NK-92MI (human NK cell line) and PC3 (human prostate cancer cell line) TrampC1 (mouse prostate cancer cell line) were purchased from the American Type Culture Collection (ATCC). NK-92MI cells were cultured in Minimum Essential Medium alpha (Invitrogen) supplemented with 2 mM L-glutamine (Invitrogen), 1.5 g/L sodium bicarbonate (Sigma-Aldrich), 0.2 mM inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Invitrogen), 0.02 mM folic acid (Sigma-Aldrich), 12.5% fetal bovine serum, and 1% penicillin/streptomycin. PC3 cells were grown in standard RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). TrampC1 cells were cultured in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 0.005 mg/ml bovine insulin and 10 nM dehydroisoandrosterone, 90%; fetal bovine serum, 5%; Nu-Serum IV, 5%. Mouse primary NK cells were purified from spleen and characterized by flow cytometry analysis.
Cytotoxicity assay of NK cells
1 × 105 cells of PC-3 were pre-stained with Cell Trace CFSE (Invitrogen) and then co-cultured with NK-92MI cells at 10:1 effector: target (E: T) ratio. After 4 h of co-incubation, cells were harvested and stained with 7-AAD (Invitrogen) for the discrimination of live and dead cells. To assess the degranulation of NK cells, co-cultured cells were stained with anti-human CD107a-APC (BD Biosciences) for 1 h at 4 °C, then analyzed on a flow cytometer (BD Bioscience).
Cell viability test
Cell viability was quantified by the colorimetric assay by using CCK-8 reagents (Dojindo Inc.). A total of 1 × 104 cells in 100 μL of complete media were plated in each well of a 96-well cell culture plate. After overnight cultivation, 100 μL of media, containing 0 to 160 μg/mL concentration of ferumoxytol, was added to each well and incubated for 72 h. Then, CCK-8 reagents were treated by the manufacturer’s instructions. After 4 h of additional incubation, the absorbance of the plate was measured by spectrophotometer (BioTek Inc.) at 450 nm (OD).
NK-92MI cells were treated with ferumoxytol for 24 h, then washed with cold DPBS and FACS buffer (BD Bioscience). Cells resuspended in FACS buffer were blocked with Fc blocker (BD Bioscience) for 15 min at 4 °C, and then incubated with dye-conjugated antibodies for 1 h at 4 °C. Antibody-labeled samples were fixed with 4% paraformaldehyde. Antibodies used for flow cytometry were purchased from Biolegend. Flow cytometry analysis was performed using BD fortessa flow cytometer (BD Bioscience).
Enzyme-linked immunosorbent assay
1 × 104 target cells were cocultured with effector cells at an effector cell: target cell (E: T) ratio of 10: 1 in round-bottom 96-well culture plates for 4 h. Cell-free supernatants were assayed for cytokine secretion by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol. Human and mouse IFN-γ ELISA kits were purchased from BioLegend.
Confocal laser scanning microscopy
For confocal fluorescence microscopy, target tumor cells were plated at least 16 h before use on confocal dish (MatTek). After attached target cells, CFSE stained NK92-MI cells were added and co-incubated for 4 h. The co-incubated cells were treated with Hoechst 33,342 for visualize nucleus and fixed with 4% paraformaldehyde. Images were taken with a Nikon A1R spectral confocal laser scanning system on an inverted microscope (Nikon) with an argon-krypton laser as excitation source. The green and red fluorescence of C11-BODIPY581/591 was acquired simultaneously using double wavelength excitation (laserlines 488 and 568 nm) and detection (emission bandpass filters 530/30 and 590/30).
Western blot analysis
PC-3 cells were seeded in the 8 cm culture dishes and cultured at 5% CO2, 37 °C overnight. The cells were treated with various amounts of ferumoxytol (Fer (0 μg), Fer (50 μg), Fer (100 μg), and Fer (200 μg)), respectively, for 12 h. No treatment of PC-3 cells was used as control. The cell lysates were collected and analyzed by Western blotting according to the manufacturer’s instructions. Antibodies were used listed as below: GPX4 Rabbit mAb, Cell Signaling, catalog number: 59735S, dilution 0.1–0.5 µg/mL; SLC7A11 rabbit mAb, Cell Signaling, catalog number: 12691S, dilution 0.1–0.5 µg/mL; Goat Anti-Rabbit IgG H&L (HRP), Abcam, catalog number: ab205718, dilution 1:2000; β-Actin (8H10D10) Mouse mAb (HRP Conjugate), cell signaling, catalog number:12262, dilution 1:1000.
Intra-tumoral injection of NK cells
All experimental procedures were approved and reviewed by the Institutional Animal Care and Use Committee of Northwestern University (IS00002377). A total of 2 × 106 Tramp C1 cells in 100 μL of PBS were subcutaneously injected into the right flank of C57BL/6 mice (male, 8–10 weeks old). After three weeks, tumor bearing mice with 100 to 150 mm3 volume were treated with NK cells and Ferumoxytol by intra-tumoral injection. MRI studies were performed using a Bruker 7.0 T ClinScan (Bruker BioSpin, Ettlingen, Germany) high-field small animal MRI system with a commercial mouse coil (Bruker Biospin).
In vivo therapeutic efficacy
All experimental procedures were approved and reviewed by the Institutional Animal Care and Use Committee of Northwestern University. PC3 cells (2 × 106 cells per mouse) in DPBS were injected into nude mice (male, 8–10 weeks old). After two weeks, tumor bearing mice were treated randomly with NK-92MI cells and Ferumoxytol. All mice were treated total 4 times in every 2–3 days. Tumor sizes were traced twice a week for 4 weeks.
Prostate cancer mouse model and T cell Characterization
A total of 1 × 106 Tramp C1 cells in 100 μL of PBS were subcutaneously injected into the right flank of C57BL/6 mice (male, 10 weeks old). After three weeks, tumor bearing mice with 100 to 150 mm3 volume were collected and treated with NK cells and Ferumoxytol by intra-tumoral injection. Tumor sizes were traced twice a week for 3 weeks. Then, mice were terminated, and T cell distributions (CD3, CD4, CD8, IFN-γ, CD25, FoxP3, CD45, IL17A, BD Biosciences) of tumor and spleen were analyzed by flow cytometry. Gating strategy is showing in Additional file 1: Fig S4.
Histology and Immunohistochemistry
To assess the distribution of NK cells, tumors were harvested from the mice and fixed for 24 h in 4% paraformaldehyde. The tissue Sects. (5 μm) were stained with hematoxylin and eosin (H&E). Immunohistological analysis was performed using a terminal transferase dUTP nick-end labeling (TUNEL) assay (Promega) or antibodies including anti-mouse NK1.1 (Thermo). All slides were scanned using Hamamatsu K.K. Nanozoomer 2.0 and analyzed by ImageJ software to determine the positive cells per field of view.
All statistical analyses were performed using the GraphPad Prism 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Graphical data are presented as means ± standard deviation. Statistical significance of differences between two groups was determined using Student’s unpaired t-test. P-values less than 0.05 were considered statistically significant.