Design, Synthesis and Biological Evaluation of 68Ga-DOTA-PA1 for

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Design, Synthesis and Biological Evaluation of 68Ga-DOTA-PA1 for Lung Cancer: A Novel PET Tracer for Multiple Somatostatin Receptors Imaging Fei Liu, Teli Liu, Xiaoxia Xu, Xiaoyi Guo, Nan Li, Chiyi Xiong, Chun Li, Hua Zhu, and Zhi Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00963 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Molecular Pharmaceutics

68

Design, Synthesis and Biological Evaluation of Ga-DOTA-PA1 for Lung Cancer: A Novel PET Tracer for Multiple Somatostatin Receptors Imaging

Fei Liu,† Teli Liu,† Xiaoxia Xu,† Xiaoyi Guo,† Nan Li,† Chiyi Xiong,‡ Chun Li,‡ Hua Zhu,*,† and Zhi Yang*,† †

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing),

Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, P.R. China ‡

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center

Houston, Texas 77030, USA

ABSTRACT Most of the radiolabeled somatostatin analogues (SSAs) are specific for subtype somatostatin receptor 2 (SSTR2). Lack of ligands targeting other subtypes of SSTRs, especially SSTR1, SSTR3 and SSTR5, limited their applications in tumors of low SSTR2 expression, including lung tumor. In this study, we aimed to design and synthesize a positron emission tomography (PET) radiotracer targeting multi-subtypes

of

SSTRs

for

PET

imaging.

PA1

peptide

and

its

conjugate

with

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator or fluorescein isothiocyanate (FITC) at the N-terminal of the lysine position were synthesized.

68

Ga was chelated to DOTA-PA1 to

obtain 68Ga-DOTA-PA1 radiotracer. The stability, lipophilicity, binding affinity, and binding specificity of

68

Ga-DOTA-PA1 and FITC-PA1 were evaluated by various in-vitro experiments. Micro-PET

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imaging of

68

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Ga-DOTA-PA1 was performed in nude mice bearing A549 lung adenocarcinoma, as

compared with

68

Ga-DOTA-(Tyr3)-Octreotate (68Ga-DOTA-TATE). Histological analysis of SSTRs

expression in A549 tumor tissues and human tumor tissues was conducted using immunofluorescence staining and immunohistochemical assay.

68

Ga-DOTA-PA1 had high radiochemical yield and

radiochemical purity of over 95% and 98%, respectively. The radiotracer was stable in vitro in different buffers over a 2-h incubation period. Cell uptake of 68Ga-DOTA-PA1 was 1.31-, 1.33- and 1.90-fold of that of

68

Ga-DOTA-TATE, which has high binding affinity only for SSTR2, after 2 h incubation in

H520, PG and A549 lung cancer cell lines, respectively. Micro-PET images of

68

Ga-DOTA-PA1

showed that PET imaging signal correlated with the total expression of SSTRs, instead of SSTR2 only, which was measured by western blotting and immunofluorescence analysis in mice bearing A549 tumors. In summary, a novel PET radiotracer,

68

Ga-DOTA-PA1, targeting multi-subtypes of SSTRs,

was successfully synthesized and was confirmed to be useful for PET imaging. It may have potential as noninvasive PET radiotracer for imaging SSTRs-positive tumors. KEYWORDS: somatostatin analogues (SSAs), somatostatin receptors (SSTRs), positron emission tomography imaging (PET), lung cancer

INTRODUCTION Approximately 70-90% of neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs, including SSTR1, SSTR2, SSTR3 and SSTR5), except SSTR4. Radiolabeled somatostatin analogues (SSAs) can detect the SSTRs overexpressed on NETs cells, and this strategy has achieved widespread acceptance in early detection of tumor, tumor staging evaluation, and therapy monitoring.1 Octreotide and its analogs are the first and most widely used SSTR2 agonists used in NETs clinical diagnosis.

111

In-pentetreotide (OctreoScan, Mallinckrodt Pharmaceuticals) is the first nuclear

imaging peptide approved by FDA, which is still considered a gold standard for NET staging since 1994. With the emergence of novel radionuclides, especially the high resolution PET nuclides (68Ga, 64

Cu), a new class of SSAs labeled with positron emitters for PET/CT imaging becomes the current

new standard for NETs diagnosis, staging, and guided therapy. However, high false-negative rate remains a challenge for early detection of NET. For example,

68

Ga labeled SSAs PET imaging may

miss the primary and metastasis lesions in grade 2 (G2) and high-grade (G3) gastroenteropancreatic neuroendocrine tumors (GEP-NETs), probably due to low and heterogeneous SSTR expression in those tumors. SSTR2 antagonists may increase the detection level of low-expression of SSTR2 in NETs. Ginj et al.2 firstly showed that radiolabeled SST antagonists are superior to agonists for tumor targeting in animal models. Wild et al.3 compared the therapeutic effect of

177


Lu-DOTA-JR11 (antagonist) and

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177

Lu-DOTATATE (agonist) in 4 NET patients, with the former showing 1.7-10.6 times higher tumor

uptake than the latter, indicating the clinical feasibility of using antagonist for NETs therapy. Recently, Dalm et al.3, 4 reported detection of SSTR2 lesions in breast cancer, using antagonistic PET radiotracers. The limitation of PET imaging of NETs using octreotide and its analogs, and related antagonists, is that the compounds target single-receptor (SSTR2).5-8 As shown by Mansi et al.9 some NETs or insulinoma were rarely detectable due to the low expression of SSTR2. Reubi et al.10 investigated the incidence and density of SSTR1-5 subtypes, using about 100 frozen samples of NETs including the intestine, pancreas and lung. Although the majority of the tested tumors expressed SSTR2, 8/26 and 9/28 samples in insulinomas, and bronchial carcinoids did not express SSTR2. Only 2 samples expressed SSTR4. If the SSTR1,2,3,5 expression was taken as a whole, all except one (glucagonomas) samples express these receptors. The extensive monitoring of SSTR1,2,3,5 multi-receptors by PET imaging is one of the strategies which could substantially decrease the false-negative rate of NETs. Moreover, some other tumors, such as lung cancers,11 melanomas,12 colorectal cancers,13 breast cancers,14 also express large numbers of SSTRs, and PET imaging of SSTRs expression in these tumors may be useful in clinical precision diagnosis and therapy of other tumors. Among the five SSTRs subtypes expressed in NETs, DOTA-(Tyr3)-Octreotate (DOTA-TATE) has high binding affinity only for SSTR2.5,

6, 8

SOM230

(pasireotide), a multi-receptors targeted ligand widely used in the treatment of acromegaly and Cushing’s disease, has high affinities for four SSTR subtypes SSTR1, SSTR2, SSTR3 and SSTR5,15, 16 and displays a 30-, 5- and 39-fold higher affinity and 30-, 11-, 158-fold higher functional affinity for SSTR1, SSTR3 and SSTR5 than octreotide.17 The multi-receptors binding property of pasireotide may make it possible for PET imaging of not only NETs patients who may or may not respond to radiotracers based on octreotide, but also other tumor patients overexpressing SSTRs. We selected 68Ga as the position emission radionuclide in our designs of PET radiotracers.

68

Ga is generator-derived

isotope that is readily available, can provide high quality PET images, and has been widely used in clinical diagnosis and differential diagnosis. 68Ga-labeled SSAs have been used in the management of NETs patients.18-21 A meta-analysis of the diagnostic performance of 68Ga-labeled SSAs using PET/CT showed that it was one of the first diagnostic choices for suspected thoracic or GEP-NETs.22 68

Ga-DOTA-TATE, approved by US Food and Drug Administration (FDA) for clinical use in 2016,23

has fast blood clearance, provides high PET/CT imaging resolution, targets SSTR2, and has been widely used in clinical applications.24 In this study, we evaluated 68Ga-labeled PA1, a conjugate of 68Ga and pasireotide, a SSA with the structure of cyclo[HyPro-Phe-D-Trp-Lys-Tyr(Bzl)-Phe] named PA1.25 We report the radiolabeling of



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this tracer and the micro-PET imaging in the lung cancer model overexpressing SSTRs.

EXPERIMENTAL SECTION Materials, Cell Culture and Animal Model. All amino acid derivatives and coupling reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DOTA-TATE and monoclonal mouse anti-β-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). Radioimmunoprecipitation assays (RIPA) lysis buffer, 4',6-diamidino-2-phenylindole (DAPI) and 3,3’-diaminobenzidine (DAB) substrate kit were acquired from Themo Fisher Scientific (Waltham, MA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE was purchased from Kangweishiji Biotechnology Co., Ltd (Beijing, China). The polyclonal rabbit anti-human anti-SSTR1 (ab2366), monoclonal rabbit anti-human anti-SSTR2 (ab134152), monoclonal rabbit anti-human anti-SSTR3 (ab137026) and monoclonal rabbit anti-human anti-SSTR5 (ab156864, ab109495) antibodies were obtained from Abcam (Cambridge, UK). The horseradish peroxidase-labeled goat anti-mouse IgG, horseradish peroxidase-labeled goat anti-rabbit IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and biotinylated goat anti-rabbit IgG were acquired from Zhongshan Beijing Golden Bridge Biotechnology Co., Ltd (Beijing, China). Unless stated specifically, other chemicals were procured from Jinjinle Chemical Co., Ltd (Shanghai, China). All chemicals used were of either high performance liquid chromatography (HPLC, Agilent Technologies, California, CA, USA) or analytical grade and were used without further purification. Human lung squamous carcinoma cell line (H520), human lung adenocarcinoma cell line (A549) and human colorectal cancer cell lines (HT-29 and HCT116) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human pulmonary giant cell carcinoma cell line (PG) and human gastric cancer cells (BGC823 cells) were obtained from Peking University Cancer Hospital. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco BRL Life Technologies, Carlsbad, CA, USA). The cells were cultured at 37 °C with 5% CO2 and the culture medium was replaced under the supplier’s instructions. Healthy female BALB/c mice and BALB/c nude mice aged 6-8 week were acquired from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). About 2 × 106 tumor cells mixed with Matrigel (BD Biosciences, Franklin Lake, NJ, USA) at 1:1 (v/v) and culture medium was subcutaneously co-injected into the front right flank of the BALB/c nude mice. When the tumors reached 0.5-0.8 cm in average diameter, the mice were used for in vivo studies. All animal experiments were conducted in accordance with the guidelines approved by Peking University Cancer Hospital Animal Care and Use Committee. Peptide Synthesis, Radiolabeling and Quality Control of


68/nat

Ga-DOTA-PA1. Peptide

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synthesis. The peptide PA1 was synthesized according the literature.25 Briefly, commercially available Fmoc-Tyr(Bzl)-O-CH2-Ph(3-OCH3)-O-CH2-poly-styrene resin (1.2 g, 0.72 mmol, 0.6 mmol/g SASRIN-resin) was used as starting material in a manually operated reactor and carried through a standard protocol consisting of repetitive cycles of Nα deprotection (20% piperidine/N, N-dimethylformamide

(DMF)),

repeated

washings

with

DMF,

and

coupling

(N,N'-Diisopropylcarbodiimide (DIPCI)/1-hydroxybenzotriazole (HOBT), DMF). The following amino acid derivatives were sequentially coupled: Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-4-hydroxyl-Pro-OH, and Fmoc-Phe-OH. Couplings were continued or repeated until completion. After Fmoc group of Lys was removed, the linear fragment of peptide was obtained by cleavage with 2% trifluoroacetic acid (TFA). For cyclization, the above linear fragment was dissolved in DMF, treated with 3 equiv of N,N-Diisopropylethylamine (DIPEA) and then 1.5 equiv of diphenylphosphoryl azide (DPPA), and stirred at 0-4°C until completion (~20 h). The solvent was almost completely removed in vacuum. The concentrate was diluted with ethyl acetate, washed with NaHCO3 and water, dried, and evaporated in vacuum. The protected cyclized product was obtained in ~70% yield. For side chain deprotection, the residue was dissolved at 0°C in TFA/H2O, 95:5, and the mixture was stirred in the cold for 30 min. The product was then precipitated with ether, filtered, washed

with

ether,

and

dried.

PA1

was

conjugated

to

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator to yield DOTA-PA1, and conjugated to 5-FITC to yield FITC-PA1. After purification by using HPLC, the product was lyophilized and the molecular weight was determined by electrospray ionization mass spectrometry (ESI-MS, Agilent Technologies, California, CA, USA). About 10 mg of DOTA-PA1 and 5 mg of FITC-PA1 was isolated with an overall yields of 6% and 3%, respectively. Radiolabeling and quality control of HCl from a 1850 MBq

68

68/nat

Ga-DOTA-PA1.

68

GaCl3 was eluted in 2 mL of 0.05 M

Ge/68Ga generator (Isotope Technologies Garching, Munich, Germany).

Twenty µg of DOTA-PA1 precursor (5 mg/mL in DMF solution) was added into a vial, 1 mL of 68

GaCl3 (370 MBq) solution was added subsequently into the reaction vial, and pH value of reaction

mixture was adjusted to pH = 3.5 by adding 65 µL of NaOAc (1 M). The mixture was incubated at 95°C for 10 min.

68

Ga-DOTA-PA1 was then purified using a Sep-Pak® Light C18 Cartridge (Waters,

Dublin, Ireland). The radiotracer, 68Ga-DOTA-PA1, was washed out with 1 mL of 80% ethanol/water (1:1 v/v), and the solution was filtered via a 0.2 µm sterilizing filter (Pall Corporation, New York, NY, USA) into the product vial. The final solution was diluted to 5 mL with saline before animal studies. The quality of final product was analyzed using the radio-HPLC and radio-thin layer chromatography



(radio-TLC, Washington, DC, USA). The mobile phases for radio-HPLC were 0.1% TFA in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B). For radio-TLC, 0.9% saline was used as the developing solvent. Non-radioactive

nat

Ga-DOTA-PA1 was synthesized using the same procedure as

that for radio-tracer, and it was identified by matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDI-TOF MS, Bruker, Bremen, Germany). 68Ga-DOTA-TATE was synthesized according to reported procedures.18 The partition coefficient (log P) of 68Ga-DOTA-PA1 was measured as follows. 10 mL of 1-octanol and 10 mL of water were mixed and equilibrated overnight ahead of the study. During measurement, 1.85 MBq of

68

Ga-DOTA-PA1 was dissolved in 500 µL of water saturated with 1-octanol, and equal

volume of 1-octanol saturated with water was added in the test tube. The mixture was then shaken vigorously using a Vortex mixer for 3 min (Scientific Industries, New York, NY, USA) and incubated at room temperature for 5 min. After the mixture separated into 2 layers visually again, the test tube was shaken again and centrifuged at 1500 × g for 5 min, then aliquots of 50 µL of each layer (four samples each) were collected and radioactivity counted using a gamma counter (Perkin Elmer, Waltham, MA, USA). In Vitro Studies. The SSTR1,2,3,5 expression in human lung adenocarcinoma cell line (A549), human colorectal carcinoma cell line (HT-29), human pulmonary giant cell carcinoma cell line (PG) and human gastric cancer cell line (BGC823) was detected using Western blotting analysis. In vitro cell uptake studies of

68

Ga-DOTA-PA1 and

68

Ga-DOTA-TATE were performed using 3 cancer cell lines

[A549, PG, H520 (human lung squamous carcinoma cell line)]. In vitro fluorescent microscopy assay was conducted in A549, H520 and HCT116 (human colorectal carcinoma cell line). Western blotting. The SSTRs expression in A549, HT-29, PG and BGC823 cells was measured using Western blotting analysis. First, cells in culture dishes were washed with cold PBS three times, lysed with 500 µL of RIPA lysis buffer per dish for 20 min, and the cell lysates in supernatants were collected after centrifugation at 12000 × g at 4°C for 20 min. The protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein lysates (40 µg) were loaded and separated in SDS-PAGE, and the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked using 5% nonfat milk dissolved in TBST solution for 1 h at room temperature. Membranes were then probed with polyclonal rabbit anti-human anti-SSTR1 (ab2366), monoclonal rabbit anti-human anti-SSTR2 (ab134152), monoclonal rabbit anti-human anti-SSTR3 (ab137026) and monoclonal rabbit anti-human anti-SSTR5 (ab156864) antibodies (all at 1:1000), or monoclonal mouse anti-β-actin antibody (1:2000) at 4°C overnight. After washed with TBST three times, the membranes


Page 6 of 27

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were incubated with a goat anti-rabbit (1:2000) or goat anti-mouse secondary antibody (1:40000) labeled with horseradish peroxidase for 1 h at room temperature. The immunoblot bands were detected using an enhanced chemoluminescence (ECL) detection system (GE Healthcare, Pittsburgh, PA, USA). In vitro cell uptake studies. PG, H520, and A549 cells in 500 µL of culturing medium were seeded in 24-well plates at a density of 1 × 105 cells per well overnight before the study. On the day of the study, the medium in each well was aspirated, and cells were washed with 1 mL of cold PBS three times, cells were then re-supplied with 500 µL of serum-free culturing medium, and cultured at 37 °C for 2 h. 37 kBq of 68Ga-DOTA-PA1 or 68Ga-DOTA-TATE in 20 µ L of PBS was added in each well, and the cells were incubated at 37 °C with 5% CO2 for 10, 30, 60 and 120 min. After the incubation, the medium in each well was aspirated, and cells were washed with 1 mL of cold PBS three times, and were lysed with 350 µL of 1 M NaOH. The solution in each well was collected, and counted in the gamma counter. Blocking studies were conducted to evaluate the specificity of the radiotracers. The cells were processed as previously described. 10 µg of cold PA1 or octreotide was added per well 10 min before the radiotracer was added. The cells were incubated for 60 and 120 min, then the cells were collected, and processed in the same way as described above. In vitro fluorescent microscopy. A549, H520, or HCT116 cells in 1 mL of culturing medium were seeded in 20 mm cell culture dish at a density of 3 × 105 cells per dish overnight before the study. On the day of the study, the medium in each well was aspirated, and the cells were washed three times. After addition of serum-free culturing medium, A549 and HCT116 cells were cultured for 1.5 h in the presence of 2.6 µM, 6.5 µ M, or 13 µM of FITC-PA1 away from light at 37 °C with 5% CO2. The cells were treated with 500 µL of Hoechst (5 µg/mL in H2O), and were cultured for an additional 0.5 h. After the incubation, the medium was aspirated, and the cells were washed three times, and were added with serum-free culturing medium. In vitro fluorescent microscopic imaging was obtained using a Leica DMI3000B confocal microscope (Leica Microsystems, Wetzlar, Germany). Saturation binding assay was performed using FITC-PA1 in H520 cell lines, and different final concentrations ranging from 2.6 µM to 26 µM of FITC-PA1 were used. The procedure was used for the other 2 cell lines. Quantitative region of interest (ROI) analysis of the fluorescent microscopic images was performed using Image J software (National Institutes of Health, Baltimore, MA, USA). GraphPad Prism 6 software was used to obtain the saturation binding curve by nonlinear regression analysis, and the dissociation constant (Kd) and maximum density of binding sites (Bmax) were determined. Each experiment point was conducted in quadruplicate and the whole experiment was repeated 3 times. Pharmacokinetics. 1.85 MBq of

68

Ga-DOTA-PA1 was injected intravenously into each female



BALB/c mouse (n = 5). At different predetermined intervals, 10 µL of blood was drawn from tail vein and radioactivity for collected blood samples was counted using the gamma counter. The pharmacokinetic parameters were analyzed using a two-compartmental model using Graphpad Prism 6 software. Micro-PET Imaging Studies. All animal experiments were conducted in accordance with the guidelines approved by Peking University Cancer Hospital Animal Care and Use Committee. For micro-PET imaging studies, BALB/c nude mice bearing subcutaneously inoculated A549 xenografts were administrated with 11.1 MBq of 68Ga-DOTA-PA1 or 68Ga-DOTA-TATE via tail vein injection (n = 3). Static images were collected at 1 h and 2 h post-injection. Animals were anesthetized with 2.5% isoflurane in 0.5 L/min flow of oxygen, moved to the bed of the scanner, and maintained with 1.5% isoflurane during the static images acquisition. Micro-PET imaging studies were performed on a SuperArgus PET scanner (Sedecal, Spain). The images were reconstructed using a three-dimensional ordered subsets expectation maximum algorithm (OSEM) without attenuation correction. The receptor blocking experiment was performed by co-injecting the same radioactivity of radiotracers and 500 µg of PA1 or octreotide into mice bearing A549 tumor. Region of interest (ROI) was drawn manually over the region of tumor, heart and muscle, and the uptake of the radiotracer was expressed as percentage of injected dose per gram of tissue (%ID/g). The tumor to blood (T/B) and tumor to muscle (T/M) ratios were calculated according to the uptake values. Biodistribution studies. 1.11 MBq of

68

Ga-DOTA-PA1/68Ga-DOTA-TATE in 200 µL sterile

saline was injected intravenously into each BALB/c nude mouse bearing A549 (n = 3). After 1 h and 2 h post-injection of the radiotracers, the nude mice were euthanized, blood, heart, liver, spleen, kidneys, lung, stomach, small intestine, large intestine, muscle, bone, brain and tumor, were removed, weighed and counted using gamma counter. A fraction of tissues were also processed and used for immunofluorescence staining. The biodistribution data (%ID/g) were expressed as mean ± standard deviation. For block studies, a large excess of PA1 (0.5 mg) was co-injected with 1.11 MBq of 68

Ga-DOTA-PA1, and the mice were euthanized and sacrificed at 2 h post injection. The GraphPad

Prism 6 software was used to analyze the data, including biodistribution and the ratios of radioactivity among different organs. Confocal laser scanning microscopy. The SSTRs (SSTR1, SSTR2 and SSTR5) expression levels in A549 tumor tissues were examined using immunofluorescence staining. After micro-PET imaging, one half of each A549 tumor resected from the mice was covered with the optimal cutting temperature (OCT) compound, snap frozen, and sliced into 4-µm sections. After fixed with cold acetone for 10 min and dried by exposing to air for 30 min, all slices were rinsed with PBS and blocked with 10% donkey


Page 8 of 27

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serum for 30 min. The slices were incubated with polyclonal rabbit anti-human anti-SSTR1 (ab2366), monoclonal rabbit anti-human anti-SSTR2 (ab134152), monoclonal rabbit anti-human anti-SSTR5 (ab109495) antibodies, and were FITC-labeled using goat anti-rabbit IgG for SSTRs staining. The cell nuclei were stained by DAPI. Finally, the immunofluorescence images were acquired using the Leica DMI3000B confocal microscope. Immunohistochemical staining for SSTRs Expression. To determine the expression of SSTRs (SSTR1, SSTR2 and SSTR5), we conducted immunohistochemical staining in tumor tissues from lung cancer and gastrointestinal cancer patients recruited at Peking University Cancer Hospital. The informed consents of all patients recruited into the study were obtained and the clinical trial was approved by the Ethics Committee of Beijing Cancer Hospital, Beijing, China. The specimens were fixed in formalin, embedded in paraffin and sliced at 4 µm thickness. Then the tissues were treated with 3% H2O2 for 15 min at room temperature. After microwave antigen retrieval, the slices were incubated with goat antiserum for 15 min to block non-specific antigens. After rinsing with PBS three times, the specimens were incubated with anti-SSTR1 (1:50), anti-SSTR2 (1:100) and anti-SSTR5 (1:100) antibodies at 4°C overnight, then the slides were incubated with biotinylated goat anti-rabbit IgG at room temperature for 15 min, followed by avidin-biotin-peroxidase complex incubation. After developed with DAB substrate kit, the slides were counter-stained with hematoxylin. The immunohistochemical staining score of SSTR-positive cells was determined semi-quantitatively by two pathologists blinded to the prior reports. A score of 0 (-) was regarded as no or partial staining in 30% of tumor cells; 2 (++) as moderate or scattered staining in >30% of tumor cells; 3 (+++) as strong staining in >30% of tumor cells. A total of 7 patient samples (3 samples from lung cancer patients) and (4 samples from gastrointestinal cancer patients) were used in this study. Statistical Analysis. Quantitative data is presented as mean ± standard error of mean (SEM). A one-way variance analysis was used to determine the significant differences for in vivo studies. A 95% confidence interval level was chosen to determine the significance between the groups. P value of less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software).

RESULTS Synthesis and Characterization of DOTA-PA1 and its PA1, DOTA-PA1, and

68

68/nat

Ga-conjugates. The structures of

Ga-DOTA-PA1 are presented in Figure 1A. The chemical syntheses of PA1

and DOTA-PA1 were performed using a standard solid phase peptide chemistry by using the



Page 10 of 27

Symphony peptide synthesizer (Protein Technologies). The peptides were purified using HPLC, and analyzed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) or electron spray ionization mass analysis (ESI-MS). Chemical yields of purified PA1 and DOTA-PA1 was 35%, 30%, respectively (Figure S1A and Figure S1B). The molecular weight of PA1 and DOTA-PA1 determined by ESI-MS was 976.70 and 1360.60, respectively (Figure S2A and Figure S2B); the calculated molecular weights for [M+H]+ (PA1) and [M-H]- (DOTA-PA1) were 976.16 and 1360.46, respectively.

nat

Ga-DOTA-PA1 was synthesized and characterized by MALDI-TOF and used as a

reference compound (Figure S2C). FITC-PA1 was also synthesized using the peptide synthesizer, with chemical yield of 5% and chemical purity of 96.9%. The molecular weight of FITC-PA1 was determined to be 1477.69 and 738.00 (main peak) (Figure S2D and Figure S1C), and the calculated molecular weight of FITC-PA1 was 1477.69 [M-H]- and 738.34 [M-2H]2- (main peak), respectively. After radiolabeled with 68

68

Ga and purified with C18 Sep-Pak Cartridge, the final product

Ga-DOTA-PA1 was characterized using both radio-HPLC and radio-thin layer chromatography

(radio-TLC). For radio-HPLC, the radiochemical purity was > 99% with the retention time of 8.4 min, with free

68

Ga3+ at a retention time of 4.1 min (Figure 1B). For radio-TLC, the retention factor (Rf)

value of 68Ga-DOTA-PA1 and free 68Ga3+ was 0-0.1, 0.9-1, respectively (Figure S3A and Figure S3B). The specific activity of

68

Ga-DOTA-PA1 was up to 92.5 ± 9.2 GBq/µmol.

68

Ga-DOTA-PA1 and

nat

Ga-DOTA-PA1 displayed the same retention time in radio-HPLC, the same Rf in radio-TLC. The log P value of

68

68

Ga-DOTA-PA1 was determined to be 0.18 ± 0.02, while that of

Ga-DOTA-TATE was -3.12 ± 0.16.

68

Ga-DOTA-PA1 was stable in PBS, NaOAc buffered solution

(pH = 5.5), and 5% HSA for at least 2 h, according to previously reported procedures.26


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Figure 1. 68Ga-DOTA-PA1 had high radiochemical purity and the lung cell lines expressed multi-subtypes of SSTRs. A, Synthetic scheme of 68Ga-DOTA-PA1. B, Radio-HPLC chromatogram of 68 GaCl3 (left) and 68Ga-DOTA-PA1 (right). C, Relative SSTRs (SSTR1, 2, 3, 5) expressions in A549 (left column) and PG cancer cells (right column), and representative SSTRs expressions were presented as ratios of SSTRs to β-actin expressions by Western blotting analysis (right panel). All data were represented as mean ± SEM (n = 5). In Vitro Studies of

68

Ga-DOTA-PA1 in Cell Lines. The expression level of SSTR1, SSTR2,

SSTR3 and SSTR5 in human lung squamous carcinoma cell line (A549), human colorectal cancer cell lines (HT-29), human pulmonary giant cell carcinoma cell line (PG) and human gastric cancer cells (BGC823) were measured by western blotting (Figure 1C and Figure S4), with β-actin protein as the internal standard. The relative expression levels of SSTR1, SSTR2, SSTR3 and SSTR5 were 1.26 ± 0.22,



0.22 ± 0.02, 1.26 ± 0.13 and 1.32 ± 0.04 for A549 and 0.94 ± 0.05, 0.33 ± 0.03, 1.01 ± 0.05 and 1.34 ± 0.03 for PG. These two lung cancer cell lines were selected as positive control for further evaluation of 68

Ga-DOTA-PA1 radiotracers in in vivo imaging studies. The relative expression levels of SSTR1,

SSTR2, SSTR3 and SSTR5 for HT-29 were 1.05 ± 0.01, 0.27 ± 0.05, 1.32 ± 0.14 and 1.01 ± 0.16. The relative expression levels of SSTR1, SSTR2, SSTR3 and SSTR5 for BGC823 were 1.25 ± 0.11, 0.30 ± 0.02, 1.44 ± 0.03 and 1.27 ± 0.08 (Figure S4). The cell uptake of both 68Ga-DOTA-PA1 and 68Ga-DOTA-TATE in the 3 cell lines, A549, PG, and H520, increased over time (Figure 2A). After 2 h of incubation, the uptake of 68Ga-DOTA-PA1 in A549, PG, and H520 reached 2.49 ± 0.30, 1.95 ± 0.16 and 2.23 ± 0.31 %ID/g, respectively, while that of 68

Ga-DOTA-TATE was 1.31 ± 0.03, 1.47 ± 0.11 and 1.70 ± 0.25 %ID/g, respectively (P = 0.0066 for

A549, P = 0.0007 for PG, and P = 0.05 for H520 between the 2 radiotracers) (Figure 2A). For blocking study, large excess of cold PA1 or octreotide was used as the blocking agent. The cell uptake of 68

Ga-DOTA-PA1 was 1.44 ± 0.04 %ID/g at 1 h and 2.49 ± 0.30 %ID/g at 2 h, while that of blocking

group was 0.53 ± 0.03 %ID/g (P = 0.041, block vs. non-block) at 1 h and 1.19 ± 0.43 %ID/g (P = 0.0025, block vs. non-block) at 2 h in A549 cell line. For PG cells, the uptake of 68Ga-DOTA-PA1 was 1.40 ± 0.24 (non-block) and 0.59 ± 0.02 %ID/g (blocking, P = 0.0468) at 1 h, and 1.95 ± 0.16 (non-block) and 1.60 ± 0.31 %ID/g (blocking, P = 0.1332) at 2 h. For H520 cells, the uptake of 68

Ga-DOTA-PA1 was 1.72 ± 0.18 (non-block) and 0.62 ± 0.25 %ID/g (blocking, P = 0.012) at 1 h, and

2.22 ± 0.31 (non-block) and 0.88 ± 0.16 %ID/g (blocking, P = 0.004) at 2 h. The cell uptake of FITA-PA1 in cell lines A549, H520, and HCT116 was also additionally examined using fluorescent microscopy analysis, to be located on the surfaces of cells (Figure 2). For A549 and H520 cells, the fluorescent signals of FITC-PA1 increased gradually with incubation time (Figure 2B and Figure S5). No fluorescent signal was observed for the SSTR-negative human colorectal cancer cell lines (HCT116) (Figure S6). The affinity of FITC-PA1 in H520 cells was determined by a competitive receptor binding assay experiment. The specific binding was analyzed against the total molar concentration of added FITC-PA1, with the Bmax and Kd value of 2.48 ± 0.07 nM and 17.8 ± 3.2 nM, respectively (Figure S7).


Page 12 of 27

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Figure 2. The 3 lung cancer cells showed higher radio-uptake of 68Ga-DOTA-PA1 than 68 Ga-DOTA-TATE. A, Cell uptake of 68Ga-DOTA-PA1 and 68Ga-DOTA-TATE in 3 lung cancer cells (A549, PG and H520). Uptake were expressed as the ratio of collected radioactivity count in cell to total radioactivity count. Data were represented as mean ± SEM (n = 5). B, Fluorescence microscopy images of H520 cells after incubating with 2.6 µM, 6.5 µM, 9.1 µM, 13 µM, 16.9 µM and 26 µM of FITC-PA1. Cell nuclei were stained with DAPI in blue. Scale bar = 20 µm. All the images were obtained under the same conditions. **P < 0.01, ***P < 0.001. Pharmacokinetics, Micro-PET Imaging and Biodistribution Studies. The radioactivity-time profile of 68Ga-DOTA-PA1 in blood follows a two-compartmental model described as follows (Figure S8): Ct = A * exp (-αt) + B * exp (-βt) where α and β are the two rate constants for the two compartmental model, and A and B are the relevant constants in the model.27 The equation for

68

Ga-DOTA-PA1 in BALB/c mice was Ct = 3.643 * exp

(-0.445t) + 2.744 * exp (-0.037t), with the half life of 1.6 h for the distribution phase and the half life of 18.6 h for the elimination phase, respectively. The comparative micro-PET imaging of 68Ga-DOTA-PA1 and 68Ga-DOTA-TATE was conducted in BALB/c nude mice bearing A549 tumors using the same acquisition conditions, to compare the ratios of tumor and reference organs. The tumor uptake of 68Ga-DOTA-PA1 was clearly discernible at 1 h and 2 h post-injection, with the T/B and T/M ratios of 1.35 ± 0.07 and 2.83 ± 0.04 at 1 h post-injection, and of 2.97 ± 0.01 and 3.45 ± 0.01 at 2 h after injection (Figure 3). With excess of cold



Page 14 of 27

PA1 as blocking agent, the T/B and T/M ratios decreased to 0.54 ± 0.002 and 1.22 ± 0.004 at 2 h post-injection (Figure 3). The liver accumulation of 68Ga-DOTA-PA1 was relatively high compared to 68

Ga-DOTA-TATE, possibly because

68

Ga-DOTA-PA1 was more lipophilic than

Under the same conditions, the micro-PET images of

68

68

Ga-DOTA-TATE.

Ga-DOTA-TATE did not show clear tumor

uptake, with the T/B and T/M ratios of 0.65 ± 0.06 and 1.24 ± 0.12 at 1 h, and 0.76 ± 0.01 and 1.10 ± 0.04 at 2 h, respectively. The kidney accumulation of 68Ga-DOTA-TATE was relatively high compared to that of

68

Ga-DOTA-PA1. With blocking, the radioactivity accumulation in the kidneys decreased

markedly. Compared with those of 68Ga-DOTA-TATE and 18F-FDG, the biodistribution of

68

Ga-DOTA-PA1

showed higher tumor accumulation in nude mice bearing A549 tumors (Figure 3D and Figure S9), and it also showed high uptake in the liver and kidney, consistent with the pattern of micro-PET images (Figure 3A). The tumor uptake of

68

Ga-DOTA-PA1 at 1 h and 2 h was 5.22 ± 0.19 and 3.25 ±

0.27 %ID/g, respectively (Figure 3D), and with blocking with excess of unlabeled PA1, the tumor uptake decreased significantly to 1.51 ± 0.25 at 2 h (P = 0.0063). In comparison, the tumor uptake of 68

Ga-DOTA-TATE under the same conditions was 0.56 ± 0.05 %ID/g at 2 h (Figure S9A).

Figure 3. Micro-PET images in A549 nude mice showed higher uptake of 68Ga-DOTA-PA1 than that of 68Ga-DOTA-TATE. A, Representative micro-PET images in nude mice bearing A549 after injection of 68Ga-DOTA-PA1 (upper) and 68Ga-DOTA-TATE (lower) at 1 h, 2 h vs. block at 2 h. White arrows indicate the location of tumors (n = 3). B and C, Tumor to blood (T/B), tumor to muscle (T/M) and tumor to liver (T/L) ratios of 68Ga-DOTA-PA1 and 68Ga-DOTA-TATE at 1 h and 2 h. D, Ex vivo biodistribution of 68Ga-DOTA-PA1 at 1 h and 2 h and block with PA1 at 2 h in nude mice bearing A549 lung tumors (n = 3). The data were expressed as %ID/g ± SEM. **P < 0.01. Histological Analysis of A549 Tumor Tissue and Human Tumor Tissue Slices. The immunofluorescence staining of SSTR1, SSTR2 and SSTR5 in A549 tumor tissue slices showed that


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SSTR1 and SSTR5 fluorescence signal were clearly discernible while that for SSTR2 expression only showed low background staining (Figure 4), consistent with the results of Western blotting (Figure 1C). To further examine the expression of SSTRs in human tumor tissues, we evaluated the immunohistochemical-staining of tissue slices from 7 patients (Figure 5 and Figure S10). The characteristics of patients were presented in Table 1. Representative immunohistochemistry staining revealed that SSTR5 expression was higher than SSTR2 in human lung adenocarcinoma and gastric adenocarcinoma tissues, while SSTR1 expression was also significantly higher in gastric adenocarcinoma tissues (Figure 5).

Figure 4. Fluorescence microscopic images of SSTRs expression in slides of A549 tumor tissue excised after PET imaging studies. SSTR1, SSTR2 and SSTR5 staining in cells were presented in green channel while cell nuclei stained with DAPI in blue channel. Scale bar = 20 µm. All the images were obtained under the same conditions. Table 1. Characteristics of Patients from Peking University Cancer Hospital for Immunohistochemical Analysis of SSTRs Expression Patient

Age

Gender

No.

(y)

1

57

Male

2

52

3

Histology

Clinical

Ki-67

TNM stage

index

L-ADC

T1N2M1

+ 20%

NP/-/+

Male

L-SCC

T1bN2M0

NP

NP/-/++

53

Male

L-ADC

T2N0M1

NP

NP/-/+

4

35

Male

R-ADC

T3N1M0

+ 50~70%

NP/+/+

5

69

Female

C-ADC

T1aN0M0

+

Design, Synthesis and Biological Evaluation of 68Ga-DOTA-PA1 for

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