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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 cells^2,3. 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 therapy^6,7. 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 therapy^1,9,10. 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 method^11,12,13. Radiolabeled antibodies, peptides, and small molecules are promising tracers for monitoring PD-L1 expression in cancer patients^{14,15,16,17,18,19,20,21,22,23,24,25}. The FDA has approved three PD-L1 therapeutic monoclonal antibodies: avelumab, atezolizumab, and durvalumab²⁶. Immuno-PET tracers based on these antibodies have been well documented^{27,28,29,30,31,32}. 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 tumors^26,35,36,37. 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 expression^39,40. 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)-peptides^41,42,43. 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 abilities^44,45,46.

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.

Protokół

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

1. Swell 100 mg of 4-methylbenzhydrylamine (MBHA) resin (loading capacity of 0.37 mmol/g) in 1 mL of N-methyl-2-pyrrolidone (NMP) for 30 min under mild N₂ bubbling.
2. 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.
3. 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.
4. 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·HCl) and N-hydroxysuccinimide (NHS). Incubate the stock buffer of DOTA/EDC·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.
5. 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.
6. 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 °C for 2 h.
   CAUTION: Prepare trifluoroacetic acid (TFA) in a fume hood wearing protective clothing, due to its highly corrosive nature.
7. 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).
8. Vortex the mixture to triturate the peptides and centrifuge at room temperature (10,000 × g, 5 min). Carefully pour the solvent out of the container. Repeat steps 1.7-1.8.
9. 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).
10. 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.
11. Freeze the collected HPLC eluents overnight at -80 °C and lyophilize (-50 °C and <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.

2. ⁶⁸Ga radiolabeling

NOTE ⁶⁸Ga was generated in-house at Nanjing First Hospital (Nanjing, China) using a ⁶⁸Ge/⁶⁸Ga generator.

1. Pipette 5 µL of stock buffer into a 1.5 mL polypropylene container with a screw cap. Add 200 MBq [⁶⁸Ga]GaCl₃ (400 µL) to the container.
2. 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.
3. 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.
   ​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.

3. Tracer stability test

1. Tracer stability test in PBS
   1. Add [⁶⁸Ga]DPA (10 µL, 3.7 MBq, in NaOAc) to the PBS (990 µL). Incubate at 37 °C for 1, 2, and 4 h with slight agitation.
   2. Collect 200 µL of the solution at each time point. Inject it into the radio-HPLC for analysis.
2. Tracer stability in mouse serum
   1. Add [⁶⁸Ga]DPA (10 µL, ~3.7 MBq, in NaOAc) to the mouse serum (90 µL, freshly prepared). Incubate at 37 °C for 1, 2, and 4 h with slight agitation.
   2. Collect 20 µL of the solution at each time point. Add MeCN and water (100 µL, 1:1, v/v).
   3. Centrifuge the mixture for 10 min (5,000 × g, 25 °C). Analyze the supernatant using radio-HPLC.

4. Analysis of PD-L1 expression by flow cytometry

1. 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 °C), and culture for at least 24 h without disturbing.
2. Wash the cells with 0.5 mL of PBS and add 250 μL of trypsin-EDTA (0.25%). Place the cells back in the incubator (5% CO₂, 37 °C) for 2 min.
3. 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 × g).
4. Remove the supernatant and resuspend the cells in 1 mL of PBS. Centrifuge the cells for 5 min (100 × g). Repeat this step one more time.
5. 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 °C for 1 h.
6. Centrifuge the cells for 5 min (100 × g), followed by washing with cold PBS twice. Analyze the PD-L1-positive cells using a flow cytometer and analysis software.

5. Immunocytochemistry

1. Seed the U87MG cells in glass bottom cell culture dishes (35 mm) at a density of 2.5 × 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.
2. Add 500 µL of 4% paraformaldehyde to the dishes. Place the cells at room temperature and fix for 30 min.
3. 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.
4. 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 °C.
   NOTE: After blocking the cells with BSA, do not wash with PBS.
5. 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.
6. Wash the cells 3x with PBS. Add 0.5 mL of DAPI (1 µg/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.

6. Cellular uptake and inhibition experiment

1. Cellular uptake experiment
   1. 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.
   2. 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.
   3. Incubate the cells with [⁶⁸Ga]DPA at 37 °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.
   4. Add NaOH solution (0.5 M, 300 µL per well) to lyse the cells. After 30 s, collect the viscous cell lysates in 1.5 mL tubes.
   5. Wash the plate with 0.4 mL of PBS twice. Collect the washing solution in the above 1.5 mL tube.
   6. 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.
2. Competitive binding assay
   1. 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.
   2. 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 µL).
   3. Dilute 4 µL of 10 mM BMS202 in 396 µL of PBS to obtain a concentration of 100 µM. Repeat this step to yield various concentrations of BMS202 (1 µM, 10 nM, 100 pM, and 1 pM).
   4. Add 0.5 mL of the diluted [⁶⁸Ga]DPA buffer to each well (0.37 MBq per well). Add 5 µL of the BMS202 solutions to each well (three wells for each concentration). Incubate the cells in a cell incubator at 37 °C for 120 min.
   5. Aspirate the medium using a pipette. Wash the cells with 3 x 0.5 mL of PBS.
   6. Add the NaOH solution (0.5 M, 300 µL 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.
   7. Collect the washing solution in the above 1.5 mL tube. Put the tubes into the built-in shelf of the automatic gamma counter.
   8. Follow the same procedures as step 6.1.6.

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 × min: 4 × 1, 8 × 2, 8 × 5).

1. 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 × 10⁶ cells per tumor, two tumors per mouse). Monitor tumor growth after injection until the tumor volume is 100-300 mm³.
2. 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 °C. 
3. Put the anesthetized mice in the right position on the animal bed of the PET machine. Apply ophthalmic ointment to both 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.
4. Adjust the animal bed position via the control panel. Inject the tracers (10-17 MBq/100-200 µL) intravenously through a preinstalled tail vein catheter.
5. 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's protocol. Perform dynamic scans (60 min for each mouse) on all mice in 3D list mode.
6. Define a histogram protocol and a reconstruction protocol following the manufacturer' protocol. Use Hanning'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.
7. Combine the protocols in a workflow and run the workflow. Analyze the resulting three-dimensional images using the software, following the manufacturer'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).

8. Ex vivo biodistribution

1. Administer [⁶⁸Ga]DPA (1.85 MBq/100 µL) 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 three mice via cervical dislocation after injection for 5, 30, 60, and 120 min.
   NOTE: 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 the euthanasia procedures in this experiment are all humanely performed.
2. 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.
3. 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.
4. 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).

9. Immunohistochemistry

1. Collect the glioma tissues and wash them 3x with PBS. Put the fresh tissues in 4% paraformaldehyde and fix overnight at 4 °C.
2. Embed the fixed tissues in paraffin and section them to a thickness of 10 µm. Place the sections in an incubator (60 °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.
3. Put the sections in 0.01 M sodium citrate and heat them to 92-95 °C. Maintain the temperature for 40 min to achieve antigen retrieval.
4. Add 200 µL 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.
5. Incubate the sections in primary anti-PD-L1 antibody (mAb, 1:100, diluted in 3% BSA) overnight at 4 °C.
   NOTE: After blocking with BSA, do not wash with PBS.
6. 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.
7. Wash the cells 3x with PBS. Add 200 µL 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.
8. Wash the sections 3x with PBS. Add 500 µL 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.
9. 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.

Wyniki

[⁶⁸Ga]DPA radiolabeling and stability
The model peptide, DPA, is an effective PD-L1 antagonist. DOTA-DPA was obtained with >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 synt...

Dyskusje

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 tumor^6,7. Peptides were c...

Materiały

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