1. Introduction
Pancreatic cancer (PC) is a highly malignant phenotype characterized by aggressive invasion and metastasis. Patients with PC often experience few symptoms in the early stages, and in most patients, local invasion and distant metastases are typically observed at diagnosis and therefore are unresectable [
1,
2,
3]. Currently, radical surgery at the early stages is the only opportunity for improving patient outcomes [
4]. Despite great advances in various imaging methods, the early diagnosis of PC still faces immense challenges at present.
Cancer-related biomarkers are defined as diagnostic indicators characterizing tumor biological behavior [
5]. Among them, chemokine (C-X-C motif) receptor 4 (CXCR4) and integrin α
vβ
3 are involved in several key biological processes of cancer and have been extensively investigated as molecular imaging targets. The overexpression of CXCR4 contributes to tumor growth, invasion, angiogenesis, metastasis, relapse, and drug resistance and is associated with an overall poor prognosis [
6]. Furthermore, CXCR4 is involved in tumorigenesis and progression in many cancers, especially PC. CXCR4 is considered a key factor in the malignant transformation of pancreatic intraepithelial neoplasia, and its expression level may be related to the grade of neoplasia [
7]. In addition, CXCR4 is involved in almost every aspect of PC, especially its invasion, metastasis, and chemoresistance [
8]. Integrin α
vβ
3 mediates interactions between cells and the extracellular matrix and has an important role in tumor angiogenesis [
9,
10]. The CXCL12/CXCR4 axis synergistically promotes angiogenesis by enhancing the production of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) as well as promoting integrin α
vβ
3 expression [
11]. Of note, CXCR4 and integrin α
vβ
3 had a synergistic effect on cancer metastasis [
12]. Therefore, molecular imaging probes targeting CXCR4 or integrin α
vβ
3 hold great promise for PC.
Many molecular agents have been developed based on the structures of CXCR4 antagonists (such as plerixafor (AMD3100) [
13], T140, FC131 [
14], or antibodies [
15]) or arginine-glycine-aspartate (RGD) derivatives for CXCR4 or integrin α
vβ
3-targeted diagnosis and therapy [
16,
17,
18,
19]. Although promising results were achieved by these agents, mono receptor recognition may limit their sensitivity for the detection of diseases because of the heterogeneity of tumors and their relatively low receptor expression density, especially during the early stages [
20,
21]. Therefore, it is essential to develop radiotracers that bind to multiple receptors simultaneously, allowing tumor contrast when either receptor is expressed, thus improving the detection sensitivity. The development of heterodimeric tracers that bind to two different receptors with suitable pharmacokinetics and targeting efficiency is thought to be an effective solution [
22,
23].
Over the past five years, there has been a growing interest in the development and evaluation of tracers possessing heterobivalent targeting properties for tumor diagnosis and treatment. Biomarkers including integrin α
vβ
3, GRPR (gastrin-releasing peptide receptor), MC1R (melanocortin receptor subtype 1), SSTRs (somatostatin receptors), PSMA (prostate-specific membrane antigen), VPAC
1R (vasoactive intestinal peptide receptor subtype 1), NPY(Y
1)R (neuropeptide Y receptor subtype 1) were extensively investigated as one of the targeting moieties [
24,
25,
26,
27,
28,
29,
30,
31,
32,
33], and the developed heterodimeric tracers demonstrating improved targeting affinity and efficiency compared with their corresponding single-receptor targeted tracers. Especially, BBN-RGD was further assessed in breast cancer and prostate cancer patients and exhibited promising value in diagnosis and staging [
25,
26]. As to the diagnosis of PC, NGR-RGD and BBN-RGD exhibited promising results in preclinical evaluations [
26,
34]. In addition, this heterodimeric strategy was also applied in the development of nanoparticle-based probes, displaying a unique advantage in improving tumor targeting efficiency [
35].
The aim of this study was to develop a dual receptor targeting peptide heterodimer probe, which can target CXCR4 and integrin αvβ3 simultaneously, to improve the detection sensitivity of tumors, especially PC. The in vitro and in vivo performance of the developed tracer 68Ga-yG5-RGD was investigated using a PC tumor model and the results were compared with their corresponding single-target probes, 68Ga-yG5 and 68Ga-RGD.
2. Materials and Methods
2.1. Reagents and Instruments
Gallium-68 was eluted from a commercial
68Ga/
68Ge-generator (Isotope Technologies Garching GmbH, Garching, Germany) with 0.05 M HCl. Peptide c(RGDyK) was customized by Chinapeptide (Shanghai, China). Endo-BCN-PEG
4-NHS ester was purchased from Broadpharm (San Diego, CA, USA). The 2,2′,2″-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid (
p-SCN-Bn-NOTA) was purchased from Macrocyclics (Dallas, TX, USA). NOTA-Bn-thioureido-cyclic(Arg-Gly-Asp-
d-Tyr-Lys) (NOTA-c(RGDyK)), NO2A
tBu-N
3, and BCN-PEG
4-c(RGDyK) were synthesized according to published methods [
24]. All other chemicals were purchased from J&K Chemicals (Beijing, China), Adamas Reagent Co., Ltd., (Shanghai, China), or Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise. The chemicals were used directly without further purification. Radioactivity of all samples was quantified by an automatic gamma counter (2470 WIZARD; PerkinElmer, Waltham, MA, USA). TransPET Discoverist 180 small animal PET/CT (Raycan Technology Co., Ltd., Suzhou, China) and lnliView-3000B small animal PET/SPECT/CT (Novel Medical, Beijing, China) were used to visualize mice with xenografted tumors. High-performance liquid chromatography (HPLC) was performed on LC-20AT (Shimadzu Corporation, Tokyo, Japan) equipped with an SPD-20A UV/VIS detector and a flow count radiation detector (Bioscan, Washington, DC, USA). Radiochemical yield and radiochemical purity of the tracers were determined by HPLC using a 4.6 × 250 mm Luna C
18 column (Phenomenex, CA, USA). The column was eluted at a flow rate of 1 mL/min at 25 °C with a gradient of 5–90% MeCN/H
2O with 0.1% trifluoroacetic acid (TFA) in 10 min.
2.2. Synthesis of NOTA-yG5 and NOTA-yG5-RGD
Cyclic peptide Cyclo(D-Tyr-N-Me-D-Lys-Arg-2-Nal-Gly), denoted as yG5, was an analogy of peptide FC131, designed as a new CXCR4 antagonist. The detailed evaluation of yG5 was published in an issued patent [
36] and the results are considered published as an article elsewhere. Peptide yG5 was prepared using Fmoc-based solid phase peptide synthesis method and then conjugated with
p-SCN-Bn-NOTA to obtain NOTA-yG5 after purification (
Scheme S1). High-resolution mass spectrometry (HRMS) (Bruker SolariX 7.0T, Bruker Daltonik, Bremen, Germany):
m/
z calcd for C
57H
75N
13O
12S [M + 2H]
2+ 583.7767, Found 583.7756.
To construct CXCR4/integrin α
vβ
3 heterodimeric ligand, bifunctional chelator NO2A
tBu-N
3 was applied to conjugate yG5 with c(RGDyK) using our previously developed method [
23,
26]. Briefly, NO2A
tBu-N
3 was first conjugated to peptide yG5 to obtain N
3-NOTA-yG5 after TFA deprotection and HPLC purification. The heterodimeric tracer Cyclo(D-Tyr-N-Me-D-Lys-Arg-2-Nal-Gly)-NOTA-click-PEG
4-c(RGDyK) (denoted as NOTA-yG5-RGD) was then obtained via copper-free click chemistry by reacting N
3-NOTA-yG5 with BCN-PEG
4-c(RGDyK) [
24] at a molar ratio of 1:1 and purifying using HPLC (
Scheme S1). High resolution mass spectrometry (HRMS) (Bruker SolariX 7.0T, Bruker Daltonik, Bremen, Germany):
m/
z calcd for C
101H
146N
24O
26: [M + 2H]
2+ 1056.5499; found 1057.0540.
2.3. Radiolabeling
In brief, NOTA-yG5-RGD (4 nmol), NOTA-c(RGDyK) (4 nmol) or NOTA-yG5 (4 nmol) was separately added to 150 µL sodium acetate buffer (0.25 M). Subsequently, 500 μL of [68Ga]GaCl3 (0.05 M HCl, 296~370 MBq) was added, vortex mixing for 30 s. The final pH of the mixed solution was about 4.0. The reaction mixture was then heated to 100 °C for 5 min. The resultant products were analyzed by analytical radio-HPLC to determine the radiochemical yield and purity.
2.4. Stability and Octanol/Water Partition Coefficient (logP)
The 68Ga-yG5-RGD (1.85 MBq, 5 μL) or 68Ga-yG5 (1.85 MBq, 5 μL) was separately added to 300 μL of PBS or normal human fresh serum, incubated for 2 h at 37 °C. After incubation, the PBS sample was subjected to HPLC for radiochemical purity under the conditions described above. An equal volume of anhydrous acetonitrile was added to serum sample, mixed, and then centrifuged at 13,000 g for 10 min to fully precipitate protein. Following this, the supernatant was collected and authenticated by HPLC.
Two normal BALB/c nude mice (female, 8 weeks) were used to evaluate in vivo metabolic stability of the two probes. The 68Ga-yG5-RGD or 68Ga-yG5 of about 74 MBq per mouse was intravenously injected via the tail vein. Two hours later, mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium solution (0.1 mL/20 g). Urine samples were collected by gentle bladder massage. After adding an equal volume of acetonitrile, the mixture was centrifuged, and the supernatant was analyzed by HPLC.
68Ga-yG5-RGD, 68Ga-yG5, or 68Ga-RGD (740 kBq each) were separately added to a mixture of 1.0 mL n-octanol and 1.0 mL PBS, and then the mixtures were thoroughly vortexed for 5 min. Each sample was then centrifuged at 8000 rpm for 4 min. Each sample (100 μL) in organic phase and aqueous phase was collected separately in tubes, 5 tubes for each phase. Radioactive counts per tube of samples were measured by gamma counter, and logP values were calculated by the following formula: logP = log10 (counts in n-octanol/counts in PBS).
2.5. Cell Culture
The BxPC3 human pancreatic cancer cell line was kindly provided by the Laboratory of Pancreatic Surgery at the Huazhong University of Science and Technology Affiliated Union Hospital (obtained from Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China). The MX-1 human breast cancer cell line was kindly provided by Basic Medical School of Huazhong University of Science and Technology. BxPC3 and MX-1 cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator.
2.6. Western Blot Assay
Briefly, the BxPC3 and MX-1 cells were separately cultured in 6-well plates at the density of 2 × 105 cells/well. After the cells adhered to the wall, the total protein was extracted. Protein concentrations were determined by BCA protein assay kit. After the preparation of 10% SDS-PAGE separation gel and concentrated gum, 40 μg total protein samples and marker were separately loaded into each well of the SDS-PAGE gel for separation by electrophoresis. Following electrophoresis, proteins were transferred onto PVDF membranes. The membrane was later incubated with anti-Integrin αν, anti-Integrin β3, and anti-CXCR4 rabbit monoclonal polyclonal antibodies (1:1000 dilution) overnight at 4 °C. Incubation was for 1 h at room temperature with secondary antibody (1:20,000 dilution) and development was by enhanced chemiluminescence (ECL). The densities of the bands were quantified by Image J Software (version 1.45, NIH, Bethesda, MA, USA).
2.7. In Vitro Cell Assays
BxPC3 cells in the logarithmic growth phase were harvested, counted, and seeded onto 24-well plate with 2 × 105 cells/well and cultured overnight. The original culture medium was discarded and fresh DMEM (serum and antibiotic-free) was then added to continue culturing for 0.5 h. Subsequently, each well was separately added 68Ga-yG5-RGD, 68Ga-yG5, or 68Ga-RGD (74 kBq/well, 50 μL) and incubated for 30 min, 1 h, and 2 h (three replicate wells/each time point). At the end of incubation, the supernatant and the cell lysate were separately collected and radioactivity was measured by gamma counter. The cell uptake was expressed as the percentage of total applied radioactivity. Cell uptake study experiment was also performed using MX-1 as a control group for 68Ga-yG5-RGD. The detailed experimental procedure was the same as above.
BxPC3 was used for cell blocking experiments. One group was taken as a control and added only 68Ga-yG5-RGD (74 kBq/well, 50 μL, 1 pmol). Other groups were incubated with the blocking agents (AMD3100, RGD, AMD3100 + RGD unlabeled, 1000 pmol/10 μL, and 68Ga-yG5-RGD (74 kBq/well, 50 μL). The rest of the procedures were carried out as described above.
2.8. Animal Models
All animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology (2021-S2875; 1 February 2021). Female BALB/c nude mice (5–6 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and were raised in a specific pathogen-free barrier system. For subcutaneous tumor xenograft model, 1 × 107 cells of BxPC3 or MX-1 in 100 μL PBS were injected into the right flanks of nude mice. Animal models were used for follow-up experiments when tumors reached 8~10 mm in diameter.
Written informed consent from all patients was obtained and the experiments were approved by the institutional review board of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (2021-0799-01; 2 November 2021). For the establishment of patient-derived xenograft (PDX) models, tumor tissues from pancreatic cancer patients were obtained via surgical resection. Collected tumor tissues were immediately placed in a sterile tube containing ice-chilled culture medium with 1% penicillin/streptomycin, 5 ug/mL tetracycline, and 10 ug/mL ciprofloxacin. Anesthetize the mouse by exposure to 2~2.5% vaporized isoflurane in 100% air using an induction chamber, then transfer it to the surgical table and place it in a prone position and maintain anesthesia with 1.5~2% vaporized isoflurane through the nose cone. The tumor tissues were then minced into 1~3 mm3 pieces and subcutaneously implanted into both flanks of BALB/c nude mice using a trocar. The PDX models were used for PET/CT scans when tumors reached a diameter of about 8~10 mm.
2.9. Small Animal PET/CT Static Scan
For comparison of in vivo performance of the probes, each group of BxPC3 tumor xenograft models (n = 4) was separately injected via tail vein with 68Ga-yG5-RGD, 68Ga-RGD, and 68Ga-yG5 (5.55~7.4 MBq, 150 μL/per mouse). Ten-minute static PET scan was acquired, followed by 6-min CT scan at 30 min, 1 h, and 2 h post-injection. Image data were reconstructed after attenuation correction. A region of interest (ROI) was drawn on the attenuation-corrected image to calculate the uptake value. In order to better assess the in vivo performance of 68Ga-yG5-RGD, pancreatic cancer-PDX models were also used for imaging.
For testing of in vivo targeting specificity of 68Ga-yG5-RGD, nude mice bearing BxPC3 tumors (n = 4) were co-injected with 68Ga-yG5-RGD (5.55~7.4 MBq, 150 μL/per mouse) and blockers (AMD3100, RGD, or AMD3100 + RGD, 10 mg/kg each) via tail vein, and PET/CT scan was conducted at 30 min after injection.
MX-1 tumor xenograft models (n = 4) were set as controls. Additionally, the detailed operation procedure was as described above. Briefly, nude mice bearing MX-1 tumor were injected with 68Ga-yG5-RGD (5.55~7.4 MBq, 150 μL/per mouse) and PET/CT scan was conducted at 30 min, 1 h, and 2 h after injection.
2.10. Biodistribution Studies
The group setting was kept consistent with PET/CT scan. Each group of mice (n = 4) was injected via tail vein with probes (5.55~7.4 MBq, 150 μL) or co-injected with probes and blockers (10 mg/kg). Mice were euthanized by cervical dislocation under anesthesia 2 h post-injection. Subsequently, tissues of interest were collected, washed, weighed, and counted by a gamma counter. After decay correction, uptake of tissues was expressed as the percentage of injected dose per gram of tissue (%ID/g).
2.11. Immunohistochemical Analysis
BxPC3, MX-1, and PDX tumor samples were collected, fixed, paraffin-embedded, and then cut into 5 µm sections. After antigen repair, endogenous peroxidase activity was blocked using a solution of 2.5% hydrogen peroxide in methanol. The tissue sections were incubated with 10% normal goat serum to reduce nonspecific binding. Incubate sections with primary antibody (anti-CXCR4, diluted 1:500; anti-αvβ3, diluted 1:100; anti-CD31 1:100) overnight at 4 °C. The next day, the sections were incubated with a universal protein block (DAKO) for 25 min at room temperature. Sections were visualized with DAB (Dako) and observed under a light microscope. Subsequently, the sections were counterstained with hematoxylin. Additionally, the sections were dehydrated with gradient ethanol, sealed with neutral gum, and then detected under the microscope.
2.12. Statistical Analysis
All quantitative data are expressed as a mean ± standard deviation. Data were plotted and statistically analyzed using GraphPad Prism software (version 8.0; GraphPad Software Inc., La Jolla, CA, USA). Comparisons between groups were performed using Student’s t-test for independent samples. p < 0.05 was considered indicative of a significant difference.
4. Discussion
Recently, the use of radiotracers targeting CXCR4 or integrin α
vβ
3 for tumor imaging has been widely investigated [
17,
18]. However, monomeric peptides usually accumulate in tumors with a relatively low concentration because of their moderate affinity for their targets and the heterogeneity of tumors, resulting in unsatisfied tumor detection sensitivity [
22]. Therefore, to improve the sensitivity of tracers for tumor imaging, we used a polyvalent strategy to develop a heterodimeric tracer,
68Ga-yG5-RGD, which targeted CXCR4 and integrin α
vβ
3 simultaneously for PC imaging. Compared with monospecific tracers, the dual-receptor targeting tracer
68Ga-yG5-RGD had enhanced tumor-targeting efficiency and prolonged tumor retention, suggesting their high clinical translation potential.
The preparation and radiolabeling process for 68Ga-yG5-RGD was simple and achieved a high radiochemical yield and purity, and there was no requirement for extra purification steps. The 68Ga-yG5-RGD also exhibited excellent in vitro and in vivo stability, with less than 1% metabolic-free Gallium-68 observed. It should be noticed that a shoulder peak was found in the main peak of the 2 h urine sample, which might be due to the metabolism of the tracer or the increased tube circles that pass through the detector cell to improve the detecting sensitivity of the gamma detector. The cellular uptake of 68Ga-yG5-RGD was considerably higher than that of 68Ga-yG5 or 68Ga-RGD in BxPC3 cells that express CXCR4 and integrin αvβ3, demonstrating the higher target efficiency of dual-targeted tracers than single-targeted tracers. The uptake of 68Ga-yG5-RGD in BxPC3 cells was notably blocked by an excess of unlabeled AMD3100, RGD, or AMD3100 + RGD, suggesting a favorable targeting specificity towards the two receptors, CXCR4, and integrin αvβ3.
The in vivo performance of 68Ga-yG5-RGD was evaluated by small animal PET/CT in BxPC3 tumor-bearing nude mice as well as PDX models. Within the observation time, high levels of radioactivity were observed in the liver and kidneys after the injection of 68Ga-yG5-RGD, which indicated 68Ga-yG5-RGD was metabolized and excreted mainly via the liver and kidneys. The outline of the BxPC3 tumor was more clearly visualized by 68Ga-yG5-RGD compared with 68Ga-yG5 or 68Ga-RGD. The ROI analysis showed that the uptake of 68Ga-yG5-RGD by BxPC3 tumors was markedly higher than that of 68Ga-yG5 or 68Ga-RGD at all time points studied. The quantitative biodistribution studies showed that the uptake of 68Ga-yG5-RGD by BxPC3 tumors was statistically higher than that of its monomeric counterparts, which was in concordance with the imaging results. Moreover, the tumor-to-blood and tumor-to-muscle ratios of 68Ga-yG5-RGD were higher than those of 68Ga-yG5 and 68Ga-RGD. This improved tumor-targeting efficiency might be attributed to the dual CXCR4 and integrin αvβ3 binding ability of 68Ga-yG5-RGD in BxPC3 tumors. The increased molecular weight of the peptide heterodimer and the introduction of short polyethylene glycol groups may result in its prolonged blood circulation, which may also contribute to its enhanced tumor uptake. Similarly, 68Ga-yG5-RGD also showed well in vivo tumor targeting performance in PDX-tumors. It should be noticed that the RGD unit contributes a major role in improving the tumor-to-nontumor ratios, and the yG5 unit improved the tumor uptake values and prolonged the retention in the tumor. A synergistic effect may be expected when designing and developing a heterodimeric tracer.
To evaluate the in vivo dual-receptor targeting specificity of 68Ga-yG5-RGD, blocking studies were performed in BxPC3 tumor-bearing nude mice. A substantial reduction of radioactivity was observed in BxPC3 tumors when they were blocked by excess amounts of unlabeled AMD3100 or RGD, indicating the specific binding between 68Ga-yG5-RGD and its targets. Of note, the lowest uptake of 68Ga-yG5-RGD by BxPC3 tumors was observed when it was administered intravenously with the co-injection of AMD3100 and RGD. Interestingly, the yG5 motif of 68Ga-yG5-RGD could still bind to CXCR4 when blocked by RGD, and the RGD motif could still bind to integrin αvβ3 when blocked by AMD3100. These blocking results suggested that 68Ga-yG5-RGD achieved dual-receptor targeting specificity. In addition, when compared with the control groups, the uptake of 68Ga-yG5-RGD by MX-1 tumors was similar to or even lower than that of muscles, further confirming its targeting specificity. Immunohistochemical analysis also confirmed that the levels of the receptors expressed on BxPC3, and MX-1 tumors were consistent with the uptake of the tracers.
Although promising results were achieved by this heterodimeric tracer, it should be noted that the accumulation of radioactivity in the liver was relatively high, which is unfavorable for detecting primary lesions of PC. The tumor-to-liver ratios of
68Ga-yG5-RGD and
68Ga-yG5 were relatively lower than that of
68Ga-RGD (no statistical difference), which might be related to the strong hydrophobic menaphthyl group and the positive charge of the arginine amino acid in the yG5 sequence, as well as better in vivo pharmacokinetic characteristics of RGD probes. In addition, normal liver tissues express multiple chemokine receptors, including CXCR4 [
17], which might also explain the high accumulation of radioactivity related to
68Ga-yG5-RGD and
68Ga-yG5 in the liver. Compared with
68Ga-yG5, the hydrophilicity of
68Ga-yG5-RGD was markedly improved via PEGylation and the introduction of an RGD moiety, assessed by their log
p values and the decreased intestinal uptake and hepatobiliary excretion of
68Ga-yG5-RGD in vivo. However, further modification of the structure of
68Ga-yG5-RGD is still needed to improve its in vivo pharmacokinetics, including extending the PEGylation and modifying the structure of the CXCR4 antagonist [
37,
38]. Another potential solution would be to pre-inject a small amount of CXCR4 antagonist to reduce liver uptake, thus improving tumor uptake [
39]. Last but not least, the cell line BxPC3 used in the study showed moderate expression of both CXCR4 and integrin α
vβ
3, which accounted for a part of the reason for suboptimal tumor imaging. For further experiments after the chemical improvement of the radiotracers, we will establish cell lines with high expression of CXCR4 and integrin α
vβ
3 by stable transfection to better evaluate the performance of the tracers for pancreatic cancer imaging. Meanwhile, cell lines with low, moderate, and high expression of targets will be used to better evaluate the specificity of the heterodimer for the different targets.
Studies have reported that CXCR4 and integrin α
vβ
3 are synergistically involved in cancer metastasis [
12,
40]. Therefore,
68Ga-yG5-RGD may have prospective applications in the detection of tumor metastatic foci. Moreover, although the BxPC3 cell line selected in our study has a moderate expression of CXCR4 and integrin α
vβ
3, BxPC3 tumors could still be visualized clearly by
68Ga-yG5-RGD because of its high tumor uptake. Many human cancers have high expressions of CXCR4 and integrin α
vβ
3 [
6,
11], suggesting
68Ga-yG5-RGD may be useful for a broad range of applications and have good prospects for clinical translation. Furthermore, with improved in vivo pharmacokinetics of this heterodimeric tracer, yG5-RGD could also be labeled by therapeutic radionuclides, such as
177Lu, providing an alternative method for the treatment of PC.