Preparation of AND logic NPs
PEG-b-P(SC7A-thioethylamine·HCl) was synthesized following the established protocols21. Completely different stimuli-responsive polymers had been synthesized as described in Supplementary Figs. 1–3. To organize NPs from every copolymer, 5 mg of the copolymer was first dissolved in 1 ml of PBS buffer at pH 5.0. Sodium hydroxide (0.1 M) was then added dropwise below steady stirring till the pH reached 7.4, permitting micelle formation. The ensuing NPs had been characterised utilizing dynamic gentle scattering (Zetasizer, Malvern Devices) outfitted with a He–Ne laser (λ = 633 nm, the place λ is wavelength of the laser gentle).
In vitro drug launch take a look at
PHM NP options had been incubated at 37 °C for twenty-four h in PBS at pH 6.5 or pH 7.4, with or with out NQO1/NADH. The discharge of MSA-2 was quantified utilizing HPLC with a C18 column. The cellular section consisted of solvent A as water with 0.08% trifluoroacetic acid and solvent B as acetonitrile with 0.08% trifluoroacetic acid. As well as, PHM NP options at pH 6.5 had been incubated with numerous circumstances, together with GSH, NADH, NQO1, NQO1/NADH or NQO1/NADH with dicoumarol (NQO1 inhibitor), at 37 °C for twenty-four h.
Cell line
THP1-Lucia ISG cells had been bought from InvivoGen (catalogue quantity thpl-isg). B16F10 cells had been bought from ATCC (catalogue quantity CRL-6475). 4T1 cells had been offered by S. Huang, Massey Most cancers Middle, Virginia Commonwealth College. LL/2 cells had been offered by Z. ‘James’ Chen, College of Texas (UT) Southwestern Medical Middle (UT Southwestern). DC2.4 cells had been offered by D. J. Siegwart, UT Southwestern. Raw264.7 cells had been offered by Z. ‘James’ Chen. IMCD3 cells had been offered by Y. Xun, UT Southwestern.
Sort I IFN reporter assay
THP1-Lucia ISG cells (InvivoGen) had been seeded at 1 × 105 cells per effectively in 96-well plates (180 µl per effectively) on day 1 and differentiated with phorbol 12-myristate 13-acetate (ultimate 20–50 ng ml−1; 20 µl per effectively) for 3 h at 37 °C, 5% CO2. Cells had been gently washed as soon as with pre-warmed PBS and replenished with 200 µl pre-warmed full development medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin, pH 7.4). On day 4, cells had been washed as soon as with pre-warmed PBS and 180 µl contemporary medium was added, adopted by 20 µl NP inventory resolution (ultimate quantity 200 µl). Plates had been incubated for twenty-four h at 37 °C below hypoxia (1% O2, 5% CO2). Supernatants (10 µl per effectively) had been transferred to white opaque 96-well plates, and QUANTI-Luc 4 reagent (InvivoGen) was ready per the producer’s directions, loaded into the luminometer injector and disbursed for instant luminescence measurement. Sign was recorded as relative gentle models (RLU), with background (medium-only) subtracted and knowledge expressed as fold-induction over untreated management.
Enzyme-linked immunosorbent assay
DC2.4 cells had been seeded at a density of 5 × 105 cells per effectively in 6-well plates and allowed to stick in a single day. Cells had been then handled with PHM NP (100 µg ml−1) for the indicated time durations. At every time level, cell tradition supernatants had been collected, centrifuged to take away particles and analysed utilizing the mouse CXCL10/IP-10/CRG-2 DuoSet enzyme-linked immunosorbent assay (ELISA) equipment and the mouse IFNβ DuoSet ELISA equipment (R&D Programs) in keeping with the producer’s directions. The absorbance at 450 nm was measured utilizing a microplate reader, and cytokine concentrations had been calculated primarily based on commonplace curves generated from recombinant mouse proteins.
NQO1 exercise assay
NQO1 enzymatic exercise was quantified by monitoring the discount of methyl crimson as a colorimetric substrate. In short, methyl crimson (25 µg ml−1) and NADH (1 mM) had been incubated with serial concentrations of recombinant mouse NQO1 protein (40 µg ml−1, 20 µg ml−1, 10 µg ml−1, 5 µg ml−1, 1 µg ml−1, 0.5 µg ml−1, 0.1 µg ml−1 and 0 µg ml−1) in 1× PBS at 37 °C. The lower in absorbance at 430 nm was recorded utilizing a microplate reader to generate a typical calibration curve. For intracellular quantification, DC2.4 cells had been cultured below normoxic (20% O2) or hypoxic (1% O2) circumstances for twenty-four h. Cell lysates had been ready from 3 × 105 cells in 10 µl of lysis buffer, and intracellular NQO1 ranges had been decided primarily based on the calibration curve.
Micelle stability and mobile uptake mechanism
For micelle stability evaluation, PHM NPs (1 mg ml−1) had been dispersed in PBS (pH 7.4) or serum-containing medium and incubated at 37 °C. The particle measurement and zeta potential had been monitored over time by dynamic gentle scattering to guage colloidal stability.
To analyze the mobile uptake mechanism, DC2.4 cells had been pretreated with endocytosis inhibitors for 30 min earlier than NP publicity: chlorpromazine (10 µg ml−1) to inhibit clathrin-mediated endocytosis by disrupting clathrin-coated pit formation; nystatin (50 µg ml−1) to dam caveolae/lipid raft-dependent internalization by depleting membrane ldl cholesterol; and amiloride (1 mM) to suppress macropinocytosis by inhibiting Na+/H+ alternate. Cells had been then incubated with PHM NP/PSC7A-Cy5 (0.1 mg ml−1, mass ratio = 7:3) for the indicated interval, washed and imaged utilizing confocal laser scanning microscopy. The fluorescence depth of internalized NPs was quantified by movement cytometry, and relative uptake amongst therapy teams was in contrast. Statistical significance was evaluated utilizing one-way evaluation of variance (ANOVA).
Mice
STING−/− and Batf3−/− mice had been bought from the Jackson Laboratory, whereas C57BL/6 WT and BALB/c mice had been obtained from Charles River Laboratories. All mice had been saved below particular pathogen-free circumstances in a barrier facility with a 12 h gentle–12 h darkish cycle and fed commonplace chow (2916, Teklad International). The experimental teams consisted of randomly chosen feminine littermates, roughly 6–8 weeks outdated, of the identical pressure. All procedures had been performed in accordance with the moral pointers and protocols accredited by the AAALAC-accredited Institutional Animal Care and Use Committee at UT Southwestern Medical Middle below protocol quantity 2017-102331.
Tumour fashions and therapy protocols
To determine the lung metastasis fashions, C57BL/6 mice had been intravenously injected with both 1 × 106 LL/2 lung most cancers cells or 1 × 105 B16F10 melanoma cells on day 0. The metastatic breast most cancers mannequin was generated in Balb/c mice by injecting 1 × 106 4T1 most cancers cells into the mammary fats pad. For the LL/2 lung most cancers mannequin, tumour-bearing C57BL/6 mice had been handled through i.v. injection of AND logic NPs (100 µl per mouse), MSA-2 or PBS (management) on day 5. Lungs had been collected on day 14 and glued in Bouin’s resolution for twenty-four h. Within the B16F10 melanoma mannequin, mice had been handled on day 7, and lungs had been collected on day 17 following fixation in formalin for twenty-four h. For the 4T1 breast most cancers mannequin, mice had been handled on days 9, 11 and 15. Major tumour volumes had been monitored, and lungs had been collected on day 24 after fixation in Bouin’s resolution for twenty-four h. In a subset of experiments, aPD1 (200 µg) was administered intraperitoneally 1 day earlier than PHM NP therapy and repeated 3 occasions throughout the research. 4T1 tumour volumes had been measured utilizing calipers, with size (L) and width (W) recorded. Volumes had been calculated utilizing the system V = L × W × W/2. Based on the institutional animal care pointers, the maximal permitted tumour measurement was 1,500 mm3, at which level animals had been euthanized.
Security evaluations
Mouse serum was collected 6 h post-injection of PHM NP, MSA-2, PSC7A or PBS for cytokine evaluation. The samples had been analysed utilizing the BD cytometric bead array (CBA) mouse irritation equipment (catalogue quantity 552364) and movement cytometry to quantify inflammatory cytokines. For liver and kidney perform assessments, serum samples had been collected 24 h post-treatments. The samples had been saved at 4 °C and subsequently despatched to the UTSW Metabolic Phenotyping Core for evaluation.
Pharmacokinetic evaluation
C57BL/6 mice bearing LL/2 lung metastases had been intravenously administered dye-labelled PHM NP, formulated as a hybrid NP consisting of PHM NP and PSC7A-ICG at a mass ratio of seven:3. Every PSC7A polymer was conjugated with ~3 ICG molecules, and the full injected NP dose was 20 mg kg−1. At predefined time factors post-injection, 50 µl of blood was collected from every mouse (n = 5 per group). Plasma was separated by centrifugation, diluted fivefold with PBS buffer containing 5 mM EDTA (pH 6.0), and fluorescence was quantified utilizing the Pearl Trilogy Small Animal Imaging System (LI-COR). Fluorescent NPs had been quantified at 800 nm (for ICG). Information had been introduced as the proportion of the injected dose (% ID), with plasma collected at 5 min post-injection representing the utmost dose (100% ID). Pharmacokinetic profiles had been analysed utilizing nonlinear regression and a two-phase decay mannequin with GraphPad Prism software program v10.5.0.
Biodistribution evaluation
To evaluate organ distribution, C57BL/6 mice bearing LL/2 lung metastases and Balb/c mice bearing 4T1 orthotopic stable tumours (~120 mm3) had been intravenously injected with dye-labelled STING NPs as described above. Mice had been euthanized 24 h post-injection, and tissues, together with metastatic lung, TDLNs, liver, lungs, coronary heart, kidney and spleen, had been collected and weighed. Tissues had been mechanically dissociated and homogenized in lysis buffer (2% Triton X-100, 100 mM HEPES, 5 mM EDTA, pH 7.1) utilizing tissue grinder tubes (Precellys Lysing Kits). Homogenates had been centrifuged at 500 × g for 3 min, and the supernatants had been transferred to black 96-well plates for fluorescence quantification utilizing the Pearl Trilogy Small Animal Imaging System (LI-COR). Fluorescent NPs had been measured at 800 nm (ICG), and NP concentrations had been calculated utilizing tissue-specific commonplace curves generated from untreated mice. Organ uptake was reported as the proportion of injected dose per gram of tissue.
Cell tropism research
For cell tropism research, C57BL/6 mice bearing LL/2 lung metastases and Balb/c mice bearing 4T1 orthotopic stable tumours (~120 mm3) had been intravenously injected with Cy5-labelled STING NPs. Mice had been euthanized 24 h post-injection, and tissues, together with lung metastases, TDLNs and spleens, had been collected and processed into single-cell suspensions. Cells had been stained with fluorochrome-conjugated antibodies, together with CD45 PerCP (clone 30-F11, BioLegend), MHC-II AF700 (clone M5/114.15.2, Invitrogen), CD11c BV605 (clone N418, BioLegend), CD11b PB (clone M1/70, BioLegend), CD4 FITC (clone RM4-5, BioLegend), CD8a PE (clone 53-6.7, BioLegend), CD3e BV786 (clone 145-2C11, BD Biosciences), F4/80 PE (clone BM8, Miltenyi Biotec), NK1.1 PE-Cy7 (clone PK136, BD Biosciences) and B220 APC-Cy7 (clone RA3-6B2, BioLegend). The LIVE/DEAD Fixable Aqua Lifeless Cell Stain Package (Invitrogen, L34966) was used to evaluate cell viability. Information had been acquired utilizing BD LSRFortessa or Beckman CytoFLEX movement cytometers and analysed with CytExpert v2.4 and FlowJo v10.10.0. All gating methods are proven in Supplementary Fig. 20.
APC activation and T cell priming research
For APC and T cell evaluation, C57BL/6 mice bearing B16F10-OVA lung metastases had been intravenously injected with PHM NPs at schematic time. Mice had been euthanized 1 or 5 days post-injection, and tissues, together with lung metastases, TDLNs and spleens, had been collected and processed into single-cell suspensions. Cells had been stained with fluorochrome-conjugated antibodies, together with CD45 PerCP (clone 30-F11, BioLegend), MHC-II AF700 (clone M5/114.15.2, Invitrogen), CD11c BV605 (clone N418, BioLegend), CD11b PB (clone M1/70, BioLegend), F4/80 APC-Cy7 (clone BM8, BioLegend), CD80 PE-Cy7 (clone 16-10A1, BioLegend), CD86 APC (clone GL-1, BioLegend), CD206 PE (clone), CD4 PB (clone GK1.5, BioLegend), CD8a AF700 (clone QA17A07, BioLegend), CD3e BV786 (clone 145-2C11, BD Biosciences), H-2Kb OVA tetramer PE (SIINFEKL, MBL Life Sciences), CD62L PE-Cy7 (clone MEL-14, BioLegend), CD44 APC-Cy7 (clone IM7, BioLegend), GrzmB APC (clone QA16A02, BioLegend) and IFNγ PE (clone XMG1.2, BioLegend). The LIVE/DEAD Fixable Aqua Lifeless Cell Stain Package (Invitrogen, L34966) was used to evaluate cell viability. Information had been acquired utilizing BD LSRFortessa or Beckman CytoFLEX movement cytometers and analysed with CytExpert v2.4 and FlowJo. All gating methods are proven in Supplementary Figs. 20–23.
mIHC evaluation
Formalin-fixed, paraffin-embedded LL/2 lung tissues had been collected for mIHC staining. mIHC was carried out utilizing the Opal 7-Colour Handbook IHC Package (Akoya Biosciences, NEL811001KT) in keeping with the producer’s protocol. Multispectral photos had been acquired utilizing the Akoya Biosciences Vectra Polaris system at ×20 magnification. For every slide, ten randomly chosen fields had been imaged. The ensuing multispectral photos had been processed and analysed utilizing the Halo v3.6.4134.464 picture evaluation software program. Particular person cells had been recognized utilizing a nuclear segmentation algorithm primarily based on DAPI staining, with a mobile masks utilized round every nucleus. This allowed for the quantification of floor marker expression on the single-cell stage. Staining brokers are as follows: 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific), phospho-STING (72971, Cell Signaling), anti-mouse/rat XCR1 antibody (148202, BioLegend) and CD8α (98941, Cell Signaling).
Western blot
All reagents had been obtained from Bio-Rad, and first antibodies included anti-NQO1 (N5288, Sigma, 1:800) and β-actin (A2228, Sigma-Aldrich, mouse monoclonal antibody, 1:5,000). Cells had been lysed in SDS pattern buffer containing protease and phosphatase inhibitors, adopted by heating for protein denaturation. The lysates had been centrifuged, and the supernatant was loaded onto a 4–15% Mini-PROTEAN gel (Bio-Rad). Electrophoresis was carried out at 50 V for 20 min, adopted by 100 V for 60 min. Proteins had been transferred to a PVDF membrane utilizing 100 V for 60 min on ice. After switch, membranes had been blocked for 1 h at room temperature in both 5% non-fat milk or BSA (for phosphorylated proteins). Membranes had been then incubated in a single day at 4 °C with major antibodies. Secondary antibodies (goat anti-mouse or goat anti-rabbit IgG, HRP-linked; BioLegend, 1:3,000) had been utilized for 1 h at room temperature. Protein bands had been visualized utilizing the GelDoc Go Gel Imaging System (Bio-Rad).
In vivo immune cell depletion experiments
For NK cell depletion, mice had been intraperitoneally injected with 500 μg of anti-NK1.1 antibody (clone PK136, BioXcell) on day 4 following LL/2 inoculation. Upkeep doses of 250 μg of anti-NK1.1 antibody had been administered each 3 days. For CD8+ or CD4+ T cell depletion, mice obtained 200 μg of anti-CD8a antibody (clone YTS169.4, BioXcell) or anti-CD4 antibody (clone GK1.5, BioXcell) by intraperitoneal injection on day 4 after LL/2 cell injection. Upkeep doses of the identical antibodies (200 μg) had been administered each 3 days.
Statistical analyses
Statistical analyses had been performed utilizing Microsoft Excel and GraphPad Prism (model 9.0). Information are introduced because the imply ± commonplace error of the imply (s.e.m.), except in any other case specified. Acceptable publish hoc statistical assessments had been utilized all through. For usually distributed datasets, one-way ANOVA adopted by Tukey’s or Bonferroni’s multiple-comparison assessments was used for comparisons amongst three or extra teams, and two-way ANOVA with Bonferroni’s publish hoc correction or main-effect-only fashions was utilized for longitudinal tumour development curves. For pairwise comparisons, two-tailed unpaired Scholar’s t-tests had been used. Survival evaluation was performed utilizing the Mantel–Cox take a look at.
Reporting abstract
Additional info on analysis design is on the market within the Nature Portfolio Reporting Abstract linked to this text.
