Cell-Membrane Immunotherapy Based on Natural Killer Cell

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Cell-Membranes Immunotherapy Based on Natural Killer Cell-Membranes-Coated Nanoparticles for Effective Inhibition of Primary and Abscopal Tumor Growth Guanjun Deng, Zhihong Sun, Sanpeng Li, Xinghua Peng, Wenjun Li, Lihua Zhou, Yifan Ma, Ping Gong, and Lintao Cai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05292 • Publication Date (Web): 16 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Cell-Membranes Immunotherapy Based on Natural Killer Cell-Membranes-Coated Nanoparticles for Effective Inhibition of Primary and Abscopal Tumor Growth

Guanjun Deng, †, ‡, § Zhihong Sun, †, § Sanpeng Li, †, ‡ Xinghua Peng, †, ‡ Wenjun Li, † Lihua Zhou, † Yifan Ma, † Ping Gong, *, † and Lintao Cai *, †

† Guangdong

Key Laboratory of Nanomedicine, Shenzhen Engineering Laboratory of Nanomedicine

and Nanoformulations, CAS Key Lab for Health Informatics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. ‡ University §

of Chinese Academy of Sciences, Beijing 100049, China.

These authors contributed equally to this work.

* Correspondence authors: Ping Gong (email: [email protected]) and Lintao Cai (email: [email protected]).

Abstract: Developing effective immunotherapies with low toxicity and high tumor specificity is the ultimate goal in the battle against cancer. Here, we reported a cell-membranes immunotherapy strategy that was able to eliminate primary tumors and inhibited distant tumors by using NK cell-membranes-cloaked photosensitizer TCPP-loaded nanoparticles (NK-NPs). Proteomic profiling of NK cell-membranes was performed through shotgun proteomics, and we found that NK cell-membranes enabled the NK-NPs to target tumors and could induce or enhance pro-inflammatory

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M1-macrophages polarization to produce antitumor immunity. The TCPP loaded in NK-NPs could induce cancer cells death through photodynamic therapy and consequently enhanced the antitumor immunity efficiency of the NK cell-membranes. The results confirmed that NK-NPs selectively accumulated in the tumor and were able to eliminate primary tumor growth and produce an abscopal effect to inhibit distant tumors. This cell-membranes immunotherapeutic approach offers a strategy of tumor immunotherapy.

Keywords: cell-membranes immunotherapy, NK cell-membranes, M1-macrophages, photodynamic therapy, antitumor immunity.

Immunotherapy following surgery, chemotherapy and radiotherapy has become standard in cancer therapy. Cancer immunotherapy was the largest scientific breakthrough in 2013.1-3 Multiple therapeutic strategies have been developed to treat cancer. Some of these approaches such as cytokine immunotherapy, blocking immunotherapy, chimeric antigen receptor T-cell therapy (CAR-T) and adoptive immunotherapy of T cells have achieved exciting outcomes in preclinical and clinical trials.4-8 However, these therapeutic strategies are not suitable for all cancers and have some limitations in clinical application due to tumor complexity, patient heterogeneity and the systemic immunotoxicity of these methods.9-11 Therefore, the development of effective, durable and tumor-specific immune responses without systemic toxicity remains a challenge. Natural killer (NK) cells are a type of innate immune cell and the first line of defense against


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infection and cancer.12 As potent effector cells, NK cells have attracted considerable attention in tumor immunotherapy research for several reasons.13 First, unlike T lymphocytes, NK cells can eliminate target cells spontaneously without previous antigen-specific stimulation and restriction of the major histocompatibility complex.14 Second, NK cells regulate the immune response by secreting a variety of cytokines such as tumor necrosis factor (TNF-).15-17 Third, NK cells promote the maturation of antigen-presenting cells (APCs) and thus activate T cells to kill tumor cells.18 More specifically, in antitumor immunotherapy, NK cells can induce pro-inflammatory M1-macrophages polarization and target tumor cells via proteins (e.g., RANKL or DNAM-1) present in the NK cellmembranes.19-27 Therefore, NK cell-membranes can elicit M1-macrophages polarization to generate cell-membranes immunotherapy and provide a cell-membranes immune inducer for stimulating the immune system during tumor immunotherapy. Photodynamic therapy (PDT) is a minimal-invasive cancer treatment. On the one hand, PDT kills tumor cells directly by utilizing photosensitizers to generate reactive oxygen species (ROS) under light irradiation,28,

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but on the other hand, the dying tumor cells can generate

damage-associated molecular patterns (DAMPs) (such as CRT exposure, HMGB1 release and secreted ATP) by PDT-induced immunogenic cell death (ICD), which activate antigen-presenting cells (APCs) to stimulate the production and proliferation of tumor-specific effector T cells.30-32 However, because of the lack of inducers that can elicit the immune response, the tumor immune response induced by PDT alone is insufficient, as it fails to provide significant clinical benefit and lacks durable immunity.33 Thus, to promote a strong, systemic antitumor immune response, PDT is usually combined with immune inducers during antitumor therapy. In this study, we found that NK cell-membranes could elicit tumor-specific immune responses


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by targeting cancer cells and inducing M1 macrophages polarization. Accordingly, we designed NK cell-membranes-decorated nanoparticles (NK-NPs) to improve the efficacy of NK cell-membranes immunotherapy and ultimately achieve desired treatment efficacy at the animal level (Scheme 1). On the one hand, proteomic profiling of NK cell-membranes was performed through shotgun proteomics and we found that it enabled the NK-NPs to target tumors and could induce or enhance the polarization of M1 macrophages to generate antitumor immune response. On the other hand, the 4,4′,4′′,4′′′-(porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (TCPP) loaded in NK-NPs not only directly eradicated primary tumor cells through photodynamic therapy (PDT), but also could trigger dying tumor cells to generate damage-associated molecular patterns by PDT-induced immunogenic cell death (ICD) for activating antigen-presenting cells (APCs) and consequently enhanced the antitumor immunity efficiency of the NK cell-membranes. Our results demonstrated that NK-NPs selectively accumulated in the tumor after intravenous injection of NK-NPs. Furthermore, NK-NPs-mediated PDT could enhance the NK cell-membranes immunotherapy that was able to eradicate primary tumor growth and produce an abscopal effect to inhibit distant untreated tumors. This study is hoped to facilitate the development of NK cell-membranes immunotherapy for cancer.

Results Preparation and characterization of the NK-NPs The NK-NPs were prepared by assembling TCPP-loaded mPEG-PLGA polymeric nanoparticles (T-NPs), isolating natural killer cell-membranes (NKCMs) from human NK-92 and then cloaking the T-NPs with the NKCMs by extrusion (Scheme 1). Transmission electron microscopy (TEM) images showed that the NK-NPs exhibited a typical core-shell structure and were spherical in shape with


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good monodispersity and an average particle size of 80 ± 1.5 nm (Figures 1a and S1). Dynamic light scattering (DLS) analysis showed that the average hydrodynamic diameter of the NK-NPs was 85 ± 1.2 nm with a polydispersity index (PDI) of 0.105, which is slightly larger than that of the parental T-NPs (Figures 1b and S2). The surface charge of the NK-NPs (-11.8 ± 0.8 mV) was similar to that of the parental T-NPs. The ultraviolet-visible absorption and fluorescence spectra of the NK-NPs and T-NPs showed absorption and emission peaks at 418 and 650 nm, respectively, for both NPs, which was consistent with the absorption and emission peaks of TCPP (Figures 1c and 1e). The protein profiles of the NKCMs and NK-NPs were determined by SDS-PAGE electrophoresis. The protein composition of the NKCMs was mostly retained in the NK-NPs, but no protein signal was detected in the T-NPs (Figure 1d), suggesting that the T-NPs were successfully coated with the NKCMs. The hydrodynamic size distribution of the NK-NPs was nearly constant over 20 days, and the particle stability of the NK-NPs was higher than that of T-NPs, indicating that the NKCMs enhanced the nanoparticle stability (Figure S3). As shown in Figure 1f, TCPP leakage from the NK-NPs was reduced compared to that from the T-NPs, suggesting that the NKCMs improved the biocompatibility and safety of the NPs. As determined by the DCFH-DA assay, ROS generation by the NK-NPs upon irradiation with light at 660 nm was similar to that of free TCPP at the same concentration (Figure 1g). Characterization of the NK cell-membranes proteins The biomimetic properties of NK-NPs depended mainly on the surface proteins in NKCMs, which had various functions, such as tumor targeting, cancer immunosurveillance, etc. So it was very important to analyze the type and quantity of proteins in NK-NPs. Proteomic profiling of the human NKCMs coating on the NK-NPs resulted in the identification of 868 distinct proteins (Figure 2a,


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Supplementary Table 1 and 2) with low-molecular-weight proteins (10 - 60 kDa) representing 66.71% via shotgun proteomics method. The majority of the proteins (61%) were associated with the natural killer cell plasma membrane (Figure 2b). The NKCMs proteins were classified as integral and lipid-anchored (23%), cytoskeletal and/or junctional (38%), peripheral (29%), and membrane vesicular or secreted proteins (10%) (Figure 2c). The presence of proteins from other cellular compartments (such as ribosomes and mitochondria) was attributed to the dynamic trafficking of proteins between internal organelles and the cell surface. Classification of the proteins according to function identified proteins involved in response stimulus (24%), transport (20%), signaling (11%), immunity (11%), developmental processes (11%), cell adhesion (9%) and lipid metabolism (9%) (Figure 2d). NKCMs proteins critical for inducing pro-inflammatory M1-macrophages polarization such as immunity-related GTPase family M protein (IRGM1), cannabinoid receptor 1 (CB1), galectin-12, ras-related protein Rab10 (RAB 10) and RANKL were identified in the NK-NPs through shotgun proteomics and flow cytometry (Figure 2e). 23-27 These proteins were further detected in the NK-NPs surface by western blotting (Figure S4). Tumor-targeting proteins such as DNAM-1 and NKG2D were also identified on the surface of the NK-NPs. Thus, the NK cell-membranes coating on the NK-NPs can target tumors and elicit pro-inflammatory M1-macrophages polarization to generate cell-membranes immune responses. Targeting efficacy of the NK-NPs It has been reported that NK cells recognize tumors via interactions between tumor cell-expressed ligands and NK cell-membranes receptors such as NKG2D and DNAX accessory molecule-1 (DNAM-1).34 To determine the potential mechanisms responsible for human NK-NPs-mediated tumor-specific targeting, NKG2D and DNAM-1 protein expression was detected


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in the human NK-NPs by western blotting. The detection of these proteins was also used to measure the quality of the purified NKCMs and the effective decoration of the NK-NPs by the NKCMs. By contrast, the β-actin and Na+/K+-ATPase 1 signals were used as internal references, and CD56 served as a standard NKCMs antigen. The protein signals of NKG2D and DNAM-1 as well as CD56 were clearly observed in the NK cells, NKCMs and NK-NPs. In addition, the β-actin signals were readily observed in NK cells but were nearly absent in purified NKCMs and NK-NPs, while the Na+/K+-ATPase 1 signals were detected in NK cells, NKCMs and NK-NPs, indicating the high purity of the extracted NKCMs and no interference from cellular actin in the decoration of the NK-NPs (Figure 3a). These results demonstrated the successful decoration of NK-NPs with NKCMs, which endowed the NK-NPs with targeting-tumor capabilities. To confirm the human NK-NPs’ targeting-tumor ability, cellular internalization of human NK-NPs was investigated by incubating the nanoparticles with several different cell lines, including tumor and normal cells. The TCPP fluorescence intensity of the NK-NPs in MCF-7 tumor cells was not only obviously stronger than that of MCF-10A normal cells but was also significantly higher than that of T-NPs in tumor cells (Figure S5). Furthermore, the differential cellular uptake of the NK-NPs in thirteen different cell lines was quantitatively investigated by flow cytometry (BD FACSCalibur Becton, Dickinson and Company, USA). The results displayed that the TCPP fluorescence intensity of NK-NPs in tumor cells was not only approximately thirty-two times stronger than that of normal cells, but also it was generally five times higher than that of T-NPs in tumor cells as well as NK-NPs have the highest uptake efficiency in a series of tumor cells (Figure 3b, 3c and S6), confirming the activated tumor-targeting ability of the NK-NPs. We next examined the biodistribution of the human NK-NPs in 4T1 tumor-bearing BALB/c mice


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by TCPP fluorescence imaging. The in vivo images showed that the human NK-NPs accumulated in the tumor tissue during the first 24 h and then gradually decreased with time, while the T-NPs exhibited lower distribution in the 4T1 tumors throughout the experiment (Figures 3d and 3e). Ex vivo tissue fluorescence imaging showed that human NK-NPs and T-NPs were both rapidly metabolized in most organs 24 h after injection, while the TCPP fluorescence intensity in the tumors of the human NK-NPs group was approximately six times higher than that of the T-NPs group (Figures 3f and 3g), suggesting that NK-NPs exhibited higher tumor accumulation due to the tumor-targeting ability of the NKCMs. Figure S7 showed that the first apparent half-life (i.e. 50% of the particles were cleared) was 8 h for human NK-NPs and 5.5 h for the T-NPs. indicating that the NKCMs improved the blood circulation time of NK-NPs. NK-NPs caused pro-inflammatory M1-macrophages polarization Based on literatures and proteomic analysis, the NK cell-membranes (NKCMs) proteins such as IRGM1, CB1, Galectin-12, RAB-10 and RANKL, are able to interact with macrophages surface receptors (e.g. toll-like receptor 4 or tumor necrosis factor receptor) interactions to induce or enhance pro-inflammatory M1-macrophages polarization (Figure 4a) and thus activate NF-κB pathway or ERK1/2 pathway for producing pro-inflammatory cytokines.23-27 To determine whether human NK-NPs (Human-derived NK cell membranes-coating nanoparticles) induce pro-inflammatory macrophages polarization (M1-type macrophages), human-derived THP-1 were treated with human NK-NPs, human NKCMs or T-NPs for 24 h. The iNOS and CD86 served as markers for M1-type macrophages, while CD206 acted as a marker for M2-type macrophages. Differences in the mRNA levels of markers for M1- and M2-type macrophages were analyzed by quantitative real-time polymerase chain reaction (RT-PCR). The results showed that macrophages treated with human


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NK-NPs and human NKCMs but not T-NPs significantly increased expression of the M1 macrophages markers iNOS/CD86 and decreased expression of the M2 macrophages marker CD206 compared to those of the control group (Figure 4b), indicating that human NKCMs enabled the human NK-NPs to induce pro-inflammatory M1-macrophages polarization. Moreover, the level of M1 macrophages in the human NK-NPs treatment was higher than that treatment with human NKCMs, because NKCMs was coated on the outer layer of NK-NPs to increase the interaction area between NKCMs proteins (such as IRGM1, CB1, Galectin-12, RAB-10 and RANKL) and macrophages. Furthermore, an ELISA assay showed that macrophages treated with human NK-NPs, human NKCMs but not T-NPs significantly increased production of M1 macrophages-related cytokines (TNF-α, IL-6 and IL-12) (Figure 4c-e) and decreased production of the M2 macrophages-related cytokine interleukin-10 (IL-10) compared to those of the control group (Figures 4f). We further determined whether human NKCMs proteins (such as IRGM1, CB1, Galectin-12, RAB-10 and RANKL) induced pro-inflammatory M1-macrophages polarization. The THP-1 cultured in 6-well plates and treated with human-derived IRGM1, CB1, Galectin-12, RAB-10, RANKL (PeproTech, USA) and the mixture of these proteins, respectively. Figure 4g showed that human-derived IRGM1, CB1, Galectin-12, RAB-10, RANKL and the mixture of these proteins could induce M1 macrophages polarization, respectively. Moreover, the amount of M1 macrophages in the mixture of these proteins treatment group was higher than that of the single protein treatment group. We repeated this experiment in murine-derived NK cell membranes-coating nanoparticles (murine NK-NPs) wherein human NK-NPs, human NKCMs and THP-1 cells were replaced with murine NK-NPs, murine NKCMs and mouse bone marrow-derived macrophages (mBMMs),


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respectively. Figure S8a-f demonstrated that murine NK-NPs and murine NKCMs could induce M1 macrophages polarization, and Figure S8g showed that murine NKCMs proteins (such as IRGM1, CB1, Galectin-12, RAB-10, RANKL and the mixture of these proteins) could induce M1 macrophages polarization. These results, which were consistent with experimental observation, indicated that murine NKCMs enabled the murine NK-NPs to induce pro-inflammatory M1-macrophages polarization and the level of M1 polarization induced by murine NK-NPs was similar to human NK-NPs. That NKCMs induced M1 macrophages polarization had been observed in vitro. We further investigated whether NKCMs induce M1 macrophages polarization in vivo, murine NK-NPs were fabricated. We harvested the tumors of mice three day post murine NK-NPs, murine NKCMs and T-NPs treatments to detect pro-inflammatory M1-macrophages by flow cytometry. The iNOS and CD86 served as markers for M1-type macrophages. In comparison to that in the control group, the percentage of M1-macrophages in the tumors of murine NK-NPs-treated mice showed a dramatic ~5.5-fold increase, while T-NPs treatment demonstrated no obvious induction M1 macrophages polarization (Figure S9). Moreover, the level of M1 macrophages in murine NK-NPs treatment were higher than that in the murine NKCMs treatment, because the EPR effect of nanoparticles enhanced the concentration of the murine NK-NPs in the tumor. These results demonstrate that the pro-inflammatory M1-macrophages polarization causes NK cell-membranes immunotherapy. NK-NPs- mediated immunity for antitumor and eliciting abscopal effect After verifying their ROS generation capability in aqueous solution, intracellular ROS generation by the NK-NPs was investigated via confocal laser scanning microscopy (CLSM) using DCFH-DA. Negligible green fluorescence was observed in blank cells as well as in human NK-NPs-


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and T-NPs-treated cells in the absence of irradiation (Figures S10a). In contrast, bright green fluorescence was observed in human NK-NPs- and T-NPs-treated cells upon irradiation with light, demonstrating that both human NK-NPs and T-NPs generate ROS in cells selectively upon irradiation. Moreover, the amount of ROS generated in the human NK-NPs-treated cells was approximately five times higher than that of the T-NPs-treated cells because the tumor-targeting proteins of the NKCMs enhanced the cellular uptake of the NK-NPs by the tumor cells (Figure S11). Next, the in vitro photodynamic toxicity of the human NK-NPs was evaluated. Flow cytometry demonstrated that the human NK-NPs failed to evoke apoptosis in the absence of irradiation but induced high levels of apoptosis upon laser irradiation. Moreover, increased apoptosis was observed in the human NK-NPs group compared to that in the other groups (Figure S10b). Furthermore, the photodynamic toxicity of the human NK-NPs was explored by detecting markers of apoptosis by western blotting. As shown in Figure S10c, in comparison to that in the control group, the expression of pro-apoptotic proteins, such as caspase-3, caspase-8 and Bax, was clearly observed in both the human NK-NPs- and T-NPs-treated cells upon irradiation with light, suggesting that the photodynamic toxicity of human NK-NPs and T-NPs evokes apoptosis. The expression of pro-apoptotic proteins in the human NK-NPs-treated cells was also much higher than that of the other groups. The expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in the human NK-NPs-treated group was much lower than that in the other groups. These results confirmed that human NK-NPs were superior at inducing cell damage upon irradiating with light at 660 nm. We repeated this experiment in murine NK-NPs wherein human NK-NPs was replaced with murine NK-NPs, and it was found that murine NK-NPs also could induce cell damage upon irradiating with light at 660 nm and the level of cell damage induced by murine NKCMs was similar to human


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NKCMs (Figure 5a-c). Cell viability assays were performed using 4T1 tumor cells, and the results indicated that human NK-NPs / murine NK-NPs and T-NPs treatment did not cause any obvious toxicity to cells in the absence of irradiation, while concentration-dependent toxicity was observed with both nanoparticles upon laser irradiation at 660 nm. Moreover, the photodynamic toxicity of the human NK-NPs / murine NK-NPs-treated cells was enhanced compared to that of the T-NPs-treated cells (Figure S12). The cytotoxicity of the human NK-NPs / murine NK-NPs in the 4T1 tumor cells under 660 nm light irradiation was further explored using confocal laser scanning microscopy. The tumor cells were labeled with calcein-AM and propidium iodide (PI) to identify live and dead / late apoptotic cells, respectively. All cells displayed green fluorescence in the control group, while red fluorescence was only observed in cells treated with human NK-NPs / murine NK-NPs and T-NPs upon irradiation with light at 660 nm; further the red fluorescence intensity of the human NK-NPs / murine NK-NPs group was the strongest of all the treatment groups (Figure S13). These results demonstrate that both human and murine NK-NPs are non-toxic in the absence of light irradiation but have good photodynamic toxicity upon irradiation with light at 660 nm. Thus, the presence and absence of light can be used to control the photodynamic toxicity of the NK-NPs. Given the robust immunological responses elicited by PDT-enhanced NK cell-membranes immunotherapy, we explored whether it could inhibit the growth of tumors at distant sites. A bilateral subcutaneous 4T1 tumor-bearing mice model was used to determine whether PDT-enhanced NK cell-membranes immunotherapy could induce a stronger antitumor immune response that would be effective against pre-existing tumors. Murine NK-NPs were fabricated and murine NK-NPs were systemically injected via the tail vein, but only primary tumors (1#) were irradiated with light at 660 nm, while the distal tumors (2#) were not directly treated and served as an artificial abscopal tumor


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model (Figure 5d). As shown in Figures 5e and 5f, T-NPs-based PDT and murine NK cell-membranes immunotherapy alone had minimal effect on the growth of either primary or distal tumors. However, murine NK-NPs-mediated PDT enhanced murine NK cell-membranes immunotherapy that completely eradicated the irradiated primary tumors, demonstrating a synergistic effect. Importantly, significantly retarded distal tumor growth was also observed, demonstrating an abscopal effect. To further evaluate the therapeutic efficacy from PDT-enhanced NK cell-membranes immunotherapy in mice, survival time was monitored following the various treatments. We found that 50% of the mice survived for at least 60 days after treatment with murine NK-NPs plus 660 nm light irradiation therapy, and the mice in the murine NK-NPs groups died within 51 days (Figure 5g). In contrast, the mice in the PBS, T-NPs and T-NPs plus light irradiation groups died within 27 - 33 days. We also determined that the efficacy of T-NPs-based PDT was lower than that of murine NK-NPs-based PDT in the primary tumor. These results demonstrated that murine NK-NPs-mediated PDT effectively inhibited distal tumor growth and prolonged mouse survival due to PDT-enhanced murine NK cell-membranes immunotherapy. Mice body weight was monitored throughout the experiment and served as a measurement of treatment-induced toxicity. As shown in Figure 5h, the body weight of mice treated with murine NK-NPs under light irradiation showed no obvious differences compared to that of the control group, demonstrating that those treatments were reasonably well tolerated. Furthermore, to investigate systemic toxicity, we examined several major organs collected from the mice 72 h post murine NK-NPs injection. Histological examination did not reveal any remarkable damage or differences in inflammation between the murine NK-NPs group and the control group (Figure S14). In all, NK-NPs-mediated PDT could enhance NK cell-membranes immunotherapy to produce a stronger tumor-specific


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immune response. NK-NPs mediated PDT-induced immunogenic cell death and activated systematic antitumor immune response The inhibition of distal tumor growth in 4T1 tumor-bearing mice treated with the combination of PDT and NK cell-membranes immunotherapy suggested that an effective systemic antitumor immune response was produced. Dying tumor cells generate DAMPs by PDT-induced immunogenic cell death (ICD), and the DAMPs then activate tumor-specific immune responses that can further enhance NK cell-membranes immunotherapy efficacy. DAMPs such as CRT exposure, HMGB1 release and secreted ATP were generated through PDT-induced ICD. CRT acts as an “eat me” signal, driving the engulfment of dying cancer cells and, in parallel, the uptake of tumor antigens by DCs; the release of both HMGB1 and ATP also triggers an immunogenic signal downstream of DCs by mediating antigen uptake that enables the optimal presentation of tumor antigens to T lymphocytes.35 We investigated NK-NPs under laser irradiation to determine whether they induced immunogenic phenotypes in tumor cells by examining the levels of CRT exposure, HMGB1 release and secreted ATP. After treatment with human NK-NPs or irradiation, cells were stained with Alexa Fluor 488-CRT antibody and then analyzed by CLSM. Both human NK-NPs and T-NPs treatment failed to induce CRT exposure in 4T1 tumor cells in the absence of irradiation, while both nanoparticles evoked CRT exposure on the surface of tumor cells after laser irradiation at 660 nm compared to that of the control group. Moreover, the amount CRT exposure induced by the human NK-NPs on the tumor cell surface was much higher than that of T-NPs, as the NKCMs coating on the NK-NPs improved the cellular uptake of the NPs (Figure S15a). Extracellular ATP and HMGB1 that is released from dying tumor cells were measured by an ATP detected kit and ELISA,


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respectively (Figures S15b and c). The extracellular ATP and HMGB1 levels in cells treated with human NK-NPs did not notably change in the absence of irradiation compared to that of the control group, but both DAMPs (secreted ATP and HMGB1 release) were increased in cells treated with human NK-NPs plus irradiation compared to those of the other treatment groups. We repeated this experiment in murine NK-NPs wherein human NK-NPs was replaced with murine NK-NPs, and it was found that murine NK-NPs-mediated PDT also could induce immunogenic cell death, and the level of PDT-induced immunogenic cell death mediated by murine NKCM was similar to human NKCM-mediated PDT (Figure 6a-c). In all, these data indicated that both human and murine-derived NK-NPs-mediated PDT could induce immunogenic cell death to strengthen antigen-presenting process for further enhancing NK cell-membranes immunotherapy efficacy. Dendritic cells (DCs) are a key type of antigen-presenting cell that is responsible for activating naïve T cells. Upon exposure to antigens, premature DCs devour and then process the antigens during their migration to nearby lymph nodes, where they then present the major histocompatibility complex-antigen (MHC) to T cell receptors (TCRs) for T cell activation. To investigate the level of DCs maturation after NK-NPs and NK-NPs plus light irradiation treatment in 4T1 tumor-bearing mouse model, murine NK-NPs were fabricated. DCs were separated from the tumor-draining lymph nodes of BALB/c mice via flow cytometry, where upregulation of the co-stimulatory molecules CD80 and CD86, typical markers of DCs maturation, was used to identify mature DCs. The results from flow cytometry indicated that murine NK-NPs and murine NK-NPs plus light significantly enhanced the level of DCs maturation, while T-NPs treatment demonstrated no obvious stimulation of DCs maturation (Figures 6d and 6e). Moreover, the level of DCs maturation of murine NK-NPs group was 3 and 2.5 times that of PBS and T-NPs plus light


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irradiation group, respectively. However, the level of DCs maturation of murine NK-NPs plus light irradiation group was only 1.2 times that of murine NK-NPs group, which suggested that PDT enhance NK cell-immune response. In all, these results demonstrated that the successful transfer of NKCMs proteins to NK-NPs was adequate for exerting their favorable immune system effects. We also investigated the immune cells in the distal tumor 72 h after treating with murine NK-NPs and murine NK-NPs plus light irradiation in a bilateral subcutaneous 4T1 tumor-bearing mouse model. Murine NK-NPs were fabricated. It is well known that cytotoxic T lymphocytes (CD3+ CD4- CD8+) kill cancer cells and helper T cells (CD3+ CD4+ CD8-) play a vital role in regulating the immune response.36 We harvested the distal tumors of mice 3 days post-PDT treatment to detect the tumor-infiltrating CD8+ (CD3+CD4-CD8+) and CD4+ (CD3+CD4+CD8-) T cells by flow cytometry. In comparison to that in the control group, the percentage of helper T cells (CD4+ T cells) in the distal tumors of murine NK-NPs and murine NK-NPs-treated mice after laser irradiation showed a remarkable increase to 26.27% and 32.23%, respectively, (Figure 6f). The percentage of CD8+ T cells in the distal tumors of murine NK-NPs and murine NK-NPs-treated mice after laser irradiation showed a dramatic ~5 and 6-fold increase, respectively (Figure 6g). In addition, the percentage of infiltrating T cells (CD4+ or CD8+) in the murine NK-NPs group was also significantly higher than that in the T-NPs plus light irradiation group. Importantly, the serum concentrations of TNF-, IL-6 and IL-12 in the murine NK-NPs and murine NK-NPs plus irradiation group were much higher than those in the other treatment groups (e.g., the serum TNF- in mice 72 h after treating with murine NK-NPs plus light irradiation was more than nine times that of T-NPs plus light group) (Figure 6h, 6i and S16). Moreover, the pro-inflammatory cytokines levels in the murine NK-NPs plus irradiation group were higher than that in the murine NK-NPs groups due to


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PDT improved NK cell-membranes immune response. In summary, these results confirmed that NK-NPs-mediated immunogenic PDT could enhance NK cell-membranes immunotherapy to induce a higher level of tumor-specific immunity. Discussion This study focused on creating a biomimetic nanostructure cloaked with cell-membranes to mimic the membrane’s cellular functions and capture the various inherent capabilities of the molecules residing on the cell-membranes extracellular surface. Biomimetic nanoparticle vesicles have been designed to mimic platelets, red blood cells and leukocytes.37-41 These biomimetic nanostructures maintain many biological properties such as ligand recognition and targeting as well as prolonged blood circulation. In contrast to the existing biomimetic nanostructures, NK-NPs not only have the ability to target tumor cells but also can elicit pro-inflammatory M1-macrophages polarization to produce antitumor immunity, as the NK cell-membranes proteins has tumor-targeting and

triggers

M1-macrophages

polarization.

NK-NPs

could

induce

pro-inflammatory

M1-macrophages polarization through the interactions of NK cell-membranes proteins (e.g. RANKL or CB1) and macrophages surface receptors (e.g. tumor necrosis factor receptor or toll-like receptor 4) for producing cell-membranes immunotherapy (Figure 4a, 4b S8a and S8b). The specific induction of M1-type macrophages remains challenging, and only a few methods (e.g., antibodies against CSF-1R) have been successful to date. NK-NPs could activate STAT3 or NF-κB pathway for producing pro-inflammatory cytokines owing to NK cell-membranes proteins (e.g. IRGM1, RAB-10 and Galectin-12), and the pro-inflammatory cytokines (TNF-α, IL-6 and IL-12) of NK-NPs group was at least five times higher than that of the control group (Figure 4c-e and S8c-e). Furthermore, NK-NPs promote DCs activation and initiate an adaptive immune response. DCs maturation plays an


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important role during antigen presentation (Figure 6e). In contrast to other DCs maturation nanoparticles, NK-NPs do not require the addition of immunostimulants such as CpG oligonucleotides and resiquimod, as the NK-NPs themselves could serve as effective immunostimulants. Thus, NK-NPs provide an effective approach for inducing M1-type macrophages polarization for generating cell-membranes immunotherapy. Based on nanoparticles that mimic NK cells (NK-NPs), our study provides a PDT-enhanced cell-membranes immune strategy to generate nanoparticles with vaccine-like functions in situ without delivery of exogenous antigens or immunologic adjuvants. Conventional tumor vaccines use specific proteins as tumor antigens; however, therapeutic responses vary considerably due to patient heterogeneity in antigen expression on the tumor surface. In contrast, in situ tumor vaccines exploit dying tumor cells to generate DAMPs via PDT-induced ICD which activate an immune-response against a broad spectrum of tumors. To enhance the immune response in conventional tumor immunotherapy, it was necessary to add immunostimulants (e.g., imiquimod); however, these immunostimulants were exogenous compounds with high toxicity. In this study, we prepared and characterized a biocompatible, endogenous NK cell-membranes-containing nanoparticle that induced cell-membranes immune response to improve tumor immunotherapy. Furthermore, the DAMPs generated by PDT-induced ICD amplified the NK cell-membranes immunotherapy efficacy to enhance the tumor infiltration of effector T cells (CD4+ and CD8+ T cells), resulting in highly efficient regression of both primary and distal tumors (Figures 5e, 5f, 6f and 6g). These findings highlight that NK-NPs-mediated PDT enhance the NK cell-membranes immunotherapy efficacy and offers an approach for tumor immunotherapy.


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Conclusions In summary, we have developed NK cell-membranes-cloaked nanoparticles (NK-NPs) for tumor-targeting

cell-membranes

immunotherapy

by

immunogenic

PDT-enhanced

NK

cell-membranes immunity to generate a stronger immune response. Proteomic profiling of NK cell-membranes was performed, and we demonstrated that NK cell-membranes enabled NK-NPs to target tumors and initiated M1 macrophages polarization to generate cell-membranes immunotherapy. Most importantly, NK-NPs-mediated immunogenic PDT could increase NK cell-membranes immunotherapy efficacy to produce an enhanced antitumor immunity which not only eradicated primary tumors but also inhibited the growth of pre-existing distal tumors. Thus, the engineered NK cells-like nanoparticles in this study can provide a versatile strategy for effective cell-membranes immunotherapy.

Materials and methods Materials 4,4′,4′′,4′′′-(Porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (TCPP), poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (mPEG-PLGA) and RIPA lysis buffer were purchased from Sigma-Aldrich (USA). Calcein-AM / propidium iodide (PI) and DAPI were provided by Invitrogen (USA). Penicillin-streptomycin, fetal bovine serum, DMEM medium, and trypsin EDTA were supplied by Gibco Life Technologies (USA). Antibodies specific for human CD226 and CD56 were obtained from Cell Signaling Technology (USA). NKG2D and mouse IgG were purchased from Abcam (USA). IRGM1, CB1, Galectin-12, RAB-10 and RANKL were purchased from PeproTech (USA). The MCF-10A, HepG2, A549, MCF-7, 4T1 and NK-92 cells line


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were provided by Shanghai Cell Bank, Chinese Academy of Sciences (CAS). The chemicals and reagents used in this study were of analytical grade. NK-92 culture conditions The base medium for NK-92 is alpha minimum essential medium with 2 mM L-glutamine and 1.5 mg/ml sodium bicarbonate but without ribonucleosides and deoxyribonucleosides. To make the complete growth medium, add the following components to the base medium: 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; 100-200 U/ml IL-2; adjust to a final concentration of 12.5% horse serum and 12.5% fetal bovine serum. Murine NK cells separation Isolation of NK cells from BALB/c mice spleen cells suspension using the NK Cell Isolation Kit II (Milternyi Biotec, Germany). Briefly, the collected spleen cell suspension and centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely. Resuspend cell pellet in 40 µl of buffer per 107 total cells, and add 10 µl of NK Cell Biotin-Antibody Cocktail per 107 total cells. Mix well and incubate for 5 minutes in the dark in the refrigerator (2−8 °C), and then wash cells by adding 2 ml of buffer per 107 cells and centrifuge at 300×g for 10 minutes and aspirate supernatant completely. Add 80 µl of buffer per 107 total cells and add 20 µl of Anti-Biotin MicroBeads per 107 total cells. Mix well and incubate for an additional 10 minutes in the dark in the refrigerator (2−8 °C). Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of NK cells. The unactivated murine NK cells were acquired. The murine NK cells activation and identification The medium for the activated murine NK cells was SuperCulture® medium with 2% FBS, 1% penicillin-streptomycin and 100 U/ml IL-2. The activated murine NK cells were evaluated by flow


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cytometry (BD FACSCalibur Becton, Dickinson and Company, USA). Through activation medium culture, murine NK cells activation rate reached 94.7% (Figure S17). Isolation of natural killer cell-membranes Briefly, the collected NK cells were added to cell lysis buffer containing protease inhibitors and were crushed using sonication in an ice bath for 20-30 min. The crushed cells were centrifuged (3500 g, 10 min, 4 °C), and the supernatant was collected and then centrifuged (20 000 g, 25 min, 4 °C). The supernatant was then centrifuged again (100 000 g, 50 min, 4 °C) to yield the NK cell-membranes (NKCMs) as a precipitate in the centrifuge tube. Formulation and characterization of the NK-NPs The NK-NPs were fabricated by coating T-NPs with NKCMs by a direct extrusion method. Briefly, 4 mg of 4,4′,4′′,4′′′-(porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (TCPP) in 200 μl of DMSO was mixed with a DMF solution (8 ml) containing 80 mg of poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (mPEG-PLGA). This solution was then added dropwise to 40 ml of water and stirred for 3 h to form the TCPP-mPEG-PLGA nanoparticles (T-NPs). The solution was dialyzed against with doubly distilled water for 24 h to separate free TCPP from the T-NPs. The purified NKCMs vesicles from 1 × 108 human NK cells / murine NK cells were coated onto the T-NPs cores by directly co-extruding the vesicles and cores through a polycarbonate membrane with a pore size of 220 nm to form the human NK-NPs / murine NK-NPs. The size distribution and surface charge of the NK-NPs and T-NPs were analyzed using a Zetasizer Nano ZS instrument (Malvern, U.K.). The particle size and morphology of the NK-NPs and T-NPs were analyzed via TEM (FEI Tecnai G2 F20 S-Twin, USA). The absorption spectra of the NK-NPs were obtained by UV/vis spectrometry (Lambda25, Perkin-Elmer, USA), and the


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fluorescence spectra of the NK-NPs were obtained using fluorescence spectroscopy at 520 nm excitation (F920, Edinburgh Instruments, Ltd., UK.). Protein detection in the NK-NPs The protein profiles of human NKCMs, T-NPs and human NK-NPs were examined by Coomassie Brilliant Blue staining and the presence of specific protein markers was detected by western blotting. All samples were prepared in loading buffer (Sigma-Aldrich, USA) at equivalent concentrations. The SDS-PAGE gels were transferred onto polyvinylidene difluoride membranes (Millipore, USA) and probed with antibodies specific for human DNAM-1, CD56 and NKG2D, along with the appropriate HRP-conjugated secondary antibodies. Protein analysis of the NK-NPs The human NKCMs coating on the NK-NPs was analyzed using peptide-level LC/MSE according to the following steps. The samples were dissolved in SDT buffer solution at pH 7.6 and then treated with 5 mM dithiothreitol at 60 °C for 30 min, followed by 15 mM iodoacetamide at 25 °C. The lipids were removed by methanol/chloroform extraction. The protein concentration was measured using the Bradford protein assay, and then, trypsin digestion was performed at 37 °C for 12 h. A Waters Corp. NanoAcquity UPLC system coupled with a Synapt HDMS (G1) mass spectrometer was used to separate the peptide mixtures using a reverse phase C18 column. Samples were injected into the mass spectrometer in positive ion (ESI) mode, and the LC system consisted of a trapping C18 column (80 µm x 20 mm Symmetry, 5 µm particle size) and an analytical C18 column (75 µm x 250 mm BEH130, 1.7 µm particle size). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The flow rate of the mobile phase was 0.3 µl/min and the column temperature was set to 35 °C. Peptide separation was conducted


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using a gradient of 5-60% solvent B in solvent A for 120 min. Mass spectrometry was performed in the data independent (parallel-ion fragmentation) MSE mode at a capillary voltage of 3 kV with alternating low (6 V) and ramped high collision energies (15 V - 45 V) at a scan rate of 1.2 s per scan. Glu-fibrinopeptide B acted as an internal calibrant. All data were collected using a time of flight (TOF) detector. We employed ProteinLynx Global Server (PLGS v2.4; Waters Corp) software to identify and quantify the proteins using both the IdentityE and ExpressionE algorithms included in the software. As long as the protein IDs reached above the 95% confidence interval, they were reserved. The data for all the identified proteins from the NKCMs of NK-NPs were submitted for bioinformatics analysis, and the proteins were classified according to their biological function and cellular component. The proteins were classified based on UniProt/GO (http://www.uniprot.org/) (http://www.geneontology.org/) information and manual searching in the literature. Cellular uptake of NK-NPs Cells (MCF-7 or MCF-10A) were seeded into 8-well plates (Thermo Fisher Scientific, USA) at a density of 5×104 cells per well in DMEM containing 10% FBS and incubated for 24 h (37 °C, 5% CO2). The cells were then treated with various concentrations of human NK-NPs or T-NPs for 3 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde (200 μl) for 10 min at 4 °C. The cells were washed twice with cold PBS, and the cell nuclei were stained with DAPI (10 μg/ml in PBS) for 10 min and then rinsed with PBS to remove any free dye. The cells were observed through a confocal microscope (CLSM, Leica TCSNT1, Germany) at the excitation wavelengths of 408 nm (emission bandpass: 430-520 nm) and 633 nm (emission bandpass: 650-700 nm). To measure cellular uptake by flow cytometry, normal and tumor cells were cultured at a density


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of 5×105 cells per well in 6-well plates. After cells reached 70-80% confluence, they were treated with different concentrations of human NK-NPs or T-NPs for 3 h. Excess nanoparticles were removed from the cells by PBS washing. The cells were trypsinized and then analyzed by flow cytometry (BD FACSCalibur, Becton, Dickinson and Company, USA). In vitro cytotoxicity and cell apoptosis For cell viability studies, 4T1 tumor cells were seeded overnight in 96-well plates followed by incubation with 100 μl of medium containing nanoparticles at different concentrations for 24 h. In the NIR laser irradiation group, nanoparticles containing medium were irradiated at 660 nm (100 mW/cm2) for 10 min. All the samples were then further incubated for 6 h before determining the cell viability using the CCK-8 assay according to the instructions given by the manufacturer (Dojindo Molecular Technologies, USA). To detect apoptosis, 4T1 tumor cells were seeded in 6-well plates at a density of 1×106/well. Cells were treated with nanoparticles for 24 h, and then irradiated with a 660 nm laser (100 mW/cm2) for 10 min. As a control, the same formulations were added without light irradiation. After incubating at 37 °C for 6 h, the cells were digested by trypsin and then repeatedly washed with PBS. The cells were collected in the tubes and resuspended in annexin-binding buffer (100 μl/tube). Alexa Fluor 488 annexin V (5 μl) and 1 μl of a 100 μg/ml PI working solution were added to 100 μl of the cell suspension, and the solution was incubated for 30 min at 25 °C. After the incubation period, 400 μl of annexin binding buffer was added to each sample, the samples were gently mixed on ice, and the samples were then immediately analyzed by flow cytometry (Becton, Dickinson and Company, USA). Cytokine detection in vitro and in vivo


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Mouse bone marrow-derived macrophages (mBMMs) were generated from the bone marrow of 8-week-old BALB/c mice according to the laboratory existing methods. mBMMs / THP-1 cells were cultured in 6-well plates (2 × 106 cells per well) with murine NK-NPs, murine NKCMs and T-NPs / (human NK-NPs, human NKCMs and T-NPs) for 24 h. After incubation, the culture supernatants were collected and the TNF-α, IL-6, IL-12p40 and IL-10 levels were analyzed with cytokine-specific ELISA kits (BD Biosciences, USA), according to the vendor’s protocols. Cytokine concentrations were quantified with a VersaMax apparatus at OD450. Serum samples were collected from mice after various treatments. TNF-α, IL-6 and IL-12p40, were analyzed using ELISA kits according to vendors’ protocols. Cytokine concentrations were quantified with a VersaMax apparatus at OD450, according to the manufacturer’s recommendations. Immune cell analysis in the distal tumors and spleens To analyze immune cells in the distal tumors, the distal tumors were harvested from the different treatment groups and digested using 1500 U/ml collagenase (Sigma-Aldrich, USA), 1000 U/ml hyaluronidase (Sigma-Aldrich, USA) and DNaseI (Sigma-Aldrich, USA) at 37 °C for 30 min. Cells were filtered via nylon mesh filters and washed with PBS containing 1% FBS. To analyze the immune cells in the spleen, spleens were harvested from the different treatment groups and prepared by applying gentle pressure with the homogenizer without digestive enzyme. Then, samples were lysed with RBC lysis buffer to remove the red blood cells (RBCs). Single cell suspensions were incubated with anti-CD3-FITC (Biolegend, USA), anti-CD8a-APC (Biolegend, USA), and anti-CD4-PerCP (Biolegend, USA) antibodies. Cells were then washed with PBS containing 1% FBS and analyzed using flow cytometry (BD FACSCalibur Becton, Dickinson and Company, USA). To analyze DCs maturation, single cell suspensions were stained with anti-CD11c FITC (eBioscience,


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USA), anti-CD86 PE (eBioscience, USA) and anti-CD80 APC (eBioscience, USA) and then sorted by flow cytometry. To analyze T cells infiltration, tumors were harvested from mice after the various treatments and were stained. The data analysis was carried out using FCS express software. ICD marker detection The extracellular ATP level was quantified using an ATP assay kit. Briefly, 4T1 tumor cells were seeded in six-well cell culture plates and then incubated with human NK-NPs / murine NK-NPs and T-NPs for 3 h. After the various treatments, the culture supernatants were collected and centrifuged at 12 000 × g for 10 min. The supernatants were transferred into tubes to test for ATP. The luminescence from a 20 μl sample was assayed using a multi-scan spectrum (Thermo Fisher Scientific, USA) together with 100 μl of ATP detection buffer. A standard curve for ATP was generated from known amounts of ATP. The ATP levels were then normalized according to the protein concentration. ELISA assays were carried out to test the expression of HMGB1 in 4T1 tumor cells. Cells were seeded at 3×106 cells per well in six-well cell culture plates. After 24 h incubation, cells were treated with human NK-NPs / murine NK-NPs and T-NPs for 3 h. HMGB expression was detected using an ELISA assay kit (Aviva Systems Biology, USA). Immunofluorescence assays were carried out to determine the expression of calreticulin in 4T1 tumor cells. Cells were seeded in an 8-well plate (Thermo Fisher Scientific, USA) and cultured overnight. Cells were incubated with human NK-NPs / murine NK-NPs and T-NPs for 3 h. The expression of calreticulin was detected using calreticulin antibodies (Cell Signaling Technology, USA) in an immunofluorescence assay. M1- and M2-macrophages associated gene expression detection


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The expression of M1- and M2-macrophages associated genes was analyzed by real-time PCR (RT-PCR). The THP-1 cells / mBMMs were cultured in 6-well plates (5×106 cells per well) and treated with human NK-NPs, human NKCMs and T-NPs / (murine NK-NPs, murine NKCMs and T-NPs). After 24 h incubation, THP-1 cells / mBMMs were harvested, and the total RNA was extracted with TRIzol (Invitrogen, USA). Real-time PCR was performed on a CFX96 Touch™ sequence detection system (Bio-Rad, USA) using the appropriate primers and a DyNAmo HS SYBR Green qPCR kit (Thermo Fisher Scientific, USA). Cycling conditions were as follows: initial denaturation at 95 °C for 15 min, followed by 40 cycles for denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. The mRNA expression levels were measured by the comparative Ct method. Animals and the tumor model Female BALB/c mice (18-22 g, 6 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (China). Experiments were performed with permission from the Guidance Suggestions for the Care and Use of Laboratory Animals, and the procedures were approved by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. BALB/c mice were maintained under aseptic conditions in a small animal isolator and were housed in groups of five in standard cages with free access to food and water and a 12 h light/dark cycle. BALB/c mice were subcutaneously injected with 4T1 tumor cells (1×106) in the flank region. The bilateral subcutaneous 4T1 tumor model was used in this study. BALB/c mice were injected s.c. with 1×106 4T1 tumor cells in the right flank (primary tumor) and 2×105 4T1 tumor cells in the left flank (distant tumor). Tumors on the right side were designated “primary tumors” and underwent PDT treatment while those on the left side were designated “distal


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tumors” and did not undergo PDT treatment. In vivo imaging and biodistribution analysis When the 4T1 tumor size reached 150 mm3, the BALB/c mice were randomly divided into two groups. Mice were administered human NK-NPs or T-NPs (100 μl, containing 350 μg/ml TCPP) via intravenous injection. The fluorescence signals of TCPP were obtained by an ex/in vivo imaging system (CRI Maestro, USA) (ex: 520 nm; filter: 650 nm). The mice were euthanized 24 h post-injection, and the major organs (heart, liver, spleen, lung, kidneys) and tumors were collected for semiquantitative biodistribution analysis using the ex/in vivo imaging system (CRI Maestro, USA). In vivo antitumor efficacy and the abscopal effect using a bilateral 4T1 tumor model BALB/c mice were injected s.c. with 1×106 4T1 tumor cells in the right flank (primary tumor) and 2×105 4T1 tumor cells in the left flank (distant tumor). When the primary tumor size reached ~100 mm3, mice were randomly divided into five groups (ten mice per group): PBS, T-NPs, murine NK-NPs, T-NPs plus irradiation, murine NK-NPs plus irradiation. Twenty-four hours after intravenous injection, mice were anesthetized, and the primary tumors were irradiated with a 660 nm laser at a power density of 100 mW/cm2 for 30 min. The primary and secondary tumor volumes and mice body weights were monitored every day. Mice with primary or distant tumor sizes exceeding 1000 mm3 were euthanized according to the animal protocol.

Supporting Information The supporting information is available free of charge on the ACS publications website. Supplementary figures S1-S17 showing characterization of T-NPs, cell uptake of nanoparticles,


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pharmacokinetics of NK-NPs, the detection of pro-inflammatory M1-macrophages polarization proteins, murine NK-NPs causing M1 macrophages polarization in vitro and in vivo, human NK-NPs mediated PDT in vitro, the determination of intracellular ROS, cell viability after treatment, biosafety assessment and evaluation, human NK-NPs-mediated PDT-induced ICD and the analysis of activated murine NK cells. Supplementary table 1 showed NK cell membranes coating on NK-NPs-protein peptide identification list and supplementary table 2 showed NK cell membranes coating on NK-NPs- protein annotation.

Acknowledgements This work was supported by Key International S&T Cooperation Project (2015DFH50230), the National Natural Science Foundation of China (31571013, 81671758, 51502333, and 81501580), Guangdong Natural Science Foundation of Research Team (2016A030312006), Shenzhen Science and

Technology

Program

(JSGG20160331185422390,

JCYJ20160429191503002,

JCYJ20170818162522440, JCYJ20170818154843625 ).

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Figure

Scheme 1: Schematic illustration of NK cell-membranes-cloaked nanoparticles for PDT-enhanced cell-membranes immunotherapy. Extracted NK cell-membranes were coated onto photosensitizer TCPP-loaded polymeric nanoparticles by extrusion. NK cell-membranes enabled the NK-NPs to elicit pro-inflammatory M1-macrophages polarization in tumor for generating cell-membranes immunotherapy. And NK-NPs could elicit dying tumor cells to generate DAMPs (CRT exposure, secreted ATP and HMGB1 release) through PDT-induced immunogenic cell death (ICD) for enhancing NK cell-membranes immunotherapy effect. Specificity, immunogenic PDT enhanced NK cell-membranes immunotherapy which significantly improved infiltration of effector T cells (CD4+ T cells and CD8+ T cells) in tumors for highly efficient inhibition of both primary tumors and abscopal tumors.


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Figure 1: Characterization of the NK-NPs. (a) TEM images of NK-NPs. (b) Size distribution of NK-NPs. (c) UV/vis absorption spectra of TCPP, T-NPs and NK-NPs. (d) Protein profiles of the NKCMs and NK-NPs determined by SDS-PAGE electrophoresis. (e) Fluorescence spectra of TCPP, T-NPs and NK-NPs. (f) The particle stability of NK-NPs. (g) The generation of ROS by TCPP, T-NPs and NK-NPs based on the fluorescence intensity changes of DCFH-DA under 660 nm light irradiation (100 mW/cm2).


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Figure 2: Analysis of the human NKCMs proteins in NK-NPs. (a) Schematic representation of the plasma membrane protein identification procedure in human NK-NPs. (b) The number of total proteins and plasma membrane-associated proteins identified in the human NK-NPs. (c) A pie chart of the proteins identified in human NK-NPs classified according to UniProt/GO information and the literature. The peripheral proteins were adhered to the plasma membrane surface while integral proteins penetrated the plasma membrane. Lipid-anchored proteins were covalently bound to the plasma membrane. Secreted proteins circulated between the exterior and the interior of the cell via vesicle-mediated secretory pathways. Cytoskeletal proteins were connected to the plasma membrane. (d) Functional characterization of the plasma membrane proteins identified in the human NK-NPs, which involved in response stimulus, transport, signaling, immunity, developmental processes, adhesion and lipid metabolism. (e) Flow cytometry analysis validates the presence of proteins on the surface of human NK-NPs, which involved in inducing pro-inflammatory M1-macrophages polarization. Both DNAM-1 and NKG2D had tumor-targeting (red words).


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Figure 3: Tumor-targeting efficacy and biodistribution of human NK-NPs. (a) Tumor-targeting proteins DNAM-1 and NKG2D in NK cells, human NKCMs, and human NK-NPs as determined by western blotting. The protein signals of β-actin and Na+/K+-ATPase 1 served as controls. CD56 served as a common NK cell-membranes antigen. (b) Cellular uptake by three pairs of tumor (MCF-7, A498 and A549) and normal cells (MCF-10A, HEK-293 and MRC-5) was measured by flow cytometry. (c) Flow cytometry profiles of ten tumor cell lines incubated with T-NPs and human NK-NPs for 3 h. (d) Time-lapse NIR fluorescent images of 4T1 tumor-bearing BALB/c mice in vivo. Human NK-NPs exhibited an obvious tumor-targeting effect, (Red circle indicaed tumor sites). (e) NIR fluorescence intensities around the tumors quantified at the indicated time points. (f) Ex vivo NIR fluorescent images of the major organs (heart, liver, spleen, lung and kidney) and tumors 24 h after injecting T-NPs or human NK-NPs. Human NK-NPs demonstrated excellent tumor accumulation. (g) Semiquantitative biodistribution of T-NPs and human NK-NPs in 4T1 tumor-bearing BALB/c mice as determined by the averaged fluorescence intensity in the organs and tumors. Data are expressed as the mean ± s.d. (n = 3); ((*) p < 0.05, (**) p < 0.01).


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Figure 4: Human NK-NPs caused pro-inflammatory M1-macrophages polarization in THP-1 cells (in vitro). (a) Schematic illustration of human NKCMs proteins inducing M1 macrophages polarization. (b) Gene signs of M1 macrophages activation in vitro. (c), (d) and (e) Pro-inflammatory cytokines levels in THP-1 cells treated with human NK-NPs, human NKCMs and T-NPs. (f) Anti-inflammatory cytokine level in THP-1 cells treated with human NK-NPs, human NKCMs and T-NPs. (g) Human NKCMs proteins such as IRGM1, CB1, Galectin-12, RAB-10, RANKL and the mixture of these proteins induced M1 macrophages polarization in THP-1 cells (in vitro). Figure S8 (This figure in the supporting information) showed that murine NK-NPs caused pro-inflammatory M1-macrophages polarization in mBMMs cells (in vitro), and it was found that murine NKCMs enabled the murine NK-NPs to induce pro-inflammatory M1-macrophages polarization and the level of M1 polarization induced by murine NK-NPs was similar to human NK-NPs. ( (*) p < 0.05, (**) p < 0.01).


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Figure 5: The antitumor immunity and abscopal effect of murine NK-NPs. (a) The generation of ROS by T-NPs and murine NK-NPs based on the fluorescence intensity changes of DCFH-DA in cells upon 660 nm light irradiation (100 mW/cm2). (b) Flow cytometry analysis showing that T-NPs and murine NK-NPs induce apoptosis upon irradiation. (c) Apoptosis protein markers were induced by murine NK-NPs-mediated PDT as detected by western blotting. (d) Schematic illustration of our experimental design. A bilateral subcutaneous 4T1 tumor model was used in this experiment. Tumors on the right side were designated ‘primary tumors’ and underwent PDT treatment while those on the left side were designated ‘distal tumors” and did not undergo PDT treatment. (e) Growth curves for the primary tumors. (f) Growth curves for the distal tumors. (g) Morbidity-free survival of the different treatment groups in the bilateral 4T1 tumor model. (h) Body weight changes of mice under the different conditions. (ten mice per group). ((*) p < 0.05, (**) p < 0.01).


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Figure 6: The mechanisms for the murine NK-NPs-mediated immunogenic PDT-enhanced NK cell-membranes immune response for tumor immunotherapy (a) Confocal images showing the CRT exposure on tumor cells after treatment with T-NPs or murine NK-NPs plus 660 nm laser irradiation (100 mW/cm2). (b) Extracellular secreted ATP from tumor cells was measured using an ATP detection kit. (c) HMGB1 released from tumor cells was measured by ELISA. (d) Schematic illustration of mechanism of DCs maturation facilitated by the major ICD-associated DAMPs from dying tumor cells. Tumor cells undergoing ICD produced DAMPs (such as CRT exposure, HMGB1 release and secreted ATP), which promoted the engulfment of tumor antigens by immature DCs, followed by stimulating DCs maturation. (e) In vivo maturation of DCs (CD80+ CD86+) from tumor-draining lymph nodes in BALB/c mice following intravenous injection of T-NPs or murine NK-NPs (three mice per group). (f) Proportions of tumor-infiltrating CD4+ T cells. (g) Proportions of tumor-infiltrating CD8+ T cells. (h) and (i) Pro-inflammatory cytokines (TNF- and IL-6) levels in the sera of mice treated with murine NK-NPs-mediated PDT from day 0, day 1, day 3 and day 7. ((*)p < 0.05, (**) p < 0.01).


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A Table of Contents (TOC) Graphic

TOC: Schematic illustration of NK cell-membranes-cloaked nanoparticles for PDT-enhanced cell-membranes immunotherapy. NK cell-membranes were coated onto photosensitizer TCPP-loaded polymeric nanoparticles (NK-NPs). NK cell-membranes enabled the NK-NPs to elicit M1 macrophages polarization in tumor for generating cell-membranes immunotherapy. And NK-NPs could elicit dying tumor cells to generate DAMPs (CRT exposure, secreted ATP and HMGB1 release) through PDT-induced immunogenic cell death (ICD) for enhancing NK cell-membranes immunotherapy effect. Specificity, immunogenic PDT enhanced NK cell-membranes immunotherapy which significantly improved infiltration of effector T cells (CD4+ T cells and CD8+ T cells) in tumors for highly efficient inhibition of both primary tumors and abscopal tumors.


Cell-Membrane Immunotherapy Based on Natural Killer Cell

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