Synthesis and Evaluation of the First 68Ga-Labeled C-Terminal Hydroxamate-Derived Gastrin-Releasing Peptide Receptor-Targeted Tracers for Cancer Imaging with Positron Emission Tomography

Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.


Introduction
Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor.It is expressed in some normal tissues and organs such as the pancreas, gastrointestinal tract, and nervous system, and regulates a series of physiological functions including stimulation of the contraction of smooth muscle cells and secretion of gastrointestinal hormones [1].GRPR is also found overexpressed in several solid malignancies, such as prostate, breast, colon, and lung cancers, and is involved in the malignant neoplasm's development by coupling with phospholipase C and activating protein kinase C [2][3][4][5][6][7].Owing to its overexpression in malignant tissues, GRPR is a promising target for cancer imaging and therapy.
In the past two decades, many radiolabeled GRPR-targeted ligands have been developed, and some of them have been translated into the clinic for cancer diagnosis and radioligand therapy [8][9][10][11][12].[ 68 Ga]Ga-SB3 (SB3: DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; Figure 1A; pABzA-DIG: p-aminomethylaniline-diglycolic acid), developed by Maina et al., is one of the most popular radiolabeled GRPR antagonists, and has been validated in the clinic for detecting prostate and breast cancer lesions [13].Although [ 68 Ga]Ga-SB3 enabled clear visualization of prostate and breast cancer lesions in PET images, a very high pancreas uptake was observed in patients [13].In addition to [ 68 Ga]Ga-SB3, a high pancreas uptake is observed by other clinically validated GRPRtargeted radioligands including derivatives of AMBA, RM2 and NeoB [9,10,12].The high uptake in pancreas not only affects lesion detection in or adjacent to the pancreas, but also significantly limits the maximum tolerated dose for targeted radioligand therapy application [1,13].
Most of the reported GRPR-targeted agonist and antagonist sequences contain a Cterminal amide.However, it seems that replacing the C-terminal amide with a C-terminal hydroxamate is tolerable, as a C-terminal hydroxamate-derived GRPR-targeted antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), reported by Devin et al., was shown to bind GRPR with a high affinity (K i = 5.8 nM) [14].To the best of our knowledge, no GRPR-targeted radioligands containing a C-terminal hydroxamate have ever been reported.Therefore, the goal of this study was to evaluate the potential of C-terminal hydroxamatederived GRPR-targeted radioligands for cancer imaging, and document the extent of their uptake in the pancreas.
In the past two decades, many radiolabeled GRPR-targeted ligands have been developed, and some of them have been translated into the clinic for cancer diagnosis and radioligand therapy [8][9][10][11][12].[ 68 Ga]Ga-SB3 (SB3: DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; Figure 1A; pABzA-DIG: p-aminomethylaniline-diglycolic acid), developed by Maina et al., is one of the most popular radiolabeled GRPR antagonists, and has been validated in the clinic for detecting prostate and breast cancer lesions [13].Although [ 68 Ga]Ga-SB3 enabled clear visualization of prostate and breast cancer lesions in PET images, a very high pancreas uptake was observed in patients [13].In addition to [ 68 Ga]Ga-SB3, a high pancreas uptake is observed by other clinically validated GRPR-targeted radioligands including derivatives of AMBA, RM2 and NeoB [9,10,12].The high uptake in pancreas not only affects lesion detection in or adjacent to the pancreas, but also significantly limits the maximum tolerated dose for targeted radioligand therapy application [1,13].
Most of the reported GRPR-targeted agonist and antagonist sequences contain a Cterminal amide.However, it seems that replacing the C-terminal amide with a C-terminal hydroxamate is tolerable, as a C-terminal hydroxamate-derived GRPR-targeted antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), reported by Devin et al., was shown to bind GRPR with a high affinity (Ki = 5.8 nM) [14].To the best of our knowledge, no GRPR-targeted radioligands containing a C-terminal hydroxamate have ever been reported.Therefore, the goal of this study was to evaluate the potential of C-terminal hydroxamate-derived GRPR-targeted radioligands for cancer imaging, and document the extent of their uptake in the pancreas.We used the chemical structure of [ 68 Ga]Ga-SB3 (Figure 1A) as a template since it has been used to successfully detect prostate and breast cancer lesions in the clinic [13].Additionally, the only difference between the GRPR-targeted sequence of [ 68 Ga]Ga-SB3 (D-Phe-

B.
C. We used the chemical structure of [ 68 Ga]Ga-SB3 (Figure 1A) as a template since it has been used to successfully detect prostate and breast cancer lesions in the clinic [13].

PET Imaging
The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post injection using [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02050 (Figure 4).All [ 68 Ga]Ga-SB3, [ 68 Ga]Ga-LW02075, and [ 68 Ga]Ga-LW02050 were excreted primarily through the renal pathway, as a very high uptake in urinary bladder was observed.A high pancreas uptake was also observed by both [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02075, while the pancreas was nearly invisible in the PET image of [ 68 Ga]Ga-LW02050.Co-injection with 100 µg of nonradioactive Ga-LW02050 significantly reduced the uptake of [ 68 Ga]Ga-LW02050 in the PC-3 tumor xenograft to close to the background level.

PET Imaging
The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post injection using [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02050 (Figure 4).All [ 68 Ga]Ga-SB3, [ 68 Ga]Ga-LW02075, and [ 68 Ga]Ga-LW02050 were excreted primarily through the renal pathway, as a very high uptake in urinary bladder was observed.A high pancreas uptake was also observed by both [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02075, while the pancreas was nearly invisible in the PET image of [ 68 Ga]Ga-LW02050.Co-injection with 100 µg of nonradioactive Ga-LW02050 significantly reduced the uptake of [ 68 Ga]Ga-LW02050 in the PC-3 tumor xenograft to close to the background level.
Consistent with the PET images, the co-injection of nonradioactive Ga-LW02050 (100 µg) reduced the average uptake of [ 68 Ga]Ga-LW02050 in PC-3 tumor xenografts by 92%, from 5.38 %ID/g down to 0.42 %ID/g at 1 h post-injection (Figure 6 and Table S4).Furthermore, the average uptake of [ 68 Ga]Ga-LW02050 in the pancreas was also reduced by 81% (0.53 %ID/g down to 0.10 %ID/g) by the co-injection of nonradioactive Ga-LW02050.A blocking study was also performed for [ 68 Ga]Ga-LW02075.Similarly, co-injecting 100 µg of nonradioactive Ga-LW02075 reduced the average uptake of [ 68 Ga]Ga-LW02075 in the PC-3 tumor xenografts by 54% (3.97 %ID/g down to 1.83 %ID/g).In addition, a significant reduction in the average pancreas uptake of [ 68 Ga]Ga-LW02075 was observed.The average pancreas uptake of [ 68 Ga]Ga-LW02075 declined by 91% from 17.8 %ID/g to 1.55 %ID/g (Table S4).
Consistent with the PET images, the co-injection of nonradioactive Ga-LW02050 (100 µg) reduced the average uptake of [ 68 Ga]Ga-LW02050 in PC-3 tumor xenografts by 92%, from 5.38 %ID/g down to 0.42 %ID/g at 1 h post-injection (Figure 6 and Table S4).Furthermore, the average uptake of [ 68 Ga]Ga-LW02050 in the pancreas was also reduced by 81% (0.53 %ID/g down to 0.10 %ID/g) by the co-injection of nonradioactive Ga-LW02050.A blocking study was also performed for [ 68 Ga]Ga-LW02075.Similarly, co-injecting 100 µg of nonradioactive Ga-LW02075 reduced the average uptake of [ 68 Ga]Ga-LW02075 in the PC-3 tumor xenografts by 54% (3.97 %ID/g down to 1.83 %ID/g).In addition, a significant reduction in the average pancreas uptake of [ 68 Ga]Ga-LW02075 was observed.The average pancreas uptake of [ 68 Ga]Ga-LW02075 declined by 91% from 17.8 %ID/g to 1.55 %ID/g (Table S4).

Discussion
Inspired by the potent GRPR antagonist sequence, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, reported by Devin et al., we synthesized LW02075 (Figure 1B) by replacing the C-terminal ethylamide in SB3 (Figure 1A) with a C-terminal hydroxamate.The design of LW02050 (Figure 1C), replacing the pABzA-DIG linker in LW02075 with a more hydrophilic Pip linker, was based on data from our previously reported GRPR-targeted tracers, [ 68 Ga]Ga-ProBOMB1 and [ 68 Ga]Ga-ProBOMB2 [15,16].Compared to [ 68 Ga]Ga-ProBOMB1 containing a pABzA-DIG linker, [ 68 Ga]Ga-ProBOMB2 containing a Pip linker showed a comparable high tumor uptake but a significantly lower uptake in normal organs/tissues, practically in the pancreas.Accordingly, in this study, we also used a Pip linker instead of the pABzA-DIG linker to synthesize LW02050 for comparison.

Discussion
Inspired by the potent GRPR antagonist sequence, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, reported by Devin et al., we synthesized LW02075 (Figure 1B) by replacing the C-terminal ethylamide in SB3 (Figure 1A) with a C-terminal hydroxamate.The design of LW02050 (Figure 1C), replacing the pABzA-DIG linker in LW02075 with a more hydrophilic Pip linker, was based on data from our previously reported GRPR-targeted tracers, [ 68 Ga]Ga-ProBOMB1 and [ 68 Ga]Ga-ProBOMB2 [15,16].Compared to [ 68 Ga]Ga-ProBOMB1 containing a pABzA-DIG linker, [ 68 Ga]Ga-ProBOMB2 containing a Pip linker showed a comparable high tumor uptake but a significantly lower uptake in normal organs/tissues, practically in the pancreas.Accordingly, in this study, we also used a Pip linker instead of the pABzA-DIG linker to synthesize LW02050 for comparison.

Discussion
Inspired by the potent GRPR antagonist sequence, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, reported by Devin et al., we synthesized LW02075 (Figure 1B) by replacing the C-terminal ethylamide in SB3 (Figure 1A) with a C-terminal hydroxamate.The design of LW02050 (Figure 1C), replacing the pABzA-DIG linker in LW02075 with a more hydrophilic Pip linker, was based on data from our previously reported GRPR-targeted tracers, [ 68 Ga]Ga-ProBOMB1 and [ 68 Ga]Ga-ProBOMB2 [15,16].Compared to [ 68 Ga]Ga-ProBOMB1 containing a pABzA-DIG linker, [ 68 Ga]Ga-ProBOMB2 containing a Pip linker showed a comparable high tumor uptake but a significantly lower uptake in normal organs/tissues, practically in the pancreas.Accordingly, in this study, we also used a Pip linker instead of the pABzA-DIG linker to synthesize LW02050 for comparison.
The average K i (GRPR) of Ga-LW02075 was comparable with that of Ga-SB3 (1.39 ± 0.54 vs. 1.20 ± 0.31 nM, Figure 2).This indicates that replacing the C-terminal ethylamide in Ga-SB3 with a hydroxamate does not affect their GRPR binding affinity.In contrast, the average K i (GRPR) of Ga-LW02050 (8.53 ± 1.52 nM) containing the Pip linker was significantly inferior to those of Ga-SB3 and Ga-LW02075.This finding is unexpected as the Pip linker is also very popular for the design of GRPR-targeted radioligands, including the clinically validated [ 68 Ga/ 177 Lu]Ga/Lu-RM2 [10,11,17].In addition, in our previous studies, we showed that the binding affinity of Ga-ProBOMB1 containing a pABzA-DIG linker was comparable to that of Ga-ProBOMB2 containing a Pip linker (K i (GRPR) = 3.97 ± 0.76 vs. 4.58 ± 0.67 nM) [15,16].
The observations from PET images were consistent with the obtained biodistribution data (Figures 4 and 5 and Table S4).A good in vivo GRPR-targeting capability of [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02050 was confirmed as PC-3 tumor xenografts were clearly visualized in the PET images, while [ 68 Ga]Ga-LW02075 showed a lower uptake in PC-3 tumor xenografts.Although the GRPR binding affinity of Ga-LW02075 was significantly better than that of Ga-LW02050 (K i = 1.39 ± 0.54 vs. 8.53 ± 1.52 nM, p = 0.0016), [ 68 Ga]Ga-LW02075 showed a lower tumor uptake (3.97 ± 1.71 vs. 5.38 ± 1.00 %ID/g).One possible explanation for these observations is the better hydrophilicity of [ 68 Ga]Ga-LW02050 resulting from the incorporation of the more hydrophilic cationic Pip linker.All [ 68 Ga]Ga-SB3, [ 68 Ga]Ga-LW02075, and [ 68 Ga]Ga-LW02050 were excreted primarily through the renal pathway with extremely high radioactivity accumulated in the urinary bladders.as observed from the PET images.A moderate hepatobiliary excretion of [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02075 was also observed, as both tracers showed higher uptake values in the liver, small intestine, and large intestine when compared with those of [ 68 Ga]Ga-LW02050.This is likely due to the incorporation of the cationic Pip linker in [ 68 Ga]Ga-LW02050, leading to a lower extent of hepatobiliary excretion.The pancreas was also clearly visualized in the PET images of [ 68 Ga]Ga-SB3 and [ 68 Ga]Ga-LW02075, but not in the PET image of [ 68 Ga]Ga-LW02050.This was also confirmed by the obtained biodistribution data: [ 68 Ga]Ga-SB3 showed the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [ 68 Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the uptake of [ 68 Ga]Ga-LW02050 in the pancreas was only 0.53 ± 0.11 %ID/g.The minimal uptake of [ 68 Ga]Ga-LW02050 in the liver, intestine, and pancreas might also contribute to the higher uptake in PC-3 tumor xenografts compared to that of [ 68 Ga]Ga-LW02075.
The good tumor uptake of [ 68 Ga]Ga-LW02050 and its minimal uptake in normal organs/tissues result in excellent tumor-to-background imaging contrast (Figure 4).Our data support [ 68 Ga]Ga-LW02050 as a promising PET tracer for detecting GRPR-expressing lesions, even for those adjacent to or in the pancreas.In addition, our findings suggest that LW02050 is a promising pharmacophore for the design of GRPR-targeted radiopharmaceuticals, especially when labeled with αor β-emitters for therapeutic application to minimize toxicity to the pancreas.
Blocking studies were performed for both [ 68 Ga]Ga-LW02075 and [ 68 Ga]Ga-LW02050 by the co-injection with 100 µg of their respective nonradioactive standard (Figures 4 and 6, and Table S4).Consistent with the observation from the PET image, the tumor uptake of [ 68 Ga]Ga-LW02050 reduced by 92%, confirming its specific uptake in PC-3 tumor xenografts.The significant reduction in [ 68 Ga]Ga-LW02050 in the pancreas (by 81%) is in agreement with the fact that the pancreas is the highest GRPR-expressing normal organ [1][2][3].For [ 68 Ga]Ga-LW02075, there was 54% reduction in PC-3 tumor uptake and 91% reduction in pancreas uptake by the co-injection of 100 µg of nonradioactive Ga-LW02075.This suggests that [ 68 Ga]Ga-LW02075 might have a higher binding affinity or be more selective toward the mouse GRPR expressed by the mouse pancreas than the human GRPR expressed by the PC-3 tumor xenografts.

General Methods
SB3 and its nonradioactive Ga-complexed standard were synthesized following previously reported procedures [18].All other reagents and solvents were purchased from commercial sources.LW02075 and LW02050 were synthesized on solid phase using an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer.Agilent (Santa Clara, CA, USA) HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (220 nm), and a Bioscan (Washington, DC, USA) NaI scintillation detector were used for the purification and quality control of LW02075, LW02050, and their nat Ga/ 68 Ga-complexed analogs.HPLC purification was performed using a semipreparative column (Luna C18, 5 µ, 250 × 10 mm), and quality control was conducted with an analytical column (Luna C18, 5 µ, 250 × 4.6 mm) purchased from Phenomenex (Torrance, CA, USA).A Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier was used for lyophilizing the collected HPLC eluates.An AB SCIEX (Framingham, MA, USA) 4000 QTRAP mass spectrometer system with an ESI ion source was used for MS analysis. 68Ga was eluted from an ITM Medical Isotopes GmbH (Munich, Germany) generator and purified via a DGA resin column from Eichrom Technologies LLC (Lisle, IL, USA).C18 Sep-Pak cartridges (1 cm 3 , 50 mg) were purchased from Waters (Milford, MA, USA).The radioactivity of 68 Ga-labeled peptides was measured via a Capintec (Ramsey, NJ, USA) CRC ® -25R/W dose calibrator.The radioactivity values of the biodistribution samples were counted on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Synthesis of Nonradioactive Ga-Complexed Standards
The nonradioactive Ga-complexed standards of LW02075 and LW02050 were prepared by incubating the DOTA-conjugated precursor (1 eq.) with GaCl 3 (5 eq.) in NaOAc buffer (pH 4.5, 0.1 M, 500 µL) at 80 • C for 15 min.The reaction mixture was then purified via HPLC after cooling down to room temperature.The collected HPLC eluates containing the desired nonradioactive Ga-complexed standard were lyophilized via a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier.The HPLC conditions, retention times, isolated yields, purities, and MS confirmations of Ga-LW02075 and Ga-LW02050 are provided in Table S2 and Figures S3 and S4.

Synthesis of 68 Ga-Labeled Tracers
The Ga-68 radiolabeling experiments were conducted following procedures reported in the literature [15,16].Purified 68 GaCl 3 in 0.5 mL water was added into a G4 glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and 10 µL of the precursor solution (1 mM).The radiolabeling reaction was performed with 1 min microwave heating in a Monowave200 (Anton Paar, Graz, Austria) microwave reactor at 100 • C, followed by HPLC purification.The eluate fraction containing the desired radiolabeled product was collected and diluted with 50 mL of water.The diluted product solution was passed through a C18 Sep-Pak cartridge which was pre-washed with ethanol (1 mL) and water (2 mL).The 68 Ga-labeled product was eluted with ethanol (0.4 mL) and diluted with DPBS, which contained 0.1% ascorbic acid for both PET imaging and biodistribution studies.Quality control was performed on HPLC using the analytical column.The HPLC conditions and retention times are listed in Table S3.

Fluorometric Calcium Release Assay
The antagonist/agonist characteristics of Ga-LW02075 and Ga-LW02050 were determined following previously published procedures [15,16].A 96-well clear-bottomed black plate was seeded with 5 × 10 4 PC-3 cells/well in 100 µL growth media 24 h prior to the assay.A calcium-sensitive dye (FLIPR Calcium 6 assay kit from Molecular Devices, San Jose, CA, USA) dissolved in the loading buffer (100 µL/well) was added into the plate followed by 1 h incubation at 37 • C.Then, the plate was transferred into a FlexStation 3 microplate reader (Molecular Devices, San Jose, CA, USA) to record the fluorescent signals.

In Vitro Competition Binding Assay
Inhibition constants (K i ) of Ga-SB3, Ga-LW02075, and Ga-LW02050 toward GRPR were measured by in vitro competition binding assay following previously published procedures [16,19,20].The assays were conducted in triplicate with varied concentrations (10 µM to 1 pM) of Ga-SB3, Ga-LW02075, and Ga-LW02050.Briefly, 2 × 10 5 cells/well of PC-3 cells were seeded in 24-well poly-D-lysine plates 24 h prior to the assay.The growth medium was replaced with 400 µL of reaction medium (RPMI 1640 containing 2 mg/mL bovine serum albumin (BSA) and 20 mM HEPES), followed by 1 h incubation at 37 • C. The tested ligands in 50 µL reaction medium and 0.01 nM [ 125 I-Tyr 4 ]Bombesin in 50 µL reaction medium were added into the wells, and then incubated at 37 • C with moderate agitation for 1 h.PC-3 cells were harvested by trypsinization after two rounds of DPBS washing, and counted on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.Data analysis was performed using nonlinear regression with GraphPad (San Diego, CA, USA) Prism 10 software (Version 10.1.1).

Ex Vivo Biodistribution and PET/CT Imaging
Imaging and biodistribution studies were performed on male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG) mice following previously published procedures [21].The experiments were conducted following the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia.The mice were sedated (2.5% isoflurane in oxygen) and injected with 5 × 10 6 PC-3 cells (100 µL; 1:1 PBS/Matrigel) subcutaneously behind the left shoulder.The tumorbearing mice were used for PET/CT imaging and biodistribution studies when the tumor grew to 5-8 mm in diameter.
PET/CT imaging experiments were performed on a Siemens (Knoxville, TN, USA) Inveon micro PET/CT scanner.Each tumor-bearing mouse was injected with ~3-6 MBq of 68 Ga-labeled tracer through a lateral caudal tail vein under anaesthesia.At around 50 min post injection, a 10 min CT scan was obtained first for localization and attenuation correction after segmentation for reconstructing the PET image, followed by a 10 min static PET scan.
For biodistribution studies, the mice were injected with the radiotracer (~2-4 MBq), as described above.Blocking of [ 68 Ga]Ga-LW02075 and [ 68 Ga]Ga-LW02050 was conducted by co-injection with 100 µg nonradioactive Ga-LW02075 and Ga-LW02050, respectively, through a lateral caudal tail vein under anaesthesia.At 1 h post injection, the mice were sedated and euthanized by CO 2 inhalation.Blood was collected via cardiac puncture, and organs/tissues of interest were collected, weighed, and counted on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Statistical Analysis
Statistical analyses were conducted with Student's t-test using the Microsoft (Redmond, WA, USA) Excel software (Version 16.84) for the data obtained from biodistribution studies.Comparison of the biodistribution data of [ 68 Ga]Ga-LW02050 and [ 68 Ga]Ga-SB3 was conducted with unpaired two-tailed test, and the comparison of the biodistribution data with/without co-injection of the nonradioactive standard was performed by unpaired one-tailed test.Statistically significant differences were considered present when the adjusted p value was <0.05.

Figure 1 .
Figure 1.Chemical structures of (A) SB3, (B) LW02075, and (C) LW02050.The differences in the chemical structures between SB3 and the C-terminal hydroxamate-derived LW02075 and LW02050 are shown in blue.

Figure 1 .
Figure 1.Chemical structures of (A) SB3, (B) LW02075, and (C) LW02050.The differences in the chemical structures between SB3 and the C-terminal hydroxamate-derived LW02075 and LW02050 are shown in blue.