Materials and reagents. DMEM, RPMI-1640 cell culture medium, fetal bovine serum (FBS) and 0.25 % trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (USA). Penicillin-streptomycin was purchased from Macgene (China). UNC2025·2HCl was purchased from Topscience (China). Mal-PEG4-NHS was purchased from QIYUE BIOLOGY (China). Ovalbumin (OVA), PE-conjugated OVA, Cy5-conjugated OVA, FITC-conjugated anti-E. coli LPS antibody and anti-phospho-MerTK antibody were purchased from Bioss (China). Anti-MerTK, anti-b-Actin and Goat-Anti-Rabbit IgG (HRP) antibodies were purchased from Abcam (UK). FITC-conjugated anti-mouse CD11c antibody was purchased from Proteintech (China). APC anti-mouse CD11b, FITC-conjugated anti-mouse CD80, PE-conjugated anti-mouse CD86, APC-conjugated anti-mouse MHC-II, PE-conjugated anti-mouse MHC-I, PE-conjugated anti-mouse CD69, APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD8, PE-conjugated anti-mouse CD4, PE-conjugated anti-mouse IFN-γ, PE-conjugated anti-mouse CD44, Pacific Blue-conjugated anti-mouse CD62L antibody, Mouse IL-6, IL-12p40, TNF-α, IFN-γ ELISA Kit were purchased from BioLegend (USA). PE-conjugated anti-mouse CD206 antibody and flow cytometry staining buffer were purchased from eBioscience (USA). Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Beyotime (China). Mouse high mobility group protein B1 (HMGB1) ELISA Kit and the Annexin V-FITC/DAPI Apoptosis Detection Kit were purchased from Elabscience (China). Micro BCA Protein Assay Kit and Prestained Protein Ladder were purchased from Thermo Scientific (USA). Coomassie brilliant blue, Cell Counting Kit-8 (CCK-8) and SDS-PAGE loading buffer were purchased from Solarbio (China). Hoechst 33342 was purchased from Life Technologies (USA). Recombinant murine IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were purchased from PeproTech (USA).
Cell lines and animals. CT26 (mouse colon cancer cell line) and B16F10 (murine melanoma cancer cell line) were kindly obtained from the Institute of Process Engineering (China). RAW264.7 (murine macrophage cell line) was purchased from Peking Union Medical College Hospital. All cell lines were maintained in a 37 °C humidified chamber with 5 % CO2. CT26, RAW264.7 and B16F10 cells were cultured in DMEM medium containing 10 % FBS and 1 % penicillin-streptomycin.
BALB/c mice and C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All animal experiments were performed under the guidance of the Beijing Animal Ethics Association and the Ethics Committee of Beijing Institute of Technology (approval ID: 2019-0010-M-2020019).
Preparation of bacterial outer membrane vesicles (OMVs). Escherichia coli MG1655 in LB medium was incubated in a shaking incubator at 220 rpm at 37 °C overnight. Then, bacterial cells were removed by centrifuging at 10000 g for 10 min at 4 °C (Sorvall ST16R, Thermo Scientific). The obtained supernatant was filtered by 0.45 μm filters (Vacuum Filter System, Corning) and concentrated by centrifugal filters with a molecular weight cutoff (MWCO) of 100 kDa (Millipore). The concentrated supernatant was filtered again through 0.22 μm pore size filters (Millipore) to remove any remaining debris or bacteria. Then, the concentrated medium was centrifuged at 150000 g for 3 h at 4 °C (XPN 100, Beckman). The supernatant was removed and the pellet was resuspended in PBS and stored at -80 °C. The protein concentration of OMVs was determined by Micro BCA Protein Assay Kit.
Preparation of U@OMVs and mU@OMVs. To obtain U@OMVs, UNC2025·2HCl molecules were encapsulated into OMVs through electroporation. In brief, 10 µg UNC2025·2HCl and 10 µg OMVs were gently suspended in the electroporation cuvette on ice. Electroporation was operated under conditions of 100 V, 200 Ω, and 100 μF by the BTX ECM 630 and GenePulser electroporators (Bio-Rad). After that, residual UNC2025·2HCl were removed by ultrafiltration at 5000 g for 10 min and washed for three times. The loading efficiency and encapsulation efficiency of U@OMVs were determined by UV-vis spectrum. To obtain mU@OMVs, 200 µL U@OMVs (containing 10 µg protein) were mixed with 10 µg Mal-PEG4-NHS solution and incubated at room temperature for 3 h. Excess Mal-PEG4-NHS was removed by ultracentrifugation at 150000 g for 3 h at 4 °C. The pellet containing mU@OMVs was resuspended in PBS and the content of UNC2025·2HCl in the supernatant was determined by UV-vis spectrum to ensure the variety of loading efficiency and encapsulation efficiency.
Characterization of OMVs, U@OMVs and mU@OMVs. The sample morphology of OMVs, U@OMVs or mU@OMVs was observed by the transmission electron microscope (TEM) (120 kV, Tecnai Spirit). The hydrodynamic sizes and zeta potentials were measured by dynamic light scattering (DLS) (Malvern Instruments). The optical properties were characterized by the UV-vis absorbance spectra (Multiskan Sky, Thermo Scientific).
In vitro antigen capture test. A 500 µL OMVs, U@OMVs or mU@OMVs solution (containing 50 µg protein) was mixed with 50 µg of OVA in a phosphate buffer (pH 6.8) and incubated for 4 h under gentle shaking at room temperature. The OVA-adsorbed vesicles were separated from free OVA by ultracentrifugation at 150000 g for 3 h at 4 °C. The pellet was resuspended with 100 µL PBS and their protein concentrations were determined by BCA assay. Then, samples were mixed with loading buffer, and heated at 99 °C for 15 min to denature the proteins. After SDS-PAGE electrophoresis, proteins were stained with coomassie brilliant blue for 2 h and then imaged to further analyze the proteins in these vesicles.
In vitro cytotoxicity. Cytotoxicities of free UNC2025 and mU@OMVs were determined by CCK-8 assays. Briefly, CT26, RAW264.7 or B16F10 cells were seeded in 96-well microplates at a density of 1.5 × 104 cells/well and incubated overnight at 37°C. Then, free UNC2025 or mU@OMVs with different concentrations was added to the wells. After 24 h treatment, cells were incubated with CCK-8 solution for another 1 h. Finally, the absorbance at 450 nm for each well was measured by a UV-spectrophotometry (Multiskan Sky, Thermo Scientific).
Cellular uptake of OMVs, U@OMVs and mU@OMVs. RAW264.7 cells (1 × 105 cells/well) were seeded in a 24-well plate and treated with 100 ng/mL IL-4 to polarize M0 to M2 macrophages for 48 h. Then, the marker of M2 macrophages (CD206) was labeled with PE-conjugated anti-mouse CD206 antibody. Finally, the labeled cells were detected by flow cytometer (Bioscience FACSAria, BD).
To determine the cellular uptake, OMVs were prelabeled with FITC-conjugated LPS antibody and incubated at 37 °C for 1 h. The free antibody was removed by centrifugal filters with MWCO of 300 kDa (Pall). U@OMVs and mU@OMVs was prepared as previously mentioned using prelabeled OMVs. Then, OMVs, U@OMVs or mU@OMVs (30 µg/mL) was added to M2 macrophages and incubated for 4 h, 3 h, 2 h, 1 h, and 0.5 h, respectively. Afterwards, parts of the cells were stained with Hoechst 33342 (100 µM in PBS) for 15 min at 25 °C. The final M2 macrophages were detected by flow cytometer (Bioscience FACSAria, BD) or confocal microscopy (Eclipse-Ti2, Nikon), respectively.
Western Blot analysis of the inhibition of MerTK phosphorylation. RAW264.7 cells (2 × 105 cells/well) were seeded in a 12-well plate and treated with 100 ng/mL IL-4 to polarize M0 to M2 macrophages for 48 h. Then, free UNC2025 or mU@OMVs with indicated dosages was added to M2 macrophages and incubated for 4 h, 2 h, 1 h, and 0.5 h, respectively.
Protein samples extracted from the above cells were separated with SDS polyacrylamide gel electrophoresis and blotted onto nitrocellulose (NC) membrane (Millipore). The membrane was blocked with 5% BSA for 3 h and incubated with anti-MerTK antibody (1:1000), anti-phospho-MerTK (1:2000), or anti-β-Actin antibody (1:10000) overnight at 4 °C. After washing with PBST (phosphate-buffered saline with Tween20) for three times, the membrane was further incubated with a diluted secondary antibody Goat-Anti-Rabbit IgG (HRP) (1:10000) for 2 h. Then the membrane was washed by PBST for three times and visualized with ECL reagent (Thermo Scientific) by Chemiluminescence system (Bio-Rad).
In vitro efferocytosis assay. Apoptosis of B16F10 tumor cells (AC) was induced by 5 µM doxorubicin at 37 °C for 6 h. Exposure of phosphatidylserine on cell surface was assessed using the Annexin V-FITC/DAPI Apoptosis Detection Kit. Apoptotic B16F10 tumor cells were then labeled with 5 µM CFSE at 37 °C for 20 min. M2 macrophages were pre-incubated with PBS, OMVs (3.6 µg/mL), UNC2025 (1 µg/mL), U@OMVs (containing 1 µg/mL UNC2025), mU@OMVs (containing 1 µg/mL UNC2025) 2 h prior to adding CFSE-labeled apoptotic B16F10 cells. After 6 h, 12 h, and 18 h incubation, macrophages in mixed cells were labeled with APC-conjugated anti-CD11b antibody. Then, cells were collected and analyzed by flow cytometer (Bioscience FACSAria, BD) or confocal microscopy (Eclipse-Ti2, Nikon). The supernatants from co-culture were collected to examine the HMGB1 level by ELISA.
The protein concentration of the above supernatants of M-AC + mU@OMVs groups after the 18 h culture was determined by Micro BCA Protein Assay Kit. Then, a 1 mL mU@OMVs solution (containing 200 µg protein) was mixed with 800 µg of supernatants in a phosphate buffer (pH 6.8) and incubated under gentle shaking at room temperature. The proteins-adsorbed mU@OMVs was separated from free proteins by ultrafiltration at 5000 g for 10 min and washed for three times. The obtained proteins-adsorbed mU@OMVs was collected, and a proteomics experiment was performed to determine the adsorbed peptide/protein on the mU@OMVs.
Dendritic cellular uptake of OVA-adsorbed mU@OMVs. Murine bone marrow-derived dendritic cells (BMDCs) from marrow cavities of femurs and tibias of C57BL/6 mice were cultivated in plates with a medium containing 20 ng/mL GM-CSF and 20 ng/mL IL-4 for 7 days. Then, BMDCs were seeded in a 24-well plate at a density of 1 × 106 per well.
mU@OMVs solution was mixed with PE-labeled OVA in a phosphate buffer (pH 6.8) and incubated for 4 h under gentle shaking at room temperature. For uptake studies, BMDCs were incubated with free PE-labeled OVA or mU@OMVs pre-mixed with PE-labeled OVA (concentration of OVA=10 µg/mL) for 12 h at 37 ºC. Then, BMDCs were labeled with FITC-conjugated anti-CD11c antibody. The Uptake was measured by the mean fluorescence intensity (MFI) of the FITC signal in CD11c+ cells via flow cytometry (Bioscience FACSAria, BD). For uptake imaging, DCs were also stained with Hoechst 33342 and then imaged with a confocal microscope (Eclipse-Ti2, Nikon).
Analysis of BMDCs maturation and T cell activation. Murine bone marrow-derived dendritic cells (BMDCs) from marrow cavities of femurs and tibias of C57BL/6 mice were cultivated in plates with a medium containing 20 ng/mL GM-CSF and 20 ng/mL IL-4 for 7 days. Then, BMDCs were seeded in a 24-well plate at a density of 5 × 105 per well.
The supernatants of necrotic B16F10 cells obtained above were incubated with OMVs, U@OMVs or mU@OMVs for 4 h under gentle shaking at room temperature, and respectively added into BMDCs for 24 h (final concertation of OMVs=5 µg/mL). Then, BMDCs were collected and stained with fluorescent antibodies of CD11c, CD80, CD86, MHCI and MHCII individually, and measured by flow cytometry (Bioscience FACSAria, BD). For the quantitative analysis of cytokines, the supernatants of BMDCs were collected and analyzed using IL-6, IL-12p40, and TNF-α ELISA kits.
Splenocytes were isolated from the spleens of C57BL/6 mice (aged 6−8 weeks) and CD8+ T cells were isolated using a CD8+ no-touch isolation kit (Miltenyi Biotec) according to the manufacturer’s guidelines. Afterwards, T cells were coincubated with above treated BMDCs for 24 h at a ratio of 1:4. Then, T cells were collected and labeled with APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD8 as well as PE-conjugated anti-mouse CD69, and analyzed by flow cytometer (Bioscience FACSAria, BD).
In vivo apoptosis and immune activation assay. C57BL/6 mice were subcutaneously incubated with B16F10 cells (1 × 106). When tumor volumes reached 100 mm3, mice were randomly divided into five groups and peritumorally injected with 100 µL of PBS, UNC2025, OMVs, U@OMVs or mU@OMVs every 3 days for two times (OMVs dose: 10 μg per mouse; UNC2025 dose: 2.7 μg per mouse). After 48 h, mice were sacrificed, and the draining lymph nodes (DLNs), spleens and tumor tissues were surgically collected for further investigation. Sera were collected for quantitative analysis of IL-6, IL-12p40, TNF-α, IFN-γ and HMGB1. Tumors were fixed in 4 % paraformaldehyde solution and sectioned into slices for the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the cleaved-Caspase 3 (c-Casp3) staining. For immune cell analysis, DLNs, spleens and tumors were cut into small pieces and then homogenized in cold PBS to form the single-cell suspension. The obtained cells were divided into several parts to analyze different immune cell types, respectively. For DC maturation analysis, cells from DLNs were stained with CD11c, CD80, CD86, MHC-II and MHC-I antibodies. For CD8+ T cells analysis, cells from spleens were stained with CD3, CD4 and CD8 antibodies. For cytotoxic T lymphocytes (CTL) analysis, cells from tumors were stained with CD3, CD8 and IFN-γ antibodies. Then, stained cells were washed with PBS three times and measured by flow cytometer (Bioscience FACSAria™ III, BD).
Antigen delivery of mU@OMVs in vivo. 5 μg Cy5-labeled OVA were mixed with 10 μg OMVs, U@OMVs or mU@OMVs, and incubated for 4 h under gentle shaking at room temperature. C57BL/6 mice were subcutaneously incubated with B16F10 cells (1 × 106). When tumor volumes reached 200 mm3, mice were randomly divided into five groups and intratumorally injected with 100 µL of PBS, free Cy5-labeled OVA, mixture of OMVs and Cy5-labeled OVA, mixture of U@OMVs and Cy5-labeled OVA or mixture of mU@OMVs and Cy5-labeled OVA, respectively (OVA dose: 5 μg per group, multipoint injection). After 24 h, the fluorescence of DLNs was monitored by an in vivo imaging system (IVIS Spectrum, PerkinElmer).
In vivo antitumor activity. To establish a bilateral tumor model, 1 × 106/100 μL B16F10 tumor cells in PBS were subcutaneously transplanted into the right flank of C57BL/6 mice as the primary tumor. After 11 days, to form the abscopal tumor, 1 × 106 B16F10 were subcutaneously injected into the left flank. When the primary tumor volume reached about 100 mm3, mice were randomly divided into five groups, and peritumorally injected with 100 µL of PBS, UNC2025, OMVs, U@OMVs or mU@OMVs every 3 days for two times at primary tumors (OMVs dose: 10 μg per mouse; UNC2025 dose: 2.7 μg per mouse). The length (L) and width (W) of the subcutaneous tumors and the body weights were measured every other day after the first administration. The tumor volumes were calculated by the formula of (L × W2)/2. When the tumor volume was larger than 1500 mm3, the mice were euthanized. At the end of therapy, some mice were sacrificed and the main organs (heart, liver, spleen, lung and kidneys) as well as bilateral tumors were harvested, fixed in 4 % paraformaldehyde solution and sectioned into slices for the hematoxylin and eosin (H&E) or TUNEL staining. For safety evaluation, the serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), lactate dehydrogenase (LDH) and creatinine (CREA) at the endpoint of the experiment were analyzed.
For CT26 tumor inhibition, BALB/c mice were subcutaneously incubated with CT26 tumor cells (1 × 106). When tumors reached about 100 mm3, mice were randomly divided into five groups, and peritumorally injected with 100 µL of PBS, UNC2025, OMVs, U@OMVs or mU@OMVs every 3 days for two times (OMVs dose: 10 μg per mouse; UNC2025 dose: 2.7 μg per mouse). The tumor volumes were calculated by the formula of (L × W2)/2. The body weight was measured every 2 days after the first administration.
Evaluation of metastasis and recurrence. To construct the lung metastasis model, 1 × 106/100 μL B16F10 tumor cells were transplanted into the flank of the mice. When tumors reached about 100 mm3, mice were randomly divided into five groups, and peritumorally injected with different formulations like before. After 24 h, the mice were intravenously injected with B16F10 cells (3 × 105). After another 15 days, the mice were killed, and their lungs were excised. Lung metastasis nodules were then manually counted, and lung tissue sections were subjected to H&E staining.
To construct the recurrence model, BALB/c mice were subcutaneously transplanted with 1 × 106 CT26 tumor cells. When tumors reached about 100 mm3, mice were randomly divided into five groups, and peritumorally injected with different formulations like before. On day 15, tumors in different groups were removed by surgery. On day 19, the mice were challenged by subcutaneous 1 × 106 CT26 tumor cells. Tumor growth was evaluated every four days. On day 60, splenocytes were isolated and the percentage of memory T cells (CD3+ CD8+ CD44+ CD62L−) was detected by flow cytometry (Bioscience FACSAria, BD).
Statistical analysis. Data were reported as mean ± standard error from three independent trials unless otherwise indicated. Comparison between groups was analyzed with Student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was determined with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and n.s., not significant.
Data availability
All the other data supporting the findings of this study are available within the article and its Supplementary Information file and from the corresponding author upon reasonable request.