Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth

Aug 1, 2018 - To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel...
0 downloads 0 Views 2MB Size
Download PDF
Subscriber access provided by UNIV OF DURHAM

Communication

Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating MET Signaling yingwei wu, Qi Fan, Feng Zeng, Jinyu Zhu, Jian Chen, Dandan Fan, Xinwei Li, Wenjia Duan, Qinghua Guo, Zhonglian Cao, Karen Briley-saebo, Cong Li, and Xiaofeng Tao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b01879 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating MET Signaling Yingwei Wu[a]#, Qi Fan[a]#, Feng Zeng[b]#, Jinyu Zhu[a], Jian Chen[b], Dandan Fan[b], Xinwei Li[b], Wenjia Duan[b], Qinghua Guo[b], Zhonglian Cao[b], Karen Briley-Saebo[c], Cong Li*[b] and Xiaofeng Tao*[a] Department of Radiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200011, China [b] Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai 201203, China [c] The Ohio State University Wexner Medical Center, 410 W. 10th Ave. Columbus, Ohio 43210 #These authors share equal first authorship. *Corresponding authors: Cong Li, Email: [email protected]; Xiaofeng Tao, Email: [email protected]. [a]

Abstract Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide (KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased three orders of magnitude to 1.32 × 1010

M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma

U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET (pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and most importantly, potentially reduced drug resistance. Keywords: Glioma, MET, cancer-targeted therapy, nanoinhibitor

Introduction Glioma, the most common malignant primary brain tumor, is characterized by infiltrative growth, high

recurrence, and poor prognosis. The current standard treatment of glioma includes maximal safe resection followed by concomitant radiotherapy and/or chemotherapy based on patient characteristics. Even with these aggressive treatments, the prognosis for glioma patients remains dismal 1. The compromised therapeutic efficacy in glioma patients can be due to a variety of reasons. First, the infiltrative nature of glioma cells and diffuse tumor margins make complete resection of glioma nearly impossible to avoid irreversible damage to eloquent areas of the brain. Second, the alkylating agent temozolomide (TMZ) is one of the few clinically approved first-line chemotherapeutics for glioma treatment1. However, the majority (60−75%) of glioma patients cannot benefit from TMZ because of the overexpression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that removes the cytotoxic TMZ metabolite O6-methylguanine2. In addition, third, due to the weak T-cell infiltration into tumor parenchyma and the presence of the blood-brain barrier (BBB), glioma patients hardly benefit from immunological checkpoint therapy. For example, in the latest phase III study, no difference in median overall survival was observed in patients treated with or without Nivolumab, a clinically approved PD-1 monoclonal antibody3. Thus, it is critical to develop new strategies for improving the therapeutic efficacy in glioma patients. Compared to chemotherapy, targeted therapy against specific molecules and signaling pathways shows higher tumor specificity, a broader therapeutic window, and less cytotoxicity4. Receptor tyrosine kinases (RTKs) are transmembrane cell surface proteins that play critical roles in the transduction of extracellular signals to the cytoplasm5. Aberrant signaling of RTK has been shown to play a key role in tumor initiation, progression, invasiveness, and metastasis6. Therefore, RTK pathways are attractive targets for the development of novel cancer treatment strategies. As an RTK member, the interaction between mesenchymal-epithelial transition factor (MET) and its endogenous stimulatory ligand, hepatocyte growth factor (HGF), triggers uncontrolled cell growth and survival, angiogenesis, and metastasis7. The MET/HGF interaction activates the Ras/Raf/MEK/ERK, phosphoinositide-3 kinase

ACS Paragon Plus Environment

Page 2 of 20

Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

(PI3K), and Rac1-Cdc42 pathways, which promote proliferation, survival, and mobility of cancer cells, respectively. Importantly, abnormal MET signaling was correlated with poor prognosis, an elevated occurrence of metastasis, and increased drug resistance in patients8. Frequent signaling activation and genomic amplification of MET were found in 29% of glioma patients9. Additionally, the median survival of patients with MET overexpression was 11.7 months, which is significantly lower than 14.3 months for patients with no or little MET expression10. Thus, blocking the MET/HGF axis is a rational therapeutic approach for glioma patients. Specific binding between HGF to MET can be blocked by HGF analogues, anti-HGF antibodies, anti-MET extracellular domain antibodies as well as chemical tyrosine kinase inhibitors (TKIs) that are in clinical trials11. For example, anti-HGF antibody Rilotumumab in combination with Bevacizumab, a humanized monoclonal antibody approved for recurrent glioma treatment by blocking angiogenesis, showed a therapeutic response in phase II trials in glioma patients11. However, monoclonal antibodies are cost-prohibitive or result in immunogenicity. Moreover, small molecule MET inhibitors, such as cabozantinib, demonstrated better antiglioma activity than bevacizumab12 but developed drug resistance by mitigating MET endocytosis and increasing its copy number, resulting in upregulated MET phosphorylation after drug withdrawal13. Therefore, there is a critical need for the development of novel strategies blocking MET activation with high efficiency, minimal rebound effect, and affordability. Due to the high receptor binding affinity, good biocompatibility, affordability and low immunogenicity, peptides are widely applied in tumor imaging. Compared with antibodies (~150 kDa), peptides are considerably smaller (1−2 kDa) and do not accumulate in the reticuloendothelial system or elicit an immune response after repeated administrations14. In contrast to small molecular inhibitors, peptides are more convenient to modify on the scaffold by increasing the overall receptor binding affinity and agonistic effect15. Even though MET-targeted peptides such as MET-pep1 and cMBPs were developed for tumor imaging and gene delivery 16, their therapeutic efficacies to block MET signaling were not reported. A multivalent effect is defined as the simultaneous binding event between multiple ligands and multiple receptors17. It creates much stronger interaction than corresponding monovalent systems due to a “collective” affinity (avidity)18.The binding affinity and/or specificity of ligands can be increased by conjugating multiple copies with the nanoparticles because of their high surface area to volume ratio. For example, the avidity of folate-coated nanoparticles to the folate receptor overexpressed in

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

epithelial cancer cells increased up to 170 000 fold compared to that of free folic acid19. Additionally, compared to the free peptide, nanoparticles modified with 20 cyclic RGD peptides showed 38-fold binding affinity enhancement to endothelial cells expressing αvβ3 integrin 20. In addition to boosting the receptor binding affinities, the multivalent effect also strengthens the functional bioactivities21. Conjugation of multiple angiopep-2 peptides on the nanoparticles significantly increased the brain uptake by overcoming the BBB via receptor-mediated transcytosis22. Similarly, by adjusting the number of adenosine 2A receptor agonistic domains functionalized on the nanoparticles, it was possible to tune BBB opening time-window by temporarily compromising inter-endothelial tight junctions23. More relevant to the current study, nanoparticles conjugated with multiple copies of antibody24 or nanobody25 were more effective in inhibiting RTK signaling than the monovalent antibody or nanobody with an identical stoichiometric concentration. Herein, we developed a MET-targeting nanoinhibitor by conjugating multiple copies of the cMBP peptide on the surface of PAMAM-NH2 G4 dendrimer via polyethylene glycol (PEG) linkages. This multivalent peptide-functionalized nanoinhibitor showed high therapeutic efficacy by blocking MET-driven downstream signaling.

Results and Discussion Overactivation of MET in tumor margins of glioma patients and glioma xenografts Activation of the MET receptor by its natural ligand HGF triggers a tightly regulated signal transduction cascade. However, tumors hijack the HGF/MET signaling pathway resulting in proliferation, invasion, and metastasis. Hence, blockage of MET transduction is a promising approach for cancer therapy. The intracellular signaling cascades activated by MET mainly include the PI3K– AKT and RAS–MAPK pathways that are actively involved in survival and proliferation/invasion of cancer cells (Figure 1A). Immunoblotting analysis showed 1,290%, 20%, and 50% higher expression of MET, AKT and ERK1/2, respectively, in U87MG cells than in the normal rat astrocytes (RA) (Figure 1B and S1). While the phosphorylated MET (pMET), AKT (pAKT) and ERK (pERK) were barely detected in RA cells, the levels of above proteins in U87MG cells were 12.6, 2.1 and 4.1 times higher, respectively, than those in RA cells. A similar phenomenon was also observed when using immortalized human oral epithelial cells (HIOEC) as a control. As shown in Figure 1B, Figure S1C-D, even though the expression levels of MET, AKT and ERK1/2 in HIOECs were higher than those in RA cells, the immunities of pMET, pAKT and pERK1/2 were still significantly lower than those of U87MG cancer cells. Furthermore, upregulation of both total and phosphorylated MET signaling proteins in the

ACS Paragon Plus Environment

Page 4 of 20

Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

glioblastoma cell line strongly implies the existence of an autocrine loop that supports the transformation, proliferation, and survival of the cancer cells26. Considering the specific and remarkable activation of MET protein transducers in cancer cells, a satisfactory therapeutic response is anticipated by blocking MET signaling. Immunofluorescence imaging in glioma margins from GBM patients and orthotopic U87MG xenografts showed overexpression of pMET (Figure 1C, E, and S2). Notably, pMET was predominately located in the luminal side of the tumor vasculature, which was verified by the colocalization of pMET and CD31, a biomarker of vascular endothelium. MET has been shown to regulate tumor angiogenesis by promoting the survival, proliferation, and migration of endothelial cells27. Crosstalk between MET and VEGFR signaling pathways has been reported as a major reason leading to tumor progression and resistance to therapy7. Anti-MET treatment had a synergistic effect on antiangiogenesis therapy as shown by a higher therapeutic efficacy of the simultaneous blockage of VEGF/VEGFR and HGF-MET than the treatment with VEGFR inhibitors alone28. The predominant location of MET in vascular endothelium in glioma margins implied an important role of MET in glioma angiogenesis and invasion29. A similar distribution pattern of pMET was also found in the U87MG glioma xenograft. In contrast, the pMET signal was barely observed in control brain tissues from patients as well as tissues from normal mouse brain (Figure 1C, E, and S2). Similarly, pMET expression in orthotopic U87MG xenograft was also significantly higher than that in contralateral hemisphere (Figure S3). The pMET expression as measured by immunofluorescence staining in the tumors from GBM patients and tumor xenografts was 28.7 and 5.0 times higher than that in corresponding normal brain tissues (Figure 1D, F). Synthesis and characterization of MET nanoinhibitor G4 PAMAM dendrimer was chosen as a scaffold for the nanoinhibitor because of its globular architecture, uniform molecular weight, optimal circulation time, and well-defined reactive groups on the particle surface30. cMBP peptide was chosen as the targeting ligand due to its high binding affinity to MET and application in tumor imaging 31. cMBP peptides were conjugated to the dendrimer through PEG linkers, which not only improved the biocompatibility of the nanoinhibitor but also minimized the steric hindrance of the hyperbranched polymer thus increasing the binding affinity of the ligands. The synthetic procedure of nanoinhibitor and control Den-PEG nanoparticles without the modified peptides is displayed in Figure 2A. Briefly, treatment of bismaleimide-functionalized PEG derivative (Mal-PEG2kMal) with cMBP peptide yielded Mal-PEG-cMBP. In the preparation of Den-cMBP5, G4 dendrimer was

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

first partially PEGylated by the reaction with mPEG-SCM, then the PEGylated dendrimer was further treated with Mal- PEG–cMBP to give Den-cMBP5. In the development of Den-cMBP10, the dendrimer was treated with Mal-PEG–cMBP directly. The obtained Den-cMBP5 and Den-cMBP10, which, on an average, contained 5 and 10 cMBP peptides conjugated on the dendrimer. Treatment of N-hydroxysuccinimidyl (NHS) ester-functionalized PEG derivative mPEG2k-NHS with the G4 dendrimergenerated control nanoparticles, Den-PEG. The hydrodynamic diameters of Den-cMBP10, Den-cMBP5, and Den-PEG were determined to be 15.1, 12.2., and 9.6 nm, respectively, by dynamic light scattering (DLS) (Figure 2B, Figure S4). Transmission electron microscopic (TEM) images showed a scattered distribution of Den-cMBP10 with an average diameter of 11.5 nm (Figure 2C). The physical parameters of the nanoparticles are listed in Table 1. The molar ratios of dendrimer/PEG/cMBP peptide in DencMBP10, Den-cMBP5, and Den-PEG were measured as 1/10/10, 1/13/5, and 1/14/0, respectively, by 1H

NMR (Figure S5−S7). Mono-functionalized intermediate Mal-PEG-cMBP was characterized by 1H

NMR (Figure S8). The molecular weight of the nanoinhibitor was 51 kDa as determined by a gelfiltration column (Figure S9). Binding affinity KD between MET and free cMBP peptide, Den-cMBP5, or Den-cMBP10 were determined as 3.964 × 10-7, 1.271 × 10-9 M, and 1.316 × 10-10 M, respectively, by the surface plasmon resonance (SPR) (Figure 2D). Compared to the free peptide, the MET binding affinities of Den-cMBP10 and Den-cMBP5 increased 332 and 32 times, respectively. Both free peptide and nanoinhibitor demonstrated similar MET binding kinetics with the fast association and deassociation rates. However, the response unit (RU) of MET to the nanoinhibitor was significantly higher than to the free peptide. The RU of 0.16 nM nanoinhibitor was similar to that of 630 nM free peptide, which further verified the remarkably increased binding avidity of the nanoinhibitor17. The substantially increased MET binding affinity of the dendrimer-based nanoinhibitor compared to that of the free peptide could be explained by the multivalent effect that is a well-studied mechanism to increase the biological potency of nanoparticles through the enhanced ligand-receptor interaction32. First, there are 64 primary amines distributed on the G4 dendrimer periphery, which provide a mechanism to functionalize numerous receptor targeting ligands with less steric hindrance 17. Second, flexibility and deformability of the polymer branches in the dendrimer facilitate the reorganization and reorientation of the prelabeled ligands according to the distribution pattern and density of the targeted receptors17,

19.

Third, the extended PEG linkers not only improve the biocompatibility of the

nanoparticle but also minimize the steric hindrance of the hyperbranched polymer to the targeting ligands17,

33.

Overall, due to the transformable backbone topology, the availability of multiple

functionalization positions and the flexibility of the PEG linker, the dendrimer-based nanoinhibitor

ACS Paragon Plus Environment

Page 6 of 20

Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

shows remarkably increased binding affinity to the MET receptor. Nanoinhibitor specifically mitigates cancer cell proliferation by down-regulating MET signaling Cellular uptake of Den-cMBP10 increased consistently with the incubation time but was almost completely blocked by preincubation with free cMBP peptide, while the uptake of Den-PEG was not affected (Figure S10). These results demonstrated the MET-assisted cellular uptake of Den-cMBP10. Figure 3A shows the white light microscopic images of U87MG cells after incubation with a stoichiometric peptide concentration of free cMBP peptide or Den-cMBP10 for 24 h. The morphology of the cells did not change after incubation with free cMBP peptide, whereas most cells perished with morphological shrinkage and detachment from the plate after treatment of Den-cMBP10. The scratch wound assay showed that while cMBP peptide-treated U87MG cells refilled more than 63% of the wound space after 24 h incubation, only 18% of the wound area was covered following the addition of Den-cMBP10, verifying the significantly increased inhibitory effect of the nanoinhibitor on the cancer cells (Figure 3B, S11, S12A−B). Additionally, Den-cMBP10 inhibited cancer cell migration in a concentration-dependent manner (Figure S11, S12A−B). After a 24 h-incubation with 3.88×10-5, 1.94×10-4 or 3.88×10-4 M Den-cMBP10 peptide, the open scratch area was measured to be 35.3, 55.0, and 82.0%, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay showed that 3.1% TUNEL-positive U87MG cells (apoptotic cells) were observed after treatment with the stoichiometric cMBP peptide concentration of 3.88×10-4 M), whereas 27% TUNEL-positive cells were detected after incubation with 3.88×10-4 M Den-cMBP10 for 24 h (Figure 3C, S12C). Cytotoxicity studies showed that the viability of U87MG cells decreased remarkably as a function of Den-cMBP10 concentration (Figure 3D). In contrast, the cell viability was unchanged even the when the concentration of cMBP peptide was increased to 3.88×10-4 M. Interestingly, DencMBP10 was not cytotoxic to normal rat astrocytes (RA). The cell viability was greater than 73.4%, even at 24 h posttreatment with Den-cMBP10 with the highest concentration (3.88×10-4 M stoichiometric cMBP peptide) (Figure S12D). Notably, no obvious cytotoxic effects were observed in either U87MG cells or RA cells even when the concentration of Den-PEG reached its maximum (3.88×10-5 M) (Figure S12E), which indicates that the cytotoxicity of the nanoinhibitor resulted from the blockage of the MET signaling pathway but not from the dendrimer itself. The specificity of DencMBP10 to restrain the proliferation of cancer cells but not the normal cells implies that a therapeutic response can be achieved by blocking MET signaling. Immunoblotting studies demonstrated the down-regulation of MET and its downstream proteins as

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a function of Den-cMBP10 dosage (Figure 3E, S12, S13). Interestingly, in contrast to the moderate reductions of MET, AKT, and ERK1/2, more pronounced attenuations of the phosphorylated proteins were observed. Figure S12F shows the ratio between the phosphorylated protein and total proteins in U87MG cells after treatment with Den-cMBP10. The ratios of pMET/MET, pAKT/AKT and pERK/ERK decreased significantly following treatment with Den-cMBP10 in a nanoinhibitor concentrationdependent manner. For example, the ratio of pMET/MET decreased by 14.7%, 63.3%, and 87.0%, respectively, in the presence of 3.88×10-5, 1.94×10-4, and 3.88×10-4 M Den-cMBP10, whereas the ratio of pAKT/AKT decreased by 18.0%, 69.7%, and 79.3%, and the ratio of pERK/ERK decreased by 38.7, 73.0, and 88.0%, respectively. The significant attenuation of the key proteins in the MET signaling pathway verified that the nanoinhibitor abrogated the proliferation of U87MG cells by blocking the MET signaling pathway. Notably, neither the total protein level nor the phosphorylated protein level was changed in RA cells, even with an increase in the nanoinhibitor concentration from 3.88×10-5 to 3.88×10-4 M (Figure S14). The low expression of MET in RA cells and the almost undetectable effect of Den-cMBP10 on RA cell viability implied that Den-cMBP10 conferred its therapeutic effect on glioma cells by blocking the over-activated MET signaling pathway. Nanoinhibitor restrains the growth of glioma xenograft by down-regulating MET signaling Figure 4A presents the time course of in vivo studies in which the MR images were collected at 7, 15, and 21 days post tumor implantation and in which the therapeutic nanoinhibitor was administered intravenously (i.v.) once every other day over a period of 18 days. T2-weighted MR images show the U87MG glioblastoma xenograft at selected time points post administration of PBS, free cMBP peptide, Den-cMBP10, or small molecule inhibitor PF-04217903 (Figure 4B). The volume of the tumor xenograft increased exponentially in the control group and was measured as 0.482, 13.015, and 101.633 mm3 at 7, 15, and 21 days, respectively, postinoculation. cMBP peptide did not show an obvious therapeutic response, and the tumor volumes were determined as 0.260, 10.963, and 94.667 mm3 at 7, 15, and 21 days, respectively. In contrast, both nanoinhibitor and PF-04217903 substantially inhibited tumor development. The volumes of tumors treated with Den-cMBP10 were recorded as 0.084, 3.033, and 13.667 mm3, which decreased 82.6, 76.7, and 86.6%, respectively, compared those treated with the control. Similarly, 67.6, 80.5, and 87.4% tumor volume reduction was recorded after the administration of PF-04217903 (Figure 4B−C). Compared to the PBS-treated mouse models with a median survival of 22 days after tumor implantation, the free cMBP peptide achieved only a marginal benefit with a median survival of 24 days (P = 0.4120 as shown by Kaplan-Meier survival curves). Significant

ACS Paragon Plus Environment

Page 8 of 20

Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

therapeutic responses were recorded after injecting Den-cMBP10 or PF-04217903, in which the median survivals were extended to 35 days (P = 0.0334 and P = 0.0033, respectively) (Figure 4D). Notably, no apparent systemic toxicity was detected by monitoring mouse body weights before and after the administration of Den-cMBP10 (Figure S15). Immunofluorescence studies showed the pMET levels in the U87MG tumor xenograft decreased 62.3 and 71.0% at 2 h post i.v. injection of Den-cMBP10 or PF-04217903. In contrast, less than a 22.0% decrease in the pMET level was observed after administering free cMBP peptide (Figure 5A−B). Immunoblotting studies show the expression levels of MET signature proteins and their phosphorylated forms in U87MG tumor xenografts at 24 h post administration of PBS, cMBP peptide, Den-cMBP10, or PF-04217903 (Figure 5C). Although Den-cMBP10 and PF-04217903 resulted in substantial reductions in pMET, pAKT, and pERK1/2, no obvious protein level variation was observed after the treatment with the cMBP peptide. The ratio between pMET, pAKT, pERK1/2 and the corresponding total proteins decreased to 44.2%, 62.2%, and 32.3% at 24 h post administration of Den-cMBP10 (Figure 5D). Similar results were observed after PF-04217903 treatment in which the ratio of pMET/MET, pAKT/AKT, and pERK/ERK decreased to 47.6%, 48.3%, and 51.7%, respectively (Figure S16). The correlated pMET/MET ratio and tumor growth rate indicated that both Den-cMBP and PF-04217903 conferred the therapeutic efficiency by blocking MET signaling. Antibodies and chemical TKIs are the two major types of therapeutics for the MET-targeting therapy34. Although both strategies show promising efficacies, the application of antibodies is limited by their high cost, poor tumor penetration, immunogenicity, and a long time for clearance16,

35.

Chemical TKIs also suffer from limitations, including the poor selectivity for RTKs and compromised therapeutic efficacy due to the short circulation time. Compared to antibodies, peptides are inexpensive, easier for mass production, less immunogenic, and more permeable in tissues 14,

16.

Importantly, peptides are convenient to modify on the nanoscaffold to enhance the receptor targeting specificity through the multivalent effect24. The remarkably enhanced therapeutic response of the nanoinhibitor compared to that of the monomeric peptide could be explained by the (1) three orders of magnitude enhancement in MET-binding affinity, (2) optimized circulation time facilitating intratumoral delivery by the enhanced permeability and retention (EPR) effect36, and (3) improved stability of the peptides shielded by the branched structure of the dendrimer37.To validate the above statements, we performed stability and pharmacokinetics by radiolabeling both free cMBP peptide and Den-cMBP10 with 125I isotope via a chloramine-T assay (Figure S17A, B). The radiochemical purity of

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cMBP-125I and Den-cMBP-125I was determined as 95.1% and 94.2%, respectively, by paper chromatography (Figure S17C). For the pharmacokinetic study, two groups of SD rats (n = 3 for each group) were intravenously injected with 18 μCi of cMBP-125I or Den-cMBP-125I. Blood samples were collected from the tail vein at indicated time points, and the radioactivities were measured using a solid scintillation counter. Based on the noncompartmental analysis of the blood clearance in rat, the DencMBP-125I exhibited 2.1-fold higher area under curve (AUC), relative to the cMBP-125I over the 24 h interval tested. As a result, the cMBP-125I exhibited an elimination blood half-life that was approximately 2 times greater than that of the nanoinhibitor, as shown in Figure S18A, Table S1. For the stability study, cMBP-125I and Den-cMBP-125I were incubated with fresh rat serum at 37 °C for the indicated time points. The radiochemical purity of cMBP-125I was 35.1% at 1.0 h post incubation and only 15.2% peptide remained intact after a 24 h-incubation. In contrast, the radiochemical purity of Den-cMBP-125I was measured as 92.3% after incubation for 1 h, and it was above 86% at 24 h (Figure S18B, C). The above study verifies the higher stability of the nanoinhibitor than the free peptide in rat serum. Our recent study demonstrated an uncompromised integrity of BBB in the invasive margin of the glioma patients regardless of tumor malignancies38. Given the high expression level of MET on both vascular endothelium and cancer cells in glioma margins, we suspected that the nanoinhibitors could first target the tumor neovasculature. The positive charge on the nanoinhibitors facilitates BBB traversing by adsorptive transcytosis39. After crossing the BBB, the nanoinhibitors further bind to the glioma cells and block MET signaling. Thus, the enhanced MET binding affinity, BBB permeability, and resistance to degradation could explain the increased therapeutic response of the nanoinhibitors compared to that of the free peptides. Antibodies and small molecule TKIs achieve their therapeutic effects through different mechanisms40. Although antibodies stoichiometrically compete with HGF at the extracellular binding site of MET, the TKIs competitively antagonize occupancy of the ATP at the intracellular binding site of MET41. Both mechanisms abrogate MET dimerization, autophosphorylation, downstream signal transmission and proliferation of cancer cells13. However, both antibodies and TKIs face the same challenge of drug resistance during the targeted therapy42. For example, MET-addicted cancer cells showed resistance to monoclonal antibody (MV-DN30) treatment due to the dramatically upregulated MET expression that overcomes the ‘blocking’ effect of the antibody42. Additionally, withdrawal of the TKIs triggered reactivation of MET-driven proliferative pathways leading to a rebound effect pushing nonproliferating cancer cells back into the cell cycle13. Interestingly, a discontinuous, combined treatment by antibodies and chemical TKIs could bypass resistance to anti-MET targeted therapies13.

ACS Paragon Plus Environment

Page 10 of 20

Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

For example, the rebound effects occurring after TKI washout could be overcome by MET antibody13. Similarly, antibody resistant cells were found to be sensitive to TKI therapy42. Moreover, there are a number of studies that suggested acquired resistance to epidermal growth factor receptor (EGFR) TKI occurring as a result of secondary EGFR mutations or parallel activation of MET. Approximately 5−22% of nonsmall cell lung cancer (NSCLC) with secondary resistance to EGFR TKIs had evidence of amplification

of

MET

oncogene43.

MET

amplification

would

activate

EGFR-independent

phosphorylation of ErbB3 and the downstream PI3K/AKT pathway, resulting in acquired resistance to EGFR-TKIs44. Available data suggest that the inhibition of both MET and EGFR pathways might overcome resistance and improve clinical outcomes45. In this respect, the nanoinhibitor may offer an alternative strategy to be managed as combined therapy with either MET TKIs or other targeted therapy such as EGFR-targeted TKIs to relieve drug resistance.

Conclusion This study reported a novel nanoinhibitor in which multiple copies of MET-targeting peptide were conjugated on a PAMAM G4 dendrimer. This nanoinhibitor not only showed three orders of magnitude higher MET binding affinity than the free peptide but was also more efficient in suppressing MET signaling than the free peptide in both cancer cell cultures and glioma xenografts. The nanoinhibitor remarkably restrained the growth of glioma xenografts by specifically abrogating MET signaling. In addition to the monoclonal antibody and small molecular TKIs, the peptide-functionalized nanoinhibitor provides a new choice for MET-targeted therapy. Furthermore, the synergistic effect of this nanoinhibitor with antibodies or TKIs may not only enhance the therapeutic response but also holds promise to minimize tumor resistance after long-term treatment.

ASSOCIATED CONTENT Supporting Information Experimental Section, Quantification of MET downstream levels, Immunofluorescence images of tissues from GBM patients and U87MG glioma xenografts, hydrodynamic diameter histogram and zeta potential distribution, 1H NMR spectrum of Den-PEG, Den-cMBP5, Den-cMBP10 and Mal-PEG-cMBP, HPLC spectra of G4 PAMAM dendrimer and Den-cMBP10, Cellular uptake of Den-cMBP10 and DenPEG, scratch wound assay images of U87 monolayer, TUNEL assay for cell apoptosis, cell viability,

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

expression of MET and downstream proteins in RA cells, body weight changes according to various treatments, procedures of radiochemical purity measurement, blood clearance and stability for DencMBP10 and free cMBP.

Acknowledgments The work was supported by the National Basic Research Program of China (973 Program, 2015CB75500), the National Natural Science Foundation of China (Nos. 81771963 to Y.W.W.; 81771901, 81471709 to X.F.T.; 81571741 and 81771895 to C.L.), the Foundation of Shanghai Municipal Commission of Health and Family Planning (No. 2014zyjb0007 to X.F.T.), Clinical Research Program of 9th People's Hospital, Shanghai Jiao Tong University School of Medicine (No. JYLJ033 to Y.W.W.) and Fudan-SIMM Joint Research Fund (No. 20173001 to C.L.).

Abbreviations MET, mesenchymal-epithelial transition factor; MRI, magnetic resonance imaging; cMBP, c-Met binding peptide; U87MG, Uppsala 87 Malignant Glioma; Akt(PKB), protein kinase B; ERK, extracellular regulated protein kinases; SOS, son of sevenless; HGF, hepatocyte growth factor; PI3K, phosphoinositide-3 kinase; Grb2, growth factor receptor-bound protein 2; GAB1, GRB2associated binding protein 1; TMZ, temozolomide; MGMT, O6-alkylguanine-DNA alkyltransferase; BBB, blood-brain barrier; RTKs, Receptor tyrosine kinases; MEK, Mitogen-activated protein kinase; TKIs, Tyrosine-kinase inhibitors; PEG, polyethylene glycol; PAMAM, Poly(amidoamine); RA, rat astrocytes; GBM, glioblastoma multiforme; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; EGFR, epidermal growth factor receptor; NSCLC, nonsmall cell lung cancer; CD31, cluster of differentiation 31; NHS, N-hydroxy-succinimidyl; DLS, dynamic light scattering; TEM, transmission electron microscope; SPR, surface plasmon resonance; RU, response unit; TUNEL, Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; EPR, enhanced permeability and retention

ACS Paragon Plus Environment

Page 12 of 20

Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

References

1. Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher, B.; Taphoorn, M. J. B.; Belanger, K.; Brandes, A. A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.; Mirimanoff, R. O., Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. New Engl J Med 2005, 352 (10), 987-996. 2. Bobola, M. S.; Silber, J. R.; Ellenbogen, R. G.; Geyer, J. R.; Blank, A.; Goff, R. D., O6Methylguanine-DNA Methyltransferase, O6-Benzylguanine, and Resistance to Clinical Alkylators in Pediatric Primary Brain Tumor Cell Lines. Clin. Cancer Res. 2005, 11 (7), 2747. 3. Renfrow, J. J.; Weller, M.; Strowd, R. E., Meeting Update-World Federation of Neuro-Oncology Societies (WFNOS) Meeting 2017. Neuro Oncol 2017, 19 (11), 1457-1459. 4. Amit, A.; Eric, M. S., Role of Tyrosine Kinase Inhibitors in Cancer Therapy. J Pharmacol Exp Ther. 2005, 315 (3), 971-979. 5. Pawson, T., Regulation and targets of receptor tyrosine kinases. Eur J Cancer 2002, 38 Suppl 5 (Suppl 5), S3. 6. Chen, J., Regulation of tumor initiation and metastatic progression by Eph receptor tyrosine kinases. Adv Cancer Res 2012, 114 (2), 1-20. 7. Gherardi, E.; Birchmeier, W.; Birchmeier, C.; Woude, G. V., Targeting MET in cancer: rationale and progress. Nat Rev Cancer 2012, 12, 89. 8. Goyal, L.; Muzumdar, M. D.; Zhu, A. X., Targeting the HGF/c-MET Pathway in Hepatocellular Carcinoma. Clin. Cancer Res. 2013, 19 (9), 2310. 9. Kong, D. S.; Song, S. Y.; Kim, D. H.; Joo, K. M.; Yoo, J. S.; Koh, J. S.; Dong, S. M.; Suh, Y. L.; Lee, J. I.; Park, K., Prognostic significance of c-Met expression in glioblastomas. Cancer 2009, 115 (1), 140. 10. Kong, D.-S.; Song, S.-Y.; Kim, D.-H.; Joo, K. M.; Yoo, J.-S.; Koh, J. S.; Dong, S. M.; Suh, Y.-L.; Lee, J.-I.; Park, K.; Kim, J. H.; Nam, D.-H., Prognostic significance of c-Met expression in glioblastomas. Cancer 2009, 115 (1), 140148. 11. Adjei, A. A.; Peters, S., MET: a promising anticancer therapeutic target. Nature Reviews Clinical Oncology 2012, 9 (6), 314. 12. Sennino, B.; Naylor, R. M.; Tabruyn, S. P.; You, W. K.; Aftab, D. T.; McDonald, D. M., Abstract A13: Reduction of tumor invasiveness and metastasis and prolongation of survival of RIP‐Tag2 mice after inhibition of VEGFR plus c‐ Met by XL184. Mol. Cancer Ther. 2009, 8 (Supplement 1), A13-A13. 13. Pupo, E.; Ducano, N.; Lupo, B.; Vigna, E.; Avanzato, D.; Perera, T.; Trusolino, L.; Lanzetti, L.; Comoglio, P. M., Rebound effects caused by withdrawal of MET kinase inhibitor are quenched by a MET therapeutic antibody. Cancer Res. 2016, 76 (17), 5019. 14. Burggraaf, J.; Kamerling, I. M.; Gordon, P. B.; Schrier, L.; de Kam, M. L.; Kales, A. J.; Bendiksen, R.; Indrevoll, B.; Bjerke, R. M.; Moestue, S. A., Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-Met. Nat. Med. 2015, 21 (8), 955-61. 15. Hersel, U.; Dahmen, C.; Kessler, H., RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials 2003, 24 (24), 4385-4415. 16. Zhao, P.; Grabinski, T.; Gao, C.; Skinner, R. S.; Giambernardi, T.; Su, Y.; Hudson, E.; Resau, J.; Gross, M.; Vande Woude, G. F., Identification of a met-binding peptide from a phage display library. Clinical Cancer Research An Official Journal of the American Association for Cancer Research 2007, 13 (20), 6049-55. 17. Myung, J. H.; Gajjar, K. A.; Saric, J.; Eddington, D. T.; Hong, S., Dendrimer-mediated multivalent binding for the enhanced capture of tumor cells. Angew. Chem. 2011, 50 (49), 11769-72. 18. Mourez, M.; Kane, R. S.; Mogridge, J.; Metallo, S.; Deschatelets, P.; Sellman, B. R.; Whitesides, G. M.; Collier, R. J., Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 2001, 19 (10), 958-61. 19. Hong, S.; Leroueil, P. R.; Majoros, I. J.; Orr, B. G.; Baker, J. R.; Banaszak Holl, M. M., The Binding Avidity of a Nanoparticle-Based Multivalent Targeted Drug Delivery Platform. Chem Biol 2007, 14 (1), 107-115. 20. Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.; Josephson, L., Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006, 49 (20), 6087-93. 21. Kitov, P. I.; Bundle, D. R., On the nature of the multivalency effect: a thermodynamic model. J. Am. Chem. Soc. 2003, 125 (52), 16271. 22. Gao, X.; Qian, J.; Zheng, S.; Xiong, Y.; Man, J.; Cao, B.; Wang, L.; Ju, S.; Li, C., Up-regulating blood brain barrier permeability of nanoparticles via multivalent effect. Pharm. Res. 2013, 30 (10), 2538-2548. 23. Gao, X.; Qian, J.; Zheng, S.; Changyi, Y.; Zhang, J.; Ju, S.; Zhu, J.; Li, C., Overcoming the Blood–Brain Barrier for Delivering Drugs into the Brain by Using Adenosine Receptor Nanoagonist. Acs Nano 2014, 8 (4), 3678. 24. Riley, R. S.; Day, E. S., Frizzled7 Antibody-Functionalized Nanoshells Enable Multivalent Binding for Wnt Signaling Inhibition in Triple Negative Breast Cancer Cells. Small 2017, 13 (26). 25. Heukers, R.; Altintas, I.; Raghoenath, S.; De, Z. E.; Pepermans, R.; Roovers, R. C.; Haselberg, R.; Hennink, W. E.; Schiffelers, R. M.; Kok, R. J., Targeting hepatocyte growth factor receptor (Met) positive tumor cells using

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

internalizing nanobody-decorated albumin nanoparticles. Biomaterials 2014, 35 (1), 601. 26. Martens, T.; Schmidt, N.-O.; Eckerich, C.; Fillbrandt, R.; Merchant, M.; Schwall, R.; Westphal, M.; Lamszus, K., A Novel One-Armed Anti-c-Met Antibody Inhibits Glioblastoma Growth In vivo