www.acsnano.org
In Situ Manipulation of Dendritic Cells by an Autophagy-Regulative Nanoactivator Enables Effective Cancer Immunotherapy Downloaded via UNIV OF SOUTHERN INDIANA on July 17, 2019 at 09:16:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Yi Wang,†,‡,∇ Yao-Xin Lin,†,§,∇ Jie Wang,†,‡ Sheng-Lin Qiao,†,‡ Yu-Ying Liu,∥ Wen-Qian Dong,∥ Junqing Wang,§ Hong-Wei An,†,⊥ Chao Yang,† Muhetaerjiang Mamuti,†,‡ Lei Wang,† Bo Huang,∥ and Hao Wang*,†,‡ †
CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, P.R. China ‡ Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P.R. China § Center for Nanomedicine and Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States ∥ National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, P.R. China ⊥ Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100149, P.R. China S Supporting Information *
ABSTRACT: Cellular immunotherapeutics aim to employ immune cells as anticancer agents. Ex vivo engineering of dendritic cells (DCs), the initial role of an immune response, benefits tumor elimination by boosting specific antitumor responses. However, directly activating DCs in vivo is less efficient and therefore quite challenging. Here, we designed a nanoactivator that manufactures DCs through autophagy upregulating in vivo directly, which lead to a high-efficiency antigen presention of DCs and antigen-specific T cells generation. The nanoactivator significantly enhances tumor antigen cross-presentation and subsequent T cell priming. Consequently, in vivo experiments show that the nanoactivators successfully reduce tumor growth and prolong murine survival. Taken together, these results indicate in situ DCs manipulation by autophagy induction is a promising strategy for antigen presentation enhancement and tumor elimination. KEYWORDS: nanoparticles, autophagy, dendritic cell, antigen presentation, cancer immunotherapy
C
early role of DCs in the immune response process revealed that targeting DCs could provide a promising tool for eliminating tumors by boosting the specific antitumor response. Additionally, the combination of DC vaccination and chemotherapeutic drugs has been tested in phase II trials in prostate cancer, lung cancer, colorectal cancer, and so on (NCT00345293, NCT00103116, NCT00103142). Generally, widespread use of DCs in immunotherapy in the clinic has initiated a specific immune response in cancer therapy.9 However, the longlasting and potent immune responses from this approach were
ancer immunotherapy aims to increase the capacity and specificity of patients’ natural immune systems to attack cancer. Given its natural capacity to elimilate exgenous components, it has received a great deal of attention in basic biomedical research communities,1−3 and techniques to enhance specific immune responses have subsequently been widely exploited. For instance, program death-1/program death-ligand 1(PD-1/PD-L1) has been developed to block immune checkpoint,4,5 and chimeric antigen receptor T cell immunotherapy (CAR-T) has been used to sensitize and activate T cells.6 Provenge, a dendritic cell (DC)-based cancer vaccine, was approved by the FDA in 2010 for prostate cancer treatment.7,8 DCs act as the antigen presentation cells and perform continuous surveillance in the immune system. The © XXXX American Chemical Society
Received: January 7, 2019 Accepted: July 1, 2019 Published: July 1, 2019 A
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
Cite This: ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 1. Schematic and characterization of self-assembled nanoactivators. (A) Schematic of nanoactivtors inducing autophagy for crosspresentation and priming T cells. (B) Representative TEM images of NP-B-OVA nanoactivators in PBS buffer solution (scale bar, 100 nm); inset: enlarged TEM images of NP-B-OVA nanoparticles (scale bar, 20 nm). (C) Hydrodynamic size and ζ potential of NP-B-OVA nanoparticles in PBS buffer (n = 3). (D) Titration curve of NP-B-OVA with 0.1 M HCl; inset: swollen NP-B-OVA nanoactivator at pH 5.5 (scale bar, 200 nm).
limited in vivo due to the magnitude of the T cells activation.10 Thus, stimulation and activation of antigen-specific immune responses in vivo are of critical importance and have proven to be useful as a supplement to other therapeutic approaches. Autophagy as a conserved metabolic process occurs in the cell in response to stress. It is important for cell survival due to its “self-digestion” property that cells undergo autophagy to clear some non-essential organelles or degrade proteins to maintain homeostasis.11 Explicitly, autophagy is the process of substance degradation within the cell. Recent reports have revealed potential relationships between autophagy and immune responses.12 The autophagy pathway plays a fundamental role in multiple immune responses, contributing to antigen presentation and specific T cell activation for tumor elimination.13 Extracellular antigens are captured and trafficked to cytoplasm, where lysosomes become involved in digestion and the autophagy process.14 Therefore, rational manipulation of the autophagy process provides a synergistic and effective method to harness the potential of the immune system. This method could be applied as a powerful tool for translationcapable cancer immunotherapy. Nanomaterials that have specific physicochemical properties show excellent efficacies in autophagy modulation,15,16 antigen delivery,17−19 and immunotherapy.20−23 Thus, rationally designed nanomaterials for application in immune modulating strategies could provide multifunctional and spatiotemporal control over immune cells. Ultimately, this could improve
anticancer activities by creating better safety profiles. Herein, we have developed a strategy to design and synthesize selfassembled nanoparticles (named nanoactivator) with autophagy-inducing ability for boosting DC associated immune responses and leading to enhanced antigen cross-presentation. Using this strategy allowed bone-marrow-derived dendritic cells (BMDCs) to mature without complex engineering and operation, including in vitro culture, stimulation, and subsequent transfer to patients. Instead, administrating nanoactivators and manufacturing DCs in vivo directly lead to highefficiency antigen presention of DCs and antigen-specific T cells generation. DCs can phagocytose nanoparticles within 1 h, thereby inducing autophagy helpful to antigen processing. Antigens are then digested and exported to the cell surface within a minimum of an additional 2 h. Efficient antigen presentation of BMDCs treated with nanoactivators increases significantly, in comparison to those cells treated with nanoparticles that are unable to induce autophagy. These nanoactivators exhibit satisfactory therapeutic efficacy in mice, which demonstrates an autophagy-assisted strategy for DCbased immunotherapy.
RESULT AND DISCUSSION Synthesis and Characterization of Self-Assembled Nanoactivators. The nanoactivators were constructed using a poly(β-amino ester) polymer with covalently conjugated B
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 2. Induction of autophagy by nanoactivators. (A) Bio-TEM images of DC2.4 cells with indicated incubations for 12 h. N, nuclei. Yellow arrows, double-membrane vesicles (autophagosomes). Scale bar, 2 μm. White box, region of interests, 3-MA, 5 mM. RAPA, 50 nM, pretreated with autophagy regulator for 0.5 h. (B) Confocal images of GFP-LC3 transgenic DC2.4 cell 12 h post-indicated stimuli. Scale bar, 5 μm. (C) Vesicles quantification of DC2.4 cells 12 h post-indicated treatments from TEM images of DC2.4 cells 12 h post-indicated treatments. Error bars, mean ± SD, n = 10. *p < 0.05, **p < 0.01, one-way ANOVA for indicated comparison.
Furthermore, ζ potential measurements showed that all of the nanoparticle surfaces were negatively charged (Figure 1C). Both the nanoparticles’ hydrodynamic diameter and ζ potential were further examined in cell culture medium utilizing the DLS measurements (Table S1). The little change of the nanoparticles’ size properties indicated their stability in a more physiologically relevant environment. Additionally, the titration curve demonstrated the pH-responsive property of NP-BOVA, which showed a buffer region at a pH around 5.5 (Figure 1D). The buffering capacity increased the possibility of endosomal escape and participation in the intracellular processes. More importantly, we observed the swollen morphology of NP-B-OVA in pH 5.5 acidic conditions, which could benefit functional peptide exposure and delivery (Figure 1D, inset). Induction of Autophagy by Nanoactivator. The cellular cytotoxicity of NP-B-OVA to dendritic cells DC2.4 and tumor cells B16-F10-OVA27 were first determined by CCK-8 assay. As shown in Figure S4a,b, a high dose of NP (>200 μg/mL) showed a little cytotoxicity to the DC2.4 cells because of a high dose of pH-sensitive nanoparticles, which would induce excessive lysosomal pressures and finally cause autophagic cell death as we previously studied.28 Thus, we chose a polymer concentration at 70 μg/mL that with 5 μM Bec1 and 2.5 μg/mL OVA. At this concentration, NP-B-OVA could induce autophagy and activate an immune response17,24 without significant cytotoxicity toward DC2.4 cells and B16F10-OVA tumor cells (Figure S4c,d). To identify the ability of autophagy induction by NP-B-OVA, biological transmission electron microscopy (bio-TEM) was used as the gold-standard method to monitor the intracellular autophagic structures of DC2.4 cells that were cultured in complete medium (C), NP-
peptides on both terminals of the backbone, which entered DCs and induced autopahgy, autophagy process facilitate antigen presentation, and subsequent T cells activation (Figure 1A). The polymers were modularly synthesized using a Michael addition reaction with a hydrophobic monomer (HDDA), a pH-responsive monomer (DBPA), and a hydrophilic amino-terminated polyethylene glycol (PEG-NH2) as previously reported.15 The autophagy-inducing peptide beclin1 (NH2-CGTNVFNATFHIWHSGQFGT-COOH, referred to as B)24 and antigen peptide OVA257−264 (NH2-CSIINFEKLCOOH, referred to as OVA) derived from ovalbumin25 were efficiently conjugated to the backbone of the polymer using thio-end click chemistry via a one-pot and facile synthesis method.26 The polymer conjugated with scrambled beclin1 (NH2-CKIFGSLAFL-COOH, referred as B(S)) and OVA was constructed as a control that is unable to induce autophagy. The chemical structure of the copolymer-peptide conjugates was identified by 1H NMR (Figures S1−S3, peptide information see data files S1−S6). Generally, the copolymerpeptide conjugation composite were constituted of equally equivalent functional peptides on average. The 1H NMR analysis revealed that the degree of polymerization (DP) of the polymer was about 40, and the molecular weight (MW) was 25,000 Da, respectively (Figure S3). The copolymers assemble into nanoparticles during dialysis becuase of their amphiphilic structures. Therefore, copolymers P, P-B(S)-OVA and P-BOVA were formed into NP, NP-B(S)-OVA, and NP-B-OVA, respectively. We observed their morphology and hydrodynamic diameter using both dynamic light scattering (DLS) and transmission electron microscopy (TEM). The hydrodynamic size of NP-B-OVA was ∼41 nm, which corresponded to the measurement diameters in TEM images (Figure 1B). C
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 3. Nanoactivator-mediated autophagic flux detection. (A) Confocal images of DC2.4 cells after incubated with NP-B-OVA-Cy5 for 0.5 h and stained with LysoTracker Yellow (upper panel), GFP-LC3 transgenic DC2.4 cell 12 h post NP-B-OVA-Cy5 incubation (middle panel), DC2.4 cells 12 h post NP-B-OVA-Cy5 incubation, and stained with endogenous p62 (lower panel). White arrow, merged signals. Scale bar, 5 μm, n = 3. (B) Western blot for LC3B II and p62 from DC2.4 cells post 2 h or 12 h with indicated treatment. (C) Co-localization organelles ER and Golgi complex with NP-B-OVA-Cy5 after incubation for 1.5 h (upper panel) and 3 h (lower panel); white dashed boxes indicate the co-localization signals; blue, ER-tracker; green, Golgi complex; red, NP-B-OVA nanoparticles, n = 3. (D) Confocal images of DC2.4 cells 2 h post NP-B-OVA-Cy5 incubation and stained with indicated organelles probes. Scale bar, 5 μm, n = 3.
B(S)-OVA, and NP-B-OVA for 12 h, respectively. Bio-TEM images showed an increase of small vesicles (autophagosome, yellow arrow) in the NP-B-OVA-treated cells (Figure 2A). Meanwhile, we measured the expression of GFP-LC3 by laser scanning confocal microscopy (LSCM). LC3 is an autophagy marker that undergoes post-translation modification during autophagy.29 DC2.4 cells were transfected with a GFP-LC3 plasmid at first, followed by treatment with NP-B-OVA for 12 h. The results demonstrated that GFP-LC3 dots in DC2.4 cells treated with NP-B-OVA increased significantly, which corresponded to the TEM observation results (Figure 2B). By quantitatively analyzing vesicles in DC2.4 cells, there is an approximately 2.5 times increase in comparison to the nonautophagic NP-B(S)-OVA group. Using 3-methyladenine (3MA, autophagy inhibitor) to block autophagy would significantly decrease the number of vesicles by NP-B-OVA
induction. On the contrary, when cells were co-treated with NP-B(S)-OVA and rapamycin (RAPA, autophagy inducer), the number of vesicles was increased, more than two times that of NP-B(S)-OVA treated cells (Figure 2C). Fluoresence intensity analysis of GFP-LC3 dots indicated a similar result as Bio-TEM images. NP-B-OVA treated cells presented a significant increase of GFP-LC3 fluoresence, and the fluorescence decreased upon pretreatment with 3-MA (Figure S5). Collectively, these results indicated that NP-B-OVA enables autophagy induction in DCs. Nanoactivator-Mediated Crosstalk between Autophagy and Antigen Presentation. To understand the detailed autophagic processes induced by NP-B-OVA nanoactivators, we adopted LSCM to monitor the co-localization of NP-BOVA with autophagy structures. NP-B-OVA was labeled with Cy5 (NP-B-OVA-Cy5), and the nanoactivator uptake process D
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 4. Nanoactivators enhance antigen cross-presentation by inducing autophagy. (A) Flow cytometry analysis of the OVA and MHC I complexes (Kb-SIINFEKL) expression in BMDC with different nanoparticles treatment for 12 h by PE-H-2Kb antibody. (B) The mean value analysis of Kb-SIINFEKL. Error bars, mean ± SEM, n = 3. **p < 0.01, one-way ANOVA for indicated comparison. (C) Cytokines IL-2 and IL-10 production by BMDC 12 h post-indicated treatments. Error bars, mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA for indicated comparison. (D) Expression of co-stimulatory molecules CD86 and (E) CD80 in BMDC, n = 3. (F) Schematic illustration of T cell proliferation assay. (G) Proliferation ratios and indices of each generation of CD8+ T cells isolated from a OT-I mouse during 12 h of incubation time with BMDC which was pretreated with different nanoparticles.
by DC2.4 cells was observed at first. Sucrose, β-cyclodextrin, and amiloride were adopted for clathrin-dependent endocytosis, caveolae-dependent endocytosis, and macropinocytosis inhibition, respectively. Low-temperature (4 °C) treatment was adopted as a negative control due to its inhibition of the energy-dependent uptake process. The results displayed a reduced fluorescence of NP-B-OVA-Cy5 when DCs were treated with sucrose, β-cyclodextrin, or cultured at 4 °C, which implied that the vast majority of nanoactivators entered cells through clathrin- and caveolae-dependent endocytosis rather than macropinocytosis (Figure S6a). Next, DC2.4 cells were stained with LysoTracker Yellow after 0.5 h incubation with NP-B-OVA-Cy5. As shown in Figure 3A, merged signals from
NP-B-OVA-Cy5 and LysoTracker Yellow were detected, indicating NP-B-OVA entered cells via endocytosis and might escape from lysosomes. This lysosomal-dependent process might assist in nanoparticle disassembly and Bec1 exposure due to the pH-responsive property of grafted copolymers,30 which benefited further autophagy induction. Subsequently, we observed co-localization of NP-B-OVA-Cy5 with autophagosome markers (GFP-LC3), providing further evidence that NP-B-OVA induced autophagy by contributing to autophagosome formation. NP-B-OVA-Cy5 was also colocalized with p62, a cargo recognition molecule found in the autophagic downstream pathway.31,32 Additionally, DC2.4 cells were transfected with a GFP-mCherry-LC3 plasmid, allowing E
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
probes. After incubating with NP-B-OVA-Cy5 for 2 h, we observed merged signals of NP-B-OVA-Cy5, autolysosome, and ER (Figure 3D), which demonstrated the possibility of autolysosome fusion with the ER for antigen delivery. Nanoactivators Enhance Antigen Cross-Presentation by Inducing Autophagy. We next sought to demonstrate the correlation between increased autophagy and antigen cross-presentation enhancement. Specific MHC I complexes, a combination of MHC I molecules loaded with antigen peptides, generated from BMDCs and expressed on the cell surface, were mesured to evaluate the level of antigen presentation. We used the H-2Kb antibody which is specific to the OVA antigen peptide and MHC I complex (KbSIINFEKL) to evaluate the efficacy of antigen crosspresentation induced by nanoactivator. As shown in Figure 4A, DCs that were cultured with NP-B-OVA yielded higher levels of the Kb-SIINFEKL complex than those treated with NP-B(S)-OVA. Interestingly, it showed the highest levels of Kb-SIINFEKL complex when the cells were co-cultured with NP-B(S)-OVA and RAPA. In contrast, Kb-SIINFEKL complex expression decreased significantly upon treatment with NP-BOVA and 3-MA. BMDCs with ATG5 appear to be knockdown at first but then, through nanoactivator stimulation, exhibted a reversed Kb-SIINFEKL complex expression compared to NP-B-OVA treatment. These results demonstrated that the increased Kb-SIINFEKL complex expression might relate to autophagy upregulation, and it was decreased once autophagy was blocked by inhibitors or siATG5 (Figure 4B). Apart from DC surface marker evaluation, we measured secretion of cytokines to verify DC maturation. We measured cytokines IL-2 and IL-10,17 which are representative helper cytokines in Th1- and Th2-mediated immune responses, respectively. The supernatant of BMDCs with these treatments was collected for ELISA analysis, and the results showed that NP-B-OVA treated DCs had a noticeable increase in IL-2 secretion with a concomitant decrease in IL-10. However, NPB(S)-OVA showed lower levels of IL-2 and higher levels of IL10 (Figure 4C). Taken together, these results confirmed that although RAPA as an immunosuppressant inhibits T cell proliferation and applied to transplant rejection,34 it was able to induce autophagy in DCs and enhanced cellular antigen processing, thereby also further promoting antigen presentation and Th1 cytokines secretion. The results of MHC I complex expression and cytokine secretion revealed that the nanoparticle-mediated induction of autophagy benefited DC antigen presentation and CD8+ T cell response. We further evaluated the T cells activation efficacy presented by BMDC after being pretreated with NP-B-OVA nanoactivators. Co-stimulatory molecules are crucial for an immune response and serve to assist T cell recognition of specific MHC I complexes.35 Given this, we first evaluated the respective cross-presentation efficiencies of co-stimulatory molecules on BMDCs. No significant difference was observed among these groups (Figure 4D,E), indicating that the nanoparticles enhanced antigen presentation without significantly impacting co-stimulatory molecules. The splenic T cells isolated from OT-I mice would express T cell receptors that could recognize the Kb-SIINFEKL complex.36 BMDCs were prestimulated by nanoparticles for 12 h and then co-cultured with T cells that isolated from OT-I mice for another 72 h. These T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) at first in order to monitor cell proliferation (Figures 4F and S10).37 Analysis results, including the ratio of each
us to visually observe the autophagy process induced by the NP-B-OVA nanoactivator (Figure S6b). These results demonstrated that NP-B-OVA nanoactivators might be endocytosed by DCs and escape from lysosomes, which would be beneficial for functional peptide exposure and initiate subsequent processes. To validate the NP-B-OVA-mediated autophagy flux, we next detected the expression changes of LC3B II and p62 at different time points by Western blot. Results presented an increase in expressions of LC3B II and p62 after treated with NP-B-OVA at 2 h. However, LC3B II and p62 expressions were decreased upon addition of 3-MA or knock-down molecules in an autophagy process by siATG5 (Figure 3B). Interestingly, we observed a slight increase in p62 expression in combination treatment with NP-B-OVA and chloroquine (CQ, autophagy inhibitor which leads to inhibition of both fusion of autophagosome with lysosome). Additionally, LC3B II and p62 expressions were observed in 12 h. As shown in Figures 3B and S7, an increase of LC3B II was observed in NP-B-OVA, but p62 expression was significanly decreased compared to those additionally treated with CQ. Nevertheless, another autophagy inhibitor bafilomycin A1 (Baf A1) exhibited similar inhibition to CQ during the NP-B-OVA nanoactivatormediated autophagy flux (Figure S8). Those p62 expression changes suggested that NP-B-OVA might induce complete autophagic process, but more proteins were synthesized than those that had been digested in the early time. Lysosome impairment assays were conducted to exclude the possibility that the nanoactivator facilitated lysosome escape process had influenced this organelle digestion function. LysoSensor Green DND189 is an agent able to detect acidic organelles like lysosome in a pH dependent way. We found that the fluorescence intensity almost had no change of NP-BOVA treatment group in comparison to the control group that was treated with completed medium (Figure S9a). In addition, cathepsin activity assays have been performed, and the relative fluorescence of NP-B-OVA treated cells was comparable to those cells with normal culture, indicating no change of cathepsin activity (Figure S9b). These results showed that there was almost no damage to lysosomes and they retained the ability to digest molecules in cytoplasm. Collectively, all of these results indicated that NP-B-OVA induced autophagy and the autophagic flux was finally accomplished, which might assist subsequent antigen presentation. To confirm autophagy-mediated antigen loading and presentation, we tracked the intracellular transportation of antigens according to the classical MHC I pathway.33 The endoplasmic reticulum (ER) and Golgi complex were labeled with specific probes after 1.5 h incubation of NP-B-OVA-Cy5. Here, we observed merged signals from NP-B-OVA-Cy5 and the ER sensors. An additional 1.5 h later, merged signals of NP-B-OVA-Cy5 and the Golgi complex were increased (Figure 3C). These results demonstrated that the NP-BOVA could induce antigen degradation over a short time. The antigens were delivered to the ER as soon as 1.5 h and subsequently transported to the cell surface, which might have been mediated by the Golgi complex through carring the newly synthesized MHC I complex after ER translocation. For a better understanding of the interaction between autophagy and antigen presentation, we used LSCM to observe the colocalization of NP-B-OVA-Cy5 with autolysosomes and ER. Autolysosomes were tracked with specific p62 immunofluorescence staining, and the ER was labeled with ER tracker F
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 5. In vivo antitumor efficacy of nanoactivator. (A) Tumor volume changes of B16-F10-OVA bearing mice with indicated treatments, n = 6***p < 0.001; one-way ANOVA for indicated comparison. (B) Percentages of cross-presentation DCs in lymph node 7 d after indicated treatments. (C) Percentages of antigen-specific CD8+ T cells isolated from the spleen 7 d after indicated treatments. (D) Percentage of CD8+ T cells of tumor after treatment. Upper panel: Subcutaneous injection; lower panel: intravenous injection. (E) Quantitative analysis of tumor-infiltrated CD8+ T cells with subcutaneous injection or intravenous injection, n = 3. (F) Tumor volume changes of TC-1 bearing C57/BL6 mice with indicated treatments. ***p < 0.001; one-way ANOVA for comparison, n = 6. (G) Survival curves of TC-1 bearing mice with indicated treatments, Kaplan−Meier survival analysis, n = 6. (H) Illustration of superior therapeutic efficacy of NP-B-E7 nanoactivator compared to FCA adjuvant and E7 peptide formulation.
and intravenous injection would have a greater benefit to its general distribution to other tissues. Tumor therapy experiments were carried out on wild-type C57BL/6 mice which were injected with B16-F10-OVA cells subcutaneously on the right limb. After 7 days, the tumor-bearing mice were subcutaneously or intravenously injected with normal saline (C), NP, NP-B(S)-OVA, and NP-B-OVA while the tumors were palpable. We monitored tumor volume and body weight to assess the therapeutic effects of each given treatment. There is no significant difference in weight change among these mice with indicated treatement (Figure S12). NP-B-OVA with subcutaneous and intravenous injections both showed satisfactory therapeutic effects and markedly reduced tumor growth compared to other groups (Figure 5A). To validate that the tumor immunotherapeutic effects resulted from enhanced antigen presentation and T cells activation by nanoactivators, lymph nodes were isolated for detecting of autophagy induction in vivo in DCs, and immunofluorescence images of lymph nodes indicated effective
generation and the proliferation index, revealed that there was a 1.5-fold increase in the proliferation index of CD8+ T cells stimulated by NP-B-OVA in comparison to the NP-B(S)-OVA group (Figure 4G). These results further demonstrated the enhanced antigen presentation of the NP-B-OVA nanoactivator and demonstrated the ability of nanoactivators for subsequent T cell generation. These results also collectively demonstrated NP-B-OVA enhanced antigen presentation of BMDCs in addition to antigen-specific T cell activation via autophagy. Prominent in Vivo Antitumor Activities of Nanoactivators. Due to the promising results of BMDC cross presentation with the NP-B-OVA nanoactivator treatment in vitro, we further explored their immunotherapeutic effects in vivo using B16-F10-OVA bearing mice. The sequential biodistribution of nanoparticles was monitored by optical imaging ex vivo (Figure S11).38 The results indicated that subcutaneous injection to the murine footpad might be associated with NP-B-OVA-mediated lymph node draining, G
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
tumor regression. However, the moderate efficiency of antigen presentation limits widespread in vivo T cell activation, which hinders the development of DC-based immunotherapy for use in clinical trials. Moreover, luxuriant DC-based methods for cancer immunotherapy lack controlled, comparative, and parallel studies in DC acquisition. Although the success of certain approaches has demonstrated the availability and practicality of DC-based therapies,13 the expertise and expense required for the cell-based therapy through complex DC culturing and engineering have restricted its use as a viable immunotherapy. Thus, our strategy meets the requirements of DCs-based immunotherapy by up-regulating autophagy, which is an essential process during antigen cross-presentation. In our strategy, antigen cross-presentation is enhanced while increasing intracellular autophagy. Antigen presentation efficacy is highly increased and stimulated T cells. Specifically, exogenous antigens were phagocytized, and ingested antigens enter an endosome-lysosomes-dependent pathway. Ingested antigens were then delivered into the cytoplasm as a cytosolic antigen through ER-mediated transportation. Lastly, those antigens were returned to the ER, loaded into newly synthesized MHC I molecules and exported to the cell surface to induce subsequent T cell activation and proliferation. Effective processing of antigens and presenting them to T cells is essential in initiating an specific antitumor immune response. We proposed the strategy could be extended with abundant biomedical materials and supplemented with other functions based on the enhanced antigen cross-presentation pathway. To achieve the best results with cancer immunotherapy, combining it with other techniques or drugs might be more helpful. For example, combining with interleukins would promote the activity of T cells or immune checkpoint blockage and inhibit negative immuno-regulative molecules. Upregulating autophagy for in vivo DC-based cancer immunotherapy simplified the procedure of DC activation and further benefited antigen-specific T cell proliferation.We also look to precise control of the dosage of autophagy-inducing peptide and antigens peptide to fully understand and optimize the therapeutic efficacy of cancer treatment, and in combination with others treatment methods.
induction of autophagy by the NP-B-OVA nanoactivator treatment, compared to those mice treated with NP-B(S)-OVA nanoparticles (Figure S13). Percentages of cross-presentation DC and antigen-specific T cells were isolated for quantitative analysis. The percentages of cross-presentation DC cells were increased ∼50% compared to NP-B(S)-OVA groups (Figure 5B). Meanwhile, antigen-specific CD8+ T cells had increased 2-fold in those mice treated with NP-B-OVA compared to those treated with NP-B(S)-OVA nanoparticles (Figure 5C). The frequencies of tumor-infiltrated cytotoxic CD8+ T cells of mice with variable treatments were enumerated. We observed a 2-fold increase of CD8+ T cells in NP-B-OVA treated mice when compared to those treated with NP-B(S)-OVA (Figure 5D,E). Immunofluorescence staining verified that increased and dispersed CD8+ T cells were distributed in the solid tumor (Figure S14a). Pathology was observed using hematoxylineosin (H&E) staining, which represented the pathological mophology of tumors and the inhibiting effects of NP-B-OVA on B16-F10-OVA tumors (Figure S14b). Collectively, the enhanced antigen presentation boosted subsequent immune responses such as T cell proliferation, thereby promoting immune responses that benefited tumor regression. Acute toxicities of the nanoparticles were assessed through hematology analysis of mice. H&E staining was used to identify the pathological features of major organs (Figures S15). We analyzed the alanine transaminase (ATL), aspartate transaminase (AST), alkaline phosphatase (ALP), and blood urea nitrogen (BUN) of mice (Table S2), and all of these indexes are normal in comparison with reference range. All of the results implied no significant acute toxicity at the experimental dosage of the nanoparticles after treated with a nanoactivator. To further demonstrate applications of nanoactivators in anticancer therapeutics, we synthesized another nanoactivator (NP-B-E7 nanoactivator) with HPV16 E749−57 peptide (NH2CRAHYNIVTF-COOH, referred to as E7) antigen peptide modification (Figure S16). A TC-1 tumor bearing mouse model was established to evaluate the therapeutic effects of the E7-nanoactivator. Tumor volume of mice and their survival time were monitored. Additionally, mice immunized with Freund’s complete adjuvant (FCA) and E7 showed minor inhibition of tumor development. However, the NP-B-E7 nanoactivator significantly inhibited tumor growth compared to FCA and E7 formulation (p < 0.05) (Figure 5F). The survival time of mice treated with NP-B-E7 nanoactivators was significantly prolonged compared to those mice treated with FCA and E7 peptide (p = 0.035). Nevertheless, NP-B-E7 nanoactivators exhibited complete therapeutic effects in two mice as their xenografted tumors disappeared (Figure 5G). The results illustrated the effectiveness of nanoactivators and their superiority compared to adjuvants in TC-1 cancer therapy (Figure 5H).
MATERIALS AND METHODS Copolymers Synthesis. The copolymers were constructed with hydrophobic monomer 1,6-hexanediol diacrylate (HDDA), pHsensitive monomer 3-(dibutylamino)-1-propylamine (DBPA), and PEG-NH2 (amino-terminated polyethylene glycol, MW = 2 kDa) in a molar ration of 1.2:0.9:0.1. They were dissolved in DMSO (2 mL) and then aerated with N2 with constant stirring (15 min). After that, the mixture was heated to 50 °C under stirring for 7 days in a light-protected space. The solution contained product was dialyzed (MWCO: 2000 Da) against water for 12 h. The dialyzed solution was lyophilized, and a pale-yellow solid was finally obtained, which was the copolymers. For the synthesis of peptide-conjugated copolymers, peptide and copolymers were added in a molar ratio of 2:1 into 2 mL of DMSO. After aeration of the mixture with N2 for 15 min, it then reacted for 3 days at room temperature before it was dialyzed against deionized water (MWCO: 3500 Da). The copolymers assembled into copolymer nanoparticles during the dialyzing process. The chemical structures of the peptides, copolymers, and copolymer-peptide conjugations were identified by NMR measurements. Bone-Marrow Derived Dendritic Cell Isolation. Bone-marrowderived dendritic cells were isolated according to the Lutz et al. reported method.39 C57/BL6 mice were sacrificed, and the legs were separated. We flushed out the bone marrow into centrifuge tubes with
CONCLUSION In general, techniques boosting specific immune responses for cancer therapy have attracted significant attention as a means of tumor elimination. To this end, DCs have been widely investigated because of their central role in immune regulation. For example, they can phagocytose exogenous antigens and initiate the specific adaptive immune response by priming T cells. This activation yields effector cytotoxic T cells through a cross-presentation pathway. As such, DCs arising from the patient’s own immune system could be harnessed for effective H
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano iced RPMI-1640 by syringe, then performed lysis on the red blood cells and centrifuged them 1−2 times at 1500 rpm (5 min). We then resuspended the cell pellet in RPMI-1640 and counted the cells, diluted the cells into RPMI-1640 (10 mL) with GM-CSF (20 ng/ mL), and plated them in Petri dishes at a density of 2 × 105 cells per plate. We then added an additional RPMI-1640 (10 mL) with GMCSF (20 ng/mL) at day 3 and removed half of the media (10 mL) at day 6. Cells were harvested on day 8. In Vivo Antitumor Activity. The B16-F10-OVA cells were injected subcutaneously into the right lateral hind hip of 4-week old female C57/BL6 mice (1 × 106 B16-F10-OVA cells). Mice were divided into four groups (n = 6) in random 7 days post-tumor injection and were treated via NS, NP, NP-B(S)-OVA, NP-B-OVA (100 μL, ∼3.25 mg/kg for NP, ∼3.5 mg/kg NP-B(S)-OVA and NPB-OVA) through tail vain injection. Drugs were intravenously or subcutaneously administered to mice every other day. For subcutaneous injection, drugs were injected via footpads (50 μL, ∼3.25 mg/kg for NP, ∼3.5 mg/kg NP-B(S)-OVA and NP-B-OVA). Tumor size and weight of mice were recorded during the process. Mice were sacrificed when tumors reached approximately one-tenth of the mice weight. Statistical Analysis. Results were presented as mean ± SEM or mean ± SD. Replicates were adopted as indicated. ANOVA was carried out for various group comparison, and multiple comparison was carried out by Turkey’s post-test when the results presented significance. Survival analysis was executed with a log-rank test.
FACS technique support. This work was supported by the National Natural Science Foundation of China (21374026, 51573032), the National Science Fund for Distinguished Young Scholars (51725302), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), CAS Key Research Program for Frontier Sciences (QYZDJ-SSW-SLH022), Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541), CAS Interdisciplinary Innovation Team, and K.C. Wong Education Foundation.
REFERENCES (1) Gu, L.; Mooney, D. J. Biomaterials and Emerging Anticancer Therapeutics: Engineering the Microenvironment. Nat. Rev. Cancer 2016, 16, 56−66. (2) Couzin-Frankel, J. Cancer Immunotherapy. Science 2013, 342, 1432−1433. (3) Wang, Y.; Lin, Y. X.; Qiao, S. L.; An, H. W.; Ma, Y.; Qiao, Z. Y.; Rajapaksha, R. P.; Wang, H. Polymeric Nanoparticles Promote Macrophage Reversal from M2 to M1 Phenotypes in the Tumor Microenvironment. Biomaterials 2017, 112, 153−163. (4) Wang, C.; Wang, J.; Zhang, X.; Yu, S.; Wen, D.; Hu, Q.; Ye, Y.; Bomba, H.; Hu, X.; Liu, Z.; Dotti, G.; Gu, Z. In Situ Formed Reactive Oxygen Species−Responsive Scaffold with Gemcitabine and Checkpoint Inhibitor for Combination Therapy. Sci. Transl. Med. 2018, 10, No. eaan3682. (5) Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.; Leming, P. D.; Spigel, D. R.; Antonia, S. J.; Horn, L.; Drake, C. G.; Pardoll, D. M.; Chen, L.; Sharfman, W. H.; Anders, R. A.; Taube, J. M.; et al. Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. N. Engl. J. Med. 2012, 366, 2443−2454. (6) Porter, D. L.; Levine, B. L.; Kalos, M.; Bagg, A.; June, C. H. Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia. N. Engl. J. Med. 2011, 365, 725−733. (7) Burch, P. A.; Croghan, G. A.; Gastineau, D. A.; Jones, L. A.; Kaur, J. S.; Kylstra, J. W.; Richardson, R. L.; Valone, F. H.; VukPavlovic, S. Immunotherapy (Apc8015, Provenge®) Targeting Prostatic Acid Phosphatase Can Induce Durable Remission of Metastatic Androgen-Independent Prostate Cancer: A Phase 2 Trial. Prostate 2004, 60, 197−204. (8) Cheever, M. A.; Higano, C. S. Provenge (Sipuleucel-T) in Prostate Cancer: The First FDA-Approved Therapeutic Cancer Vaccine. Clin. Cancer Res. 2011, 17, 3520−3526. (9) Kandalaft, L. E.; Powell, D. J., Jr.; Chiang, C. L.; Tanyi, J.; Kim, S.; Bosch, M.; Montone, K.; Mick, R.; Levine, B. L.; Torigian, D. A.; June, C. H.; Coukos, G. Autologous Lysate-Pulsed Dendritic Cell Vaccination Followed by Adoptive Transfer of Vaccine-Primed Ex Vivo Co-Stimulated T Cells in Recurrent Ovarian Cancer. Oncoimmunology 2013, 2, e22664. (10) Sabado, R. L.; Balan, S.; Bhardwaj, N. Dendritic Cell-Based Immunotherapy. Cell Res. 2017, 27, 74−95. (11) Rubinsztein, D. C.; Codogno, P.; Levine, B. Autophagy Modulation as a Potential Therapeutic Target for Diverse Diseases. Nat. Rev. Drug Discovery 2012, 11, 709−730. (12) Shibutani, S. T.; Saitoh, T.; Nowag, H.; Munz, C.; Yoshimori, T. Autophagy and Autophagy-Related Proteins in the Immune System. Nat. Immunol. 2015, 16, 1014−1024. (13) Deretic, V.; Saitoh, T.; Akira, S. Autophagy in Infection, Inflammation and Immunity. Nat. Rev. Immunol. 2013, 13, 722−737. (14) Patterson, N. L.; Mintern, J. D. Intersection of Autophagy with Pathways of Antigen Presentation. Protein Cell 2012, 3, 911−920. (15) Wang, Y.; Lin, Y. X.; Qiao, Z. Y.; An, H. W.; Qiao, S. L.; Wang, L.; Rajapaksha, R. P. Y. J.; Wang, H. Self-Assembled AutophagyInducing Polymeric Nanoparticles for Breast Cancer Interference In Vivo. Adv. Mater. 2015, 27, 2627−2634.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b00143. Data file S1: The MS-ESI spectra of B peptide (TIF) Data file S2: The HPLC spectra of B peptide (TIF) Data file S3: The MS-ESI spectra of OVA peptide (TIF) Data file S4: The HPLC spectra of OVA peptide (TIF) Data file S5: The MS-ESI spectra of B(S) peptide (TIF) Data file S6: The HPLC spectra of B(S) peptide (TIF) Materials, methods of transfection, RNA extraction and laser scanning confocal microscopy imaging, characterization of peptide and copolymers, and toxicity assessment of nanoactivators in vitro and in vivo (PDF)
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Yi Wang: 0000-0003-3077-2899 Sheng-Lin Qiao: 0000-0001-5006-5257 Hao Wang: 0000-0002-1961-0787 Author Contributions ∇
Y.W. and Y.-X.L. contributed equally. Y.W., Y.-X.L., and H.W. conceived the project and wrote the manuscript. Y.W., Y.-X.L., and H.W. planned and designed the experiments. Y.W., Y.X.L., J.W., J.-Q.W., and C.Y. performed the experiments. S.L.Q., H.-W.A., M.M., and L.W. provided advice for both the project and data discussion. Y.-Y.L., W.-Q.D., and B.H. provided assistance with the mouse model. All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS The authors are grateful to Pengcheng Jiao (Center of Biomedical Analysis, Tsinghua University) for assistance in I
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano (16) Lin, Y.-X.; Qiao, S.-L.; Wang, Y.; Zhang, R.-X.; An, H.-W.; Ma, Y.; Rajapaksha, R. P. Y. J.; Qiao, Z.-Y.; Wang, L.; Wang, H. An In Situ Intracellular Self-Assembly Strategy for Quantitatively and Temporally Monitoring Autophagy. ACS Nano 2017, 11, 1826−1839. (17) Li, H.; Li, Y.; Jiao, J.; Hu, H. M. Alpha-Alumina Nanoparticles Induce Efficient Autophagy-Dependent Cross-Presentation and Potent Antitumour Response. Nat. Nanotechnol. 2011, 6, 645−650. (18) Fang, R. H.; Zhang, L. Nanoparticle-Based Modulation of the Immune System. Annu. Rev. Chem. Biomol. Eng. 2016, 7, 305−326. (19) Yang, R.; Xu, J.; Xu, L.; Sun, X.; Chen, Q.; Zhao, Y.; Peng, R.; Liu, Z. Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose Modification for Effective Anticancer Vaccination. ACS Nano 2018, 12, 5121−5129. (20) Meng, C.; Zhi, X.; Li, C.; Li, C.; Chen, Z.; Qiu, X.; Ding, C.; Ma, L.; Lu, H.; Chen, D.; Liu, G.; Cui, D. Graphene Oxides Decorated with Carnosine as an Adjuvant to Modulate Innate Immune and Improve Adaptive Immunity In Vivo. ACS Nano 2016, 10, 2203−2213. (21) Chen, Q.; Wang, C.; Zhang, X.; Chen, G.; Hu, Q.; Li, H.; Wang, J.; Wen, D.; Zhang, Y.; Lu, Y.; Yang, G.; Jiang, C.; Wang, J.; Dotti, G.; Gu, Z. In Situ Sprayed Bioresponsive Immunotherapeutic Gel for Post-Surgical Cancer Treatment. Nat. Nanotechnol. 2019, 14, 89−97. (22) Wang, C.; Ye, Y.; Hu, Q.; Bellotti, A.; Gu, Z. Tailoring Biomaterials for Cancer Immunotherapy: Emerging Trends and Future Outlook. Adv. Mater. 2017, 29, 1606036. (23) Chao, Y.; Xu, L.; Liang, C.; Feng, L.; Xu, J.; Dong, Z.; Tian, L.; Yi, X.; Yang, K.; Liu, Z. Combined Local Immunostimulatory Radioisotope Therapy and Systemic Immune Checkpoint Blockade Imparts Potent Antitumour Responses. Nat. Biomed. Eng. 2018, 2, 611−621. (24) Shoji-Kawata, S.; Sumpter, R.; Leveno, M.; Campbell, G. R.; Zou, Z.; Kinch, L.; Wilkins, A. D.; Sun, Q.; Pallauf, K.; MacDuff, D.; Huerta, C.; Virgin, H. W.; Helms, J. B.; Eerland, R.; Tooze, S. A.; Xavier, R.; Lenschow, D. J.; Yamamoto, A.; King, D.; Lichtarge, O.; et al. Identification of a Candidate Therapeutic Autophagy-Inducing Peptide. Nature 2013, 494, 201−206. (25) Rotzschke, O.; Falk, K.; Stevanovic, S.; Jung, G.; Walden, P.; Rammensee, H. G. Exact Prediction of a Natural T-Cell Epitope. Eur. J. Immunol. 1991, 21, 2891−2894. (26) Qiao, Z. Y.; Lin, Y. X.; Lai, W. J.; Hou, C. Y.; Wang, Y.; Qiao, S. L.; Zhang, D.; Fang, Q. J.; Wang, H. A General Strategy for Facile Synthesis and In Situ Screening of Self-Assembled Polymer-Peptide Nanomaterials. Adv. Mater. 2016, 28, 1859−1867. (27) Yang, J.; Sanderson, N. S. R.; Wawrowsky, K.; Puntel, M.; Castro, M. G.; Lowenstein, P. R. Kupfer-Type Immunological Synapse Characteristics Do Not Predict Anti-Brain Tumor Cytolytic T-Cell Function In Vivo. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4716−4721. (28) Lin, Y.-X.; Wang, Y.; Qiao, S.-L.; An, H.-W.; Zhang, R.-X.; Qiao, Z.-Y.; Rajapaksha, R. P. Y. J.; Wang, L.; Wang, H. pH-Sensitive Polymeric Nanoparticles Modulate Autophagic Effect via Lysosome Impairment. Small 2016, 12, 2921−2931. (29) Mizushima, N.; Yoshimori, T.; Levine, B. Methods in Mammalian Autophagy Research. Cell 2010, 140, 313−326. (30) Qiao, Z.-Y.; Hou, C.-Y.; Zhang, D.; Liu, Y.; Lin, Y.-X.; An, H.W.; Li, X.-J.; Wang, H. Self-Assembly of Cytotoxic Peptide Conjugated Poly(Β-Amino Ester)S for Synergistic Cancer Chemotherapy. J. Mater. Chem. B 2015, 3, 2943−2953. (31) Kirkin, V.; McEwan, D. G.; Novak, I.; Dikic, I. A Role for Ubiquitin in Selective Autophagy. Mol. Cell 2009, 34, 259−269. (32) Porgador, A.; Yewdell, J. W.; Deng, Y.; Bennink, J. R.; Germain, R. N. Localization, Quantitation, and In Situ Detection of Specific Peptide−MHC Class I Complexes Using a Monoclonal Antibody. Immunity 1997, 6, 715−726. (33) Neefjes, J.; Jongsma, M. L. M.; Paul, P.; Bakke, O. Towards a Systems Understanding of MHC Class I and MHC Class II Antigen Presentation. Nat. Rev. Immunol. 2011, 11, 823−836.
(34) Mukherjee, S.; Mukherjee, U. A Comprehensive Review of Immunosuppression Used for Liver Transplantation. J. Transplant. 2009, 2009, 701464. (35) Banchereau, J.; Steinman, R. M. Dendritic Cells and the Control of Immunity. Nature 1998, 392, 245−252. (36) Didierlaurent, A. M.; Collignon, C.; Bourguignon, P.; Wouters, S.; Fierens, K.; Fochesato, M.; Dendouga, N.; Langlet, C.; Malissen, B.; Lambrecht, B. N.; Garcon, N.; Van Mechelen, M.; Morel, S. Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01 Depends on Activated Dendritic Cells. J. Immunol. 2014, 193, 1920−1930. (37) Quah, B. J.; Warren, H. S.; Parish, C. R. Monitoring Lymphocyte Proliferation In Vitro and In Vivo with the Intracellular Fluorescent Dye Carboxyfluorescein Diacetate Succinimidyl Ester. Nat. Protoc. 2007, 2, 2049−2056. (38) De Jong, W. H.; Hagens, W. I.; Krystek, P.; Burger, M. C.; Sips, A. J. A. M.; Geertsma, R. E. Particle Size-Dependent Organ Distribution of Gold Nanoparticles after Intravenous Administration. Biomaterials 2008, 29, 1912−1919. (39) Lutz, M. B.; Kukutsch, N.; Ogilvie, A. L. J.; Rossner, S.; Koch, F.; Romani, N.; Schuler, G. An Advanced Culture Method for Generating Large Quantities of Highly Pure Dendritic Cells from Mouse Bone Marrow. J. Immunol. Methods 1999, 223, 77−92.
J
DOI: 10.1021/acsnano.9b00143 ACS Nano XXXX, XXX, XXX−XXX
