Cell line 4T1-luc murine breast cancer cells were originally provided by Dr. Gary Sahagian at Tufts University and have been maintained in high-glucose DMEM (ATCC® 30–2002™) (ATCC)-supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) (three items above from Atlanta Biologicals). 4T1-luc cells, passage numbers between 2–7 were thawed for expansion, and cells with passage numbers between 10–20 were used in the described ex vivo or in vivo experiments. Cells were tested periodically to ensure no mycoplasma contamination was present.
Mice and tumor models Female Balb/c mice (8–10 weeks of age) were purchased from Jackson Laboratory and housed in the ODU animal facility accredited by the AAALAC. 4T1-luc tumor was initiated by an inoculation of 1 x 106 live 4T1-luc cells in the left posterior mammary fat pad in female Balb/C mice. The size of tumor was assessed by digital calipers twice a week. Tumor volume was determined using the following formula: V = πab2/6, where (a) is the longest diameter and (b) is the shortest diameter perpendicular to (a). All experimental protocols were approved by Old Dominion University Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC). And all experiments were performed in accordance with relevant guidelines and regulations.
Reagents and antibodies The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit including 3% H2O2 for the establishment of standard curve (Cat#A-22188), Invitrogen™ 96-well microplates for fluorescence-based assays (Cat#M33089), dihydroethidium (DHE) (Cat #D1168), Hoechst 33342 (Cat #H3570), and MitoSOX™ Red mitochondrial superoxide indicator (Cat #M36008) were purchased from Invitrogen. Trolox (Cat #SC-200810) was obtained from Santa Cruz Biotechnology. Lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Cat#L4391), Rosmarinic acid (RA) (Cat #536954-5G), N-acetyl cysteine (NAC) (Cat #A9165), Vitamin C (Cat #A4403), and sodium pyruvate (Cat #P5280) were purchased from Sigma-Aldrich. WST-1 (Cat # 11644807001) for cell viability assays was obtained from Roche Applied Science. Rat anti-mouse CD3 pacific blue (Cat #100334), CD4 FITC (Cat #100406), CD8 Percp (Cat #100732), CD44 APC (Cat #103012), CD62L PE/Cy7 (Cat #104418), IFN-γ PE (Cat #505807), IL-17 PE/Cy7 (Cat #506921), CD103 PE (Cat #121406), CD86 Pacific blue (Cat #105021), I-A/I-E FITC (Cat #107605), CD40 APC (Cat #124611), anti-mouse CD16/32 (Cat#156603), hamster anti-mouse CD11c PerCP (Cat #117326), hamster anti-mouse CD80 PE/Cy7 (Cat #104734) and mouse anti-mouse H-2Kd/H-2Dd PE (Cat #114708) were purchased from BioLegend.
In vitro NPS treatment In vitro NPS treatment for cancer cells was described in our previous publication (44). Briefly, a custom-made nanosecond pulse generator was used to generate 60 nanosecond (ns) electric pulses with various pulse frequencies and applied electric fields of interest. 4T1-luc cells, 100 µl at a concentration of 5 x 106 cells/ml in a 0.1 cm-gap cuvette were pulsed with NPS; pulse duration of 60 ns, frequency of 1 Hz, applied electric field of 5 kV (or 50 kV/cm), and pulse number of 10 to 150 dependent on experimental design.
In vivo NPS treatment and the secondary tumor challenge In vivo NPS treatment was detailed in our published paper (5). Briefly, mice with tumors (6–8 mm) were randomly grouped according to tumor volume and treated with NPS (100 ns, 50kV/cm, 3 Hz and 1000 pulses). Animals with tumor free over 7 weeks were challenged orthotopically in the right posterior mammary fat pad with 0.5 x 106 live 4T1-luc tumor cells. Tumor growth was monitored twice weekly by caliper measurements.
Tissue harvesting and processing for the analysis of immune cells Nine days after NPS treatment, mice were euthanized and draining lymph nodes were collected. Draining lymph nodes from tumor bearing mice without NPS treatment were used as control. Single cell suspicions were prepared to analyze immune cells including CD3, CD4, CD8 and tissue-resident marker CD103. To examine effect memory and central memory T cells, animals with tumor free over 3 months after NPS treatment were euthanized. Spleens were harvested. Spleens of tumor bearing mice were used as control. Single cell suspensions were prepared from spleens then stained with CD3, CD4, CD8, CD44 and CD62L antibodies.
To quantify IFN-γ producing T cells, intracellular staining was carried out. Splenocytes (2 x 106/ml) 1 ml per well were incubated with media or plate bound low endotoxin/azide free LEAF anti-CD3 Ab (0.5µg/ml in DPBS) in a 24-well plate. cells were incubated for 6 hours and monensin added for the final 4 hours.
Detection of H 2 O 2 using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit A protocol provided by the manufacturer was modified and adopted to quantify the concentration of H2O2 released from cells. Briefly, 4T1-luc cells were treated with various parameters of NPS as described above, following which 25,000 cells were seeded into individual wells of a 96-well plate pre-filled with 50 µl cell culture media per well. AmplexR Red reagent/HRP working solution, 50 µl, was added to each microplate well and placed in the chamber of PLUOstar Omega fluorescent microplate reader with the Atmospheric Control Unit that enables us to set temperature at 37°C and CO2 at 5%. This setting allowed for measuring the samples continuously at designated time points and with minimal disturbance of cells. The measurement of H2O2 was done by top-reading with cell adhered to the bottom of the plate wells without mixing. Fluorescence intensity was measured at the excitation of 550 nm and emission of 590 nm. Cell culture medium without cells treated with NPS (100 pulses) was used as the background control. Serial dilutions of H2O2 (concentrations from 10 µM to 0.03125 µM) were used to establish a standard curve. In some cases, the fluorescence intensity or relative fluorescence units (RFU) were used to indicate the relative levels of H2O2.
Measurement of ROS by DHE DHE (10 µM) was added into 4T1-luc cell suspensions followed by incubation at 37°C and 5% of CO2 for 15 minutes. Next, the cells were treated with NPS as described above following which 25,000 cells were then taken out of the NPS cuvette and seeded into individual wells of a 96- well plate pre-filled with 100 µL of cell culture media (without phenol red) per well. Superoxide anion (O2¯) converts DHE to ethidium which was monitored by the plate reader over time. The fluorescence intensity was measured as described above with a fluorescence excitation wavelength at 520 nm and emission wavelength at 600 nm. The difference here is that no superoxide standard curve was established, so the relative fluorescence units (RFU) was used as the relative level of ROS.
Live cell imaging using MitoSOX™ Red mitochondrial superoxide indicator Briefly, 4T1-luc cells with or without NPS treatment were placed into a 6-well plate with 0.5 x 106 cells per well. Cells were then incubated at 37°C, 5% CO2 overnight. After gentle removal of culture medium 1.0 ml of working solution containing 5 µM MitoSOX™, which was made by a 1:1000 dilution from the 5 mM stock solution in DMSO into culture media, was added into each well and the plate was incubated at 37°C for 10 minutes in the dark. The incubation time for MitoSOX loading cells was adopted according to manufacturer’s instructions. The MitoSOX™ working solution was then removed and replaced with nuclear staining buffer, 0.5 µg/ml Hoechst 33342 in DMEM without phenol red. Cells were imaged immediately under a fluorescence microscope (Olympus BX51). The DAPI filter was used to detect nuclear staining and the TRITC filter was used to detect oxidized MitoSOX™ Red in the cells.
Cell viability assay WST-1 cell viability assay was described previously (44). Briefly, 10 µL (5 x 106/ml) of cell suspension after exposure to NPS with or without ROS blockers was placed into a clear-flat-bottom 96-well plate filled with 90 µL complete medium per well. All ROS blockers including Trolox (1 mM), RA (100 µM), Vitamin C (0.5 mM), NAC (3mM) and Sodium pyruvate (10 mM), and with their corresponding concentrations previously screened for their ability to block ROS generation were examined by cell viability assays. Cells were incubated at 37°C and 5% CO2. Following an 18-hour incubation 10 µl of WST-1 reagent was added to each well. Cells were incubated with WST-1 for 2 hours and then measured by MultiSkan MCC/340 microplate reader (Fisher Scientific, Hampton, NH) with a test wavelength of 450 nm and a reference wavelength of 630 nm. Cell viability (%) was calculated using the formula: Treated sample (OD450-OD630)/control (OD450-OD630) × 100. 4T1-luc cells without NPS exposure but otherwise treated the same way as those exposed to NPS were used as the control.
Generation and activation of bone marrow-derived DCs (BMDCs) BMDCs were prepared from harvested bone marrow cells by 8 days of culture and differentiation in the presence of 20 ng/ml GM-CSF (R&D). BMDCs (2 x 105) were then incubated with 4T1-luc cells (2 x 105) treated with a lethal dose of NPS (100 pulses, 60 ns, 50 kV/cm and 1 Hz) alone or with the addition of Trolox (1 mM) in a 24-well plate at 37°C, 5% of CO2 for 2 days. BMDCs in the presence of either media alone or LPS (5 µg/ml), but without 4T1-luc cells, were used as negative and positive controls, respectively. Cells were harvested to analyze cell surface activation markers (MHC-I/II, CD40 and CD80) by flow cytometry.
Vaccination and tumor challenge Mice were shaved and subcutaneously (SC) inoculated with 3 x 106 NPS treated 4T1-luc cells with or without a ROS blocker in 100 µl sterile saline. Control animals were inoculated SC with the same quantity of cells lysed with 3 freezing/thawing cycles. There were 4 groups: control (Ctrl), NPS treated cells (NPS), NPS treated cells with preincubation with Trolox 1 mM (NPS + Trolox) or sodium pyruvate 10 mM (NPS + SP). All 20 mice (n = 5 per group) were challenged with 0.5 x 106 4T1-luc live cells in 50 µl sterile saline in the left posterior mammary fat pad 10 days later. Tumor growth was monitored twice weekly by caliper measurements. Animals with complete tumor rejection were followed for at least 4 months, and those with tumor growth were euthanized when the volume of tumor reached 1.5 cm3 unless euthanasia was required earlier due to other criteria described for experimental endpoints in the approved IACUC protocol.
Flow cytometry analysis 2 x 105 BMDCs were incubated with an antibody cocktail (anti-MHC-I/II, CD40 and CD80, each at 1 µg per million cells) in 100 µL FACS buffer (2% FBS DPBS) at room temperature for 30 minutes. Cells were then washed with 2 ml FACS buffer twice and resuspended in 0.5 ml FACS buffer for flow cytometric analysis by MACSQuant® Analyzer 10 (Miltenyi Biotec). Cells stained with isotype antibodies were used as negative controls. All stained cells were run through a flow cytometer. Live cells were gated in a forward scatter (FSC) versus side scatter (SSC) plot then analyzed for cell surface biomarkers.
For intracellular staining, 2 x 106 splenocytes were prepared by pre-incubation with purified anti-CD16/32 (Fc block), followed by surface labeling of cells with anti-CD3 pacific blue, anti-CD4 FITC and anti-CD8 PerCP followed by intracellular staining using mAbs anti-IL-17A PE-Cy7 and anti-IFN-γ PE after fixation and permeabilization with fixation and permeabilization buffer. Samples were analyzed on a flow cytometer (FACSAria, BD Biosciences)
Statistical analysis Values were presented as the mean ± standard deviation (SD) or standard error (SE). Student’s t-test was utilized to compare quantitative data including tissue-resident memory, effector and central memory T cells between two groups. One Way ANOVA (3 or more groups) was utilized to analyze the quantitative data including cell viability and the level of ROS (H2O2 concentration or RFU). To compare the dynamic change of ROS among different treatment groups, the accumulated ROS or area under curve was calculated and analyzed. If One Way ANOVA showed statistical significance, then Pairwise Multiple Comparison Procedures (Holm-Sidak method) would be done to compare various pairs of groups. Chi-square was employed to analyze the vaccine effect or the rate of protection. If Chi-square for multiple groups showed statistical significance, then Pearson Correction and Chi-square between two groups would be done to compare two groups. Animal survival will be analyzed with Kaplan–Meier Survival LogRank analysis. Statistical significance is assumed at p < 0.05. All statistical analysis was completed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA).