Materials
Distearoyl Phosphatidylcholine (DSPC) (S50960, Shanghai Yuan Ye), Phosphatidylcholine (PC) (Y46792, Shanghai Yuan Ye), Cholesterol (Chol) (S11040, Shanghai Yuan Ye), DSPE-PEG-2000 (S25991, Shanghai Yuan Ye), DSPE-PEG-mannose (SJ6784, Shanghai Yuan Ye). HPV16 E744 − 62 (44QAEPDRAHYNIVTFCCKCD62) was purchased from GL Biochem. MnC4H6O4•4H2O was purchased from Aladdin. Cell Counting Kit-8 (CCK8 kit) was acquired from Glpbio (GK10001, USA). Unless otherwise specified, all chemicals were obtained from commercial sources and used without further purification. DMEM medium, RPMI 1640 medium, penicillin/streptomycin (P/S), 0.25% trypsin-EDTA, phosphate buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Gibco (USA).
Mice and cell lines
All cells were purchased from American Type Culture Collection (ATCC, USA). C57BL/6 mice (female, 4 weeks old) were obtained from the Guangdong Provincial Experimental Animal Center (Guangzhou, China) and STING−/− mice were gifted form Dr. Cunbao Liu from the Institute of Medical Biology, Chinese Academy of Medical Sciences (IMBCAMS). All the mice were raised in Specific Pathogen Free (SPF) animal facilities. All animal experiments were conducted in accordance with the guidelines provided by Guangzhou Medical University IACUC under the ethics approval GY-2022-188.
Preparation of EM@LNP vaccines
In briefly, liposomes were fabricated via the thin-film hydration technique. For the blank liposome devoid of loading any payload, DSPC, PC, cholesterol, DSPE-PEG2k, DSPE-PEG-Mannose was dissolved in chloroform/methanol solvent (V: V = 1:1). This mixture was then subjected to rotary evaporation under vacuum to remove the solvent for 1 h, resulting in a dried lipid film. The dried lipid film was subsequently rehydrated using PBS for 1 h. Then, monodispersed liposomes were obtained by extruding the suspension using an Avanti mini-extruder equiped with 200 nm polycarbonate filters. EM@LNP (liposome incorporating E7 and Mn2+), E7@LNP (liposome incorporating E7) nanoparticles containing drugs were accomplished by incorporating the respective ingredients into the PBS during the hydration step, following the same procedures as described above.
EM@LNP was freeze-dried using a freeze dryer (Scientz-10ND, Ningbo) according to different pre-freezing programs as shown in Table 1. The as-prepared liposomes were designated as EM@LNPfresh. Afterwards, the EM@LNP was characterized with a JEM-1400PLUS transmission electron microscope (TEM) for morphology examination, and the Malvern zetasizer (Nano-ZS90) for dynamic light scattering measurement. The encapsulation efficiencies of E7 and Mn2+ were determined by bicinchoninic acid (BCA) assay kit (20201ES76, Yeasen Bio.) and inductively coupled plasma mass spectrometry (ICP-MS), respectively.
In vitro cellular uptake
In vitro cell uptake was studied by confocal laser scanning microscopy (CLSM) and flow cytometry. For CLSM, DC2.4 cells were plated in glass-bottom dishes at a concentration of 1 × 104 cells. Then, free E7 (FAM-E7), EM@LNPfresh.or EM@LNP were added to cells. After a series of incubation durations, cells were stained with Hoechst 33,342 (C1025, Beyotime) following the manufacturers’ instructions. The cells were then visualized using a Zeiss LSM 800 confocal microscope. For flow cytometry, DC2.4 cells were seeded on 6-well plates, and then treated with FAM-E7. Post-incubation at various time intervals, cells were collected and rinsed twice in preparation for analysis using a CytoFLEX Flow Cytometer (Beckman, USA).
Viability assay and activation of BMDCs
As previously described, bone marrow-derived DCs (BMDCs) were obtained from the tibia and fibula of female C57BL/6 mice, inoculated into 96-well plates at 1 × 105 cells/well, and incubated with different concentrations of EM@LNP for 24 h, respectively. Finally, the cell viability was detected using CCK8 cell viability kit.
The activation of DCs was evaluated by examining the expression levels of cytokines and cell surface markers. The BMDCs were incubated with PBS, LNPs, free E7, free E7 + Mn2+, E7@LNP and EM@LNP for 24 h. The cytokine concentrations in the supernatants were measured with enzyme-linked immunosorbent assay (ELISA) according to a previously described method. Antibodies were provided by Elabscience Ltd., surface molecular expression of DCs was analyzed by flow cytometry using Pcy5.5-CD11c, PE-CD80, APC-CD86, APC-H-2Kb (MHC I), and FITC-CD40 antibodies. Data acquisition was performed using a CytoFLEX flow cytometer (Beckman, USA) and analysis was conducted using FlowJo software. The gating strategies applied are depicted in Supplementary Figures S4–5.
EM@LNP vaccine activated cGAS-STING pathway in BMDCs
BMDCs were treated separately with PBS, LNPs, free E7, free E7 + Mn2+, E7@LNP, EM@LNPfresh. and EM@LNP. Following treatment, cells were lysed using RIPA lysis buffer (20101ES, Yeasen Bio.) containing protease and phosphatase inhibitors (ST505, Beyotime). Protein extraction from cells was achieved by centrifugation at 12,000 rpm for 15 min. Protein concentration was determined using the BCA protein assay kit (20201ES, Yeasen Bio.). Subsequently, equal amounts of protein were separated on a 12% SDS-PAGE gel by electrophoresis (20326ES, Yeasen Bio.), and then transferred to a PVDF membrane (1620177, BioRad). The membrane was blocked for 1 h in a TBST buffer containing 5% blotting grade (P0216, Beyotime). After blocking, the membrane was incubated overnight at 4 °C with primary antibodies against Phospho-STING (72971, CST), Phospho-TBK1 (5483, CST), Phospho-IRF-3 (29047, CST), STING (13647, CST), TBK1 (38066, CST), and IRF-3 (4302, CST). The PVDF membrane was washed three times for 30 min each and incubated at room temperature with HRP-conjugated secondary antibodies (33101ES60, 33201ES60, Yeasen Bio.) for 2 h. Chemiluminescence was used to develop the protein bands with a gel imaging system (AI600), applying 200 µL of ECL chemiluminescent reagent (P0018S, Beyotime) was applied to the top of the membrane. Stripping buffer (P0025B, Beyotime) and the process was repeated. GAPDH (GB12002, Servicebio) was served as an internal loading control.
In vivo immunization and the flow cytometry assay of antigen-specific T cell response
C57BL/6 mice were immunized via subcutaneous injection with different formulations of vaccines on days 1 and 7: (1) PBS, (2) E7, (3) E7 + Mn2+, (4) E7@LNP and (5) EM@LNP equivalent to the dose of E7 at 0.1 µg/mouse. On day 14, tumor challenge was conducted by subcutaneous inoculation with TC-1 tumor cells (1 × 106) on the right flank. Tumor sizes and body weight were tracked every two days. Tumor volume was calculated as Volume = (length× width× width)/2. On day 29, mice were euthanized, and lymphocytes from lymph nodes and spleen were extracted using PBS. These cells were then subjected to a lymphocyte separation process followed by analysis via flow cytometry.
Biodistribution and biosafety study
To assess the in vivo antigen stability and the pattern of EM@LNP migration to the draining LNs, C57BL/6 mice were subcutaneously (s.c.) injected with PBS, free FAM-E7, FAM-EM@LNP. The IVIS spectrum imaging system (PerkinElmer, USA) injected the FAM-labeled E7 signal at the immunization site under the conditions of the excitation filter of 470 nm and emission filter of 535 nm. Six hours after immunization, major organs including heart, liver, spleen, lung, kidney, and inguinal lymph nodes were obtained from sacrificed mice for ex vivo FAM fluorescence imaging.
In vivo tumor models and combination iCBT
C57BL/6 mice were subcutaneously injected with TC-1 cells (1 × 106) on the right flank. On day 7, mice were randomly divided into 6 groups (n = 6) and vaccinated with (1) PBS, (2) αPD-1, (3) E7 + Mn2+, (4) EM@LNP, (5) E7 + Mn2+ + αPD-1, (6) EM@LNP + αPD-1 equivalent to the dose of E7 at 1 µg/mouse and αPD-1 (i.p. 100 µg/mouse). Vaccines were subcutaneous injection on days 8 and 15. Again, mouse αPD-1 (BE0146, BioXcell) were administered intraperitoneally on days 9 and 16, followed by monitoring tumor volume and body weight monitoring as above. Mice were euthanized if the tumor volume exceeded 2,000 mm3. On day 24, mice were sacrificed, tumors, spleens, and LNs were collected for the following analysis.
Gene expression in the tumor microenvironment
Tumor RNA was isolated with TRIeasyTM reagent (10606ES, Yeasen Bio.). cDNA synthesis was performed using a cDNA synthesis kit (11142ES, Yeasen Bio.). β-actin was used as the reference gene. The expression of target genes was evaluated using RT-qPCR with StepOne Plus instrument (BI, USA) and SYBR Green reagent (11203ES, Yeasen Bio.). Primer sequences can be found in Supplementary Table S1. Tumor protein was analyzed as described in 2.6.
Postsurgical tumor model and combination immunotherapy
A density of 1 × 106 TC-1 cells were subcutaneously injected into C57BL/6 mice on right flank, 11 days after inoculation, anesthetized the mice and the tumors were resected with sterile instruments, leaving about 20 mm3 of residual tumor tissue. Three days after surgery, tumor volume and body weight were monitored as above. On day 17, mice were randomly divided into 4 groups (n = 6) and treated with (1) PBS, (2) αPD-1, (3) EM@LNP, (4) EM@LNP + αPD-1 equivalent to the dose of E7 at 1.5 µg/mouse. Vaccinations were done by subcutaneous injection on days 17 and 24. For ICB combination therapy group, mouse αPD-1 was administered intraperitoneally on days 18 and 25. Mice were euthanized when the tumor volume exceeded 2,000 mm3.On day 33, mice were sacrificed, tumors, spleens, and LNs were collected for the following immune analysis.
STING-/- mice tumor models and combination iCBT
STING knockout mice were subcutaneously injected with TC-1 cells (1 × 106) on the right flank. On day 7, mice were randomly divided into 6 groups (n = 6) and vaccinated with (1) PBS, (2) αPD-1, (3) E7@LNP + αPD-1, (4) EM@LNP + αPD-1 equivalent to the dose of E7 at 1.5 µg/mouse. Vaccines were subcutaneously injected on days 8 and 15. Again, mouse αPD-1 (BE0146, BioXcell) were administered intraperitoneally on days 9 and 16, followed by monitoring tumor volume and body weight monitoring as above. Tumor volume was calculated as (length× width× width)/2. On day 24, mice were sacrificed, tumors, spleens, and LNs were collected for the following analysis.
Flow cytometric analysis of tumor immune microenvironment
Tumors, lymph nodes, and spleens were harvested and processed for assessment of the anti-tumor immune response via flow cytometry using the CytoFLEX platform. In brief, the collected tissues were dissociated into single-cell suspensions, and red blood cells were lysed using a red blood cell lysis buffer (R1010, Solabio). The cells were then blocked with 0.1% BSA (36101ES, Yeasen Bio.) in PBS followed by a 1-hour incubation with specific antibodies at room temperature. For the characterization of T cells and DCs in tumors and spleens, staining was performed using anti-mouse CD3-Pcy5.5 (F1013J, Elabscience), anti-mouse CD4-FITC (F1097C, Elabscience), anti-mouse CD8a-PE (F1104D, Elabscience), anti-mouse CD11b-FITC (F1081C, Elabscience), anti-mouse Gr-1-PE (F1120D, Elabscience), anti-mouse IFN-γ-APC (F1101E, Elabscience), anti-mouse Foxp3-PE (F1238D, Elabscience), anti-mouse CD62L-Pcy5.5 (F1011J, Elabscience) and anti-mouse CD44-APC (F1100E, Elabscience). Data acquisition was performed using CytExpert software, and analysis was conducted using FlowJo software. The gating strategies are detailed in Supplementary Figures S8–10.
Immunohistochemistry (IHC) and immunofluorescence (IF)
The tumor tissue sections were stained with hematoxylin and eosin (H&E) and TUNEL to detect histological changes. For IHC analyses on immune cell infiltration, the tissue sections were incubated with an anti-CD8 or anti-Foxp3 antibody (Servicebio). Finally, images were photographed using fluorescence microscopy (Nikon).
Statistical analysis
All quantitative values are reported as the mean ± standard error of the mean (SEM). Data were evaluated by one- or two-way analysis of variance (ANOVA) for comparison of multiple groups using GraphPad Prism 5 software. *p < 0.05, **p < 0.01, ***p < 0.001 were considered as significant difference.