Materials
Manganese Chloride (MnCl2) and Spermidine were purchased from Aladdin, (Shanghai, China). Hydrogenated soybean phospholipids (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-Polyethyleneglycol-2000 (DSPE-mPEG2000), and cholesterol (CHOL) were obtained from AVT (Shanghai, China). Cell Counting Kit-8 (CCK8), Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit, ROS Detection Kit, ATP Detection Kit were supplied from Beyotime (Shanghai, China). Calreticulin (CRT) Antibody Rabbit Polyclonal PerCP Conjugated was obtained from StressMarq Biosciences (Victoria, Canada). Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and Lipopolysaccharide (LPS) were purchased from Novoprotein (Suzhou, China). [Ru(dpp)3]Cl2 was purchased from Alfa Aesar (Shanghai, China). HIF-1α Mouse Monoclonal Antibody was obtained from Affinity Biosciences (Liyang, China). Alexa Fluor 488-anti HMGB1, APC-PD-L1, APC-CD11c, FITC-CD80, Brilliant Violet 421™ MHC-II (IA/IE) and PE-CD86, APC-CD3, FITC-CD4, PE-Cy7-CD8α, PE-Foxp3, FITC-CD11b, PE-Cy7-Gr-1, FITC-PD-1 were supplied from BioLegend (California, USA). GAPDH antibody, β-actin antibody, phospho-STING antibody, phospho-IRF3 antibody were purchased form Abmart (Shanghai, China). STING antibody, IRF3 antibody were obtained from MCE (New Jersey, United States). The Hydrogen Peroxide Assay Kit was purchased from Abcam (Cambridge, UK). ELISA kits of TNF-𝛼, IL-6, IFN-γ were purchased from Dakewe (Shenzhen, China). ELISA kit of IFN-β was obtained from Cusabio (Wuhan, China). Seahorse XF microplates and kits were purchased from Agilent (USA).
Cell lines
Murine 4T1 breast cancer cells were acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Bone marrow-derived monocytes were isolated from the femurs and tibias of female BALB/c mice and differentiated into BMDC following a standard procedure [37]. Briefly, BMDC was induced in RPMI 1640 medium containing 20 ng/mL GM-CSF and 10 ng/mL IL-4, with partial medium changes every 2 days. All cells were maintained in an incubator containing 5% CO2 at 37 ℃.
Preparation and characterization of L@Mn@SPD
Ethanol injection method was used to prepare Mn and SPD co-loaded liposomes (L@Mn@SPD) from optimized parameters of previous literature [28]. Briefly, 95.8 mg HPSC, 31.9 mg cholesterol, 31.9 mg DSPE-mPEG2000 were co-dissolved in 2 mL anhydrous ethanol. 1mL organic phase was dropwise added into 9 mL 200mM MnCl2 under 500 rpm stirring at 55 °C. Keep stirring the mixture solution for 10 min and then the liposomes were extruded through a 200 nm polycarbonate membrane using an Avanti Polar Lipids extruder (Alabaster, USA). Subsequently, liposomes were placed in a dialysis bag (MWCO: 300KD) and then placed in ultrapure water to remove ethanol and Mn2+ outside the liposomes to obtain L@Mn. SPD solution was injected into L@Mn (Mn and SPD molar ratio 1:3) under 500 rpm stirring at 55℃ for 30 min. The unencapsulated SPD was subsequently removed by dialysis, resulting in the formation of L@Mn@SPD.
The liposomes were characterized for particle size, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS, Malvern Panalytical, UK). The morphology of liposomes was photographed by transmission electron microscopy (TEM, Talos L120C, FEI, USA). The elemental mapping of liposomes was measured by Field Emission Transmission Electron Microscope (FETEM, Tecnai G2 F20 X-TWIN, FEI, USA). The concentration of manganese was determined using inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent 5110, USA). The stability of liposomes was assessed by observing variations in size in pH 7.4 PBS at specified intervals. XPS (Thermo Scientific K-Alpha, USA) was employed to evaluate the valence state of Mn. The drug-loading capacity (DL%) and encapsulation efficiency (EE%) of liposomes were calculated by ICP-OES and Mouse spermidine ELISA assay kit (COIBO BIO, China) detection. In vitro drug release was assessed by dialysis. L@Mn@SPD was placed in a dialysis bag containing 30 mL of dialysate, and 0.5 mL of the dialysate was collected at predetermined time points for analysis. Fresh dialysate (0.5 mL) was added to replace the removed volume.
Cell viability assay
The cell viability assay was conducted using the CCK8 method. 4T1 cells, BMDC and Jurkat cells were seeded in 96-well plates with a density of 1 × 104 cells per well and incubated for 12 h. Subsequently, the culture medium was substituted with RPMI 1640 containing PBS, MnCl2, SPD, and L@Mn@SPD at specified concentrations, respectively. After 24 h of incubation, the cells were washed with PBS, and a medium containing 10% CCK8 was added. Absorbance at 450 nm was measured one hour later to determine cell viability. Additionally, the cytotoxicity of L@Mn@SPD was evaluated using the Calcein-AM/Propidium Iodide assay.
Intracellular ROS generation
4T1 cells were cultured in 12-well plates at a density of 2 × 105 per well for 12 h. After removing the medium, the cells were treated with PBS, MnCl2, SPD, or L@Mn@SPD and incubated for an additional 12 h. To measure intracellular ROS levels, DCFH-DA (10 µM) was added to the cells for 30 min at 37 °C in the dark, and ROS fluorescence intensity was assessed using flow cytometry. Subsequently, the cells were stained with DAPI for 10 min and visualized using fluorescence microscopy.
Detection of CRT
4T1 cancer cells were plated in 12-well plates and incubated at 37 °C for 12 h. Then, the cells were treated with PBS, MnCl2, SPD, and L@Mn@SPD for 12 h, respectively. Afterward, the cells were collected and incubated with PerCP conjugated -CRT antibody (1:100) for 0.5 h at 4 ℃, and the CRT expression was analyzed by flow cytometry.
Detection of ATP
4T1 cells were cultured for 12 h and then treated with PBS, MnCl2, SPD, or L@Mn@SPD for an additional 12 h. Extracellular ATP levels were assessed using an Enhanced ATP Assay Kit following the manufacturer’s instructions.
Detection of HMGB1
4T1 cells were treated as above described. After a 12-hour incubation, cell samples were collected, fixed with 4% paraformaldehyde, and permeabilized using 0.5% Triton X-100. After blocking for 30 min, Alexa Fluor 488-conjugated HMGB1 antibody was incubated with cell samples for 12 h at 4 ℃. Subsequently, cells were covered with Hoechst 33,342 for further imaging using fluorescence microscopy.
O₂ packaging capability and Catalase-Like activity
To assess the O₂ packaging capability, the changes in oxygen concentration in PBS, MnCl₂, and L@Mn solutions before and after the addition of SPD were evaluated under liquid paraffin-sealed conditions. Briefly, each solution was placed in a 50 mL centrifuge tube and sealed with liquid paraffin wax. The initial oxygen concentration was measured using a dissolved oxygen meter (Rex, JPBJ-608, China). SPD was then added, and after the reaction, the change in oxygen concentration was assessed.
Catalase-like activity of L@SPD@Mn was detected by measuring O2 generation and H2O2 concentration. Firstly, different solutions were sealed with liquid paraffin, followed by the addition of 200 µL H₂O₂ (50 mM). Oxygen concentration was immediately determined and the variation in oxygen levels over time was recorded. Secondly, intracellular oxygen production through catalyzing high level H2O2 of tumor cells was evaluated by using a [Ru(dpp)3]Cl2 indicator. Intracellular O2 can effectively diminish the red fluorescence of this agent [23]. 4T1 cells were cultured in 12-well plates for 12 h and then moved into a hermetically anaerobic culture tank (MGC, Japan) consisting of an anaerobic production bag and a sealed culture tank for 24 h. The cells were treated with [Ru(dpp)3]Cl2 (10 µg/mL) for 4 h, followed by incubation with PBS, MnCl2, SPD, and L@Mn@SPD for an additional 4 h. The cells were harvested to measure fluorescence intensity using flow cytometry. Finally, the treated cells as above was covered with Hoechst 33,342 and further imaged by fluorescence microscopy. The cells collected from the hermetically sealed anaerobic culture tank were further analyzed for H₂O₂ concentration using the Hydrogen Peroxide Assay Kit.
Expression of HIF-1α protein
The expression of HIF-1α in 4T1 cells under hypoxic conditions was assessed using Western blot analysis. 2 × 105 4T1 cells were cultured for 12 h under normoxia and then incubated for 24 h in a hypoxic condition. Subsequently, the cells were incubated with PBS, MnCl2, SPD, and L@Mn@SPD, respectively. After 4 h, the cells were harvested and subjected to western blot analysis.
Expression of PD-L1
Flow cytometry was performed to detect the expression of PD-L1 in hypoxia 4T1 cells. The detected cells were planted in 12-well plates. After adhering, 4T1 cells were exposed to a hypoxic environment for 24 h, followed by incubation with PBS, MnCl2, SPD, and L@Mn@SPD, respectively. After 4 h, the cells were harvested, washed, stained with APC-labeled PD-L1 antibody, and subsequently analyzed using flow cytometry.
BMDC maturation and the STING pathway activation
4T1 cells were implanted in the 12-well plates (1 × 105 cells/well) for 12 h, then cells were treated with PBS, MnCl2, SPD and L@Mn@SPD for 24 h, respectively. On day 6 after BMDC inducing (details in the cell culture section), the collected BMDC was co-cultured with pretreated tumors cells. After 24 h, the cells were collected, washed and incubated with anti-CD16/32 (Fc Block) for 15 min at 4℃. Subsequently, the maturation status of BMDC was assessed by flow cytometry following staining with APC-CD11c, FITC-CD80, Brilliant Violet 421™ MHC-II (IA/IE) and PE-CD86. The supernatant was collected to measure IFN-β by enzyme-linked immunosorbent assay kits (ELISA) as the manufacturer’s instructions. In addition, the expression of the STING pathway-related proteins was evaluated through western blotting.
In vitro T cells stimulation and proliferation assay
Single-cell suspensions of splenocytes were prepared by grinding the spleens of BALB/c mice, filtering the tissue, lysing the red blood cells, and washing the splenocytes in a sterile environment. The splenocytes were resuspended with CFSE working solution (5 µM), incubated for 10 min and then washed. The BMDC were cocultured with the 4T1 tumor cell in 12- well plates that were pretreated with PBS, MnCl2, SPD, and L@Mn@SPD for 24 h, respectively. Then, the above splenocytes -CFSE cells were added to the 12- well plates to co-culture with pretreated BMDC and 4T1 cells for 3 days. Afterward, the cells were labeled with CD3 and CD8 antibodies and flow cytometry was used to evaluated T cells stimulation and proliferation by analyzing the ratio of CD3+CD8+CFSE+. The supernatants also were collected for determining IFN-γ level using an ELISA kit following the manufacturer’s guidelines.
Seahorse assay
The oxygen consumption rate (OCR) was measured using the Seahorse XFe24 analyzer (Agilent, USA). Jurkat cells were seeded in 12-well plates at a density of 2 × 10⁶ cells per well and incubated with PBS or L@Mn@SPD for 12 h. OCR was assessed at baseline and after sequential injections of oligomycin (1 µM), FCCP (1 µM), and rotenone/antimycin A (1 µM).
Western blot
Cells were collected and lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) with protease and phosphatase inhibitors (Thermo Fisher scientific, USA). After centrifuging at a speed of 12,000 rpm for 5 min, protein contents in the supernatant were quantified by a BCA protein assay kit (Beyotime, China) and then the proteins were boiled at 95 °C for 10 min in the presence of loading buffer. The proteins were loaded onto an SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) via electrophoresis. The membrane was treated with western blocking buffer (Beyotime, China) for 1 h on a shaker and then incubated with specific primary antibodies at 4 °C overnight, including β-actin, HIF-1α, STING, phospho-STING, IRF3, phospho-IRF3. Next, HRP-conjugated Goat anti-Rabbit IgG or anti-Mouse IgG was used as the secondary antibody, and protein bands were visualized using a ChemiDoc MP Imaging System (Bio-Rad, USA).
In vivo antitumor efficacy
Female BALB/c mice (approximately 6 weeks old) were sourced from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and housed in a sterilized, specific pathogen-free (SPF) facility at Tongji University. The BALB/c female mice were injected subcutaneously with a 5 × 105 4T1 cell suspension in the right flank. When the tumor volume reached approximately 60–100 mm³, the mice were randomly assigned to four groups and received intravenous injections of (1) PBS, (2) MnCl2, (3) SPD, or (4) L@Mn@SPD every 2 days for a total of 18 days. Tumor volume and body weight were monitored every 2 days, with tumor volume determined by the formula: V = (length) × (width)2/2. At the treatment endpoint, all mice were euthanized, and tumors were collected for weighing, Ki-67 staining, TUNEL staining, flow cytometry, and immunofluorescence analysis. TDLNs, major organs (heart, liver, spleen, lung, kidney), blood samples were also obtained for further studies.
Immune response evaluation
To investigate antitumor immune response, tumor, TDLNs and spleen were also analyzed via flow cytometry. Tumor tissues were homogenized with collagenase A (2 mg/mL) and DNase (0.2 mg/mL) and shaken 60 min at 37 °C, 150 rpm for digestion. The tissue solution was centrifugated and passed through a 70 μm cell strainer. The resulting samples were stained with fluorescence-labeled antibody to assess the infiltration of CD8+ T cells (CD3+CD8+), Tregs (CD4+Foxp3+) and the expression of PD-L1. Single-cell suspensions from TDLNs were prepared by directly tissue grinding and then stained with fluorescence-labeled antibodies against CD11c, MHCII, CD86, and CD80 to explore DC maturation. The single-cell suspension from the spleen was prepared and stained to assess the relative abundance of CD8+ T cell subsets, as well as the proportion of MDSC (CD45+CD11b+Gr1+). The levels of the cytokines IFN-γ, TNF-α and IL-6 in the tumor tissue were measured using ELISA.
Transcriptomics
The tumor tissues were collected and TRIzol method was used for total RNA extraction. All transcriptome experiments were performed by Shanghai Biochip Co., Ltd.
Biosafety analysis
For assessing systemic toxicity, the main organs were collected and analyzed using H&E analysis. For serum analysis, peripheral blood was used for biochemical assays, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CR).
Statistical analysis
All data were expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was conducted using GraphPad Prism 8.0 software. Differences between groups were assessed using Student’s t-test or ANOVA, with significance defined as a p-value less than 0.05, and non-significance (ns) indicated by p-values greater than 0.05.