Cell Culture: The macrophage cell line RAW264.7 and the bladder cancer cell line MB-49 were purchased from the American Type Culture Collection (Rockville, MD, USA). RAW264.7 cells were cultured in DMEM, MB-49 cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA). All media supplemented with 10% fetal bovine serum (Dainippon Pharmaceutical, Tokyo, Japan), at 37 °C in a humidified 5% CO2 atmosphere. M1 macrophages were induced by the addition of 100 ng/mL lipopolysaccharide (LPS) and 20 ng/ml IFN-γ. M2 macrophages were induced by the addition of 20 ng/mL IL-4 (Invitrogen, CA, USA).
Preparation of M0 NV, M1 NV and M1 NV-R848: To obtain M0 NV or M1 NV, we suspended RAW264.7 cells (M0 macrophages) or LPS/IFN-γ-treated RAW264.7 cells (M1 macrophages) in PBS at a concentration of 5×106 cells/mL. Nanometer-sized extracellular vesicles were obtained by successively extruding 11 times through polycarbonate membrane filters (Whatman) with pore sizes of 1 µm, 400 nm, and 200 nm using a microextruder (AvantiPolarLipids). The extracellular vesicles were then ultracentrifuged at 100,000 g for 2 h at 4 °C in a density gradient formed by 10 and 50% OptiPrep layers, and the extracellular vesicles obtained from the interface of the layers were further ultracentrifuged at 100,000 g for 2 h at 4 °C to obtain M0 NV or M1 NV. To obtain M1 NV-R848, 10 mg M1 NV (10 mL in PBS) was incubated with 4 mg R848 (1 mL in DMSO) (R848 purchased from DC Chemicals, Shanghai, China) and mixed for 12 h at 37 °C on a rotator. Unencapsulated R848 was removed and M1 NV-R848 was concentrated using a 30 kDa centrifugal filter. The collected M1 NV-R848 were rinsed by centrifugal filtration with an equal volume of PBS 3 times. The amount of free R848 that remained in the dialysis water was analyzed by high performance liquid chromatography (HPLC) to obtain the loading content (LC) and loading efficiency (LE) of M1 NV. The drug LC and LE for M1NV-R848 were calculated using the following equations: LC(%) =(total amount of R848 added - free R848)/ amount of M1NV *100%, LC(%) =(total amount of R848 added - free R848)/ total amount of R848 added *100%.
LC-MS/MS and Bioinformatics Analysis：Proteins were extracted from macrophages and vesicles, then subjected to trypsin digestion and quantitative analysis. The peptides were subjected to NSI source processing and analyzed by tandem mass spectrometry (MS/MS) using Q Exactive TM Plus (Thermo Scientific) coupled online to UPLC. The MS/MS data were processed using MaxQuant with Integrated Andromeda search engine (v.22.214.171.124). To analyze these data, bubble plots and KEGG pathway-based heat maps were employed in order to better identify and visualize differentially expressed proteins and possible signaling pathways.
Characterization of M0 NV, M1 NV and M1 NV-R848: The morphology and particle size of M0 NV, M1 NV and M1 NV-R848 were measured by transmission electron microscopy (TEM) (Hitachi H-7650). The dynamic light scattering (DLS) of the M0 NV, M1 NV and M1 NV-R848 was measured using a Brookhaven apparatus (Brookhaven Instruments, USA). The colloidal stability of M1 NV-R848 was investigated in PBS and serum at 37 °C by measuring their mean diameter with DLS. The amount of R848 loaded in M1 NV was determined by the reverse-phase ultrahigh-performance liquid chromatography (UHPLC) method according previous report. The percentages of release of R848 were calculated according to a formula, release percentage (%) = Mr/Mt, where Mr is the amount of released R848, and Mt is the total amount of loaded R848. Drug release kinetics of R848 from M1 NV-R848 were evaluated utilizing 20 kDa dialysis cassettes (Thermo Scientific). M1 NV-R848 were suspended in 10 mL of PBS at 37 ℃. The release system was maintained at 37 °C under shaking. The release medium was sampled with 0.5 mL each time, and UHPLC was used to determine the percentage of released R848. The sample was added back to the original release system.
mRNA Quantification of NVs and Cells: Total RNA was extracted from macrophages using 1 mL of Trizol (Qiagen Valencia, CA, USA). The total RNA concentration was determined using a NanoDrop spectrometer (ND-2000, NanoDrop Technologies, USA). Six hundred nanograms of total RNA from each sample were reverse-transcribed into cDNAs, and SYBR green-based qRT-PCR was performed using a Step One Plus real-time PCR system (Applied Biosystems, USA) with TOPreal qPCR 2X PreMIX (Enzynomics, Finland). Cycling conditions were the following: initial denaturation at 95°C for 15 min, followed by 45 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s. All of the data were analyzed using the comparative Ct method. Three samples were analyzed per group.
In Vitro Cellular Uptake of NVs after Treatment with NVs: M2 macrophages were prepared by the IL-4 treatments of RAW264.7 cells. M2 macrophages were allowed to attach to culture plates containing 10% (v/v) FBS-containing medium for 24 h. Subsequently, DiR-labeled M0 NV, M1 NV, M1 NV-R848 were added into the culture at a concentration of 50 µg/mL and incubated for 4 h. Cellular uptake of NVs was evaluated using fluorescence microscopy. Additionally, 4 h after the treatment with DiR-labeled NVs, M2 macrophages were washed with PBS and analyzed with FACS. Three samples were analyzed per group.
In Vitro Analyses of Macrophage Polarization: M2 macrophages were treated with M0 NV, M1 NV, R848 and M1 NV-R848 for 24 h. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and washed with PBS, subsequent staining was performed using primary antibodies against CD163 (Santa Cruz Biotechnology, CA, USA), the samples were then incubated in PBS containing rhodamine-conjugated secondary antibody (Jackson-Immunoresearch) for 1 h at room temperature, all samples were mounted with mounting solution containing 4 ', 6-diamidino-2-phenylindole (DAPI, VectorLaboratories, Burlingame, CA, USA) to stain nuclei and using a fluorescence microscope (Olympus, Tokyo, Japan). Moreover, qRT-PCR was performed to determine the expression of M1 (iNOS, IL-6 and TNF-α), M2-related genes (IL-10, Fizz-1 and IL-4), angiogenesis factor VEGF and metastatic factor CCL-18. To further confirm the expression of M1 marker IL-6 and M2 marker IL-4 in M2 macrophages treated with M0 NV, M1 NV, R848 and M1 NV-R848 for 24 h, cytokine secretion levels were analyzed using mouse IL-6 and IL-4 ELISA kits (R&D Systems, MN, USA) according to the manufacture instructions. Each group was repeated three times.
The establishment of orthotopic bladder cancer model: Female C57BL/6 mice (8 weeks old, weight 18-20 g) were purchased from the Model Animals Research Center, Nanjing University. All mice were used in accordance with the Institutional Animal Care Regulations and Use Committee (IACUC) of Nanjing Drum Tower Hospital, Medical School of Nanjing University. All animal experiments were approved by the Institutional Animal Care Committee of Jiangsu Province and the Ethics Committee of Nanjing Drum Tower Hospital, Medical school of Nanjing University. An orthotopic bladder cancer model was established in C57BL/6 mice with a minimally invasive method based on our previously published article. Briefly, we made a longitudinal small incision (approximately 2 mm) on the skin of the lower abdomen of the mouse, found the bladder, and carefully clamped the bladder using smooth forceps. MB-49luc cells were injected into the bladder wall using a 1-mL syringe, the bladder was returned to its original position, and the abdominal incision was closed. One week later, tumor growth in the bladder was monitored using an IVIS Spectrum computed tomography system (PerkinElmer, Waltham, MA, U.S.).
In Vivo Imaging of M1NV-R848 in orthotopic bladder cancer model: The biodistributions of M0 NV, M1 NV and M1 NV-R848 were investigated using an IVIS Spectrum computed tomography system (PerkinElmer, Waltham, MA, U.S.) after intravenous injections of M0 NV, M1 NV and M1 NV-R848 into orthotopic bladder cancer model. Briefly, 100 µg M0 NV, M1 NV and M1 NV-R848 were labeled with DiR according to the manufacturer's instructions, suspended in 100 µL of PBS, and intravenously injected via the tail vein. At 24 h after injection, the mice were sacrificed, and NIR fluorescence images of the major organs (heart, liver, spleen, lung, and kidney) and tumor were acquired at a 748 nm excitation wavelength and a 780 nm emission wavelength. Then the relative fluorescence intensities of the organs and tumor were quantified using the software Living Image 3.1.
For organ toxicity analysis, MB-49 tumor-bearing mice were intravenously injected with 100 µL PBS or 50 µg M1 NV-R848 suspended in 100 µL of PBS at day 1, 6 and 13 days. Major organs (heart, liver, spleen, lung, and kidney) were retrieved at 20 days after the first injection and fixed with 4% paraformaldehyde in PBS. The samples were embedded in OCT compound, sectioned at a thickness of 4 µm, stained using H&E and imaged using an optical microscope.
Comparison of the macrophage polarization induction effects in M0 NV, R848, M1 NV and M1 NV-R848 in vivo: The orthotopic bladder cancer model was established in C57BL/6 mice. One week later, the bladder tumor was confirmed using an IVIS Spectrum computed tomography system. Mice were intravenously injected with PBS, M0 NV (5mg/kg), M1 NV (5mg/kg), R848 (0.5mg/kg) and M1 NV-R848 (M1 NV:5mg/kg, R848 0.5mg/kg) at day 0, day 3 and day 6. At day 20, tumors were surgically excised and frozen sections of tumors were collected. IHC staining for M1 and M2 markers was performed. Sections were treated with sodium citrate buffer (10 mM Sodium citrate and 0.05% Tween 20, pH 6.0) for 10 min at 85℃ for antigen retrieval after hydration. Prior to staining, the tissue sections on slides were blocked with 5% (v/v) normal goat serum (Gibco) and incubated with primary antibodies against iNOS (Abcam), and arginase 1 (Abcam) for 18 h at 4°C. The sections were washed three times with PBS and were incubated for 1 h with rhodamine-conjugated secondary antibodies. After washing with PBS, the slides were mounted with a mounting medium (VectaMount mounting medium, Vector Labs Inc., Burlingame, CA, USA). For FACS, tumor tissues were mechanically cut into small pieces and then next enzymatically digested (DMEM containing 1.5 mg/mL collagenase I, 125 units/mL hyaluronidase, 0.1 mg/mL DNAase I) to generate single-cell suspensions. Then, the single-tumor cell suspensions were incubated with antibodies for 30 min on ice followed by FACS.
M1 NV-R848 potentiate antitumor effects of PD-L1 inhibitors (aPD-L1) in orthotopic bladder cancer model: The orthotopic bladder cancer model was established in C57BL/6 mice. One week later, the bladder tumor was confirmed using an IVIS Spectrum computed tomography system. Mice were intravenously injected with PBS, aPD-L1 (5mg/kg), M1 NV-R848 (M1 NV: 5mg/kg, R848: 0.5mg/kg) and aPD-L1 (5mg/kg) combined with M1 NV-R848 (M1 NV: 5mg/kg, R848: 0.5mg/kg) at day 0, day 3 and day 6. At day 20, Tumor tissues were removed from euthanized mice and were homogenized after grinding with a scalpel. For IHC and H&E staining, tumor tissues were fixed with formaldehyde, embedded in paraffin, and sliced at a thickness of 4 µm. The sections of the tumor tissues were stained with H&E and were examined using an optical microscope (Olympus, Tokyo, Japan). IHC staining with Ki67 was performed. Tumor tissues were enzymatically digested to generate single-cell suspensions. Then, the single-tumor cell suspensions were incubated with anti-CD8A antibody for 30 min on ice followed by FACS.
Therapeutic efficacy of NVs with or without aPD-L1 in subcutaneous xenograft model of bladder cancer: Female C57BL/6 Mice were anesthetized and MB-49 cells (5×106 cells in 100 µL PBS per mouse) were subcutaneously injected into a flank of the mice. The tumor volume (V) was estimated according to an ellipsoidal calculation, whereby V=a×b2×0.5, where a is the largest and b is the smallest diameters of the tumor ellipsoid. When the tumor size reached 70 mm3, either M0 NV (5mg/kg), M1 NV (5mg/kg), R848 (0.5mg/kg), M1 NV-R848 (M1 NV: 5mg/kg, R848: 0.5mg/kg) and anti-PD-L1 mAb (5mg/kg) (aPD-L1, BioXcell) only, or a combination of M1 NV (5mg/kg) and aPD-L1 (5mg/kg), R848 (0.5mg/kg) and aPD-L1 (5mg/kg), M1 NV-R848 (M1 NV: 5mg/kg, R848: 0.5mg/kg) and aPD-L1 (5mg/kg) were injected intravenously at day 0, day 3 and day 6. The tumor volume was determined every three days. At day 20, tumors were removed from euthanized mice, The sections of the tumor tissues were stained with H&E and were examined using an optical microscope (Olympus, Tokyo, Japan).
Statistical Analyses: Data were presented as mean ± standard deviation (SD). Statistical comparisons were performed using unpaired Student’s t-test for two group comparisons, one way analysis of variance (ANOVA) for comparisons of more than three groups using Prism 6 (GraphPad software). Differences were considered statistically significant when p<0.05.