Preparation of AND logic NPs
PEG-b-P(SC7A-thioethylamine·HCl) was synthesized following the established protocols21. Totally different stimuli-responsive polymers have been synthesized as described in Supplementary Figs. 1–3. To arrange 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 underneath steady stirring till the pH reached 7.4, permitting micelle formation. The ensuing NPs have been characterised utilizing dynamic gentle scattering (Zetasizer, Malvern Devices) geared up 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 have 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 cell 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 have been incubated with numerous situations, 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 have been bought from InvivoGen (catalogue quantity thpl-isg). B16F10 cells have been bought from ATCC (catalogue quantity CRL-6475). 4T1 cells have been supplied by S. Huang, Massey Most cancers Middle, Virginia Commonwealth College. LL/2 cells have been supplied by Z. ‘James’ Chen, College of Texas (UT) Southwestern Medical Middle (UT Southwestern). DC2.4 cells have been supplied by D. J. Siegwart, UT Southwestern. Raw264.7 cells have been supplied by Z. ‘James’ Chen. IMCD3 cells have been supplied by Y. Xun, UT Southwestern.
Sort I IFN reporter assay
THP1-Lucia ISG cells (InvivoGen) have 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 have been gently washed as soon as with pre-warmed PBS and replenished with 200 µl pre-warmed full progress medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin, pH 7.4). On day 4, cells have been washed as soon as with pre-warmed PBS and 180 µl recent medium was added, adopted by 20 µl NP inventory answer (ultimate quantity 200 µl). Plates have been incubated for twenty-four h at 37 °C underneath hypoxia (1% O2, 5% CO2). Supernatants (10 µl per effectively) have 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 rapid 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 have been seeded at a density of 5 × 105 cells per effectively in 6-well plates and allowed to stick in a single day. Cells have been then handled with PHM NP (100 µg ml−1) for the indicated time durations. At every time level, cell tradition supernatants have been collected, centrifuged to take away particles and analysed utilizing the mouse CXCL10/IP-10/CRG-2 DuoSet enzyme-linked immunosorbent assay (ELISA) package and the mouse IFNβ DuoSet ELISA package (R&D Programs) in accordance with the producer’s directions. The absorbance at 450 nm was measured utilizing a microplate reader, and cytokine concentrations have been calculated based mostly on customary curves generated from recombinant mouse proteins.
NQO1 exercise assay
NQO1 enzymatic exercise was quantified by monitoring the discount of methyl purple as a colorimetric substrate. In short, methyl purple (25 µg ml−1) and NADH (1 mM) have 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 an ordinary calibration curve. For intracellular quantification, DC2.4 cells have been cultured underneath normoxic (20% O2) or hypoxic (1% O2) situations for twenty-four h. Cell lysates have been ready from 3 × 105 cells in 10 µl of lysis buffer, and intracellular NQO1 ranges have been decided based mostly on the calibration curve.
Micelle stability and mobile uptake mechanism
For micelle stability evaluation, PHM NPs (1 mg ml−1) have been dispersed in PBS (pH 7.4) or serum-containing medium and incubated at 37 °C. The particle dimension and zeta potential have been monitored over time by dynamic gentle scattering to judge colloidal stability.
To research the mobile uptake mechanism, DC2.4 cells have 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+ change. Cells have 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 move 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 have been bought from the Jackson Laboratory, whereas C57BL/6 WT and BALB/c mice have been obtained from Charles River Laboratories. All mice have been stored underneath particular pathogen-free situations in a barrier facility with a 12 h gentle–12 h darkish cycle and fed customary chow (2916, Teklad International). The experimental teams consisted of randomly chosen feminine littermates, roughly 6–8 weeks previous, of the identical pressure. All procedures have been carried out in accordance with the moral pointers and protocols authorized by the AAALAC-accredited Institutional Animal Care and Use Committee at UT Southwestern Medical Middle underneath protocol quantity 2017-102331.
Tumour fashions and therapy protocols
To determine the lung metastasis fashions, C57BL/6 mice have 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 have been handled by way of i.v. injection of AND logic NPs (100 µl per mouse), MSA-2 or PBS (management) on day 5. Lungs have been collected on day 14 and stuck in Bouin’s answer for twenty-four h. Within the B16F10 melanoma mannequin, mice have been handled on day 7, and lungs have been collected on day 17 following fixation in formalin for twenty-four h. For the 4T1 breast most cancers mannequin, mice have been handled on days 9, 11 and 15. Major tumour volumes have been monitored, and lungs have been collected on day 24 after fixation in Bouin’s answer 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 instances throughout the examine. 4T1 tumour volumes have been measured utilizing calipers, with size (L) and width (W) recorded. Volumes have been calculated utilizing the components V = L × W × W/2. In response to the institutional animal care pointers, the maximal permitted tumour dimension was 1,500 mm3, at which level animals have 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 have been analysed utilizing the BD cytometric bead array (CBA) mouse irritation package (catalogue quantity 552364) and move cytometry to quantify inflammatory cytokines. For liver and kidney operate exams, serum samples have been collected 24 h post-treatments. The samples have 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 have 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 entire 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 have been quantified at 800 nm (for ICG). Information have 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 have 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 strong tumours (~120 mm3) have been intravenously injected with dye-labelled STING NPs as described above. Mice have been euthanized 24 h post-injection, and tissues, together with metastatic lung, TDLNs, liver, lungs, coronary heart, kidney and spleen, have been collected and weighed. Tissues have 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 have been centrifuged at 500 × g for 3 min, and the supernatants have been transferred to black 96-well plates for fluorescence quantification utilizing the Pearl Trilogy Small Animal Imaging System (LI-COR). Fluorescent NPs have been measured at 800 nm (ICG), and NP concentrations have been calculated utilizing tissue-specific customary 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 strong tumours (~120 mm3) have been intravenously injected with Cy5-labelled STING NPs. Mice have been euthanized 24 h post-injection, and tissues, together with lung metastases, TDLNs and spleens, have been collected and processed into single-cell suspensions. Cells have 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 have been acquired utilizing BD LSRFortessa or Beckman CytoFLEX move 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 have been intravenously injected with PHM NPs at schematic time. Mice have been euthanized 1 or 5 days post-injection, and tissues, together with lung metastases, TDLNs and spleens, have been collected and processed into single-cell suspensions. Cells have 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 have been acquired utilizing BD LSRFortessa or Beckman CytoFLEX move 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 have been collected for mIHC staining. mIHC was carried out utilizing the Opal 7-Colour Handbook IHC Package (Akoya Biosciences, NEL811001KT) in accordance with the producer’s protocol. Multispectral photographs have been acquired utilizing the Akoya Biosciences Vectra Polaris system at ×20 magnification. For every slide, ten randomly chosen fields have been imaged. The ensuing multispectral photographs have been processed and analysed utilizing the Halo v3.6.4134.464 picture evaluation software program. Particular person cells have been recognized utilizing a nuclear segmentation algorithm based mostly 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 have 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 have been lysed in SDS pattern buffer containing protease and phosphatase inhibitors, adopted by heating for protein denaturation. The lysates have 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 have been transferred to a PVDF membrane utilizing 100 V for 60 min on ice. After switch, membranes have been blocked for 1 h at room temperature in both 5% non-fat milk or BSA (for phosphorylated proteins). Membranes have been then incubated in a single day at 4 °C with main antibodies. Secondary antibodies (goat anti-mouse or goat anti-rabbit IgG, HRP-linked; BioLegend, 1:3,000) have been utilized for 1 h at room temperature. Protein bands have been visualized utilizing the GelDoc Go Gel Imaging System (Bio-Rad).
In vivo immune cell depletion experiments
For NK cell depletion, mice have 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 have 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) have been administered each 3 days.
Statistical analyses
Statistical analyses have been carried out utilizing Microsoft Excel and GraphPad Prism (model 9.0). Information are introduced because the imply ± customary error of the imply (s.e.m.), except in any other case specified. Applicable put up hoc statistical exams have been utilized all through. For usually distributed datasets, one-way ANOVA adopted by Tukey’s or Bonferroni’s multiple-comparison exams was used for comparisons amongst three or extra teams, and two-way ANOVA with Bonferroni’s put up hoc correction or main-effect-only fashions was utilized for longitudinal tumour progress curves. For pairwise comparisons, two-tailed unpaired Scholar’s t-tests have been used. Survival evaluation was carried out utilizing the Mantel–Cox take a look at.
Reporting abstract
Additional data on analysis design is on the market within the Nature Portfolio Reporting Abstract linked to this text.