Peptide-Functionalized Nanomaterials for the ... - ACS Publications

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Letter

Peptide functionalized nanomaterials for efficient isolation of HER2-positive circulating tumor cells Jiaxi Peng, Qiong Zhao, Wangshu Zheng, Ping Li, Wenzhe Li, Ling Zhu, Xiaoran Liu, Bin Shao, Huiping Li, Chen Wang, and Yanlian Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017

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ACS Applied Materials & Interfaces

Peptide functionalized nanomaterials for efficient isolation of HER2-positive circulating tumor cells Jiaxi Penga,b, Qiong Zhaob, Wangshu Zhengb, Wenzhe Lib, Ping Lib, Ling Zhub, Xiaoran Liud, Bin Shaod, Huiping Lid*, Chen Wangb,c* and Yanlian Yangb,c*

a

Department of Chemistry, Renmin University of China, Beijing 100872, China.

b

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS

Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China.

c

University of Chinese Academy of Sciences, Beijing, 100049, China

d

Department of Breast Oncology, Key laboratory of Carcinogenesis and Translational

Research (Ministry of Education), Peking University Cancer Hospital & Institute, 52 Fucheng Rd, Beijing100142, China

* Corresponding Authors: [email protected], [email protected], [email protected]

ABSTRACT

Detection of circulating tumor cells (CTCs) with specific antigen expression is necessary in therapeutic response monitoring and targeted therapy guidance. The existence of human

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epidermal growth factor receptor 2 (HER2) and the concentration of HER2-positive CTCs are strong indicators for patient diagnosis, prognosis, and therapeutic monitoring. Herein we report the direct isolation of HER2-positive CTCs by peptide-functionalized nanomaterials. We designed and screened out a peptide as an HER2 antibody alternative demonstrating high HER2 affinity and selectivity. This HER2 recognition peptide bound efficiently with HER2 at the ligand-binding domain. Efficient HER2-positive CTC capture and detection were demonstrated using magnetic nanoparticles (MNPs) functionalized with the HER2 recognition peptide.

Keywords: nanomaterials, peptide, circulating tumor cells (CTCs), HER2, cancer metastasis

Metastasis is a multistep process that tumor cells disseminate from a primary tumor and colonized in a distance organ. The isolation and analysis of circulating tumor cells (CTCs) may provide relevant information in metastasis progression and implication of disease prognosis and treatment choice.1-3 The extremely low abundance of CTCs in bloodstream is the major challenge for accurate enumeration and further analysis. CTCs are distinguished from surrounding leukocyte on their different properties, particularly physical properties (size, density or electrical properties), or biological properties (expression of protein markers or cancer-specific antigens). A large panel of technologies are based on those unique properties, and improvement of the CTC capture efficiency and purification are always the


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major driving factors for the technological development in both the research and industrial fields. Recently, novel approaches to CTCs enrichment were developed based on nanomaterials, nanostructured

surfaces,

and

microfluidic

devices

with

nanostructures.

Various

micro/nanostructures have been designed and used to improve the capture efficiency for target cells, such as nanopillars,4-5 nanofibers,6-7 and nanodots8-10 with surface antibody functionalization. Based on the topological enhanced adhesion together with the antibody– antigen recognition, a high isolation efficiency of up to 90–95% was achieved. Moreover, immunomagnetic-based assays have been commonly applied to CTC detection due to the quality of easy manipulation, fast magnetic response, and high recovery efficiency.11-14 For now, the most widely used method still relies on the cancer specific interaction of antibodies, like the epithelial cell adhesion molecule (EpCAM), a common antigen frequently overexpressed in solid tumors, to isolate and enrich CTCs. For example, the FDA-approved CellSearch system and other techniques, such as nanomaterials and microfluidics, were developed to increase capture efficiency and minimize blood cell contamination.15-17 However, antibody-based diagnostics and therapeutics are always confronting the bench-to-bench stability and repeatability problem, and they are always very expensive, which limits the widespread use of antibody-based methods. Thus, some other EpCAM-targeting probes, like DNA-aptamers4 or recognition peptides,12 were also tested in CTC isolation. Peptides play key roles in ligand–receptor and protein–protein interactions, and their unique properties, like small size, stability and easy to functionalize by chemical



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synthesis, makes recognition peptides tend to perform better than whole antibodies.18-21 Our previous work12 shows that the EpCAM-targeted recognition peptide efficiently and selectively binds to the EpCAM protein and can successfully isolate CTCs when functionalized onto 200nm magnetic nanoparticles (MNPs). The peptide-based nanomaterials demonstrated comparable capture efficiency (reaching above 90%) and purity (reaching above 93%) to antibody based isolation for spiked breast, prostate, and liver cancer cells from human blood. EpCAM-positive selections were limited by inability for heterogeneous CTCs, and the role of CTC detection in therapy guidance, especially in treatment regimens including targeted therapy, is not clear. Thus, a specific antigen-positive CTC detection method is necessary in therapeutic response monitoring and targeted therapy guidance. From the viewpoint of significance for diagnosis and therapeutics, human epidermal growth factor receptor 2 (HER2, also known as Neu and ErbB2) should be the first choice. HER2 is a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinase receptors,

22-23

and its

overexpression mediates cell proliferation and motility and causes metastasis-related properties such as invasion and angiogenesis. HER2 is overexpressed in about 20–30% of human breast and ovarian cancer cases. and also expressed in Wilms’ tumor, bladder cancer, pancreatic, lung, and colon tumors.24 As a typical type I transmembrane protein with an extracellular domain, HER2 is also an ideal anti-tumor therapeutic target.25-26 HER2-specific monoclonal antibodies, like trastuzumab and pertuzumab, were proven to be an effective therapy for HER2-positive metastatic breast cancer patients.24-25 The fact is, HER2 status


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changes during the disease recurrence or progression, and optimal use of HER2-targeted drugs in different stages is not yet clear. Therefore, there is an urgent need to identify factors enabling the therapy to be monitored. CTCs are the ideal marker for continuously monitoring real-time changes. HER2-positive CTCs were detected in a considerable number of patients diagnosed HER2-negative in primary tumors.27-28 Meng et al.29 assessed HER2 status of primary tumor and CTC level in 31 metastatic patients. 9 of 24 patients (37.5%) detected HER2-positive CTCs even though they were initially diagnosed as HER2-negative tumors. 4 of these 9 patients were treated with trastuzumab, and 3 patients (75%) showed partial or complete release. Thus, the reevaluation of HER2 status assessed by HER2 expression on CTCs is necessary and maybe more important than HER2 expression on primary tumor to provide a significant clinical response for HER2-targeted therapies. Most works on HER2 expression in CTCs are demonstrated by immunohistochemistry after typical CTC isolation procedures, and the standardized CellSearch assay is also used for HER2 expression in CTCs. To date, a method involving the direct isolation of HER2-positive CTCs has not yet been reported. Given this, it is feasible for the highly efficient capture and enrichment of HER2-positive CTCs

based

on

HER2-targeting

peptide-functionalized

magnetic

nanoparticles

(Pep@MNPs). In this work, a series of chemically synthesized peptide fragments based on HER2 and other proteins were collected, and a cell-based selection and screening was performed. We obtained one candidate with comparable affinity and high selectivity by flow cytometry. Computer-aided molecular docking was used to investigate the detailed



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interaction between this peptide and the HER2 protein. We also demonstrated the isolation of CTCs with selected Pep@MNPs from artificial cancer patient’s blood under a magnetic field. In brief, recognition peptide was chemical synthesized with a biotin tag, and conjugated to streptavidin-conjugated magnetic nanoparticles via biotin-streptavidin interaction. The result Pep@MNPs conjugates were washed with PBS under a high magnetic field to remove extra peptide. Then added resuspended Pep@MNPs conjugates to artificial patients’ samples, which prepared by spiking epithelial cancer cells to human blood, and finally isolated CTCs under a magnetic field with characterization by immunofluorescence.

Micro/nanostructure-based magnetic isolation is widely applied to CTC detection due to its advantages of fast magnetic response, easy manipulation, and high capture efficiency. In addition, recognition peptides can make up for the limitations of antibodies, such as instability, irreproducibility, low yield production, and high cost. Based on these features, we developed a peptide-based magnetic isolation method for the CTC method (Pep@MNPs). The process of CTC isolation by the Pep@MNPs method is illustrated below (Scheme 1). We de novo designed a series of peptides targeted to HER2 protein based on electrostatic, hydrogen bonding, and hydrophobic interactions. After a screening of recognition peptides monitored using flow cytometry on HER2–positive cancer cell lines, a candidate was selected with comparable binding affinity and selectivity. The selected recognition peptide was functionalized on 200nm MNPs via streptavidin–biotin interaction. Synthetic peptides can be easily modified with a biotin tag and functionalized to other nanomaterials.


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HER2 is overexpressed in about 20–30% of breast and ovarian cancer cases. To clarify HER2 expression in cultured cell lines, flow cytometry was used to analyze HER2 binding capacity with all four cell lines from four tumor types. Figure 2a shows the low HER2 expression in K562 and MCF7 cell lines compared with SKBR3 and SKOV3, which

is consistent with

previous reports.30 SKBR3 and MCF7 represent two types of human breast cancer cell lines with or without the overexpression of HER2. A series of chemically synthesized peptide fragments based on HER2 and other proteins were collected, and a cell-based selection and screening was performed. Among all these peptides, H2, H12, H13, and H14 were found to have a binding capacity comparable to that of antibodies in the HER2-positive SKBR3 cell line (Figure 2b), while only peptide H13 showed a low binding capacity in the HER2-negative cell line K562 (Figure 2c). Regarding HER2 binding selectivity, indicated by the ratio of binding capacity between HER2-positive (SKBR3) and -negative cell lines (K562),

it

was

also

shown

that

the

HER2

recognition

peptide

H13

(GRQLFDNPDQALLDTANDG) was selected, as it shown the best affinity and selectivity to HER2. To investigate the detailed interaction between the recognition peptide H13 and the HER2 protein, computer-aided molecular docking was conducted. Members of the ErbB family share a similar architecture. They are type I transmembrane proteins with a ligand-binding extracellular domain, a single transmembrane helix, and a tyrosine phosphorylation-rich C-terminal tail work as enzymes to facilitate downstream signaling. Leahy and colleagues26 reported human HER2 complexed with the Herceptin antigen-binding fragment (Fab).It



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reveals that HER2 keeps a fixed conformation in ligand-activated state and Fab will bind to the juxtamembrane region, revealing this pocket-like structure site is a target for anticancer therapies. In our molecular docking processing, we choose this pocket position as our analysis domain. The structure of human HER2 was obtained from the Protein Data Bank (entry 1n8z) with antigens removed. The best binding position of H13 to the HER2 extracellular domain is shown in Figure 3. Peptide H13 bound to all three domains of HER2 (domain I, II and III) and entered the narrow pocket around their interface. The binding affinity reached -6.5 kcal/mol, and several hydrogen bonds were formed, including Arg12, Thr268, Tyr387, Cys411, and Ser441. This binding position also suggests our H13 peptide may contribute to the modulation of HER2 dimerization and ligand-mediated ErbB signaling. The HER2 recognition peptide H13 was applied to isolate HER2-positive CTCs by the Pep@MNPs method. Spiked cancer cells were used as model CTCs. MNPs were functionalized with peptide H13 via streptavidin–biotin interaction, and after 30 min of incubation with artificial cancer patient blood samples, CTCs were isolated in a strong magnetic field. The three-color immunofluorescence staining of typical epithelial cancer cells captured by H13@MNPs is shown in Figure 3a, which confirmed that captured cells are typical epithelial cancer cells as spiked rather than contaminated blood cells. CTCs are identified as DAPI+/CK+/CD45– from non-specifically trapped blood cells (DAPI+/CK– /CD45+). The average capture efficiency of the isolated CTCs by H13@MNPs of SKBR3, MCF7, and SKOV3 cell lines reached up to 68.56%, 20.04%, and 79.26% (Figure 3b), respectively, which is consistent with HER2 expression in different cell lines. In particular,


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for MCF7, which expresses relatively low levels of HER2, approximately 20% of spiked CTCs could still be detected by our peptide-based isolation method. The high binding affinity of the H13 peptide and the nanomaterials’ assistance of magnetic isolation both contributed to CTC isolation and detection.

In this work, we de novo designed a series of peptide pools as a random peptide library and then selected a high affinity and selectivity candidate. Computer-aided molecular docking was used to explain the mechanism of H13 binding to the HER2 protein and provide a possible way by which H13 modulates HER2 in ligand-mediated ErbB signaling. We also demonstrated CTC isolation with selected Pep@MNPs from artificial cancer patients’ blood under a magnetic field, and the capture efficiency of SKBR3, MCF7, and SKOV3 reached up to 68.56%, 20.04%, and 79.26%, respectively. This synthetic recognition peptide can be treated as a comparable alternative to HER2 monoclonal antibodies with advantages of low price, constant properties, and easy manipulation. It is likely that this peptide-formed complex as an ADC will serve as a new weapon against HER2-overexpressed cancer. ASSOCIATED CONTENT

Supporting Information.

Detailed Experimental Section: protein expression characterized by FCM, functionalization of MNPs with recognition peptide and CTC isolation and enumeration. AUTHOR INFORMATION



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Corresponding Authors: *E-mail: [email protected], [email protected], [email protected] Notes The authors declare no competing financial interest.

Acknowledgement This work was supported by the National Natural Science Foundation of China (21273051, 31600803), Natural Science Foundation of Beijing Municipality (2162044) and the Chinese Academy of Sciences (XDA09030306). Financial support from CAS Key Laboratory of Standardization and Measurement for Nanotechnology and Key Laboratory for Biological Effects of Nanomaterials and Nanosafety are also gratefully acknowledged.

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FIGURES

Figure 1 (a) Binding capacity of anti-HER2 on K562, SKBR3, MCF7, SKOV3, A549, and PC3 cell lines. (b) HER2 binding capacity with H1 to H18 peptides was determined on SKBR3 cell line; IgG and Anti-HER2 were used as negative and positive controls, respectively. (c) Binding capacity with four selected peptides (H2, H12, H13, and H14) on K562 and SKBR3 cell lines. (d) HER2 binding selectivity compared with HER2 antibody. Binding capacity was determined by FCM.


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Figure 2 HER2-H13 complex. Surface-stick representation of HER2 extracellular domain (Domains I, II, III, and IV of HER2 extracellular domain are colored pink, green, blue, and gray, respectively) with recognition peptide H13 (shown in aqua sticks) complex shown in stereo. A zoom-in insert model is superimposed to show the interface between HER2 and H13 peptide. The protein is shown in surface form, and the peptide is shown with side chains in stick form and backbone in ribbon form. The intermolecular H- bonds are shown with gold dashed lines.



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Figure 3 (a) Fluorescent micrographs of typical epithelial cancer cells captured from human blood by MNPs functionalized with HER2-targeting peptides. Cells stained by immune-cytochemistry for CK19 (green), CD45 (red), and DAPI (blue). (b) Capture efficiency (ratio of captured to spiked cells) of H13 on SKBR3, MCF7, and SKOV3 cell lines using Pep@MNPs methods.


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SCHEMES Scheme 1 Schematic Illustration for (a) HER2 targeting peptide screening and (b) CTCs isolation by Pep@MNPs.



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Graphic for manuscript


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Peptide-Functionalized Nanomaterials for the ... - ACS Publications

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