Cell lines and animals
The murine 4T1 breast cancer cell line and the murine B16F10 melanoma cell line were obtained from the lab of Prof. Peter Siegel and the lab of Prof. Ian Watson at McGill University respectively. Cells were incubated in DMEM (Gibco, 11995065) with 10% FBS (Gibco, 12484028) and 1% penicillin/streptomycin (Gibco, 15140122) at 37 °C with 5% CO2. Female BALB/c mice (7–8 weeks) were purchased from Charles River Laboratories. DMEM (Gibco, 21013024) was used in the CAP treatment. The animal studies were conducted by protocols approved by the Institutional Animal Care and Use Committee at the University of McGill, Canada.
Generation of CAP
The CAP device, featuring a needle electrode and a grounded ring electrode connected to a high-voltage transformer, was utilized for this research (Fig. S1A) [12, 33]. The gas used for the experiment was high-purity helium (99.996%) (Praxair, HE 5.0UH-K), with a flow rate of 8.5 L/min. The apparatus was operated at a current of ~ 3.5 A and a voltage of ~ 6 V. To produce CAP-treated solutions, the CAP jet was positioned 1 cm above the liquid surface (Fig. S1A).
Preparation and characterization of RSL3@NP
RSL3@NP was prepared by dissolving 200 mg PEG-PLGA (Sigma, 764825) and 100 mg RSL3 (AdooQ Bioscience, A15865) in 9 ml acetone. 50 ml dH2O was added dropwise while stirring overnight. The sample was frozen in a -80 °C refrigerator and then lyophilized in the freezer dryer. The average particle size of RSL3@NP was measured by DLS (Brookhaven Instrument). The morphology of RSL3@NP was observed using transmission electron microscopy (TEM). The unencapsulated RSL3 was removed with a 0.45 μm filter. The encapsulation of RSL3 in NP was analyzed by a high-performance liquid chromatography (HPLC) system (UltiMate 3000 HPLC) and a C18 column (NUCLEOSIL C18, 5 μm 15CM X 4.6 MM,). The mobile phase: acetonitrile water (50:50, v/v), constant flow rate: 1.0 mL/min. 10 µl of the solution were injected into the HPLC and then detected at a wavelength of 254 nm. The RSL3 loading efficiency was calculated according to the equation below: Loading efficiency (%) = WRSL3 in NPs /WNPs ×100%, encapsulation efficiency (%) = WRSL3 in NPs/WRSL3 fed ×100%, where WRSL3 in NPs represented the amount of RSL3 loaded in RSL3@NPs detected by HPLC. WNPs represented the total weight of RSL3@NP; WRSL3 fed represented the amount of RSL3 added in preparation of RSL3@NP.
Release of RSL3 from NPs
The RSL3@NP was prepared as described before. RSL3@NP was added to the dialysis bag (MW: 3500D) and incubated with 10 ml PBS release medium (PBS containing 5% SDS) at 37 °C and 100 rpm shaking. 100 µl of releasing media was collected at 1, 2, 4, 8, 12, 32, and 48 h for detection and substituted with the same amount of fresh release medium. The released RSL3 was detected using HPLC with the conditions described before.
Western blot
Protein extraction was using RIPA (Thermo Scientific, 89900) supplemented with protease inhibitors (Thermo Scientific, A32965) followed by sonication. Subsequently, the protein samples were separated through electrophoresis on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad, 1620112) using electroblotting. The membrane was placed in 5% BSA TBST and incubated for 1 h at room temperature, then stained overnight with the antibody of GPX4 (Invitrogen, cat. no. PA5-19710) or β-actin (Invitrogen, PA516914) at 4 °C. Subsequently, the membranes were thoroughly washed and incubated with the HRP-conjugated secondary antibody (Invitrogen, 31460) for 1 h at room temperature. The bands were visualized using an ECL substrate (Thermo Scientific, 32209).
In vitro cell viability
Cancer cells (5 × 104/ml) were seeded into culture plates (96 wells) overnight. 4T1 cells were processed with the treatment of CAP (45 s), RSL3@NP (400 nM), and CAP/RSL3@NP for 24 h. 4T1 cells were processed with the treatment of CAP (45 s), RSL3 (75 nM), and CAP/RSL3 for 24 h. B16F10 cells were processed with the treatment of CAP (15 s), RSL3 (400 nM), and CAP/RSL3 for 24 h. Untreated cells were used as a control group. Cell viability was tested using the MTT assay. The cytotoxicity of RSL3@NP to T cell in vitro was analyzed by flow cytometry after staining with a LIVE/DEAD™ Viability/Cytotoxicity kit (ThermoFisher, L3224).
Intracellular ROS and RNS levels
Cancer cells (5 × 104/ml) were seeded into culture plates (24 wells) and cultured overnight. Cells were treated for 4 h and then stained with H2DCFDA (Invitrogen, D399) or DAF-FMDA (Invitrogen, D-23841) for 1 h. In the end, cell analysis was conducted using flow cytometry on the LSRFortessa system from BD.
Release of HMGB1
4T1 cells (5 × 103/well) were seeded in a 96-well plate for 24 h. 4T1 cells were treated under the different conditions of CAP (45 s), RSL3@NP (400 nM), and CAP/RSL3@NP. The culture medium was collected and analyzed with HMGB1 Elisa kit (Invitrogen, cat. no. EEL102).
Immunofluorescence
Cancer cells (5 × 104/ml) were seeded into culture plates (24 wells) and cultured overnight. The cells were treated for 12 h, and then they were fixed using 4% paraformaldehyde and permeabilized using 0.1% Triton for 10 min each. Following the blocking step in 5% BSA, cells were stained with HMGB1 primary antibody (Invitrogen, PA5-27378) and FITC secondary antibody (Invitrogen, F2765) at room temperature. Hoechst stain (Thermo Scientific, 62249) was used on the nuclei of cells. SlowFade Diamond Antifade Mountant (Invitrogen, S36972) was used to adhere coverslips to glass slides. Slides were examined by Zeiss (Observer. Z1) wide field microscopy).
The frozen sections of spleen were washed with PBS, and then blocked in 3% BSA for 1 h at room temperature. CD8a antibody (Invitrogen, cat. no. 42008182) and CD4 antibody (Invitrogen, cat. no. 50976682) were used to stain overnight at 4°C. Hoechst (Thermo Scientific, cat. no. 62249) was used to stain nuclei. The sections were preserved under SlowFade Diamond Antifade Mountant (Invitrogen, cat. no. S36972) and analyzed by widefield microscopy (Zeiss Observer. Z1).
In vitro CRT levels
Cancer cells (5 × 104/ml) were seeded into culture plates (24 wells) and cultured overnight. The next day, cells were treated under different conditions for 24 h (as described above). Afterward, cells were stained with CRT antibody (Invitrogen, PA3-900) and Alexa Fluor 555 antibody (Invitrogen, A21428). The BD LSRFortessa flow cytometry system was used for the subsequent analysis.
ATP release assay
Cancer cells (5 × 104/ml) were seeded into culture plates (96 wells) overnight. The cells were subjected to different treatments for 24 h. A kit for ATP Determination (Invitrogen, A22066) was used to measure the luminescent signal of ATP in the cells and culture medium according to the guidelines provided by the manufacturer.
Dendritic cell maturation in vitro
Bone marrow-derived dendritic cells were isolated according to an established method [43]. The maturation of DCs was assessed utilizing a transwell culture system. The upper chamber of the transwell was cultured with cancer cells, while the lower chamber was cultured with bone marrow-derived DCs. Cancer cells were processed with the treatments for 6 h, then they were co-cultured with DCs for another 24 h. Following that, DCs were collected for staining with CD80+ (BioLegend, 104723) and CD86+ (BioLegend, 105008). The samples were subsequently analyzed using a BD LSRFortessa flow cytometer. IL-6 ELISA kit (BioLegend, Cat no. 431301) was utilized to measure the IL-6 levels in the culture medium for DCs.
Macrophage polarization in vitro
Bone marrow-derived macrophage cells were prepared as previous procedure [27, 45]. The macrophage polarization experiment was tested in a transwell assay. In the transwell, bone marrow-derived macrophages were cultured in the lower chamber, and cancer cells were grown in the upper chamber. Following a 6 h treatment, bone marrow-derived macrophages were co-cultured with cancer cells for an additional 24 h period. Subsequently, macrophages were collected for staining with CD80+ (Invitrogen, 2381018) and CD206+ (BioLegend, 141717), and were subsequently analyzed using a BD LSRFortessa flow cytometer.
Preparation of injectable gel
200 mg of Pluronic F-127 (Sigma, P2443-250G) was dissolved in 1 ml CAP-treated dH2O to form CAP@gel at 4 °C. RSL3@NP@gel was prepared by loading RSL3@NP into Pluronic gel. CAP/RSL3@NP@gel was prepared by loading RSL3@NP into CAP@gel.
In vivo tumor models and treatment
To study the therapeutic effectiveness of CAP/RSL3@NP@gel, 1 × 106 4T1 cells were inoculated under the second left nipple of female BALB/c mice [39]. Mice with tumor sizes around 100 mm3 were categorized into 4 groups (n = 7–9). CAP@gel, RSL3@NP@gel (50 mg/kg), or CAP/RSL3@NP@gel were injected intratumorally into mice-bearing tumors every two days for a total of five injections. The tumor volume was assessed using a digital caliper and was computed using the subsequent formula: width2 × length × 0.5.
Immunological analysis
Tumors were collected after the last intratumoral injection. Tumor samples were cut and homogenized to form single cell suspensions with collagenase (Gibco, 17104019). Cell samples were stained with fluorescence-labeled antibodies: CD11c (BioLegend, 117310), CD86 (BioLegend, 105008), CD80 (BioLegend, 104723), F4/80 (BioLegend, 123116), CD206 (BioLegend, 141717), CD3 (BioLegend, 100204), CD4 (BioLegend, 100412), CD8 (BioLegend, 140408), Foxp3 (BioLegend, 126404). Blood samples were collected for the staining of CD3 (BioLegend, 100204), CD4 (BioLegend, 100412), and CD8 (BioLegend, 140408). The BD LSRFortessa flow cytometer was used to measure the stained cells.
Detecting cytokines in tumor tissue
Tumors were harvested and ultrasonic homogenized after the last injection. For detection of the intratumoral levels, ELISA kits of IL-12 (BioLegend, 433604), IFN-γ (BioLegend, 430801), and IL-10 (BioLegend, 431411) were utilized to measure.
Statistical analysis
Mean ± SD (standard deviation) was used to present results, except mean ± SEM (standard error of the mean) was used for the tumor growth curve. Multiple comparisons were performed using one-way ANOVA and Tukey post-hoc tests. All statistical analyses were carried out with the Prism software package (PRISM 5.0, GraphPad 2007). The threshold for statistical significance was P < 0.05.
