Vx3 Functionalized Alumina Nanoparticles Assisted Enrichment of

Subscriber access provided by MT ROYAL COLLEGE

Article

Vx3 Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy Jinjin Zhao, Ning Pan, Fang Huang, Mohanad Aldarouish, Zhifa Wen, Rong Gao, Yuye Zhang, Hong-Ming Hu, Yanfei Shen, and Li-xin Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00578 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 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 free 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 accessible to all readers and 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.

Bioconjugate Chemistry 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 30 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

Bioconjugate Chemistry

Vx3 Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy Jinjin Zhao†, Ning Pan†, Fang Huang†, Mohanad Aldarouish†, Zhifa Wen†, Rong Gao†, Yuye Zhang§, Hong-Ming Hu†┴, Yanfei Shen‡* and Li-xin Wang†* †

Department of Microbiology and Immunology, Medicine School of Southeast

University, Nanjing, Jiangsu 210009, P.R. China. ‡

Department of Bioengineering, Medicine School of Southeast University, Nanjing,

Jiangsu 210009, P.R. China. §

School of Chemistry and Chemical Engineering, Southeast University, Nanjing,

Jiangsu 210009, P.R. China. ┴

Laboratory of Cancer Immunobiology, Earle A. Chiles Research Institute,

Providence Portland Medical Center, Portland, OR 97213 USA.

*

Address correspondence to [email protected], [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 2 of 30

ABSTRACT A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as: α-Al2O3-Vx3) via a chemical linker. The functionalized α-Al2O3-Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3-Vx3-UPs (α-Al2O3/Vx3-UPs).

than

the

Laser

physical confocal

mixture

of

microscope

α-Al2O3 and Vx3-UPs images

revealed

that

α-Al2O3-Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3-Vx3-UPs to CD8+ T cells in autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3-Vx3-UPs enhanced more potent anti-tumor immune response and anti-tumor efficacy than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.


Page 3 of 30 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


INTRODUCTION During 20th century, a great effort has been devoted towards the development of cancer treatment1, 2 by surgery,3 chemotherapy,4-6 radiation therapy7, 8 and targeted therapy.9 Although these approaches have great potential to improve the survival and life quality of patients,10, 11 they are often aggravated by serious side effects.12-14 Moreover, these therapeutic approaches have been largely unsuccessful in providing long-term response against cancer, which would contribute to tumor recurrence.15 In the last few years there has been a growing interest in cancer immunotherapy based on its promising preliminary results in achieving meaningful and durable treatments responses with minimal manageable toxicity.16-18 Cancer immunotherapy has become an important modality for complementing and enhancing the current standard therapies to provide long-term management of cancer patients.19,20

During

the

immunotherapy

process,

cross-presentation

of

tumor-associated antigens (TAAs) from tumor cells which induces cytotoxic T lymphocytes (CTLs) plays pivotal role.21-27 However, the clinical successes of efficient vaccines have been underachieving. Due to the lack of skills for the effective extraction of TAAs from tumor cells, traditionally inactivated tumor cells or tumor cell lysate are used as vaccines directly.28 Nevertheless, the immunotherapy efficiency by using inactivated tumor cells or tumor cell lysate is insufficient, due to the inadequate amount of TAAs, which is thought to be one reason of inefficiency for immunotherapy of cancers.29 Therefore, it is highly desirable to develop a strategy to enrich TAAs from tumor cells for the highly efficient cancer immunotherapy.30



We previously demonstrated that, with induction of autophagy and inhibition of lysosomal/proteosomal activity in tumor cells, a broad spectrum of defective ribosomal products (DRiPs) were sequestered into autophagosomes and secreted as DRiPs-containing blebs form known as DRibbles.21, 22, 31 We also documented that these DRibbles were efficient TAAs carriers for cross-presentation by dendritic cells (DCs) and could stimulate dramatic T-cell activation, leading to an anti-tumor efficacy in several tumor models such as melanomas, lung carcinomas,22 breast carcinomas27 and liver cancer.32, 33 More importantly, we found that the anti-tumor efficacy induced by DRibbles was mainly based on their content of ubiquitinated TAAs.23 Recent studies showed that ubiquitined proteins (UPs) can be enriched from tumor cells, after blocking their proteasomal degradation pathway, using Ni-NTA agarose beads conjugated with ubiquitin binding protein Vx3 protein.34-36 We found that UPs which physically mixed with α-Al2O3 nanoparticles have the potential as a potent cancer vaccine.36, 37 However, we found that those UPs were not highly immunogenic, in addition to the time consuming of this approach. Moreover, Ni ions of Ni-NTA beads are not environmentally-friendly.38 In this study, we applied a new design for efficient enrichment of UPs from tumor cell lysate using Vx3-covalently linked-α-Al2O3 (denoted as α-Al2O3-Vx3), where α-Al2O3 served as carrier and adjuvant simultaneously, and Vx3 functioned as a linker between α-Al2O3 and UPs. Compared with our previous work, the current design is expected to possesses the following advantages: (1) By using Vx3 covalently linked α-Al2O3 nanoparticles, UPs could be simply enriched from tumor cell lysate by


Page 4 of 30

Page 5 of 30 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


centrifugation;

(2)

Compared

with

Ni-NTA

beads,

α-Al2O3

is

cheap,

environmentally-friendly and sustainable; (3) Since the α-Al2O3 is the only licensed adjuvant in clinical applications,38-40 the α-Al2O3-Vx3-UPs could be used directly as a cancer vaccine without extracting UPs from α-Al2O3, and more importantly, the presence of α-Al2O3 (as adjuvant) can enhance an efficient anti-tumor immune response.

RESULTS AND DISCUSSION Modification of α-Al2O3. To synthesize α-Al2O3-Vx3, we firstly modified α-Al2O3 nanoparticles by 3-aminopropyl triethoxysilane (APTES).41-44 Then, Vx3 protein was covalently linked to α-Al2O3-APTES using glutaraldehyde45-47 as a linker (Figure 1a). Fourier transform infrared spectroscopy (FT-IR) was used to confirm the modification of α-Al2O3 nanoparticles by APTES and glutaraldehyde. We observed a broad absorption peak centered at about 3448 cm-1 and a narrow absorption peak centered at around 1632 cm-1, representing hydrogen-bonded OH stretching vibration and the scissoring vibration of adsorbed water, respectively48 (Figure 1b). Importantly, those two absorption peaks were significantly reduced after the reaction between α-Al2O3 and APTES. Moreover, a new absorption band was observed at 2932 cm-1 and could be ascribed to aliphatic γ (CH2) groups49 (Figure 1b). These data indicated that α-Al2O3 nanoparticles were successfully modified by APTES. α-Al2O3-APTES was then modified with glutaraldehyde, which was confirmed by a small absorption band centered at 1720 cm-1 and a stronger bands centered at 2860-2960 cm-1, referred to as γ(C=O) groups50 and aliphatic ν (CH2) groups,51 respectively.



Figure 1. Modification of α-Al2O3 nanoparticles. a) Scheme for the synthesis of α-Al2O3-Vx3 nanoparticles. b) FTIR spectra of α-Al2O3, α-Al2O3-APTES and α-Al2O3-CHO nanoparticles. c) EDS of α-Al2O3, α-Al2O3-APTES and α-Al2O3-CHO nanoparticles.

Energy dispersive X-Ray spectroscopy (EDS) was further used to confirm the modification of α-Al2O3-CHO nanoparticles.52, 53 Si, C and N elements were detected in α-Al2O3-APTES but not in α-Al2O3. Much more C elements were detected on α-Al2O3-CHO after the reaction with glutaraldehyde (Figure 1c and Supporting Information, Figure S1). After the successful functionalization and modification by APTES and glutaraldehyde, α-Al2O3 nanoparticles were ready to covalently link to


Page 6 of 30

Page 7 of 30 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


Vx3 protein. Synthesis and characterization of α-Al2O3-Vx3. Vx3 protein was then mixed with α-Al2O3-CHO at different ratios at 4 ºC overnight, and employed fluorescence microscopy to detect the covalent conjugation between Vx3 protein and α-Al2O3-CHO nanoparticles. Figure 2a showed that after centrifugation, α-Al2O3-Vx3 nanoparticles were separated from the supernatant and exhibited bright green color under excitation at 488 nm,54 by which the green color refers to the enhanced green fluorescent protein (eGFP) that fused with Vx3 protein. Besides, ultraviolet light (UV) images revealed that the fluorescence intensity increased gradually with increasing ratios of Vx3 to α-Al2O3-CHO in the reaction (Supporting Information, Figure S2). In contrast, the physical mixtures of Vx3 and α-Al2O3 nanoparticles which they were prepared under the same conditions showed almost no green fluorescence on the precipitation while the supernatant was still green (Supporting Information, Figure S3a), and fluorescence microscopy image of the precipitation displayed a faint green color under excitation at 488 nm (Supporting Information, Figure S3b). These data demonstrated that, Vx3 was successfully conjugated to α-Al2O3-CHO nanoparticles. Since the amount of conjugated Vx3 protein to α-Al2O3-CHO is critical for efficient enrichment of UPs from tumor cell lysate. Our results showed that, the optimal amount of Vx3 linked to α-Al2O3-CHO was observed at the ratio of 128 µg: 1 mg (Figure 2b). So that, 128 µg: 1 mg ratio of Vx3 protein to α-Al2O3-CHO was used as a preferable strategy to enrich UPs in the following experiments.



The long-term stability of covalently linked α-Al2O3-Vx3 was determined after storage for 1, 2 and 4 weeks at 4 °C. Fluorescence microscopy analysis showed that α-Al2O3-Vx3 exhibited a bright fluorescence even after 4 weeks (Figure 2c). In contrast, the faint green fluorescence of α-Al2O3/Vx3-UPs completely disappeared after 1 week (Supporting Information, Figure S3c). These data demonstrated that, the covalently linked α-Al2O3-Vx3 was much more stable than the physical mixtures of Vx3 and α-Al2O3.


Page 8 of 30

Page 9 of 30 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


Figure 2. Vx3 was conjugated to α-Al2O3-CHO. a) Fluorescence images of α-Al2O3-CHO nanoparticles conjugated with different amounts of Vx3. b) the relationship between the amount of Vx3 conjugated onto α-Al2O3-CHO and that of Vx3 added for the reaction with 1 mg α-Al2O3-CHO. c) Fluorescence images of α-Al2O3-Vx3 stored at 4°C for 1, 2, 4 weeks after synthesis.

Enrichment of UPs by α-Al2O3-Vx3. We then prepared lysate from 4T1 tumor cells in which proteasome function was inhibited by Bortezomib and Ammonium Chloride.36 Whole cell lysate was incubated overnight with α-Al2O3-Vx3 followed by centrifugation. Western blot analysis confirmed the enrichment of α-Al2O3-Vx3-UPs by which the amount of enriched UPs was directly proportional to the increasing ratio of 4T1 cell lysate to α-Al2O3-Vx3 until maximum UPs were captured (Figure 3a). The maximal level of enriched UPs (about 160µg) was observed when the mass ratio of cell lysate to α-Al2O3-Vx3 was 300 µg: 1 mg (Figure 3b). These results demonstrated that the covalently linked α-Al2O3-Vx3 was an efficient tool for the enrichment of UPs from tumor cells.

Figure 3. UPs were enriched by α-Al2O3-Vx3. a) Western blot analysis of UPs in



precipitate after the reaction of different amounts of cell lysate with α-Al2O3-Vx3 followed by centrifugation. b) The relationship between the amount of enriched protein and that of the cell lysate reacted with 1 mg α-Al2O3-Vx3. c) Western blot analyses of UPs in supernatant (S) and precipitate (P) after 1, 2, 4 weeks the first enrichment.

Given the importance of the long-term stability of cancer vaccines in clinical applications, we stored α-Al2O3-Vx3-UPs for 1, 2 and 4 weeks at 4 °C, followed by suspension in PBS and centrifugation to detect their stability by western blot analysis. There was an abundant level of UPs in precipitates samples even after 4 weeks of storage, in contrast to supernatants which had no detectable level of UPs (Figure 3c). This result indicated that α-Al2O3-Vx3-UPs nanoparticles which were prepared by specific binding possessed a long-term stability. Transmission electron microscopy (TEM) showed that the clean surface of the single-crystalline α-Al2O3 nanoparticles (Figure 4a) were coated with an amorphous layer after conjugation with UPs (Figure 4b), which further confirmed the successful conjugation of UPs to α-Al2O3-Vx3 nanoparticles.


Page 10 of 30

Page 11 of 30 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


Figure 4. TEM images of a) α-Al2O3 and b) α-Al2O3-Vx3-UPs.

The toxicity of α-Al2O3-Vx3-UPs. To evaluate whether α-Al2O3 was still safe after modification with APTES, BALB/c mice were subcutaneously vaccinated three times at 2 days interval with PBS, α-Al2O3-Vx3 (containing 100 µg Vx3) and α-Al2O3-Vx3-UPs (containing 100 µg UPs), and were sacrificed 30 days later. H&E stain was used to identify features of liver and kidney sections. Results showed that, there was no difference regarding the features of liver and kidney between three vaccinated groups (Supporting Information, Figure S4), indicating that α-Al2O3 had no cytotoxicity and still safe after modification with APTES. Cross-presentation of α-Al2O3-Vx3-UPs. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that are critical for induction of cytotoxic



lymphocytes (CTLs) responses. Antigen cross-presentation, the process through which exogenous antigens are presented on MHC class I molecules by DCs, is crucial for the generation of tumor specific CTLs. To test whether the covalently linked α-Al2O3-Vx3-UPs could be more effectively acquired by DCs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs), α-Al2O3-Vx3-UPs or α-Al2O3/Vx3-UPs were co-incubated with DC2.4 cells for indicated times, followed by evaluation the phagocytosis using flow cytometry. As shown in Figure 5a, the percentage of GFP+ DC2.4 cells incubated with α-Al2O3-Vx3-UPs was significantly higher than that of α-Al2O3/Vx3-UPs at different time points. These results indicated that α-Al2O3, covalently linked with Vx3-UPs proteins, can be more effectively phagocytized by DCs. Subsequently, a confocal microscope was used to determine the subcellular localization of internalized α-Al2O3-Vx3-UPs and α-Al2O3/Vx3-UPs. Results showed that α-Al2O3-Vx3-UPs, but not α-Al2O3/Vx3-UPs, were co-localized with the autophagosome marker, LC3 (Figure 5b). These results are consistent with previous study that antigen conjugated to α-Al2O3 nanoparticles can be delivered into the autophagosome of DCs.48 Next, UPs were enriched from the cell lysate of EG7 tumor cells (EL-4 tumor cells transduced with OVA) and conjugated with α-Al2O3-Vx3, which we named α-Al2O3-Vx3-UPs (OVA+). The antibody specific for the peptide and MHC class I molecule (H2Kb-SIINFEKL) complexes was used to evaluate the efficiency of cross-presentation of α-Al2O3-Vx3-UPs (OVA+) by DCs.48 Flow cytometry analysis showed that the DCs loaded with α-Al2O3-Vx3-UPs (OVA+) yielded a higher level of


Page 12 of 30

Page 13 of 30 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


H2Kb-SIINFEKL complexes than the DCs loaded with α-Al2O3/Vx3-UPs (OVA+) (Figure 5c). We further examined the ability of DCs loaded with α-Al2O3-Vx3-UPs (OVA+) or α-Al2O3/Vx3-UPs (OVA+) to stimulate naïve OVA- specific CD8+ T cells in vitro. DCs loaded with α-Al2O3-Vx3-UPs (OVA+) induced a stronger proliferation of OT-I T cells from naïve TCR transgenic mice, which recognize H2Kb-SIINFEKL complexes, than DCs loaded with α-Al2O3/Vx3-UPs (OVA+) (Figure. 5d). To further examine whether autophagy affects the cross-presentation of α-Al2O3-Vx3-UPs (OVA+), a phosphoinositide 3-kinase inhibitor: 3-methyladenine (3-MA) was used to inhibit the autophagy of DCs. This chemical inhibitor nearly abolished the

cross-presentation of α-Al2O3-Vx3-UPs (OVA+), but not α-Al2O3/Vx3-UPs (OVA+) by loaded DCs (Figure. 5d). These findings suggested that when loaded onto DCs,

the covalently linked α-Al2O3-Vx3-UPs, were superior in activating CD8+ T cells as compared to the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs), and the functional autophagy pathway is required for the efficient cross-presentation of α-Al2O3-Vx3-UPs (OVA+).



Figure 5. Cross-presentation of α-Al2O3-Vx3-UPs by DCs. a) Flow cytometry analysis of α-Al2O3-Vx3-UPs or α-Al2O3/Vx3-UPs phagocytosis by DCs. b) Confocal images of the localization of α-Al2O3/Vx3-Ups (top row) or α-Al2O3-Vx3-UPs (bottom row) within DCs after staining with an antibody against LC3 (red). c) Surface expression of major histocompatibility complex class I peptide complexes (H2Kb-SIINFEKL) on DCs after loading with α-Al2O3-Vx3-UPs or α-Al2O3/Vx3-Ups. d) Flow cytometry analysis showing that the cross-presentation of α-Al2O3-Vx3-UPs by DCs, but not α-Al2O3/Vx3-UPs, was blocked by 3-MA treatment.

Anti-tumor Immune response. To investigate whether α-Al2O3-Vx3-UPs could induce an anti-tumor immune response, BALB/c mice were subcutaneously


Page 14 of 30

Page 15 of 30 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


vaccinated three times at 2 days interval with different doses (0, 10, 30, 100 µg/mouse) of α-Al2O3-Vx3-UPs. On day 12, mice were sacrificed, and their splenocytes were collected and stimulated with inactivated 4T1 tumor cells for 48 hours. Splenocytes were also cultured without stimulation (culture medium, CM) as a negative control or with anti-CD3 antibody as a positive control. ELISA results of secreted interferon-γ (IFN-γ) by stimulated splenocytes revealed that, vaccination with α-Al2O3-Vx3-UPs induced a potent immune response against 4T1 tumor cells in a dose-dependent manner. Since 30 µg of UPs induced the highest level of IFN-γ production (Supporting Information, Figure S5a), it was used as a preferable dose in the next experiment. Moreover, we found that the ratio of inactivated 4T1 tumor cells to splenocytes plays an important role in inducing the production of IFN-γ by which 1:1 ratio enhanced the secretion of the highest level of IFN-γ (Supporting Information, Figure S5b). In order to investigate whether α-Al2O3-Vx3-UPs could induce a stronger anti-tumor immune response than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs, five groups (3 mice/group) of BALB/c mice were vaccinated with α-Al2O3-Vx3-UPs, PBS, α-Al2O3-Vx3, α-Al2O3/cell lysate, or α-Al2O3/Vx3-UPs. After re-stimulation with inactivated 4T1 tumor cells, splenocytes from α-Al2O3-Vx3-UPs vaccinated mice produced the highest level of IFN-γ compared to that from other four groups (Figure 6a). It is important to mention that, splenocytes from α-Al2O3/Vx3-UPs and α-Al2O3/cell lysate vaccinated mice also produced IFN-γ,36 but its level was significantly lower than that from splenocytes of α-Al2O3-Vx3-UPs vaccinated mice.



Together, these findings demonstrated that α-Al2O3-Vx3-UPs have the potential to induce anti-tumor immune response, supporting it as a tumor vaccine candidate. The antitumor efficacy of α-Al2O3-Vx3-UPs vaccination. The ability of α-Al2O3-Vx3-UPs to induce an efficient immune response prompted us to determine their therapeutic efficacy on established murine tumor model. BALB/c mice were subcutaneously inoculated with 1×106 4T1 tumor cells. When tumors were palpable, tumor-bearing mice were divided randomly into 6 groups (6 mice/group), 5 groups were received triple subcutaneously vaccination of α-Al2O3-Vx3-UPs (containing 30 µg UPs), α-Al2O3-Vx3, α-Al2O3/cell lysate (containing 30 µg total protein), α-Al2O3/Vx3-UPs (containing 30 µg total protein) or PBS into both flanks on day 3, 5 and 7 after the first injection of tumor cells; 1 group was received a caudal vein injection with Epirubicin (40 µg/mouse) on days 3, 7 and 11. On day 14, blood sera were collected from the vaccinated mice and the level of IFN-γ was monitored by ELISA. As shown in Figure 6b, the IFN-γ level in the serum of mice vaccinated with α-Al2O3-Vx3-UPs was higher than that in the serum of mice vaccinated with α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. Moreover, tumor growth curve showed that vaccination with α-Al2O3-Vx3-UPs significantly inhibited the tumor growth compared with the other five groups (Figure 6c and Supporting Information, Figure S6). Importantly, even the physical mixture of Vx3-UPs and α-Al2O3 also had the ability to inhibit tumor growth, but their potential was obviously less than α-Al2O3-Vx3-UPs. Besides, the inhibitory effect of


Page 16 of 30

Page 17 of 30 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


Epirubicin, chemotherapeutic agents commonly used in the treatment of breast cancer, on the tumor growth was remarkably less than that of α-Al2O3-Vx3-UPs. We also found that α-Al2O3-Vx3-UPs prolonged the median survival time of tumor bearing mice (49 days for α-Al2O3-Vx3-UPs vs. 40 days for α-Al2O3/Vx3-UPs, 32.5 days for α-Al2O3/cell lysate and 33.5 days for Epirubicin) (Figure 6d).

Figure 6. Anti-tumor immune response triggered by α-Al2O3-Vx3-UPs. a) The levels of produced IFN-γ by splenocytes of vaccinated mice after re-stimulation with inactivated 4T1 tumor cells. b) IFN-γ levels in the sera of tumor burden mice after vaccination. c) Tumor growth and d) the median survival days of tumor burden mice with different treatments.



CONCLUSION These findings provide clear evidence that the covalently linked α-Al2O3-Vx3 can be used to enrich UPs from tumor cell lysate with high efficiency and long-term stability. Furthermore, the covalently linked α-Al2O3-Vx3-UPs could be effectively phagocytized by DCs and induce efficient autophagy dependent cross-presentation. More importantly, results showed that α-Al2O3-Vx3-UPs have a potential to induce potent anti-tumor immune responses and anti-tumor efficacy in the established tumor model. This work presents a novel strategy to improve cancer immunotherapy and support further studies to test the efficacy of this vaccine in clinical trials.

EXPERIMENTAL SECTION Materials. Alumina (α-Al2O3) nanoparticles (30 nm) were purchased from Aladdin (Cat number: A119402). (3-Aminopropyl) triethoxysilane, aldehyde, deubiquitinating enzymes inhibitor (PR-619) were purchased from Sigma-Aldrich (St. Louis, MO). The anti-ubiquitin antibody was obtained from Cell Signaling Technology. Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Gibco. Fetal bovine serum (FBS) was obtained from Hyclone. Penicillin-Streptomycin Solution, BCA Protein Assay Kit were purchased from Beyotime Institute of Biotechnology, China. Bortezomib was obtained from Millennium pharmaceuticals, Cambridge, MA. RIPA Lysis Buffer was purchased from Millipore. Protease inhibitors, Phosphatase


Page 18 of 30

Page 19 of 30 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


inhibitors were obtained from Roche. The mouse IFN gamma ELISA Ready SET Go!® kit was purchased from eBioscience. Animals. 6-8 weeks old BALB/c female mice were purchased from the Comparative Medicine Center, Yangzhou University (Yangzhou, China) and maintained in a specific pathogen-free condition. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Southeast University. Vx3 protein expression and purification. Vx3 protein was purified according to our previous work.36 Briefly, His-Vx3-eGFP-expressing plasmid-transferred E.coli was grown in LB medium at 37°C. After that, at the logarithmic phase 0.1 M isopropy-β-D-thiogalactoside (IPTG) was added for 16 hours at 15°C. Then, cells were harvested and centrifuged by high speed centrifugation, which was further treated with lysozyme on ice followed by sonication and centrifugation. The as-obtained supernatant containing Vx3 was poured into a column containing Ni-NTA resin and incubated at 4 ºC over-night with stirring. After releasing the waste liquid the column was washed with washing buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 10 mmol/L imidazole) for 3 times, and the purified protein was collected by washing off the column with Elution buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, 250 mmol/L imidazole). Preparation of Vx3 functionalized α-Al2O3 Nanoparticles (α-Al2O3-Vx3). α-Al2O3 nanoparticles (20 mg) and (3-Aminopropyl) triethoxysilane (APTES) (66.45 mg) were added to anhydrous ethanol (1.3 mL) and stirred at room temperature for 12 hours. The APTES-functionalized α-Al2O3 (α-Al2O3-APTES) was obtained after



centrifugation and washing with anhydrous ethanol for 3 times. Then the α-Al2O3-APTES was further modified with glutaraldehyde. Glutaraldehyde (25%, 59µL) and deionized water (530 µL) were mixed together and added drop by drop to the as-obtained α-Al2O3-APTES and stirred at room temperature for another 2 hours. After washing with deionized water, the above product was further modified with purified Vx3 by stirring the mixture at 4 °C overnight. The Vx3-functionalized α-Al2O3 (α-Al2O3-Vx3) was finally obtained by centrifugation, and the amount of Vx3 modified on α-Al2O3 was evaluated by collecting and calculating the amount of Vx3 in the supernatant. The successful conjugation of Vx3 to the modified α-Al2O3 was confirmed by images under UV light and Laser confocal microscope. To examine the stability of α-Al2O3-Vx3, it was dispersed in PBS for 1, 2 and 4 weeks, Green fluorescence was detected by Laser confocal microscope. Characterization of modified α-Al2O3. Fourier transform infrared spectroscopy (FT-IR) was used to confirm the modification of α-Al2O3 nanoparticles by APTES and glutaraldehyde. After reaction the α-Al2O3, α-Al2O3-APTES, and α-Al2O3-CHO was detected by Fourier transmission infrared spectrometer (Thermo Fisher IS10). Enrichment of UPs from cancer cells by α-Al2O3-Vx3. 4T1 tumor cells were cultured in RPMI1640 supplemented with 10% heat inactivated Fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. To enrich UPs from tumor cells, Tumor cells were treated with Bortezomib (velcade, 200 nmol/L) and Ammonium Chloride (20 mmol/L) for 9 hours. The treated cells were collected and washed with PBS for 3 times, then the precipitated cells were re-suspended in RAPI buffer


Page 20 of 30

Page 21 of 30 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


supplemented with protease inhibitors, phosphatase inhibitors and PR-619. After incubated on ice for 20 minutes, the cell lysate was centrifuged at 15000 g for 30 minutes to remove cell debris, the supernatant was collected and the total proteins concentration was quantified by the bicinchoninic acid kit according to the manufacturer’s protocol. α-Al2O3-Vx3 was added to the supernatant, and the mixture was stirred overnight at 4 ºC. The covalently linked α-Al2O3-Vx3-UPs was obtained by centrifugation, the amount of UPs conjugated to α-Al2O3-Vx3 was calculated by proteins reduction in the cell lysate. Western blotting. α-Al2O3-Vx3-UPs were mixed with SDS-PAGE loading buffer, boiled for 5 minutes and centrifuged at 15000g for 10 minutes. The supernatant was resolved by 4% to 8% SDS-PAGE. Proteins were transferred to a PVDF membrane, blocked by 5% dry milk for 2 hours and incubated with anti-ubiquitin antibodies overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody was added for 1 h. Membrane was exposed using chemiluminescent

reagents

(Thermo

fisher). To

confirm

the Stability

of

α-Al2O3-Vx3-UPs western blot analysis was used, α-Al2O3-Vx3-UPs were firstly re-suspended in PBS for 1, 2 and 4 weeks. After centrifugation the supernatant and precipitation were re-suspended in SDS-PAGE loading buffer and western blot was performed as described above. Transmission electron microscopy (TEM). Samples were prepared in PBS at a concentration of 0.1 mg/ml, for TEM, a droplet of the sample was deposited on a carbon-coated 400-mesh Cu grids and dried at 40 °C. Samples were detected using a



Page 22 of 30

HITACHI JEM-2100 operating at 120 kV and photos were captured using Gatan Imaging system. Evaluation the toxicity of α-Al2O3-Vx3-UPs. BALB/c mice were subcutaneously vaccinated three times at 2 days interval with PBS, α-Al2O3-Vx3 (containing 100 µg Vx3) and α-Al2O3-Vx3-UPs (containing 100 µg UPs), 30 days later mice was sacrificed. Liver and kidney sections were collected for H&E staining. Detection the cross-presentation of α-Al2O3-Vx3-UPs and α-Al2O3/Vx3-UPs α-Al2O3-Vx3-UPs or α-Al2O3/Vx3-UPs were incubated with DC2.4 cells for 6 h. After washing, cells were fixed with 4% formaldehyde and permeabilized with 0.2% TritonX-100 in PBS for 15 min. Cells were stained with rabbit anti-LC3 antibody and Alexa fluor 568 labelled donkey anti-rabbit secondary antibody, images were captured by confocal microscope. The percentage of GFP+ DCs was evaluated by Flow cytometry after the incubation of α-Al2O3-Vx3-UPs or α-Al2O3/Vx3-UPs with DC2.4 cells for 6 h. UPs were enriched from OVA expressing tumor cells (EG7). The antibody specific for the peptide and major histocompatibility complex class I molecule (H-2Kb-SIINFEKL) complexes was used to evaluate the efficiency of cross-presentation of OVA by DCs. The ability of DCs loaded with α-Al2O3-Vx3-UPs (OVA+) or α-Al2O3/Vx3-UPs (OVA+) to stimulate naïve OVA-specific CD8+ T cells in vitro was examined. Naïve OVA-specific CD8+ T cells were collected from OT-1 transgenic

mice

and

labeled

with

CFSE.

α-Al2O3-Vx3-UPs

(OVA+)

or

α-Al2O3/Vx3-UPs (OVA+) were incubated with DCs without or with 3-MA treatment for 12 hours. After washing, CFSE labeled OT-1 CD8+ T cells were pulsed and


Page 23 of 30 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


co-cultured for 60 hours and the percentage of divided OT-I CD8+ T cells were determined by flow cytometry analysis Detection the anti-tumor immune responses. Mice were subcutaneously vaccinated with α-Al2O3-Vx3-UPs (containing 30 µg UPs), α-Al2O3-Vx3, α-Al2O3/cell lysate (containing 30 µg total protein), α-Al2O3/Vx3-UPs (containing 30 µg UPs) and PBS on day 1. The same vaccination schedule was performed on days 3 and 5 after the first injection. On day 12, mice were sacrificed and their splenocytes were cultured with inactivated 4T1 tumor cells for 48 hours. The immune response was evaluated by IFN-γ releasing using Mouse IFN gamma ELISA Ready SET Go!® kit according to the manufacture’s instruction. Detection the anti-tumor efficacy. To establish tumor-bearing mouse, 1×106 4T1 cells were subcutaneously injected into the right flank of BALB/c female mice. Subsequently, when tumors were palpable, mice were randomly divided into 6 groups and vaccinated with α-Al2O3-Vx3-UPs (containing 30 µg UPs), α-Al2O3-Vx3, α-Al2O3/cell lysate (containing 30 µg total protein), α-Al2O3/Vx3-UPs (containing 30 µg UPs) and PBS, respectively, into both flanks of the tumor-bearing mice on days 3, 5 and 7 after the first injection of tumor cells, 1 group received triple caudal vein injection with Epirubicin (40 μg/mouse) on days 3, 7 and 11. On day 14, blood sera were collected from the vaccinated mice to detect the level of IFN-γ by ELISA. Tumor growth was assessed by measuring the perpendicular diameters three times per week. Mice were sacrificed when the tumor diameter or tumor area reached a size larger than 15 mm and 200mm2, respectively.



Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China No. 31670918, 31370895 (to L.X.W.), No. 21675022, 21305065 (to Y.F.S.), the Fundamental Research Funds for the Central Universities, the Natural Science Foundation of Jiangsu Province (BK20170084） and The Research and Innovation Program for Doctoral Graduate Students in Jiangsu (KYLX15_0178). Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Conflict of interest The authors declare they have no conflict of interest. Corresponding Authors *E-mail: [email protected], *E-mail: [email protected]. Abbreviation UPs

ubiquitinated proteins

Vx3

ubiquitin binding protein

CM

culture medium

TAAs

tumor-associated antigens

CTLs

cytotoxic T lymphocytes

DRiPs

defective ribosomal products

α-Al2O3-Vx3

Vx3 covalently linked α-Al2O3

APTES

aminopropyl triethoxysilane

FT-IR

Fourier transform infrared spectroscopy


Page 24 of 30

Page 25 of 30 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


EDS

Energy dispersive X-Ray spectroscopy

eGFP

enhanced green fluorescent protein

UV

ultraviolet light

TEM

Transmission electron microscopy

α-Al2O3/Vx3-UPs

the mixture of α-Al2O3 and Vx3-UPs

α-Al2O3/cell lysate

the mixture of α-Al2O3 and cell lysate

α-Al2O3-Vx3-UPs

UPs conjugated with α-Al2O3-Vx3

α-Al2O3-Vx3

Vx3 conjugated to α-Al2O3

REFERENCES (1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

Vaillant, O., Cheikh, K. E., Warther, D., Brevet, D., Maynadier, M., Bouffard, E., Salgues, F., Jeanjean, A., Puche, P., Mazerolles, C., et al. (2015) Mannose-6-Phosphate Receptor: A Target for Theranostics of Prostate Cancer. Angew. Chem. Int. Ed. 54, 5952-5956. Wang, H., Xie, H., Wu, J., Wei, X., Zhou, L., Xu, X., and Zheng, S. (2014) Structure-Based Rational Design of Prodrugs To Enable Their Combination with Polymeric Nanoparticle Delivery Platforms for Enhanced Antitumor Efficacy. Angew. Chem. Int. Ed. 53, 11532-11537. Yoo, T. K., Chae, B. J., Kim, S. J., Lee, J., Yoon, T. I., Lee, S. J., Park, H. Y., Park, H. K., Eom, Y. H., Kim, H. S., et al. (2017) Identifying long-term survivors among metastatic breast cancer patients undergoing primary tumor surgery. Breast Cancer Res Treat 165, 109-118. Oh, S.-J., Ryu, C.-K., Baek, S.-Y., and Lee, H. (2011) Cellular Mechanism of Newly Synthesized Indoledione Derivative-induced Immunological Death of Tumor Cell. Immune Netw 11, 383-389. Sun, T., Zhang, Y. S., Pang, B., Hyun, D. C., Yang, M., and Xia, Y. (2014) Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angew. Chem. Int. Ed. 53, 12320-12364. Zhao, Y., Luo, Z., Li, M., Qu, Q., Ma, X., Yu, S.-H., and Zhao, Y. (2015) A Preloaded Amorphous Calcium Carbonate/Doxorubicin@Silica Nanoreactor for pH-Responsive Delivery of an Anticancer Drug. Angew. Chem. Int. Ed. 54, 919-922. Pushpavanam, K., Narayanan, E., Chang, J., Sapareto, S., and Rege, K. (2015) A Colorimetric Plasmonic Nanosensor for Dosimetry of Therapeutic Levels of Ionizing Radiation. ACS Nano 9, 11540-11550. Walker, A. J., Kim, H., Saber, H., Kluetz, P. G., Kim, G., and Pazdur, R. (2017)



(9) (10)

(11)

(12)

(13)

(14)

(15)

(16) (17)

(18)

(19) (20)

(21)

Clinical Development of Cancer Drugs in Combination With External Beam Radiation Therapy: US Food and Drug Administration Perspective. Int J Radiat Oncol Biol Phys 98, 5-7. Hutchinson, L. (2011) Targeted therapies: Radiopeptide therapy improves outcomes for neuroendocrine cancers. Nature reviews. Clinical oncology 8, 384. Jeong, J., Oh, J. H., Sonke, J. J., Belderbos, J., Bradley, J. D., Fontanella, A. N., Rao, S. S., and Deasy, J. O. (2017) Modeling the Cellular Response of Lung Cancer to Radiation Therapy for a Broad Range of Fractionation Schedules. Clin Cancer Res 23, 5469-5479. Mehdizadeh, A., Bonyadi, M., Darabi, M., Rahbarghazi, R., Montazersaheb, S., Velaei, K., Shaaker, M., and Somi, M. H. (2017) Common chemotherapeutic agents modulate fatty acid distribution in human hepatocellular carcinoma and colorectal cancer cells. Bioimpacts 7, 31-39. Chen, Y., Huang, Z., Zhao, H., Xu, J. F., Sun, Z., and Zhang, X. (2017) Supramolecular Chemotherapy: Cooperative Enhancement of Antitumor Activity by Combining Controlled Release of Oxaliplatin and Consuming of Spermine by Cucurbit[7]uril. ACS Appl Mater Interfaces 9, 8602-8608. Miwa, S., Nishida, H., Tanzawa, Y., Takeuchi, A., Hayashi, K., Yamamoto, N., Mizukoshi, E., Nakamoto, Y., Kaneko, S., and Tsuchiya, H. (2017) Phase 1/2 study of immunotherapy with dendritic cells pulsed with autologous tumor lysate in patients with refractory bone and soft tissue sarcoma. Cancer 123, 1576-1584. Steverding, D., and Rushworth, S. A. (2017) Front-line glioblastoma chemotherapeutic temozolomide is toxic to Trypanosoma brucei and potently enhances melarsoprol and eflornithine. Exp Parasitol 178, 45-50. Galaal, K., Naik, R., Bristow, R. E., Patel, A., Bryant, A., and Dickinson, H. O. (2010) Cytoreductive surgery plus chemotherapy versus chemotherapy alone for recurrent epithelial ovarian cancer. Cochrane Database Syst Rev, CD007822. Mellman, I., Coukos, G., and Dranoff, G. (2011) Cancer immunotherapy comes of age. Nature 480, 480-489. Lin, S.-Y., Yeh, T.-K., Kuo, C.-C., Song, J.-S., Cheng, M.-F., Liao, F.-Y., Chao, M.-W., Huang, H.-L., Chen, Y.-L., Yang, C.-Y., et al. (2016) Phenyl Benzenesulfonylhydrazides Exhibit Selective Indoleamine 2,3-Dioxygenase Inhibition with Potentin VivoPharmacodynamic Activity and Antitumor Efficacy. J. Med. Chem. 59, 419-430. Liu, Z., Ravindranathan, R., Kalinski, P., Guo, Z. S., and Bartlett, D. L. (2017) Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy. Nat Commun 8, 14754. Ott, P. A., Hodi, F. S., Kaufman, H. L., Wigginton, J. M., and Wolchok, J. D. (2017) Combination immunotherapy: a road map. J Immunother Cancer 5, 16. Saba, N. F., Mody, M. D., Tan, E. S., Gill, H. S., Rinaldo, A., Takes, R. P., Strojan, P., Hartl, D. M., Vermorken, J. B., Haigentz, M., Jr., et al. (2017) Toxicities of systemic agents in squamous cell carcinoma of the head and neck (SCCHN); A new perspective in the era of immunotherapy. Crit Rev Oncol Hematol 115, 50-58. Li, Y., Wang, L. X., Yang, G., Hao, F., Urba, W. J., and Hu, H. M. (2008) Efficient


Page 26 of 30

Page 27 of 30 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


(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29) (30)

(31)

(32)

(33)

(34) (35)

cross-presentation depends on autophagy in tumor cells. Cancer Res 68, 6889-6895. Li, Y., Wang, L. X., Pang, P., Cui, Z., Aung, S., Haley, D., Fox, B. A., Urba, W. J., and Hu, H. M. (2011) Tumor-derived autophagosome vaccine: mechanism of cross-presentation and therapeutic efficacy. Clin Cancer Res 17, 7047-7057. Twitty, C. G., Jensen, S. M., Hu, H. M., and Fox, B. A. (2011) Tumor-derived autophagosome vaccine: induction of cross-protective immune responses against short-lived proteins through a p62-dependent mechanism. Clin Cancer Res 17, 6467-6481. Alloatti, A., Kotsias, F., Pauwels, A. M., Carpier, J. M., Jouve, M., Timmerman, E., Pace, L., Vargas, P., Maurin, M., Gehrmann, U., et al. (2015) Toll-like Receptor 4 Engagement on Dendritic Cells Restrains Phago-Lysosome Fusion and Promotes Cross-Presentation of Antigens. Immunity 43, 1087-1100. Bernhard, C. A., Ried, C., Kochanek, S., and Brocker, T. (2015) CD169+ macrophages are sufficient for priming of CTLs with specificities left out by cross-priming dendritic cells. Proc Natl Acad Sci U S A 112, 5461-5466. Kitano, M., Yamazaki, C., Takumi, A., Ikeno, T., Hemmi, H., Takahashi, N., Shimizu, K., Fraser, S. E., Hoshino, K., Kaisho, T., et al. (2016) Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node. Proc Natl Acad Sci U S A 113, 1044-1049. Yu, G., Li, Y., Cui, Z., Morris, N. P., Weinberg, A. D., Fox, B. A., Urba, W. J., Wang, L., and Hu, H. M. (2016) Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression. Sci Rep 6, 37558-37571. Gruijl, T. D., van den Eertwegh, A. J., Pinedo, H. M., and Scheper, R. J. (2008) Whole-cell cancer vaccination: from autologous to allogeneic tumor- and dendritic cell-based vaccines. Cancer Immunol Immunother 57, 1569-1577. McGranahan, T., Li, G., and Nagpal, S. (2017) History and current state of immunotherapy in glioma and brain metastasis. Ther Adv Med Oncol 9, 347-368. Zhu, Z., Liu, Z., Liu, Y., Wang, C., Li, R., Liu, H., Gu, B., Li, G., and Zhang, S. (2017) Screening and identification of the tumor-associated antigen CK10, a novel potential liver cancer marker. FEBS Open Bio 7, 627-635. Xue, M., Fan, F., Ding, L., Liu, J., Su, S., Yin, P., Cao, M., Zhao, W., Hu, H. M., and Wang, L. (2014) An autophagosome-based therapeutic vaccine for HBV infection: a preclinical evaluation. J Transl Med 12, 361-372. Su, S., Zhou, H., Xue, M., Liu, J.-Y., Ding, L., Cao, M., Zhou, Z.-X., Hu, H.-M., and Wang, L.-X. (2013) Anti-tumor Efficacy of a Hepatocellular Carcinoma Vaccine Based on Dendritic Cells Combined with Tumor-derived Autophagosomes in Murine Models. ASIAN PAC J CANCER P 14, 3109-3116. Ren, H., Zhao, S., Li, W., Dong, H., Zhou, M., Cao, M., Hu, H. M., and Wang, L. X. (2014) Therapeutic antitumor efficacy of B cells loaded with tumor-derived autophagasomes vaccine (DRibbles). J Immunother 37, 383-393. Kirkin, V., and Dikic, I. (2011) Ubiquitin networks in cancer. Curr Opin Genet Dev 21, 21-28. Sims, J. J., Scavone, F., Cooper, E. M., Kane, L. A., Youle, R. J., Boeke, J. D., and



(36)

(37)

(38)

(39) (40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

Cohen, R. E. (2012) Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat Methods 9, 303-309. Aldarouish, M., Wang, H., Zhou, M., Hu, H. M., and Wang, L. X. (2015) Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine. J Exp Clin Cancer Res 34, 34-45. Yu, G., Moudgil, T., Cui, Z., Mou, Y., Wang, L., Fox, B. A., and Hu, H. M. (2017) Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation. J Immunother 40, 155-163. Zhang, S., Luo, H., Peng, M., Tian, Y., Hou, X., Jiang, X., and Zheng, C. (2015) Determination of Hg, Fe, Ni, and Co by Miniaturized Optical Emission Spectrometry Integrated with Flow Injection Photochemical Vapor Generation and Point Discharge. Anal. Chem. 87, 10712-10718. Guy, B. (2007) The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 5, 505-517. Flach, T. L., Ng, G., Hari, A., Desrosiers, M. D., Zhang, P., Ward, S. M., Seamone, M. E., Vilaysane, A., Mucsi, A. D., Fong, Y., et al. (2011) Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity. Nat Med 17, 479-487. Yang, Y. H., and Gao, M. Y. (2005) Preparation of fluorescent SiO2 particles with single CdTe nanocrystal cores by the reverse microemulsion method. Adv. Mater. 17, 2354-2357. Li, H., Sun, Y., Elseviers, J., Muyldermans, S., Liu, S., and Wan, Y. (2014) A nanobody-based electrochemiluminescent immunosensor for sensitive detection of human procalcitonin. Analyst 139, 3718-3721. Qiu, C., Xing, Y., Yang, W., Zhou, Z., Wang, Y., Liu, H., and Xu, W. (2015) Surface molecular imprinting on hybrid SiO2-coated CdTe nanocrystals for selective optosensing of bisphenol A and its optimal design. Appl. Surf. Sci. 345, 405-417. You, M., Yang, S., Tang, W., Zhang, F., and He, P.-G. (2017) Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO2@Au Nanocomposites. ACS Appl Mater Inter 9, 13855-13864. Budnyak, T. M., Yanovska, E. S., Kichkiruk, O. Y., Sternik, D., and Tertykh, V. A. (2016) Natural Minerals Coated by Biopolymer Chitosan: Synthesis, Physicochemical, and Adsorption Properties. Nanoscale Res Lett 11, 492-503. Chung, Y., Ahn, Y., Christwardana, M., Kim, H., and Kwon, Y. (2016) Development of a glucose oxidase-based biocatalyst adopting both physical entrapment and crosslinking, and its use in biofuel cells. Nanoscale 8, 9201-9210. Peng, Y. Y., Glattauer, V., and Ramshaw, J. A. M. (2017) Stabilisation of Collagen Sponges by Glutaraldehyde Vapour Crosslinking. Int J Biomater 2017, 8947823-8947828. Li, H., Li, Y., Jiao, J., and Hu, H. M. (2011) Alpha-alumina nanoparticles induce efficient autophagy-dependent cross-presentation and potent antitumour response. Nat Nanotechnol 6, 645-650. Liu, W., Wei, F., Ye, A., Tian, M., and Han, J. (2017) Kinetic stability and membrane structure of liposomes during in vitro infant intestinal digestion: Effect of cholesterol


Page 28 of 30

Page 29 of 30 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


(50)

(51)

(52) (53)

(54)

and lactoferrin. Food Chem. 230, 6-13. Kucuk Baloglu, F., Baloglu, O., Heise, S., Brockmann, G., and Severcan, F. (2017) Triglyceride dependent differentiation of obesity in adipose tissues by FTIR spectroscopy coupled with chemometrics. J Biophotonics 10, 1345-1355. Rak, S., De Zan, T., Stefulj, J., Kosović, M., Gamulin, O., and Osmak, M. (2014) FTIR spectroscopy reveals lipid droplets in drug resistant laryngeal carcinoma cells through detection of increased ester vibrational bands intensity. The Analyst 139, 3407-3415. Kostko, O., Xu, B., Jacobs, M. I., and Ahmed, M. (2017) Soft X-ray spectroscopy of nanoparticles by velocity map imaging. J Chem Phys 147, 0139311-0139318. Shao, Y., Guo, Z., Li, H., Su, Y., and Wang, X. (2017) Atomic Layer Deposition of Iron Sulfide and Its Application as a Catalyst in the Hydrogenation of Azobenzenes. Angew Chem Int Ed Engl 56, 3226-3231. Cerezo, M. I., Linden, M., and Agusti, S. (2017) Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces. Mar Pollut Bull 118, 64-70.



Table of Contents graphic


Page 30 of 30

Vx3 Functionalized Alumina Nanoparticles Assisted Enrichment of

Recommend Documents