-8-week-old BALB/C nude mice in this study. Collect the U87MG cells following steps 4.1-4.4 and aspirate the cells into a 0.5 mL syringe. Subcutaneously inject the cells into the mice (1 &#215; 106 cells per tumor, two tumors per mouse). Monitor tumor growth after injection until the tumor volume is 100-300 mm3.</li>
	<li>Anesthetize the mice using 1%-2% (v/v) isoflurane (1 mL/min). Turn on the heating device and keep the animal bed of PET at 37 &#176;C.&#160;</li>
	<li>Put the anesthetized mice in the right position on the animal bed of the PET machine. Apply&#160;ophthalmic ointment to both&#160;eyes to prevent dryness. 
	CAUTION: Keep the mice in the prone position to avoid death during scanning. During the whole imaging process, administer isoflurane flow (1.0 mL/min) via the nose using a preinstalled tube.</li>
	<li>Adjust the animal bed position via the control panel. Inject the tracers (10-17 MBq/100-200 &#181;L) intravenously through a preinstalled tail vein catheter.</li>
	<li>Create a scanning workflow in a host computer using the referenced software (see Table of Materials) Create a study folder and set the acquisition protocol as per the manufacturer&#39;s protocol. Perform dynamic scans (60 min for each mouse) on all mice in 3D list mode.</li>
	<li>Define a histogram protocol and a reconstruction protocol following the manufacturer&#39; protocol. Use Hanning&#39;s filter with a Nyquist cutoff of 0.5 cycle/pixel to reconstruct PET dynamic images (25-30 min and 55-60 min) through filtered back-projection. Generate maximum intensity projection (MIP) images for all mice.</li>
	<li>Combine the protocols in a workflow and run the workflow. Analyze the resulting three-dimensional images using the software, following the manufacturer&#39;s protocol. 
	NOTE: Use the simulation software to select the volumes of interest. Radioactivity is decay-corrected to the injection time and presented as the percentage of the total injection dose/per gram tissue (% ID/g).</li>
</ol>
8. Ex vivo biodistribution
<ol>
	<li>Administer [68Ga]DPA (1.85 MBq/100 &#181;L) to the U87MG-bearing BALB/C nude mice through tail vein injection. Anesthetize the mice using 1%-2% (v/v) isoflurane (1 mL/min), then sacrifice&#160;three mice via cervical dislocation after injection for 5, 30, 60, and 120 min. 
	NOTE:&#160;The individuals demonstrating this procedure should be trained in the technical skills of cervical dislocation to ensure the loss of consciousness is rapidly induced. This will ensure that&#160;the euthanasia procedures in this experiment are all humanely performed.</li>
	<li>After death is confirmed, open the chest wall of the mice. Then, open the heart. Use a 1 mL syringe to draw blood; squeeze the blood from the syringe into a radioimmunoassay (RIA) tube (13 mm in diameter) for the gamma counter.</li>
	<li>Excise the major organs and tumors and place them in RIA tubes (13 mm in diameter) for the gamma counter. Major organs include the total blood, heart, thymus, liver, spleen, bone, stomach, kidneys, muscle, intestinal lymph node, small intestine, pancreas, testis, brain, and lungs. Weigh all the organs.</li>
	<li>Measure the radioactivity within the collected organs using an autogamma counter and decay-correct the values. Calculate the percentage of injected dose per gram of wet tissue (% ID/g).</li>
</ol>
9. Immunohistochemistry
<ol>
	<li>Collect the glioma tissues and wash them 3x with PBS. Put the fresh tissues in 4% paraformaldehyde and fix overnight at 4 &#176;C.</li>
	<li>Embed the fixed tissues in paraffin and section them to a thickness of 10 &#181;m. Place the sections in an incubator (60 &#176;C) and incubate for 2 h. Dewax and hydrate the sections by incubating for 10 min each in the following: xylene (twice), absolute alcohol, 95% alcohol, 90% alcohol, 80% alcohol, 75% alcohol.</li>
	<li>Put the sections in 0.01 M sodium citrate and heat them to 92-95 &#176;C. Maintain the temperature for 40 min to achieve antigen retrieval.</li>
	<li>Add 200 &#181;L of H2O2 solution (3%) to the sections and inactivate the endoperoxidases for 10 min at room temperature. Block the non-specific sites by treatment with 3% BSA for 2 h at room temperature.</li>
	<li>Incubate the sections in primary anti-PD-L1 antibody (mAb, 1:100, diluted in 3% BSA) overnight at 4 &#176;C. 
	NOTE: After blocking with BSA, do not wash with PBS.</li>
	<li>Wash the sections 3x with PBS. Incubate the sections with HRP-labeled goat anti-rabbit secondary antibody (1:500, diluted in PBS) at room temperature for 1 h.</li>
	<li>Wash the cells 3x with PBS. Add 200 &#181;L of 3,3-diaminobenzidine (DAB) working solution (solution A: solution B: solution C = 1:1:18) to the sections and incubate for 5 min in the dark.</li>
	<li>Wash the sections 3x with PBS. Add 500 &#181;L of hematoxylin solution (100%), and incubate for 5 min at room temperature. Wash the sections with water for 30 s. Put the sections into differentiation solution (75% alcohol:HCL = 99:1) for 30 s. Wash the sections using water for 1 min.</li>
	<li>Dehydrate the samples by sequentially incubating with 75% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, absolute alcohol, and xylene (twice) (10 min each). Mount all the sections using neutral resin and observe them under an optical microscope.</li>
</ol>

No competing interests are declared.

The critical steps described in this method include the efficient labeling of 68Ga to DPA and choosing a suitable time window for PET imaging, which must perfectly match the pharmacodynamic pattern of DPA in the tumor.
In contrast to IHC, PET imaging enables real-time detection of whole-body PD-L1 expression in a noninvasive manner, allowing the visualization of each positive area in a heterogeneous tumor6,7. Peptides were chosen as ligands to avoid the disadvantages of antibodies and small molecules. Antibodies with large molecular weights generally have long circulating half-lives, which causes higher toxicity to healthy organs. The clearance of small molecules is usually too rapid to attain the required tumor retention. The molecular weight of peptides ranges between that of antibodies and small molecules. This enables peptide-based radiotracers to achieve both long-term tumor retention and good tissue penetration with minimal toxicity13,49,50,51,52,53. Importantly, the utility of the D-peptide DPA, rather than the commonly reported L-peptides, confer the [68Ga]DPA with a remarkably prolonged metabolic half-life. Moreover, DPA is positively charged and hydrophilic in vivo, and hence has high solubility and can avoid nonspecific targeting in blood, facilitating the generation of PET images with high imaging quality.
Notably, successful 68Ga radiolabeling requires a specific pH and no interference from transition metal ions, such as Cu (II) and Fe (III) cations. In some cases, the Cu2+ contamination leads to low radiochemical yield. Therefore, it is critical to ensure that all the containers and pipette tips are not contaminated. In addition, in this method, U87MG was used for tumor inoculation. Although PD-L1 expression in U87MG xenografts was verified in previous studies, its expression varies across individual animals. Therefore, the absolute uptake of the tracer in the U87MG tumors varied across individual mice. To ensure effective tracer uptake in the tumors, animals with an appropriate tumor size (500 mm3 &#60; volume &#60; 100 mm3) must be selected for PET scanning.
One of the limitations of [68Ga]DPA is that the binding affinity of DPA to PD-L1 is relatively low compared to several other PD-L1 targeting peptides, such as WL12, which makes it unsuitable for tumors with relatively low PD-L1 expression26,47. Further modification of the D-peptide will improve its specific binding capacity. In addition, to enhance the imaging effect of [68Ga]DPA, the formulation of the injection strategy can be optimized, for example, by concurrently injecting unlabeled DPA before [68Ga]DPA to block the nonspecific binding sites54,55,56.
In conclusion, this study developed a noninvasive and real-time method to track PD-L1 distribution in the whole body of living animals using [68Ga]DPA as a radiotracer. The results revealed a relatively high, in vivo specifical binding affinity, favorable stability, and excellent imaging capacity of [68Ga]DPA, suggesting that [68Ga]DPA-PET is a promising approach for visualizing PD-L1-overexpressing tumors. Furthermore, this technique can also be applied to the treatment of PD-L1 positive tumors when labeling DPA with other radionuclides, such as 177Lu and 225Ac. Therefore, the DPA radiolabeling technique not only overcomes the limitation of IHC dependent diagnosis, but also provides a new option for treatment.

The discovery of immune checkpoint proteins was a breakthrough in tumor therapy, and has led to major advances in the development of immune checkpoint blockade therapy1. Programmed cell death-protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are potential drug targets with several antibodies approved by the Food and Drug Administration (FDA). PD-1 is expressed by tumor-infiltrating immune cells, such as CD4+, CD8+ T cells, and regulatory T cells. PD-L1 is one of the PD-1 ligands, which is overexpressed in a variety of tumor cells2,3. The interaction between PD-1 and PD-L1 inactivates PD-1, thus suppressing the antitumor immune response4. These findings suggest that the inhibition of PD-L1 can improve the killing effect of immune cells and eliminate tumor cells5. Currently, chromogenic immunohistochemistry (IHC) is the most commonly used approach to identify patients who are most likely to respond to immune checkpoint therapy6,7. However, due to the heterogeneous expression of PD-L1 in tumor cells, IHC results from biopsies cannot provide accurate information about PD-L1 expression in patients8. Previous studies have reported that only 20%-40% of patients gain long-term benefits from immune checkpoint blockade therapy1,9,10. There is, therefore, an urgent need to develop a new method to circumvent the false-negative results caused by the heterogeneous expression of these immune checkpoint proteins.
Molecular imaging technology, such as positron emission tomography (PET), enables real-time visualization of the whole body in a noninvasive way, and thus can outperform the conventional IHC method11,12,13. Radiolabeled antibodies, peptides, and small molecules are promising tracers for monitoring PD-L1 expression in cancer patients14,15,16,17,18,19,20,21,22,23,24,25. The FDA has approved three PD-L1 therapeutic monoclonal antibodies: avelumab, atezolizumab, and durvalumab26. Immuno-PET tracers based on these antibodies have been well documented27,28,29,30,31,32. Early-phase clinical trials have revealed limited value for clinical application, because of the unfavorable pharmacokinetics30. Compared with antibodies, peptides exhibit faster blood and organ clearance from healthy organs, and can be easily chemically modified33. Multiple peptides with high affinities for PD-1/PD-L1 have been reported2; WL12 is a reported peptide that shows specific binding to PD-L134. Radiolabeled tracers, [64Cu]WL12, [68Ga]WL12, and [18F]FPyWL12, have been reported to show high in vivo specific tumor-targeting ability, which allows for the harvest of high-quality images of PD-L1 expression in tumors26,35,36,37. Moreover, the first in-human evaluation of radiolabeled WL12 has demonstrated that [68Ga]WL12 (chelated by NOTA) has a safe and efficient potential for clinical tumor imaging38. Due to its high hydrophobicity and high uptake in the healthy liver, WL12 has limited clinical use. Other radiolabeling peptides, such as TPP1 and SETSKSF, which specifically bind to PD-L1, have also showed potential stability and specificity to visualize whole-body PD-L1 expression39,40. However, unmodified peptides are easily degraded by proteases, and are rapidly metabolized by the kidney. Dextrorotary(D)-peptides have been widely used as effective mediators, due to the poor stability of left-handed (L)-peptides41,42,43. D-peptides are hyper-resistant to proteolytic degradation and have remarkably prolonged metabolic half-lives. Compared with their L-counterparts, D-peptides mostly show specific binding abilities44,45,46.
This study designed a new method to detect PD-L1 expression, based on PET imaging of a 68Ga-labeled PD-L1-targeted D-peptide, D-dodecapeptide antagonist (DPA), in a tumor-bearing mouse model47. The stability of [68Ga]DPA was first studied in phosphate-buffered saline (PBS) and mouse serum, after which the binding affinity of [68Ga]DPA in PD-L1-overexpressing tumors was tested. Thereafter, PET imaging was performed in glioblastoma xenograft models to confirm whether [68Ga]DPA was an ideal PET tracer to monitor PD-L1 expression in tumors. The combination of PET imaging and DPA not only provides a new approach to overcome challenges associated with the heterogeneous expression of PD-L1, but also lays the basis for the development of D-peptide-based radiotracers.

<table><tbody><tr><td>1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)</td><td>Merck</td><td>60239-18-1</td><td>&lt;sup&gt;68&lt;/sup&gt;Ga&amp;nbsp; chelation</td></tr><tr><td>3,3-diaminobenzidine (DAB) Kit</td><td>Sigma-Aldrich</td><td>D7304-1SET</td><td>Immunohistochemistry</td></tr><tr><td>anti-PD-L1 monoclonal antibody</td><td>Wuhan Proteintech</td><td>17952-1-ap</td><td>Immunohistochemistry: primary antibody</td></tr><tr><td>BMS202</td><td>Selleck</td><td>1675203-84-5</td><td>Competitive binding assay: inhibitor</td></tr><tr><td>BSA</td><td>Merck</td><td>V900933</td><td>Immunofluorescent : blocking&amp;nbsp;</td></tr><tr><td>DAPI</td><td>Merck</td><td>D9542</td><td>Immunofluorescent: staining of nucleus</td></tr><tr><td>Dichloromethane (DCM)</td><td>Merck</td><td>34856</td><td>Solvent</td></tr><tr><td>DIPEA</td><td>Merck</td><td>3439</td><td>Peptide coupling</td></tr><tr><td>EDC&amp;middot;HCl</td><td>Merck</td><td>E6383</td><td>Activation of DOTA</td></tr><tr><td>FBS</td><td>Gibco</td><td>10099</td><td>Cell culture: supplement</td></tr><tr><td>FITC-conjugated anti-human IgG Fc Antibody</td><td>Biolegend</td><td>409310</td><td>Immunofluorescent: secondary antibody</td></tr><tr><td>FITC-conjugated anti PD-L1 antibody</td><td>Biolegend</td><td>393606</td><td>Flow cytometry: direct antibody</td></tr><tr><td>HCTU</td><td>Energy Chemical</td><td>E070004-25g</td><td>Peptide coupling</td></tr><tr><td>HRP labeled goat anti-rabbit antibody</td><td>Servicebio</td><td>GB23303</td><td>Immunohistochemistry: secondary antibody</td></tr><tr><td>Hydroxysuccinimide (NHS)</td><td>Merck</td><td>130672</td><td>Activation of DOTA</td></tr><tr><td>MeCN</td><td>Merck</td><td>PHR1551</td><td>Solvent</td></tr><tr><td>Morpholine</td><td>Merck</td><td>8.06127</td><td>Fmoc- deprotection</td></tr><tr><td>NMP</td><td>Merck</td><td>8.06072</td><td>Solevent</td></tr><tr><td>Paraformaldehyde</td><td>Merck</td><td>30525-89-4</td><td>Fixation of tissues</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td>Cell culture: buffer</td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Gibco</td><td>10378016</td><td>Cell culture: supplement</td></tr><tr><td>RIA tube</td><td>PolyLab</td><td>P10301A</td><td>As tissue sample container</td></tr><tr><td>RPMI-1640 medium</td><td>Gibco</td><td>11875093</td><td>Cell culture: basic medium</td></tr><tr><td>Sodium acetate</td><td>Merck</td><td>1.06264</td><td>Salt for buffer</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td>Cell culture: dissociation agent</td></tr><tr><td>U87MG cell line</td><td>Procell Life Science &amp;amp; Technology Co</td><td>CL-0238</td><td>Cell model</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;sup&gt;68&lt;/sup&gt;Ge/&lt;sup&gt;68&lt;/sup&gt;Ga generator</td><td>Isotope Technologies Munich, ITM</td><td>Not applicable</td><td>Generation of [&lt;sup&gt;68&lt;/sup&gt;Ga]</td></tr><tr><td>Autogamma counter</td><td>Perkin Elmer&amp;nbsp;</td><td>Wizard2</td><td>Detection of radioactivity</td></tr><tr><td>Confocal fluorescent microscopy</td><td>Keyence</td><td></td><td>Observation of immunofluorescent results</td></tr><tr><td>Flow cytometer</td><td>Becton Dickinson, BD</td><td>LSRII</td><td>Monitoring the PD-L1 positive cells</td></tr><tr><td>High-performance liquid chromatography (HPLC)</td><td>SHIMAZU</td><td>LC-20AT&amp;nbsp;</td><td>Purification of DPA peptide</td></tr><tr><td>PET scanner</td><td>Siemens Medical Solutions</td><td>Inveon MultiModality System</td><td>PET imaging</td></tr><tr><td>Optical microscopy</td><td>Nikon&amp;nbsp;</td><td>Eclipse E100</td><td>Observation of immunohistochemistry results</td></tr><tr><td>Solid phase peptide synthesizer</td><td>Promega Vac-Man Laboratory Vacuum Manifold</td><td>LOT#11101</td><td>Synthesis of DPA-DOTA peptide</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ASIPro</td><td>Siemens Medical Solutions</td><td>Not applicable</td><td>Analysis of PET-CT results</td></tr><tr><td>FlowJo</td><td>Becton Dickinson, BD</td><td>FlowJo 7.6.1</td><td>Analysis of the flow cytometer results</td></tr><tr><td>Inveon Acquisition Workplace (IAW)</td><td>Siemens Medical Solutions</td><td>Not applicable</td><td>Management of PET mechine</td></tr><tr><td>Prism</td><td>Graphpad</td><td>Prism 8.0</td><td>Analysis of the data&amp;nbsp;</td></tr></tbody></table>

development of a 68gallium-labeled d-peptide pet tracer for imaging programmed death-ligand 1 expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

[68Ga]DPA radiolabeling and stability 
The model peptide, DPA, is an effective PD-L1 antagonist. DOTA-DPA was obtained with &#62;95% purity and a yield of 68%. The mass of DOTA-DPA is experimentally observed at 1,073.3 ([M+2H]2+). 68Gallium is considered a suitable radionuclide to label peptides for PET imaging, and therefore was chosen for this study. To radiolabel DPA with 68Ga (half-life: 68 min), DOTA-PEG3-DPA was synthesized (Figure 1A). DOTA was used as a chelator for the 68Ga radiolabeling. To space DOTA and DPA, PEG3 was used as a linker. The [68Ga]-DOTA-PEG3-DPA (referred to as [68Ga]DPA in the following text) showed high radiochemical yield (&#62;95%) and radiochemical purity (&#62;95%) (Table 1). A tracer stability test was also performed using HPLC, and the results showed that [68Ga]DPA had great stability both in PBS and mouse serum. The 68Ga decomposition or peptide hydrolysis was not detected after a 4 h incubation at 37 &#176;C (Figure 1B).
Expression of PD-L1 in U87MG cells 
A previous study showed that an increased expression of PD-L1 correlated with poor patient survival in glioblastoma tumors, indicating that PD-L1 may be a remarkable prognostic biomarker and therapeutic target in glioblastoma48. Therefore, a human glioblastoma cell line, U87MG, was used to establish a tumor model to determine the efficacy of [68Ga]DPA in PET/CT for PD-L1 tumor imaging. The flow cytometry results suggested that approximately 60% of the U87MG cells were PD-L1 positive (Figure 2A). Moreover, immunofluorescent staining confirmed the strong expression of PD-L1 in the U87MG cells (Figure 2B). Together, these data demonstrated that the U87MG cell line was suitable for this study.
Cellular uptake and specificity of [68Ga]DPA 
The uptake of [68Ga]DPA by U87MG cells presented a time-dependent pattern. When a PD-L1 inhibitor, BMS202, was used as a blocking agent, the binding portion and the uptake of [68Ga]DPA were significantly reduced (Figure 3A). A competitive binding assay further examined the binding affinity (Ki) of BMS202 to the U87MG cells. The estimated binding affinity was 43.8 &#177; 8.6 nmol/L for BMS202 when [68Ga]DPA was used as a competitor (Figure 3B).
[68Ga]DPA PET imaging of tumor models 
PET imaging of [68Ga]DPA was performed in U87MG tumor-bearing BALB/C nude mice. [68Ga]DPA was administered through intravenous injection until the U87MG tumor grew to 100 mm3. Whole-body PET images revealed high [68Ga]DPA accumulation in the tumor after 30 and 60 min of injection, and showed the highest accumulation in the kidney and bladder (Figure 4A). To confirm whether [68Ga]DPA specifically accumulated in PD-L1-positive tumors, another mouse model bearing the PanNET cell line Bon-1 was used as a negative control. A parallel experiment demonstrated little [68Ga]DPA accumulation in Bon-1 tumors at 60 min post-injection (Figure 4B).
To clarify this difference, immunohistochemical staining was conducted to analyze PD-L1 expression in tumor tissues. The results revealed that the U87MG cells presented considerable PD-L1 expression (Figure 5A,C), but not the Bon-1 tumor (Figure 5B,C). These data were consistent with the PET results. Therefore, it is possible that the different tumor cell growth statuses resulted in different PD-L1 expression (e.g., tissue necrosis). To verify this, hematoxylin and eosin (H&#38;E) staining was performed. As expected, a similar cell morphology was observed between the two tumor tissues (Figure 5D).
Ex vivo biodistribution of [68Ga]DPA in U87MG tumors 
The ex vivo biodistribution study was also conducted using U87MG-bearing mice (Table 2). The results showed a rapid clearance in blood and most analyzed organs, including the heart, liver, lung, and muscle. The kidney accumulated the highest amount of radioactivity and showed a clearance rate of 0.12% ID/(g&#8729;min) from 5 min to 120 min. The tumor exhibited uptake of the second-highest tracer uptake at all time points. In addition, from 5 min to 60 min post-injection, the tumor presented a lower tracer clearance rate of 0.027% ID/(g&#8729;min). For blood, the clearance rate was 0.069% ID/(g&#8729;min), while for muscle, the clearance rate was 0.037% ID/(g&#8729;min).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65047/65047fig01.jpg" /> 
Figure 1: [68Ga]DPA radiolabeling and stability. (A) The chemical structure of [68Ga]DPA and the schematic representation of its binding to PD-L1 expressing tumor cells. (B) HPLC curves showing the radioactivity of [68Ga]DPA after incubation with PBS or mouse serum for 0.5, 2, and 4 h. This figure was modified from Hu&#160;et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; PBS = phosphate-buffered saline; HPLC = high-performance liquid chromatography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65047/65047fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65047/65047fig02.jpg" /> 
Figure 2: Expression of PD-L1 in the U87MG cell line. (A) The expression of PD-L1 in the U87MG cell line was measured through flow cytometry analysis. (B) The expression of PD-L1 in U87MG cells was measured by immunofluorescence staining assay. Scale bar = 100 &#181;m. This figure was modified from Hu&#160;et al.47. Abbreviations: PD-L1 = programmed death-ligand 1; SSC = side scatter; FITC = fluorescein isothiocyanate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65047/65047fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65047/65047fig03.jpg" /> 
Figure 3: Cellular uptake and inhibition of [68Ga]DPA. (A) The uptake of U87MG cells when incubated with [68Ga]DPA (0.74 MBq/mL) or [68Ga]DPA (0.74 MBq /mL) + BMS202 (100 &#181;mol/L) for different durations. (B) Competitive binding of [68Ga]DPA (0.74 MBq /mL) to the U87MG cells following incubation with BMS202. The Ki value is shown in the panel. This figure was modified from Hu&#160;et al.47. Abbreviations: DPA = dodecapeptide antagonist; %AD = administered dose (with respect to the binding portion). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65047/65047fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65047/65047fig04.jpg" /> 
Figure 4: PET imaging of [68Ga]DPA in PD-L1-overexpressing U87MG tumors. (A,B) PET-CT images showing the distribution of [68Ga]DPA in U87MG-bearing mice (A) and Bon-1-bearing mice (negative control, B) after intravenous injection (~18.5 MBq) for 30 min and 60 min. Representative maxiumum-intensity projection (MIP) (top panel) and transverse PET-CT images (bottom panel) are presented. The tumor positions are marked by white dashed circles. This figure was modified from Hu et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; PET-CT = positron emission tomography-computed tomography; MIP = maxiumum-intensity projection; p.i. = post-injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65047/65047fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65047/65047fig05.jpg" /> 
Figure 5: Immunohistochemical analysis of [68Ga]DPA-treated tumors. (A,B) Whole-section immunohistochemical images of PD-L1 in (A) U87MG and (B) Bon-1 tumors. (C) Enlarged pictures of the marked areas in A and B. (D) H&#38;E staining of U87MG and Bon-1 tumor. Scale bars = 100 &#181;m (C,D). This figure was modified from Hu&#160;et al.47. Abbreviations: DPA = dodecapeptide antagonist; PD-L1 = programmed death-ligand 1; H&#38;E = hematoxylin and eosin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65047/65047fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tracers</td>
			<td>[68Ga]DPA</td>
		</tr>
		<tr>
			<td>Radiochemical yield (%)</td>
			<td>&#62;95</td>
		</tr>
		<tr>
			<td>Molar activity (GBq &#181;mol-1)</td>
			<td>37 &#177; 8</td>
		</tr>
		<tr>
			<td>Radiochemical puritya (%)</td>
			<td>&#62;95</td>
		</tr>
	</tbody>
</table>
Table 1: Radiolabeling and quality control of [68Ga]DPA. Radiochemical yield, molar activity, and radiochemical purity of [68Ga]DPA. Data are represented as the mean &#177; SD (n = 7). This table was modified from Hu&#160;et al.47. Abbreviation: DPA = dodecapeptide antagonist. aRadiochemical purity of [68Ga]DPA was analyzed by reverse-phase HPLC under an optimized condition: 1) column: YMC-Triat-C18 (4.6 mm i.d., 150 mm, 5 mm); 2) solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% trifluoroacetic acid); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate of 1 mL/min.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="9">[68Ga]DPA</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">5 min</td>
			<td colspan="2">30 min</td>
			<td colspan="2">60 min</td>
			<td colspan="2">120 min</td>
		</tr>
		<tr>
			<td>blood</td>
			<td>3.89</td>
			<td>&#177;0.43</td>
			<td>1.55</td>
			<td>&#177;1.07</td>
			<td>0.11</td>
			<td>&#177;0.02</td>
			<td>0.05</td>
			<td>&#177;0.01</td>
		</tr>
		<tr>
			<td>heart</td>
			<td>1.19</td>
			<td>&#177;0.39</td>
			<td>0.52</td>
			<td>&#177;0.33</td>
			<td>0.06</td>
			<td>&#177;0.02</td>
			<td>0.05</td>
			<td>&#177;0.02</td>
		</tr>
		<tr>
			<td>liver</td>
			<td>1.06</td>
			<td>&#177;0.26</td>
			<td>0.59</td>
			<td>&#177;0.43</td>
			<td>0.19</td>
			<td>&#177;0.02</td>
			<td>0.16</td>
			<td>&#177;0.04</td>
		</tr>
		<tr>
			<td>spleen</td>
			<td>0.98</td>
			<td>&#177;0.14</td>
			<td>0.68</td>
			<td>&#177;0.67</td>
			<td>0.14</td>
			<td>&#177;0.06</td>
			<td>0.09</td>
			<td>&#177;0.03</td>
		</tr>
		<tr>
			<td>lung</td>
			<td>1.64</td>
			<td>&#177;0.42</td>
			<td>1.03</td>
			<td>&#177;0.9</td>
			<td>0.13</td>
			<td>&#177;0.05</td>
			<td>0.09</td>
			<td>&#177;0.02</td>
		</tr>
		<tr>
			<td>kidney</td>
			<td>19.23</td>
			<td>&#177;1.95</td>
			<td>16.13</td>
			<td>&#177;1.51</td>
			<td>11.5</td>
			<td>&#177;0.44</td>
			<td>5.2</td>
			<td>&#177;0.31</td>
		</tr>
		<tr>
			<td>stomach</td>
			<td>1.54</td>
			<td>&#177;0.1</td>
			<td>0.61</td>
			<td>&#177;0.35</td>
			<td>0.08</td>
			<td>&#177;0.01</td>
			<td>0.08</td>
			<td>&#177;0.03</td>
		</tr>
		<tr>
			<td>intestinal</td>
			<td>0.72</td>
			<td>&#177;0.27</td>
			<td>0.47</td>
			<td>&#177;0.35</td>
			<td>0.08</td>
			<td>&#177;0.02</td>
			<td>0.06</td>
			<td>&#177;0.03</td>
		</tr>
		<tr>
			<td>pancreas</td>
			<td>1.88</td>
			<td>&#177;0.28</td>
			<td>0.77</td>
			<td>&#177;0.75</td>
			<td>0.16</td>
			<td>&#177;0.03</td>
			<td>0.13</td>
			<td>&#177;0.03</td>
		</tr>
		<tr>
			<td>muscle</td>
			<td>2.21</td>
			<td>&#177;0.27</td>
			<td>0.71</td>
			<td>&#177;0.37</td>
			<td>0.18</td>
			<td>&#177;0.02</td>
			<td>0.14</td>
			<td>&#177;0.04</td>
		</tr>
		<tr>
			<td>bone</td>
			<td>2.18</td>
			<td>&#177;0.11</td>
			<td>0.85</td>
			<td>&#177;0.51</td>
			<td>0.26</td>
			<td>&#177;0.09</td>
			<td>0.14</td>
			<td>&#177;0.06</td>
		</tr>
		<tr>
			<td>brain</td>
			<td>0.19</td>
			<td>&#177;0.04</td>
			<td>0.11</td>
			<td>&#177;0.08</td>
			<td>0.03</td>
			<td>&#177;0.01</td>
			<td>0.02</td>
			<td>&#177;0.01</td>
		</tr>
		<tr>
			<td>tumor</td>
			<td>4.5</td>
			<td>&#177;0.32</td>
			<td>3.77</td>
			<td>&#177;0.27</td>
			<td>2.99</td>
			<td>&#177;0.03</td>
			<td>0.89</td>
			<td>&#177;0.19</td>
		</tr>
		<tr>
			<td>fat</td>
			<td>2.09</td>
			<td>&#177;0.49</td>
			<td>0.81</td>
			<td>&#177;0.12</td>
			<td>0.27</td>
			<td>&#177;0.07</td>
			<td>0.1</td>
			<td>&#177;0.07</td>
		</tr>
	</tbody>
</table>
Table 2: Biodistribution of [68Ga]DPA in U87MG tumor-bearing mice after administration for different durations (n = 3 per time point). This table is modified from Hu et al.47. Abbreviation: DPA = dodecapeptide antagonist.

Watch this Scientific Journal Video about Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression at JoVE.com

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

development-68gallium-labeled-d-peptide-pet-tracer-for-imaging

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.4K Views.  Nanjing Medical University. Deze methode maakt gebruik van D-peptide DPA, gelabeld met Gallium-68. De radiotracer Gallium-68 om de DPA te labelen maakt real-time visualisatie van PDL-1-expressie in het hele lichaam op een niet-invasieve manier mogelijk. Het gebruik van D-peptide geeft de radiotracer hyperresistent tegen proteolytische afbraak en verlengt opmerkelijk hun metabole halfwaardetijd.In het bijzonder kan de techniek worden toegepast om verschillende ziekten te evalueren, afhankelijk van de ontdekking va...

Video: Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Chemistry

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicine

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Immunology and Infection

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Biology

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Development of a ⁶⁸Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

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