K+ ATPase α1-targeted peptide for PET imaging of breast cancer

Accepted Manuscript Identification of a sodium pump Na+/K+ ATPase α1-targeted peptide for PET imaging of breast cancer

Qian Wang, Shi-Bing Li, Yi-Ying Zhao, Da-Nian Dai, Hui Du, Yan-Zhu Lin, Jia-Cong Ye, Jing Zhao, Wei Xiao, Yan Mei, YiTai Xiao, Shi-Chu Liu, Yan Li, Yun-Fei Xia, Er-Wei Song, GangHua Tang, Wei-Guang Zhang, Zhi-Jiang Li, Xiao-Bin Zheng, DeHai Cao, Man-Zhi Li, Qian Zhong, Zhong-Ping Chen, Chao-Nan Qian, Wei Fan, Guo-Kai Feng, Mu-Sheng Zeng PII: DOI: Reference:

S0168-3659(18)30284-0 doi:10.1016/j.jconrel.2018.05.019 COREL 9301

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

5 December 2017 3 April 2018 16 May 2018

Please cite this article as: Qian Wang, Shi-Bing Li, Yi-Ying Zhao, Da-Nian Dai, Hui Du, Yan-Zhu Lin, Jia-Cong Ye, Jing Zhao, Wei Xiao, Yan Mei, Yi-Tai Xiao, Shi-Chu Liu, Yan Li, Yun-Fei Xia, Er-Wei Song, Gang-Hua Tang, Wei-Guang Zhang, Zhi-Jiang Li, Xiao-Bin Zheng, De-Hai Cao, Man-Zhi Li, Qian Zhong, Zhong-Ping Chen, Chao-Nan Qian, Wei Fan, Guo-Kai Feng, Mu-Sheng Zeng , Identification of a sodium pump Na+/K+ ATPase α1-targeted peptide for PET imaging of breast cancer. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi:10.1016/j.jconrel.2018.05.019

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ACCEPTED MANUSCRIPT Identification of a sodium pump Na+/K+ ATPase α1-targeted peptide for PET imaging of breast cancer

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Qian Wang1# , Shi-Bing Li1# , Yi-Ying Zhao1,2# , Da-Nian Dai1 , Hui Du1 , Yan-Zhu Lin1 , Jia-Cong Ye1 , Jing Zhao1 , Wei Xiao1 , Yan Mei1 , Yi-Tai Xiao1 , Shi-Chu Liu3 , Yan Li1 , Yun-Fei Xia1 , Er-Wei Song4 , Gang-Hua Tang5 , Wei-Guang Zhang1 , Zhi-Jiang Li1 , Xiao-Bin Zheng1 , De-Hai Cao1 , Man-Zhi Li1 , Qian Zhong1 , Zhong-Ping Chen1 , Chao-Nan Qian1 , Wei Fan 1 *, Guo-Kai Feng1 *, Mu-Sheng Zeng1 * 1 State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China 2 Department of Neurosurgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China 3 Shenzhen Pingshan District People’s hospital, Shenzhen 518118, China 4 Breast Cancer Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China 5 PET-CT Center, Department of Nuclear Medicine, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510120, China *Correspondence to: Wei Fan, MD, PhD Sun Yat-sen University Cancer Center 651 Dongfeng Road East, Guangzhou 510060, People’s Republic of China E-mail: [email protected] Guo-kai Feng, MD, PhD Sun Yat-sen University Cancer Center 651 Dongfeng Road East, Guangzhou 510060, People’s Republic of China Phone: 86-20-8734-3192; Fax: 86-20-8734-3171; E-mail: [email protected] Mu-Sheng Zeng, MD, PhD Sun Yat-sen University Cancer Center 651 Dongfeng Road East, Guangzhou 510060, People’s Republic China Phone: 86-20-8734-3191; Fax: 86-20-8734-3171; E-mail: [email protected] # These authors contributed equally to this work.

ACCEPTED MANUSCRIPT Abstract The sodium pump Na+/K+ ATPase a1 subunit （ NKA a1 ） , an attractive cancer-related biomarker and therapeutic target, is closely related to the development and progression of several cancers including breast cancer.

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Currently, a NKA a1 inhibitor, UNBS1450, has already evidenced its great therapeutic potential in personalized cancer treatment. The ability of

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non-invasive imaging of NKA a1 expression would be useful for selecting

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cancer patients who may benefit from this drug. Here, we identified an S3 peptide that is specifically homed to breast cancer by phage display. All data of

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in vitro and in vivo experiments suggested the excellent targeting character of the S3 peptide. As the binding activity of the S3 phage was positively

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correlated to the level of NKA α1 expression in various breast cancer cells, NKA α1 was validated as the primary target of the S3 peptide. Based on

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immunohistochemistry staining result of 107 breast cancer patients, NKA α1

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was verified to be a novel tracking marker and a prognostic predictor for breast cancer. Importantly, we proposed and validated an S3 peptide-based radiotracer

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F-ALF-NOTA-S3 for PET (Positron Emission Tomography)

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imaging of breast cancer and other NKA α1 -overexpressing cancers, including hepatocellular carcinoma and non-small cell lung cancer, in mouse models. Our findings demonstrated the potential application of

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F-ALF-NOTA-S3 for

visualization of NKA α1-positive lesions, which provide a new approach to character tumor phenotypic imaging. Keywords: Na+/K+ ATPase a1; Positron Emission Tomography; Breast cancer; Targeting peptide; Phage display

ACCEPTED MANUSCRIPT 1.

Introduction As the global burden of cancer maintains sustained growth, molecular imaging,

defined as the characterization and quantification of biological processes at the cellular and subcellular levels in vivo, has been recognized as the key to increase the survival rate and reduce the mortality of cancer [1, 2]. Compared with traditional modalities,

including

ultrasound,

X-ray-based

mammography,

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imaging

CT

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(Computed Tomography) and MRI (Magnetic Resonance Imaging), which provide

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anatomical information for the diagnosis of cancer, molecular imaging combined with various imaging techniques has enabled the monitoring of cancer progression at the

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molecular and subcellular levels in real time [3, 4]. These abnormally expressing proteins, termed cancer biomarkers, are considered as ideal imaging and therapeutic

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targets for cancers. Oestrogen receptor (ER) [5], progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) have been employed for the

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molecular imaging of breast cancer [6]. However, because of the high heterogeneity of cancer, no single tumor biomarker is suitable to stratify all cancer subtypes. Given

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tumor treatment is developing rapidly personalized, more novel tumor biomarkers which characterize tumors molecularly are in urgent needed to select patients for

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tailored therapies [7].

The sodium pump Na+/K + ATPase (NKA) is a ubiquitous transmembrane ion transporter which is essential for cell survival, growth and differentiation [8]. NKA consists of two subunits, the α (including α1, α2, α3 and α4) catalytic subunit and the β (including β1, β1 and β3) regulatory subunit [9]. Among them, the investigation of α1 subunit provide new insights into the occurrence and development of tumors at molecular level. The aberrant overexpression and activity of NKA α1 subunit has been reported in the progression of various cancers, including breast cancer [10],

ACCEPTED MANUSCRIPT oesophageal carcinoma [11], non-small cell lung cancer [12, 13], melanoma [14], and hepatocellular carcinoma [15]. Growing evidences from large scale of epidemiology studies and in vitro experiments strongly support the possibility of cardiac glycosides which are inhibitors of the NKA (especially α1 subunit) could combat number of malignancies [16]. Thus, a biomarker based imaging tracer for stratifying NKA

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α1-positive patient is essential. Although NKA-targeted digoxin-based radiotracers

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have been developed for myocardial imaging [17, 18], none has been employed for the molecular imaging of NKA α1-positive tumors. Considering the above findings,

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the ability of non- invasive imaging of NKA α1 expression would be helpful in the

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precise diagnosing of NKA α1-overexpressing tumors and grouping NKA α1-targeted therapy benefiting patients.

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In this study, we screened a breast cancer-targeted S3 peptide using phage display. Following, we identified NKA α1 as the target of the S3 peptide by combining peptide chemical

modification

using

cross- linking

reagents,

affinity

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synthesis,

chromatography, and liquid chromatography electrospray ionisation tandem mass

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spectrometry (LC-ESI-MS/MS) as described previously [19]. Furthermore, NKA α1 is overexpressed and is associated with poor survival in breast cancer patients

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suggesting NKA α1 serve as a monitoring marker for tumor imaging. Our results indicated 18 F-ALF-NOTA-S3 based PET imaging achieved the expression of NKA α1 visualization in xenograft models with high tumor uptake and low background accumulation. The S3-based radiotracer exhibited a definite PET imaging of breast cancer and other NKA α1-overexpressing cancers, including hepatocellular carcinoma and non-small cell lung cancer. 2.

Material and methods

2.1. Cells and mice

ACCEPTED MANUSCRIPT MDA-MB-231, MCF7, BT-549, MDA-MB-468, MDA-MB-453, MDA-MB-415, SK-BR-3, HCC1937, and NCI-H460 cell lines were purchased from the American Type Culture Collection (ATCC). All breast cancer cell lines were cultured wit h DMEM containing 10% foetal bovine serum, and NCI-H460 cells were cultured with RPMI-1640 medium supplemented with 10% foetal bovine serum. All mice were

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purchased from Vital River, Charles River China (Beijing, China). The animal

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experiments were approved by the Use Committee for Animal Care. Xenografts were

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created by subcutaneously injecting 1×10 7 MDA-MB-231 or H460 cell suspension with 20% matrigel (Corning 354234, USA). Tumor growth was measured every 2

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days, and the tumor volume was calculated using the formula (ab2 /2), where a and b are the tumor length and width, respectively. When the tumor volume reached

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approximately 300 mm3 , the mice were subjected to near- infrared imaging or small-animal PET scan. The experimental hepatocarcinogenesis model was induced

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using diethylnitrosamine (DEN). The C57BL/6 mice were intraperitoneally injected into 200 mg/kg DEN (Sigma, USA) and were subjected to magnetic resonance

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imaging (MRI) every two weeks. When the liver cancer reached 30 mm3 , the mice were then subjected to small-animal PET scan.

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Knocking out NKA α1 using the CRISPR/Cas9 system. To generate the ATP1A1 knockout cell line, the CRISPR/Cas9 system was used. The sequences of guiding RNA were as follows: sgRNA#1: CGTTGGGACCATCTCGCGCC; sgRNA#2: GCAACCAGTTATGATTACAA. The virus suspensions of Cas9 and sgRNA were separately transfected into MDA-MB-231 cells stably. 2.2. Peptide screening and peptide sequencing The ph.D.-CX7CTM Phage Library kit was purchased from New England BioLabs (NEB E8121L, USA). First, MDA-MB-231 cells were prepared as a 1×106 cell

ACCEPTED MANUSCRIPT suspension in 500 µl of DMEM containing 5% bull serum albumin and were incubated with 1×1011 pfu of phage library on ice for 2 h. Thereafter, unbound phages were washed away. Specific-binding phages were transferred into the organic lower phase (Sigma, USA) and were amplified with coliER2738 (HUAYUEYANG, China) host cells. After three rounds, the phages were titrated on plates and were picked up DNA

extraction.

The

sequences

were

detected

using

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for

primers

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(sense:5’-AACGTGAAAAAATTATTATTCGCAA-3’, anti-sense:5’-ATTCCACAGA

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CAGCCCTCATAGTTA-3’), and the nucleic acids were transferred into 3% agarose gel (Biowest 111860, Spain). The inserted DNA was purified (Qiagen 28304, China),

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subjected to end repair, dA-tailed and ligated to Illumina adapters in sequence. The ligation products were size fractionated by agarose gel electrophoresis, and the

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fragments were excised for PCR amplification. The amplified fragments were sequenced using Illumina HiSeq™ 2000 by GeneDenovo Co. (Guangzhou, China).

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Finally, the repeated sequences were analyzed, and the top ten of these sequences were selected for further study.

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2.3. Enzyme-linked immunosorbent assay Cells were seeded into 96-well plates. When the cells grew to a confluence of

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70-80%, 109 isolated phages were added to each well, followed by incubation for 3~4 h at 37°C. After 5 washes with PBS containing 0.1% Tween-20 (Weijia 180810, China), the cells were fixed with 4% paraformaldehyde (MP Biomedical 150146, USA) and incubated with 1% Triton X-100 to break the membranes. Thereafter, horseradish peroxidase (HRP)-conjugated anti-M13 phage antibody (Abcam ab50370) was added to each plate at 1:3000 dilution, followed by incubation for 30 minutes. The plates were washed 5 times and then were developed with 200 µL of 3,3′,5,5′ -tetramethylbenzidine (TMB) for coloration. Finally, 2 M sulfuric acid solution was

ACCEPTED MANUSCRIPT used to stop the reaction. The results were read using an ELISA plate reader (Bio-Tek EPOCH2, USA) at 450 nm. For the competition binding assay, the synthetic peptide was added 30 min before the addition of the phage. 2.4. Immunofluorescence staining Approximately 1×105 MDA-MB-231 cells were plated on fibronectin-coated

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coverslips and were incubated with biotin-peptide at 80 µM for 4 hours at 37°C,

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washed with PBST 5 times, fixed in 4% paraformaldehyde and blocked in 5% BSA.

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The biotin label was detected using streptavidin-FITC (Thermo Fisher SA10002, USA), and the receptor for the peptide was dyed with anti-ATP1A1 antibody (Thermo

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Fisher E7-BP-M9, USA) overnight at 4°C. Thereafter, the cells were incubated with anti-rabbit AF594 secondary antibody at 1:1000. The coverslips were mounted in

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ProLong Gold anti- fade (Invitrogen P26930, USA). Fluorescence images were captured by 40× to 100× confocal microscopy (O lympus FV1000, Japan) and were

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analysed using FluoView application software FV10-ASW 3.0. 2.5. Near-infrared fluorescence imaging

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Mice bearing breast tumors were injected with an equivalent of 10 nmol (0.5 mg/kg) of Cy5-peptide via the tail vein. After 48 h of circulation, mice were

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anaesthetized with isoflurane and were imaged using IVIS ® Spectrum (Xenogen Corporation, Caliper Life Sciences, Hopkinton, MA, USA) with an excitation wavelength of 633 nm for Cy5. The whole-body images of mice were captured. Additionally, the tumors and normal organs (heart, liver, spleen, lungs, and kidneys) were dissected and imaged together. The fluorescence signal intensity was quantitatively analysed by placing a region of interest (ROI) on tissues. The ROI was manually drawn within the tumor and normal organs. 2.6. Photoreactive protein crosslinking

ACCEPTED MANUSCRIPT Sulfo-SBED (Thermo Fisher 33034, USA) was dissolved into DMSO and was incubated with CS3 peptide for 30 min at a concentration of 1 mM in the dark at RT. When MDA-MB-231 cells grew to 80% confluence, the biotinylated CS3 peptide was incubated with the washed cells for 1 hour at 37°C, and unbound peptide was then washed away. The aryl azide was photoactivated using a longwave UV lamp (365 nm) 15

minutes.

Biotinylated

(Sigma

protein

67395,

was

extracted

USA).

The

using

biotin- modified

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octyl-β-D-1-thioglucopyranoside

receptor

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for

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interacting protein could be captured by streptavidin beads. Finally, the target protein was detected by far-western blotting and silver staining (Beyotime, China).

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2.7. In-Gel Digestion and LC-ESI-MS/MS analysis

The difference band between the S3 and control group was excised, washed

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with 25 mM NH4 HCO 3 and 50% acetonitrile twice (1 h each time), and then dehydrated by the addition of 500 μL of acetonitrile. Disulfide bonds were cleaved for

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spots by incubating the samples for 60 min at 56°C with 200 μL of 10 mM DTT in 25 mM NH4 HCO 3 buffer, and alkylation of cysteines was performed by the addition of

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200 μL of 55 mM iodoacetamide in 25 mM NH4 HCO 3 buffer and incubation of the samples for 45 min at room temperature in the dark. Additionally, the spots were

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dehydrated again with 500 μL of acetonitrile after washing with 25 mM NH4 HCO 3 twice. Thereafter, Trypsin (Promega) solution was added for 37°C overnight digestion, which was stopped by the addition of 5% formic acid. The samples were reconstituted in 3 μL of 0.1% trifluoroacetic acid (TFA) prior to MS analysis. After protein digestion, the supernatant was recovered to obtain a peptide solution. Next, 10 μl of supernatant was loaded on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) using the autosampler onto a 2-cm C18 trap column. Next, the peptides were eluted onto a 10-cm analytical C18 column. Data acquisition was performed using a TripleTOF

ACCEPTED MANUSCRIPT 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). Protein identification was performed using a Mascot search engine (Matrix Science, London, UK; version 2.3.02) against the human Swiss-Prot database (20201 sequences, release 2016-5-24).

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2.8. Immunohistochemistry

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Tumors from mouse models were carefully dissected and were cut into 3- mm

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sections, which were counterstained with H&E. All paraffin sections were deparaffinized in xylene and were dehydrated in a series of graded alcohols and

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distilled water. Antigen retrieval was performed by boiling the samples in 10 mM citrate buffer (pH 6.0) for 2 min under high pressure for antigen retrieval. The

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samples were blocked with 5% bull serum albumin (BSA) and then were immunostained with the ATP1A1 antibody at a dilution of 1:150 overnight. The

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samples were washed with PBS containing 0.1 % Tween and then were incubated with the anti-rabbit monoclonal antibody at 25°C for 30 min. Finally, the signals were

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coloured using the DAB Kit (ZhongShanJinQiao, ZLI-9017, China). Images were captured by Nikon Eclipse at 10× and 20× magnification.

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2.9. PET imaging

For PET imaging, the following solutions were mixed according to the manufacturer’s instructions: 50 µg of NOTA-S3 peptide with 6 µl of AlCl3 (2 mM). The peptide is available as a freeze-dried powder freely soluble in saline solution. Thereafter, 5 µl of ethanoic acid, 324 µl of ethanol and 10 mCi of fluoride ion (~370 MBq) were added, and the mixture was boiled for 10 min and cooled gradually. The 18

F-AlF-NOTA-S3 product was captured using the C18 plus column (Waters, Sep-Pak,

USA), and the free fluoride ion was concentrated and disregarded. The columns were

ACCEPTED MANUSCRIPT washed with saline solution twice and were eluted with 400 µl of ethanol. The samples were prepared for HPLC analysis (Agilent 1200, USA). The mice were injected via the tail vein with 100 µCi of eluted

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F-AlF-NOTA-S3 imaging tracer.

After circulation for 1 hour, PET scans and image analysis were performed. For the blockade assay, 200 µg of unlabelled S3 peptide was injected 30 min before the F-S3 tracer circulation. After imaging was acquired, regions of interest (ROIs) were

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drawn on the tumor and normal organs, and the maximum uptake value was

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calculated. The injected mice were sacrificed, and the radiation intensity of organs was calculated as percentage of the injected dose per gram (%ID/g). For dynamic

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imaging (n=4 group), the imaging data was acquired as follows: 1X5s, 1X25s, 9X30s, 5X60s, 5X120s and 9X240s. Time activity curves were generated according to the

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3. Results 3.1. Identification of S3 peptide

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pharmacokinetics of the S3 tracer in the tumors and major organs.

Previously, we applied the phage-display CX7C peptide library to screen breast

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cancer-targeting peptides for the molecular imaging of breast cancer [20]. We have identified a neuroplin-1 targeted CLKADKAKC (CK3) peptide which has been

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employed for near- infrared fluorescence imaging (NIRF) and SPECT imaging of breast cancer in mouse models [20]. Here, to identify additional breast cancer-targeting peptides for the molecular imaging of breast cancer, we used high-throughput sequencing technology to sequence the phage pools from the second and third rounds of screening. We revealed six highly repeated peptide sequences, including

CNTGSPYEC,

CMARYMSAC,

CLKLGEKWC,

CLMTSQFRC,

CSISSLTHC, and CRGATPMSC, from both rounds of screening using this method (Figure 1A-B).

ACCEPTED MANUSCRIPT Among all six highly repeated phage clones, we selected the phage coding CSISSLTHC peptide (referred to as the S3 peptide) for further study because it had the highest binding ability among these phage clones (Figure 1C). The binding of S3 phage to breast cancer MDA-MB-231 cells was blocked by the corresponding synthetic S3 peptide in a dose-dependent manner (Figure 1D), suggesting the binding

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ability of S3 phage to breast cancer cells is mediated by its coding S3 peptide. To

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investigate the targeting ability of the S3 phage in vivo, mice bearing MDA-MB-231

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tumors were intravenously injected with 1 × 1010 plaque- forming units (PFU) of S3 phage. The S3 phage was allowed to circulate for 2 h and flush with

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phosphate-buffered solution. Afterwards, we recovered normal organs and tumor tissues to calculate the titres of phage according to a previous study [19]. Significantly,

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more S3 phage accumulated in the tumor tissues than in normal organs, such as the lung, heart, and brain (Figure 1E). To further investigate the in vivo tumor- imaging

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ability of S3 peptide, cyanine dye Cy5-labeled S3 peptide (Cy5-S3), Cy5- labeled CG7C peptide (Cy5-CG7C) and Cy5-labeled scrambled peptide (Cy5-scrambled)

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were synthesized. Whole-body NIRF imaging of the Cy5-S3 peptide in mice bearing the MDA-MB-231 xenograft at different times showed that the Cy5-S3 peptide was

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highly homed to tumor tissues at 24 h post- injection (pi) (Figure 1F). Moreover, compared with scrambled peptide, S3 peptide targeted the breast tumor in the sequence-specific manner (Figure S1). NIRF imaging of the collected normal organs and tumor tissues showed that Cy5-S3 was predominantly accumulated in the tumor tissue and, to a lesser extent, in the kidneys for excretion (Figure S2). Quantification of the collected normal organs and tumor tissues further confirmed that Cy5-S3 was primarily accumulated in the tumor tissues (Figure 1G-H). These findings demonstrate the in vivo tumor- homing ability of the S3 peptide in breast cancer mouse

ACCEPTED MANUSCRIPT models. Thus, this peptide may serve as a potential delivery vehicle for the nuclear

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imaging of breast cancer.

Figure. 1. Identification of breast cancer-targeted S3 peptide using phage displa y. (A) Summary of the peptide sequences recovered from phage screening. High-throughput sequencing showed that highly repeated peptide sequences comprised 1% and 11% in the second and third round of screening,

ACCEPTED MANUSCRIPT respectively. (B) Si x highly repeated peptide sequences were revealed from both rounds of screening. The proportion of each peptide is shown. (C) The binding affinities of six highly repeated phages were analysed by phage ELISA. The insertless phage was used as a negative control. Note that the CSISSLTHC peptide (referred to as the S3 peptide) had the highest binding affinities among all six highly repeated peptide sequences. The graphs represent the means ± SEM. Statistical analysis was performed using unpaired two-tailed t-test, *p < 0.05, **p < 0.01, ***p <0.001, compared with the control phage. (D) The binding of S3 phage to MDA-MB-231 cells was dose dependently inhibited by the corresponding synthetic S3 peptide. The synthetic CG7C peptide was used as a negative control. Statistical analysis was performed using one-way ANOVA, n = 3; mean ± SEM; **p< 0.01, ***p< 0.001. (E)

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In vivo bio-distribution of S3 phage in mice bearing the MDA-MB-231 xenograft. S3 phage was intravenously injected into mice and was allowed to circulate for 2 h before washing out by PBS perfusion. Xenograft and organs were removed for the determination of the phage titre. (F) Approximately 6 nmol of

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the Cy5-S3 peptide was dissolved in PBS and intravenously injected into mice bearing the MDA-MB-231 xenograft. The Cy5-CG7C peptide served as a negative control. Near-infrared fluorescence (NIRF)

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imaging was acquired after 24 h pi. (G) After whole-body NIRF imaging, the mice were sacrificed, and normal organs and tumor tissues were removed for NIRF imaging. (H) Quantification of NIRF imaging signals in the collected normal organs and tumor tissues. Statistical analysis was performed using

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3.2. The S3 peptide binds to NKA α1

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unpaired two-tailed t-test, n = 3; mean ± SEM; ***p <0.001.

To identify the target of S3 peptide, we combined peptide synthesis, chemical

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modification using cross-linking reagents, affinity chromatography, silver staining, far

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western blotting and LC-ESI-MS/MS according to a previous report [19] with some modifications (Figure 2A). The samples eluted by affinity chromatography were run under denaturing and non-reducing conditions using the Bis-Tris Electrophoresis

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System. The far western blotting and silver-stained gel analyses showed that a protein band between 95 kDa and 130 kDa was eluted from the S3 peptide-treated cells (Figure 2B and C). The gel slice containing this protein band was excised, digested with trypsin, and analysed using Triple TOF 5600-based LC-ESI-MS/MS. Protein identification was performed using the Mascot search engine against the human Swiss-Prot database. A total of 18 membrane proteins with a molecular weight between 95 kDa and 130 kDa were identified (Table S1). Among them, we focused

ACCEPTED MANUSCRIPT on NKA α1 (Protein ID sp|P05023|AT1A1) because it had the highest protein Q score and coverage rate (Figure 2D and Table S1). To validate whether NKA α1 was the target of the S3 peptide, the samples eluted from the affinity chromatography experiment were subjected to western blotting using

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an anti-NKA α1 antibody. The immunoblotting results confirmed the presence of NKA α1 in S3 peptide-treated cells (Figure 2E). Moreover, we observed that S3

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peptide co- localized with NKA α1 both in vitro (Figure 2F) and in vivo (Figure S4).

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Additionally, we knocked out NKA α1 with two different sgRNAs in breast cancer MDA-MB-231 cells using the CRISPR-Cas9 system (Figure S3A). Knocked out of

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NKA α1 dramatically lowered the S3 phage-binding ability 2.7- to 4.4-fold (Figure

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2G and Figure S3B). These findings suggest that NKA α1 is the primary target of the

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S3 peptide.

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ACCEPTED MANUSCRIPT Figure. 2. The S3 peptide binds to NKA α1. (A) Schematic diagram of the experimental design. (B) Far western blotting revealed the presence of a protein band between 95 and 130 kDa that was pulled down by the S3 peptide compared with the control peptide. (C) Silver staining gel further confirmed the presence of the protein band between 95 and 130 kDa that was pulled down by the S3 peptide but not by the control peptide. (D) The peptide sequence of the target protein was deduced by LC -ESI-MS/MS. The peptide sequence covered rate was approximately 31.18%. (E) Validation of the target protein by western blotting using an anti-NKA α1 antibody. Western blotting revealed the presence of a protein band between 95 and 130 kDa that was pulled down by the S3 peptide but not b y the control peptide. (F) Biotin-labelled S3 peptide was co-localized with NKA α1 protein in MDA-MB-231 cells. Streptavidin-Cy3-

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and Alexa Fluor 488-conjugated goat anti-mouse were added to detect the biotin-labelled peptide and NKA α1, respectively. (G) Knocking out of NKA α1 using the CRISPR-Cas9 system dramatically reduced the binding of S3 phage to MD A-MB-231 cells. Two different sgRNAs with similar results were obtained.

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Statistical analysis was performed using Student’s t-test, n = 3; mean ± SEM; ***p < 0.001.

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3.3. Positive correlation between the binding activity of S3 phage and NKA α1

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expression

We further linked the binding abilities of S3 phage to the expression levels of

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NKA α1 in different breast cancer cells. The expression levels of NKA α1 in different breast cancer cell lines were examined by western blotting (Figure 3A). The relative

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NKA α1 expression in different breast cancer cells were calculated by setting the

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expression level of NKA α1 in MCF-7 cells as a reference (Figure 3B). The binding activity of S3 phage was compared in different breast cancer cell lines (Figure 3C). We observed that the binding activity of S3 phage was positively correlated with

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NKA α1 expression in these cells with a correlation coefficient of approximately 0.9491 (Figure 3D). This finding indicates that the binding ability of S3 peptide can reflect the expression level of NKA α1 in breast cancer. Previous studies have indicated that overexpression of NKA α1 associated with modulations in cellular migration [21, 22], which are indicative of potential cancer malignant potential. Moreover, reducing NKA α1 activity remarkedly attenuate the migration ability of malignant tumor cells. Thus, in this study, we examined whether the NKA α1-targeted S3 peptide stimulates cellular aggressive biological behaviours

ACCEPTED MANUSCRIPT when it binds to breast cancer cells. We found that the S3 peptide did not affect cellular growth, migration or invasion of breast cancer under the indicated conditions (Figure S5). It targeted cancer cells without causing any side effect and satisfied all the requirements of imaging tracers. Thus, the S3 peptide demonstrated strong

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potential in translational research for early malignant disease detection.

Figure. 3. Correlation between the binding of S3 phage and NKA α1 expression. (A) NKA α1 expression in different breast cancer cells was examined by western blotting. Three repeated experiments were carried out with similar results obtained. (B) Quantification of the relative NKA α1 expression in different breast cancer cells by setting the expression level of NKA α1 in MCF-7 cells as a reference. (C) The binding ability of S3 phage in different breast cancer cells. (D) The binding of S3 phage was positively correlated with the relative NKA α1 e xpression level in different breast cancer cells. Statistical analysis was performed with Pearson's correlation analysis. The Pearson correlation coefficient was 0.9705.

ACCEPTED MANUSCRIPT 3.4. NKA α1 is a prognostic predictor for breast cancer patients Although NKA α1 has been reported to be overexpressed in breast cancer [10], the relationship between the expression level of NKA α1 and breast cancer patient outcome remains largely unknown. Here, we used immunohistochemistry to examine

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the prognostic significance of NKA α1 expression, tumor size, menopause, lymph node infiltrated, oestrogen receptor (ER) status, progesterone receptor (PR) status,

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human epidermal growth factor receptor-2 (HER-2) status, histological grade, tumor

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status and TNM staging in clinical tumor samples from the cohorts of breast cancer patients (n = 107). Representative images of human breast cancer stained with an

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anti-NKA α1 antibody showed that NKA α1 was overexpressed in breast cancer

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specimens (Figure 4A). We divided these breast cancer patients into two groups (NKA α1 low and high) by setting the median NKA α1 expression level observed in

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breast cancer specimens with the cutoff value of 8.5. Patients with high NKA α1 expression in the cancer tissues showed a poorer prognosis for overall survival

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(Figure 4B) and disease- free survival (Figure 4C). The combination with clinical data predicted that high NKA α1 expression was significantly correlated with

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HER2-positive status of patients (Table S2). We also verified the prognostic value of NKA α1 in breast cancer, HCC and lung cancer in TCGA dataset (Figure S6). These findings suggest that NKA α1 is a prognostic predictor for breast cancer patients; thus, nuclear imaging of NKA α1 expression will be helpful in predicting the outcome for breast cancer patients.

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Figure. 4. High NKA α1expression correlates with a poor prognosis for breast cancer patients. (A) Immunohistochemical staining (IHC) of NKA α1 in sections derived from breast cancer patients. Representative images showed high NKA α1 expression in breast cancer tissues but not in adjacent

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normal tissues. Scale bar=50μm. Kaplan-Meier plots of overall survival (B) and disease-free survival (C) of patients bearing breast cancer were segregated by the expression level of NKA α1. Patients were divided into two groups (low and high) based on their levels of NKA α1 expression, with a cutoff value set to 8.5. High NKA α1 expression correlated with poor overal l (B) and disease-free survival rates (C).

3.5. PET imaging with 18 F-ALF-NOTA-S3 in breast cancer mouse models The

cyclic

chelator,

the

1,4,7-triazacyclononane-1,4,7-triacetic

acid

(NOTA)-conjugated peptide, can be easily radio- labelled with 18 F-aluminium–fluoride complex (18 F-AlF) to form activity without the

18

need

F-AlF-NOTA-peptides with reasonably high specific for

further HPLC

(High

Performance Liquid

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18

18

F-AlF to form the integrin αvβ3

F-alfatide for PET imaging of lung cancer patients [24]. Here,

we synthesized the NOTA-conjugated S3 peptide, which was radiolabelled with 18

F-AlF to form a radiotracer

18

F-AlF-NOTA-S3 for PET imaging of breast cancer in

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mouse models (Figure S7A). Based on the input amount of radioactivity and amount

F-AlF-NOTA-S3 was approximately 40 % when the reaction volume maintained at

approximately 400 μl. The

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18

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of radioactivity trapped on the C-18 columns, the labelling efficiency for

F-AlF-NOTA-S3 trapped on C-18 columns was eluted

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with 400 μl of ethanol. The radiochemical purity of 18 F-AlF-NOTA-S3 was more than 95 % by analytical HPLC (Figure S7B). We also determined the serum stability of F-AlF-NOTA-S3 in vitro and in vivo by analytical HPLC. Even after incubation for

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2 h, a single peak of radioactivity was achieved in the indicated conditions, suggesting

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the excellent stability of 18 F-AlF-NOTA-S3 both in vitro and in vivo (Figure S7C-D). Mice bearing MDA-MB-231 tumors were intravenously injected with 3.7 MBq

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(100 μCi) of 18 F-AlF-NOTA-S3. PET static imaging was acquired at 30 min, 60 min and 120 min post injection, respectively. Bio-distribution studies were performed right

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after PET/CT imaging. Mice were sacrificed, and normal organs and tumor tissues were collected, weighed and measured by the gamma-counter (Table S3). 18

F-AlF-NOTA-S3 showed the highest tumor uptake and the lowest background

uptake at 60 min post injection. Thus, PET static images were acquired at 60 min post injection in the following study. Representative PET images showed that 18 F-S3 was highly accumulated in breast tumor tissues at 60 min post injection (Figure 5A). Highly radioactive accumulation of 18 F-AlF-NOTA-S3 in the kidneys and bladder at 60 min post injection suggested the major renal- urinary clearance of the imaging

ACCEPTED MANUSCRIPT tracer (Figure 5A). The

18

F-ALF-NOTA-S3 tracer also displayed excellent targeting

effect in the whole body image and 3D model image of mice bearing MDA-MB-231 (Figure S8). An in vivo blocking assay showed that, with the presence of unlabelled S3 peptide, signal intensity in the tumor region was dramatically reduced (Figure 5B), thereby confirming that S3 peptide was responsible for the tumour uptake of the

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radiotracer. Based on PET imaging in Workplace Software, the maximum value of

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tumor region uptake was calculated as 5.1378 ± 0.2577 %ID/g. Additionally, the

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calculated tumor uptake values for kidneys, bone, intestine, spleen, liver, heart, lung, muscle and brain, were 8.38 ± 0.1061, 3.44 ± 0.0942, 2.4033 ± 0.2017, 2.1 ± 0.0909,

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1.76 ± 0.051, 1.5867 ± 0.0759, 1.64 ± 0.0216, 0.9967 ± 0.0655, and 0.1667 ± 0.0125, respectively. The curvature of the malignant lesion was clearly reflected in

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tumor-to-organ ratios. At 60 min post injection, the tumor-to-skin, tumor-to-heart, tumor-to-liver and tumor-to-bone ratios were 7.74226 ± 0.498, 4.8665 ± 0.2265,

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4.3864 ± 0.1321 and 2.073 ± 0.1016, respectively. The tumor uptake of F-AlF-NOTA-S3 was effectively blocked by unlabelled S3 peptide (reduced from

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5.1378 ± 0.2577 to 2.0133 ± 0.0942 %ID/g) (Figure 5C). Decreased tracer uptake was also found in other organs such as the bones and intestines (Figure 5C).

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Unlabelled S3 peptide was responsible for these blockages in the tumor-to-organ ratios (Figure 5D). All quantification data were in agreement with the PET signal intensity. In addition, 50- min dynamic PET imaging was performed, and the time activity curves for major organs and tumor tissue uptake of

18

F-AlF-NOTA-S3

(Figure 5E and Figure S7E) and tumor/non-tumor curves (Figure 5F) were calculated. Rapid circulation and kidney clearance were further confirmed. Furthermore, the tumor-to-muscle ratio increased steadily over the 50 min period of PET imaging. These findings suggest that 18 F-AlF-NOTA-S3 tracer is feasible for use

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in detecting breast cancer in mouse models.

Figure. 5. PET/CT imaging with 18F-AlF-NOTA-S 3 in mice bearing breast cancer. (A) Representative PET/CT images of MDA-MB-231 xenograft mice after injection of 3.7 MBq of

18

F-AlF-NOTA-S3 without

(A) or with (B) a blocking dose of unlabeled dimeric S3 peptide. Next, 3.7 MBq of

18

F-AlF-NOTA-S3 was

intravenously injected into MDA-MB-231 xenograft mice and circulated for 60 min before PET/CT. Tumors were marked with red arrows. (C) Quantification of tracer uptake in normal organs and tumor tissues was calculated as percent ID per gram (mean ± SD, n = 4) at 1 hour after tracer injection. (D) Ratio of the tumor to main organs in MDA-MB-231 xenograft mice at 60 min after

18

F-AlF-NOTA-S3

tracer injection (n = 4/group). Statistical analysis was performed using Student’s t-test, n = 4; mean ± SEM; ***p< 0.001. (E) Dynamic PET imaging on a MDA-MB-231 xenograft mouse for a period of 50 min.

ACCEPTED MANUSCRIPT Time activity curves were drawn for tumor and muscle uptake of 18 F-AlF-NOTA-S3. (F) Time-dependent curves of the tumor/non-tumor ratio.

3.6. PET imaging with 18 F-AlF-NOTA-S3 in other NKA α1-overexpressing tumors In addition to its overexpression in breast cancer, NKA α1 is overexpressed in esophageal squamous cell carcinoma [11], non-small-cell lung cancer [12], melanoma [14] and hepatocellular carcinoma [15]. We proposed that the novel developed F-S3 may also be feasible as a detector of these NKA α1-overexpressing

A proof-of-principle

study was conducted;

18

F-AlF-NOTA-S3 was

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tumors.

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radiotracer

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intravenously injected into mice bearing hepatocellular carcinoma or lung cancer. In sufficient agreement with the PET imaging of breast cancer, diethylnitrosamine (DEN)

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induced hepatocellular carcinoma was clearly visible with high tumor uptake (3.1513 ± 0.1213 %ID/g; n = 4) and low background accumulation (1.3212 ± 0.1677 %ID/g; n

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= 4) (Figure 6A and B). High NKA α1 expression in hepatocellular carcinoma was confirmed through immunohistochemistry (Figure 6C). Similarly,

18

F-AlF-NOTA-S3

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was determined feasible for detecting of lung cancer with a high tumor-to-muscle ratio (3.669008 ± 0.1296%; n = 4) (Figure 6D and E). Meanwhile, IHC was used to

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measure the specific membrane positive expression of NKA α1 in H460 xenografts (Figure 6F). Thus,

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F-AlF-NOTA-S3 has exhibited potential for application in the

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precise diagnosis of NKA α1-overexpressing tumors.

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Figure. 6. PET/CT imaging with

F-AlF-NOTA-S3 in mice bearing hepatocellular carcinoma or

18

F-AlF-NOTA-S3 PET/CT imaging of a mouse bearing hepatocellular

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lung cancer. (A) Representative

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carcinoma (HCC) induced by diethylnitrosamine (DEN). The red arrow indicates HCC. (B) Quantification of the tracer uptake in HCC and adjacent liver tissues were calculated as percent ID per gram (mean ± SD, n= 3) at 1 hour after tracer injection. (C) Immunohistochemistry staining of hepatocellular carcinoma

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with the NKA α1 antibody. Scale bar = 50 μm. (D) Representative 18F-AlF-NOTA-S3 PET/CT imaging of a mouse bearing the lung cancer H460 xenograft. The red arrow indicates cancer lesion. (E)

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Quantification of tracer uptake in the H460 xenograft and muscle was expressed as percent ID per gram (mean ± SD, n= 3) at 1 h pi. (F) Immunohistochemistry staining of the H460 xenograft with the NKA α1 antibody. Scale bar = 50 μm.

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4. Discussion

Here, our findings provided proof of concept for imaging of cancer progression by monitoring the expression of NKA α1. We revealed that NKA α1 served as a tracking marker and a prognostic predictor for breast cancer patients. The S3 peptide was the first report of Na+/K+ ATPase α1-targeted peptide which could be linked with contrast agents and applied for molecular imaging of cancers. The S3-based PET imaging probe,

18

F-AlF-NOTA-S3, demonstrated excellent tumor targeting properties in

several human tumor xenograft models including breast cancer, lung cancer and

ACCEPTED MANUSCRIPT hepatocellular carcinoma. The imaging measurement values were well correlated with receptor 18

expression

levels

of

xenograft

samples.

The

application

of

F-AlF-NOTA-S3 exhibited definite visualization of NKA α1-positive lesions, which

provide a new approach to character tumor phenotypic imaging. As precision medicine and non-invasive precision diagnostics develop rapidly, it

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is necessary to explore cancer biomarkers which reflect tumors information at cellular

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and subcellular levels long before anatomical change [3, 25]. As one of the nuclear

detecting

signals

from

radiotracers

at

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imaging modality, PET imaging has been applied to human patients rapidly with picomolar

concentration.

The

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2-fluoro-2-deoxyglucose (FDG) is the most common biomarker for reflecting the hexokinase enzyme levels, and 18 F-FDG based PET tracer is universally recognized as

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a powerful tumor diagnostic tool [25, 26]. However, the “metabolic imaging” confused the malignant area with chronic inflammation because of the abnormal 18

F-FDG promotes the

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absorption in inflammatory lesions [27]. The limited use of

on cancer cells.

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study of other radiolabeled antibodies or peptides monitoring receptors overexpressed

Clinical studies on the use of peptides to perform functional characterizations of

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tumor angiogenesis have primarily investigated the use of the Arg-Gly-Asp (RGD) peptide [28]. The RGD peptide and S3 peptide share a common metabolic characteristic, whereby their low molecular weight enables their rapid clearance from the blood and normal tissues. However, excretion of peptides from the kidneys restricts its application in imaging of lesions in the urinary system. The S3 peptide is similar to the RGD peptide in its manner of imaging modalities; therefore, we evaluated the efficacy of S3 for performing functional characterizations of tumor. One study found that the tumor uptake of cyclic RGD dimer 3PRGD2 radiolabeled with

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F in U87MG tumors and MDA-MB-435 tumors was 4.38 ±0.47% ID/g and

3.15 ±0.39% ID/g, respectively [29]. In our study, the tumor uptake of MDA-MB-231 tumors was 5.14 ±0.26% ID/g, greater than that of

18

18

F-S3 in

F-3PRGD2.

These results demonstrate the advantage of using the S3 peptide to detect breast cancer. As different tracking target for peptide provide different perspective to

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visualization of tumor type, peptides vary a lot in directions of translational researches.

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NKA a1 is overexpressed in many types of cancers, including breast cancer,

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non-small-cell lung cancer, and hepatocellular carcinoma, using S3 peptide -which target NKA a1-would be more appropriate for imaging these cancers [12, 21, 30]. By

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comparison, the RGD peptide targets integrin aVβ3 and appears superior for imaging gastric cancer, colorectal cancer, and melanoma [31, 32].

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With intense research of the appearance of these biomarkers in different stages of tumor progression [33]. The NKA family, which comprises ubiquitous transmembrane

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ion transporters, has drawn our attention. The NKA α catalytic subunit has the binding sites for cations, ATP, and cardiac glycoside inhibitors, and the β regulatory subunit is

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essential for regulating normal activity of the enzyme [34, 35]. Increasing evidence suggested that NKA participates in the occurrence and development of cancers [9].

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NKA α1 promotes cancer cellular proliferation and migration through the EGFR/Src-Ras-Erk pathway and PI3K-Pdk-Akt pathways [36] . However, to date, none of NKA family subunit has been explored as a potential clinical diagnostic biomarker in the molecular imaging of cancer. Clinical data obtained by our team strongly suggested that NKA α1 was overexpressed in breast cancer lesions. Moreover, up-regulation of NKA α1 accompanied poor prognoses for patients. Thus, our novel proposed NKA α1-targeted S3-based PET radiotracer may provide a novel perspective for describing tumors and

ACCEPTED MANUSCRIPT judging prognoses for breast cancer patients. As expected, NKA α1-positive breast tumors were clearly visualized on PET images at 60 min post injection. Furthermore, 18

F-AlF-NOTA-S3 firstly highly accumulated in the kidneys and bladder suggesting

major renal- urinary clearance of the imaging tracer. When dynamically tracing of 18

F-AlF-NOTA-S3, the tumor-to-background ratio was high and maintained within a

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period of 50 min post injection. The newly designed S3-based PET tracer

cancer, the novel developed radiotracer

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demonstrated high sensitivity, stability and negligible toxicity. In addition to breast F-AlF-NOTA-S3 may also feasibly detect

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NKA α1-overexpressing hepatocellular carcinoma and lung cancer. Additionally,

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overexpression of NKA α1 has been involved in the modulation of cellular migration whereas downregulation of the expression of ATP1A1 has been validated to reduce

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the migration of malignant cells[12, 21]. These studies encourage further exploration of exploiting NKA α1-targeted imaging for detecting metastasis lesions.

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With the emergence of epidemiological data suggested that cardiac glycoside treatment unexpectedly decreases the death rate of patients with breast cancer, the

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potential new therapeutic roles for cardiac glycosides was highlight [37-39]. Cardiac glycosides also exert anticancer effects by inducing immunogenic cell death [40, 41]

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and synergizes with MEK inhibitors to promote tumor regression [42]. Currently, a striking NKA α1 inhibitor, called UNBS1450, has been developed as a novel cardiac glycoside anti-cancer drug [13, 43]. Moreover, it is being evaluated in phase I clinical trials to combat non-small cell lung cancer [19], metastatic melanoma [14] and glioma [22]. NKA α1-based identification of patients who might benefit from a specific therapy could provide options for personalized therapy. Meanwhile, preventing NKA α1-negative patients from unnecessary toxicity could

improve the overall

cost-effectiveness of healthcare delivery. Considering these facts, the development of

ACCEPTED MANUSCRIPT noninvasively imaging tracer of NKA α1 expression is of great significance. Previously, the most widely used cardiac glycoside inhibitor, digoxin, has been radiolabelled with

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I for myocardial imaging to monitor cardiac glycoside therapy

[17, 18]. The quite narrow therapeutic window of digoxin, to a great extent, limits the use of these digoxin-based radiotracers for cancer patients [44, 45]. Comparatively,

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the novel developed NKA α1-targeted peptide has advantage of small size, low

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immunogenicity and nontoxic. The S3 peptide-based imaging may offer a unique

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utility for the screening of NKA α1-positive cancer patients who may benefit from this cardiac glycoside anti-cancer therapy, clustering patient subgroups and

Conclusions

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5.

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monitoring the treatment response in the future.

Here, we identified a tumor-specific homing NKA α1-targeted S3 peptide and

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showed it was a feasible vehicle for PET imaging of breast cancer and other NKA α1-overexpressing cancers in mouse models. Our present results would encourage

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further research into expanding the application of this NKA α1-targeted peptide to characterize cancers, including disease staging of regional and distant metastases,

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monitoring of relapse, and in some cases, predicting the outcome of personalized therapy in the future.

Acknowledgements We thank Hua Zhang, Yi Yuan, Tian-Liang Xia for their assistance with the cell-based and animal experiments. We thank BGI Tech Solutions Co., Ltd. for performing the MS analysis of the proteins. This work was supported by grants from the National Key R&D Program of China (2017YFA0505600-04), the National

ACCEPTED MANUSCRIPT Natural Science Foundation of China (81602364, 81230045, 91440106, 81161120408, 81572600, 81772883, and 81520108022), the Health & Medical Collaborative Innovation Project of Guangzhou City, China (201400000001), the Science and Technology project of Guangdong Province (2017A020211010, 2014B050504004, 2015B050501005), the Guangzhou Science Technology and Innovation Commission

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(201607020038) and the Guangzhou Scientific Planning Programs（201607020038）.

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Conflict of interest

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The authors declare no competing financial interests.

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