Materials
Lipid A, monophosphoryl from Salmonella enterica serotype, tetramethyl orthosilicate, hydrochloric acid solution, sodium chloride, low molecular weight chitosan, poly-l-lysine, puromycin dihydrochloride, rhodamine B isothiocyanate mixed isomers and 10% buffered formalin were purchased from Sigma-Aldrich. CpG oligonucleotide 1826 and 2006 were purchased from Invivogen and PEI (25k linear) was purchased from Polysciences. Prolong Gold antifade mountant with DAPI, Alexa Fluor 488 alpha tubulin antibody phosphate-buffered saline (PBS) and RPMI 1640 were purchased from Thermo Fisher Scientific. Fetal bovine serum (FBS) was purchased from ATCC. EDTA trypsin solution (0.05%), penicillin/streptomycin and rhodamine or Alexa Fluor 647 phalloidin were purchased from Life Technologies. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Caisson Labs. Cell Titer-Glo 2.0 Assay was purchased from Promega. Recombinant murine granulocyte macrophage colony stimulating factor (GM-CSF) was purchased from R&D Systems. XenoLight d-luciferin potassium salt was purchased from Perkin Elmer. Reversible strainers (37 µm mesh) were purchased from STEMCELL Technologies.
Antibodies
CD11c FITC (HL3), CD11c FITC (B-ly6), CD326 (EpCAM, G8.8) APC, CD326 (MαH EpCAM IB7) eFluor 660, CD3 (17A2) APC-eFluor 780, CD4 (GK1.5) APC, CD8a (53-6.7) eFluor 450 and Alexa Flour 488, CD11b (M1/70) APC and FITC, CD11c (N418) PerCP-Cyanine5.5 and PECy7, CD40 (3/23) PE, CD44 (IM7) PerCP-Cyanine5.5, CD62L (l-selectin, MEL 14) FITC, CD152 (CTLA-4, UC10-4B9) PE and PerCp-Cy5.5, CD279 (PD-1, J43) PE-Cyanine7, FOXP3 (FJK-16s) PE, IFN gamma (XMG1.2) Alexa Fluor 488, MHC Class II (I A/I E) (M5/114.15.2) FITC, GATA-3 PerCP-eFlour 710 (TWAJ), TNFα PerCP-eFluor 710 (MP6-XT22), CD45R (B220) FITC, Fc receptor blockers (anti-CD16/CD32 (clone 2.4G2)), mouse IgG (31205), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation were purchased from eBioscience/Thermo Fisher Scientific. IL-4 APC (11B11), CD152 (CTLA-4) PerCP, TIM3 (B8.2C12) APC and CD11c FITC (N418; Amnis BMDC and mouse tissues) were purchased from BioLegend. IL-2 PE (JES6-5H4) was purchased from BD Bioscience.
Cell lines and mouse models of ovarian cancer
The BRCA1-deficient BR5-Akt cell line, generated on an FVB background, was a kind gift from Dr Sandra Orsulic (Cedars-Sinai)44. The ID8ova cell line, generated from C57BL/6 ovarian epithelial cells, and transfected to express ovalbumin constitutively, was a gift from Dr George Coukos at the University of Pennsylvania45. Both ID8ova and BR5-Akt cell lines are syngeneic models of high-grade serous epithelial ovarian cancer. To monitor tumour burden using a bioluminescent tag, the cell lines were lentivirus transduced to constitutively express firefly2 luciferase. Cell lines were cultured in DMEM containing 10% FBS and 100 units per 100 µg penicillin/streptomycin at 37 °C and 5% CO2. Trypsin-EDTA was used to collect cells.
To prepare BMDC, bone marrow was collected from the femurs of female murine C57BL/6 or FVB mice using a 27 g needle and syringe to flush the marrow from the bone. RBC were lysed with BD lysis buffer as described by the vendor. Cells were cultured in 6-well plates (3 ml per well) for 8–10 d in RPMI 1640 medium supplemented with 10% FBS, 100 mM β-mercaptoethanol, penicillin/streptomycin and 10 ng ml−1 recombinant murine GM-CSF. Half of the media was replaced every 2–3 d with fresh media and cytokines. Human DCs were enriched from ascites samples by loose adhesion to plastic cell culture dishes.
Mice were purchased from Charles River or Jackson Laboratories and housed in a specific pathogen-free facility. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico (Albuquerque, NM, USA). To generate consistent engraftment and predictable disease progression, 2 × 105 BR5-Akt-Luc2 or 1–5 × 106 ID8ova-Luc2 cells in 200 µl PBS were administered by IP injection in 6–7 week old FVB or albino C57BL/6 female mice46. Mice were killed when moribund or when weight reached 30 g due to ascites accumulation. Mice were monitored and weighed every 2–3 d. For studies that included SC tumours, female mice were injected with 200 µl PBS containing 2 × 105 BR5-Akt-Luc2 cells on the dorsal surface using isoflurane as an inhalation anaesthetic.
Cell silicification
BR5-Akt or ID8ova cells (3 × 106) as well as leukocytes obtained from RBC lysed FVB mouse spleen, or human ascites cancer cells enriched by filtration capture, were washed with PBS, followed by physiological saline (154 mM NaCl), and then suspended in 1 ml silicic acid solution containing 10 mM TMOS, 100 mM NaCl and 1.0 mM HCl (pH 3.0), with scale up as needed. Optimization of conditions for biological use evaluated cell stability and dispersion following silicification in 5–100 mM TMOS and 100 versus 154 mM NaCl. Following a 5–10 min incubation at room temperature, the cell suspension was transferred to −80 °C for 24 h. Si cells were then washed with endotoxin-free water, followed by PBS. To compare silicon content with published cell silicification techniques, cells were also silicified at room temperature in silicic acid solution containing 100 mM TMOS, 154 mM NaCl and 1.0 mM HCl (pH 3.0) for 24 h15.
Coating Si cells with cationic polymer
Si cells were made cationic using chitosan, poly-l-lysine or PEI. Si cells (3 × 106) were washed with water, followed by PBS, and then suspended in 1 ml PEI (0.2 mg ml−1), 2 mg ml−1 chitosan or 1 mg ml−1 poly-l-lysine in PBS. Following 10 min (or as indicated) rotation at room temperature, cells were washed twice with PBS and zeta potentials were evaluated.
Fluorescent PEI synthesis
PEI (5 g, 0.2 mmol) was dissolved in 5 ml ethanol and Cy3-NHS (10 mg ml−1 in dimethylformamide (DMF), 150 µl, 2 µmol) was added. The solution was rotated at 40 °C for 4 d. The mixture was concentrated using a rotavap, then 50 µl DMF was added to dissolve any unreacted dye. The mixture was centrifuged at 21,000 × g for 20 min and the isolated pellet was dissolved in ethanol and transferred to the rotavapor to remove DMF traces. After 1 h, the PEI-Cy3 was dissolved in PBS at 0.5 mg ml−1.
Adsorption of TLR ligand to Si or irradiated cells
Si cells (12 × 106) with or without polymer coating were washed with PBS and then suspended in 25 µl MPL (1 mg ml−1) in DMSO. After 10 min incubation at room temperature, Si-PEI-MPL or Si-MPL cells (or their chitosan counterparts) were washed with PBS by centrifugation at 2,000 × g for 5 min followed by suspension in PBS. Oligodeoxynucleotide 1826 adsorption followed a similar protocol using 20 µl CpG (2 mg ml−1) in endotoxin-free water for every 12 × 106 Si cells. For dual adsorption of MPL and CpG, CpG was introduced first for 10 min, followed by the addition of MPL for an additional 10 min. To quantitate ligand loading, unbound fluorescent TLR ligand was measured using a BioTek microplate reader with excitation/emission at 470/560 nm for PEI-Cy3, and excitation/emission at 488/528 nm for CpG-FITC detection. MPL was quantified by absorption at 290 nm using a ThermoScientific NanoDrop 2000. Irradiated BR5-Akt cells were incubated with PEI, CpG and MPL using the same conditions optimized for Si cells.
Dehydration and rehydration of vaccine cells
Si or irradiated cells (with or without PEI) were rinsed with PBS and then dried under vacuum at room temperature for 16 h. Samples were stored at room temperature for 14 d. Before use, cells were rehydrated in PBS with vortexing and coated with PEI, CpG and MPL.
Zeta-potential measurements
Zeta-potential measurements were performed using the Malvern Zetasizer Nano-ZS (Westborough) equipped with a He–Ne laser (633 nm) and non-invasive backscatter optics. Cells were suspended in 5 mM NaCl solution with measurements performed using the monomodal analysis tool. All reported values correspond to the average of at least three independent samples.
Optical microscopy
For bright-field imaging, cells were suspended in the water or PBS and imaged using the Nikon eclipse TS 100 inverted microscope equipped with a Nikon digital-sight DS-L3 camera.
In vitro DC internalization of fluorescent Si cells
To image DC association with Si cells, BR5-Akt cancer cells were first incubated with fluorescent lipid-coated MSN labelled with Cy3 or DyLight 488 and presenting MPL for 4–24 h. Tumour cells were then silicified using optimized conditions and surface-masked with TLR ligands (as indicated). DC were seeded onto glass coverslips in 6-well plates at a density of 5 × 105 cells per well and the next day, fluorescent Si vaccine cells were added and DC were incubated as indicated. DC were then washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, followed by overnight incubation at 4 °C. The following day, cells were washed with PBS, permeabilized with 0.1% Triton-X in PBS for 15 min, blocked with 1% BSA for 20 min, and then labelled with Alexa Fluor 647 phalloidin in 1% BSA for 1 h. After a final wash in PBS, coverslips were mounted on slides using Prolong Gold with DAPI. Images were acquired using a 63X/1.4NA oil objective in sequential scanning mode using a Leica TCS SP8 confocal microscope.
DC uptake of Si cells was quantified using an Attune NxT flow cytometer (Thermo Fisher) or the Amnis ImageStream. Human ascites cancer cells or mouse BR5-Akt cells were stained with CTFR (1:4,000; Thermo Fisher) before silicification. Si cells, surface modified with TLR ligands as indicated, were co-cultured with Cell Trace Violet- (Thermo Fisher, flow cytometry) or anti-CD11c FITC- (Amnis; 1:1,000) labelled human or mouse DCs for 1–24 h as indicated at a ratio of 5:1 (vaccine:BMDC), and then analysed by flow cytometry for double-positive cell populations or using the Amnis Imagestream. For Amnis analysis, a single cell population was gated on using a dot plot created using a bright-field laser (aspect ratio versus area), then focused bright-field cells were gated using a histogram of normalized frequency versus gradient root mean square. Lastly, a dot plot was created showing internalized and non-internalized cells (intensity of CTFR channel versus intensity of FITC channel).
T-cell killing assay
BR5-Akt cells were seeded into a 96-well plate at 1 × 103 cells per well in 200 µl cell culture medium, followed by overnight incubation at 37 °C in 5% CO2. Peritoneal T cells were purified using the Miltenyi Biotec CD8a+ T-cell isolation kit from vaccinated (day 57)/tumour-challenged or naive FVB mice. T cells were added to cancer cells at a ratio of 5:1 and the cell impermeant nuclear dye YOYO-3 iodide (1 mM in DMSO) was added at a dilution of 1:4,000. Cells were put in the IncuCyte Live Cell Imaging System (Sigma Aldrich) and imaged for 48 h with images acquired every 2 h.
IFNγ ELISpot
FVB mice were treated with PBS, Si cells or vaccine on day 0 and peritoneal cells were isolated on day 14 as previously described. The ELISpot assay was performed as described by the vendor (R&D Systems), with 2 × 105 RBC lysed peritoneal cells included per well per 100 µl complete media. Ovalbumin was added at 15 µg per well for 19 h at 37 °C. Spots were counted manually using a dissection microscope.
SEM and energy dispersive X-ray (EDX) analysis
Si tumour cells were suspended in 100% ethanol and then dropped onto 5 × 5 mm glass slides. The glass slides were then mounted on SEM stubs using conductive adhesive tape (12 mm OD PELCO Tabs). SEM and EDX images were acquired under high vacuum at 7.5k using a FEI Quanta 3D Dualbeam FIB-FEGSEM with EDAX SDD EDS detector (Thermo Fisher).
ICP-OES
ICP-OES was used to measure Si concentration in Si cells. Cells (20 × 106) were washed with water and dried under vacuum for 7 d and then mineralized in aqua regia (1:3 mixture of ultrapure HNO3 and HCl) with a Digi prep MS SCP Science block digester at 95 °C for 4 h. The digested samples were diluted and passed through 0.45 μm filter. The concentration of silicon was then measured using a Perkin Elmer Optima 5300DV ICP-OES, with a detection limit of <0.5 mg l−1. ICP-OES was calibrated with a five-point calibration curve. QA/QC measurements were also obtained to ensure quality results.
Si-cell degradation in simulated endosomal solution
Twenty million Si cells were suspended in 4 ml simulated endosomal solution containing acetate buffer (pH 5.2; Poly Scientific R&D) and 20% FBS at room temperature under rotation for 3 d. After incubation, the cells were rinsed with water and then dried under vacuum for 7 d. The silicon content of Si cells pre and post treatment was measured using ICP-OES.
Proliferation assays
Native and Si cells with or without PEI coating were assessed for cell growth using the CellTiter-Glo 2.0 assay. Briefly, cells were seeded at a density of 100,000 cells ml−1 in culture media in opaque white 96-well plates. After 24 h, CellTiter-Glo 2.0 reagent was added to each well, and following a 10 min incubation, luminescence was determined using a BioTek microplate reader. Percent cell viability was calculated relative to control, non-treated cells.
Preparation of MSN
A mixture of water (100 ml), ethanol (40 ml), sodium hydroxide (NaOH, 2 M, 0.75 ml) and cetyltrimethylammonium bromide (CTAB, 0.640 g) was heated to 70 °C under vigorous stirring (750 r.p.m.) in a round-bottom flask immersed in an oil bath. Afterwards, tetraethyl orthosilicate (1 ml) was added dropwise to the solution. The tetraethyl orthosilicate was allowed to undergo a series of hydrolysis condensation reactions for 2 h to yield silica CTAB-templated silica nanoparticles. The particles were then isolated by centrifugation (2,000 × g, 20 min) and then washed with methanol three times. The surfactant removal was performed by suspending the nanoparticles in a solution of 0.45 g l−1 ammonium nitrate in ethanol and stirring at 60 °C for 20 min. Finally, the template-free MSN were consecutively washed twice with water and ethanol, and stored suspended in ethanol. MSN (0.5 mg) were rinsed twice with water and then suspended in 1 ml PEI (0.2 mg ml−1) in PBS solution. After 10 min rotation at room temperature to allow PEI binding on the MSN surface, the MSN with PEI coating (MSN-PEI) were then rinsed with PBS twice. MSN-PEI (0.87 mg) were suspended in 20 μl CpG (2 mg ml−1) in double distilled water solution. After 10 min incubation at room temperature, 25 μl MPL (1 mg ml−1) in DMSO solution was added and incubated another 10 min. MSN-PEI-CpG-MPL particles were then centrifuged at 20,000 r.c.f. for 5 min to remove extra free ligand, and then resuspended and stored in 1 ml PBS. These conditions resulted in similar CpG and MPL dose content to vaccine cells.
In vitro DC functional studies
Bone marrow-derived or human ascites DCs were seeded in 12-well plates at a density of 1 × 105 cells per well. After 24 h, the media was removed and replaced with 2 ml of fresh complete media supplemented with 100,000 Si-PEI-CpG-MPL (or irradiated PEI-CpG-MPL) ID8ova or human cancer cells for 24 or 72 h as indicated. Alternatively, DC were incubated with Si-LPS or Si-MPL ID8ova cells for 72 h. Irradiated cells were suspended in PBS in microfuge tubes at 3 × 106 cells per ml and exposed to 100 Gy using a Faxitron Multirad Irradiator at 22rV, 15 mA, 48 Gy min−1. DCs were collected using 3 mM EDTA. The suspended cells were centrifuged, washed with PBS containing 1% BSA and labelled with fluorescent antibodies specific for CD11c and either co-stimulatory molecules or SIINFEKL-H2kb. Cells were analysed using the Becton Dickinson Fortessa or Calibur flow cytometer.
Vaccination of mice with Si tumour cells
Tumour-bearing or naive female FVB or albino C57BL/6 mice were vaccinated intraperitoneally with irradiated (100 Gy using a Faxitron Multirad Irradiator at 225 V, 15 mA, 48 Gy min−1; or a Cs-137 gamma irradiator with a dose rate of 63 cGy min−1) or Si BR5-Akt (or BR5-Akt-Luc2 for vaccine viability analysis or ID8ova) cells (with TLR ligands as indicated) using doses of 3 × 104, 3 × 105, 3 × 106 or 3 × 107 Si cells per mouse in 200 μl of PBS at the indicated schedules. Control vaccines, all containing CpG and MPL, included no antigen MSN, Si leukocytes (splenocytes from naive mice) or oxidized cell lysate. Cell lysate was prepared by incubating BR5-Akt cells in 60 µM hypochlorous acid in PBS for 1 h at 37 °C, followed by extensive PBS washing and five freeze–thaw cycles using dry ice and a 37 °C water bath7,47,48. Alternatively, mice were vaccinated subcutaneously with 3 × 106 Si BR5-Akt cells by scruffing the skin at the back of the neck and injecting an equivalent volume into the loose fold of skin. Mice that cleared all tumour cells based on IVIS Spectrum bioluminescent imaging were re-challenged with 2 × 105 BR5-Akt-Luc2 cancer cells at a later date, as indicated for each study. All control (no Tx) mice received sham PBS injections (200 μl per mouse). Single agent or combination therapy with cisplatin used IP administration of cis-diamineplatinum (II) dichloride (Sigma-Aldrich) on day 9 at 2 mg kg−1 in physiological saline.
Preparation of ascites-derived tumour vaccines
To prepare vaccine using murine tumour (ascites) cells, peritoneal fluid was collected from mice with late-stage BR5-Akt cancer. The intact peritoneal cavity was exposed and ascites, as well as two peritoneal wash samples with cold PBS, were collected using an 18 g needle and 5 ml syringe inserted in the hypogastric region and positioned towards the caecum. In addition, ascites fluid was collected from patients with a diagnosis of ovarian cancer at the time of surgical debulking in accordance with approved IRB Protocol #UNM INST 1509 entitled Single Institution (UNM) Prospective Laboratory Study of Cancer and Immune Cells in the Ascites Fluid of Ovarian Cancer Patients to Test Alternative Therapies. Human specimens were de-identified before transfer for research purposes. To isolate peritoneal cells, human or mouse ascites were centrifuged at 1,400 r.p.m. for 5 min, after which the supernatant was removed, and RBCs were removed using ACK lysis buffer. Tumour cells were enriched using a 37 µm reversible strainer. EpCAM+ populations were evaluated by flow cytometry. Cells were then silicified and surface modified as previously described.
Adoptive transfer of CD8+ T cells
Peritoneal washings were collected from tumour-bearing vaccinated (day 33 post tumour challenge) and control (tumour and vaccine naive) FVB mice as described above. Cells were enriched for CD8+ T cells using the negative selection mouse CD8a+ T-Cell Isolation MACS Cell Separation Kit (Miltenyi Biotec). Purification was confirmed post separation using flow cytometry. Cells were resuspended in cold PBS and adaptively transferred IP (2 x 105 cells per mouse) to tumour and vaccine naive FVB mice. Control mice received sterile PBS IP. Twenty-four hours after transfer of CD8+ T cells, recipient mice were challenged with IP BR5-Akt-Luc2+ tumour cells (2 × 105 per mouse). Mice were then monitored for tumour progression using luminescence on IVIS Spectrum In Vivo Imaging System (Perkin Elmer).
Imaging tumour burden
For in vivo monitoring of tumour burden, mice with BR5-Akt-Luc2 tumours were administered 150 mg luciferin per kg by IP injection, with a 10 min delay before imaging. Mice were then anaesthetized using 2.5% isoflurane, and 2D/3D bioluminescence images were acquired using the Xenogen IVIS Spectrum animal imager (Perkin Elmer). Region of interest measurements of total flux (p s−1) were acquired using Living Image Software (Perkin Elmer).
Murine tissue/cell collection
All mice were euthanized in accordance with the IACUC at the University of New Mexico. Spleens were mechanically dissociated, and RBC were eliminated using ACK or BD Pharm Lyse. Blood was collected by retro-orbital withdrawal using EDTA or heparin to prevent blood clotting. Omentum, peritoneal tumour, lungs, gut, brain and kidneys were dissected out and fixed in 10% buffered formalin. Tissues were embedded in paraffin, sectioned and stained with H&E by the University of New Mexico Health Science Center Histology and Molecular Pathology Shared Resource. Bright-field images were acquired using a dissection microscope (World Precision Instruments) equipped with a Sony CCD progressive scan colour camera.
Biodistribution of vaccine
To track vaccine cells, BR5-Akt cancer cells were first incubated with Cy3-labelled MSN for 16 h. Cells were then washed to remove free MSN and following silicification, 3 × 106 Si-PEI-CpG-MPL cells in 200 μl PBS were IP administered to FVB mice 4 d post IP tumour challenge. Twenty-four hours later, mice were euthanized, and peritoneal tissues were frozen in optimal cutting temperature compound. Following sectioning, tissues were fixed in ice-cold acetone for 15 min, labelled with anti-mouse CD11c FITC antibody and mounted in Prolong Gold mounting media containing DAPI. Images were acquired using a 63X/1.4NA oil objective in sequential scanning mode using a Leica TCS SP8 confocal microscope. To study in vivo tissue biodistribution, CTFR-labelled vaccine cells were administered to FVB mice 4 d post tumour challenge. On days 5 and 6, organs were isolated from mice (n = 2 to 3 per group) and the IVIS Spectrum was used to measure fluorescent intensities.
Immune cell phenotyping
Single-cell suspensions were first blocked with Fc receptor blockers (1 μg anti-CD16/CD32 (clone 2.4G2)) and 1 μg mouse IgG. Next, samples were surface stained with conjugated primary antibodies (1:250 dilution) at room temperature for 30 min in the dark. Samples were then stained with LIVE/DEAD Fixable Aqua Dead Cell Stain for 15 min at room temperature in the dark. For intracellular cytokine analysis, cells were stimulated using eBioscience Cell Stimulation Cocktail (500X) plus Protein Transport Inhibitors Cocktail (500X) for 4 h in RMPI complete media. Cell permeabilization for intracellular staining was done using the eBioscience FoxP3/Transcription Factor Staining Buffer Set. Phenotyping was performed on stained cells using the Attune NxT flow cytometer and analysed using FlowJo 10.6 (Becton, Dickinson and Company).
Cytokine analysis
Neat peritoneal fluid from mice bearing BR5-Akt tumours at various stages of progression, with no treatment or following treatment with dehydrated vaccine, was collected and stored at −80 °C. Samples were evaluated using a custom Milliplex MAP Mouse High Sensitivity T-Cell Panel using the Luminex MAGPIX System (Sigma Aldrich) without dilution as decribed by the vendor.
Blood metabolite measurements
Anti-coagulated blood metabolites and complete blood counts were measured on day 18 or 19 using the Vetscan VS2 Analyser and Comprehensive or Partial Diagnostic Profile discs (Abaxis) as described by the vendor.
Statistical Analysis
Measurements in this study were obtained from distinct samples. Graphpad Prism v6.0d was used to perform statistical analysis. Kaplan–Meier survival curves were analysed using log-rank Mantel–Cox, and Match SPSS and SAS tests were used for two and multiple group comparisons, respectively. For tumour burden comparisons, multiple t-tests assuming all rows were sampled from populations with the same scatter and correction for multiple comparisons using the Holm–Sidak method were used. Column statistics were analysed using unpaired two-tailed parametric t-tests with equal s.d. Graphs include means and error bars, with the latter representing s.d. or s.e.m. as indicated.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
