Preparation of AND logic NPs
PEG-b-P(SC7A-thioethylamine·HCl) was synthesized following the established protocols21. Different stimuli-responsive polymers were synthesized as described in Supplementary Figs. 1–3. To prepare NPs from each 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 under continuous stirring until the pH reached 7.4, allowing micelle formation. The resulting NPs were characterized using dynamic light scattering (Zetasizer, Malvern Instruments) equipped with a He–Ne laser (λ = 633 nm, where λ is wavelength of the laser light).
In vitro drug release test
PHM NP solutions were incubated at 37 °C for 24 h in PBS at pH 6.5 or pH 7.4, with or without NQO1/NADH. The release of MSA-2 was quantified using HPLC with a C18 column. The mobile phase consisted of solvent A as water with 0.08% trifluoroacetic acid and solvent B as acetonitrile with 0.08% trifluoroacetic acid. In addition, PHM NP solutions at pH 6.5 were incubated with various conditions, including GSH, NADH, NQO1, NQO1/NADH or NQO1/NADH with dicoumarol (NQO1 inhibitor), at 37 °C for 24 h.
Cell line
THP1-Lucia ISG cells were purchased from InvivoGen (catalogue number thpl-isg). B16F10 cells were purchased from ATCC (catalogue number CRL-6475). 4T1 cells were provided by S. Huang, Massey Cancer Center, Virginia Commonwealth University. LL/2 cells were provided by Z. ‘James’ Chen, University of Texas (UT) Southwestern Medical Center (UT Southwestern). DC2.4 cells were provided by D. J. Siegwart, UT Southwestern. Raw264.7 cells were provided by Z. ‘James’ Chen. IMCD3 cells were provided by Y. Xun, UT Southwestern.
Type I IFN reporter assay
THP1-Lucia ISG cells (InvivoGen) were seeded at 1 × 105 cells per well in 96-well plates (180 µl per well) on day 1 and differentiated with phorbol 12-myristate 13-acetate (final 20–50 ng ml−1; 20 µl per well) for 3 h at 37 °C, 5% CO2. Cells were gently washed once with pre-warmed PBS and replenished with 200 µl pre-warmed complete growth medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin, pH 7.4). On day 4, cells were washed once with pre-warmed PBS and 180 µl fresh medium was added, followed by 20 µl NP stock solution (final volume 200 µl). Plates were incubated for 24 h at 37 °C under hypoxia (1% O2, 5% CO2). Supernatants (10 µl per well) were transferred to white opaque 96-well plates, and QUANTI-Luc 4 reagent (InvivoGen) was prepared per the manufacturer’s instructions, loaded into the luminometer injector and dispensed for immediate luminescence measurement. Signal was recorded as relative light units (RLU), with background (medium-only) subtracted and data expressed as fold-induction over untreated control.
Enzyme-linked immunosorbent assay
DC2.4 cells were seeded at a density of 5 × 105 cells per well in 6-well plates and allowed to adhere overnight. Cells were then treated with PHM NP (100 µg ml−1) for the indicated time periods. At each time point, cell culture supernatants were collected, centrifuged to remove debris and analysed using the mouse CXCL10/IP-10/CRG-2 DuoSet enzyme-linked immunosorbent assay (ELISA) kit and the mouse IFNβ DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate reader, and cytokine concentrations were calculated based on standard curves generated from recombinant mouse proteins.
NQO1 activity assay
NQO1 enzymatic activity was quantified by monitoring the reduction of methyl red as a colorimetric substrate. In brief, methyl red (25 µg ml−1) and NADH (1 mM) were 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 decrease in absorbance at 430 nm was recorded using a microplate reader to generate a standard calibration curve. For intracellular quantification, DC2.4 cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions for 24 h. Cell lysates were prepared from 3 × 105 cells in 10 µl of lysis buffer, and intracellular NQO1 levels were determined based on the calibration curve.
Micelle stability and cellular uptake mechanism
For micelle stability assessment, PHM NPs (1 mg ml−1) were dispersed in PBS (pH 7.4) or serum-containing medium and incubated at 37 °C. The particle size and zeta potential were monitored over time by dynamic light scattering to evaluate colloidal stability.
To investigate the cellular uptake mechanism, DC2.4 cells were pretreated with endocytosis inhibitors for 30 min before NP exposure: chlorpromazine (10 µg ml−1) to inhibit clathrin-mediated endocytosis by disrupting clathrin-coated pit formation; nystatin (50 µg ml−1) to block caveolae/lipid raft-dependent internalization by depleting membrane cholesterol; and amiloride (1 mM) to suppress macropinocytosis by inhibiting Na+/H+ exchange. Cells were then incubated with PHM NP/PSC7A-Cy5 (0.1 mg ml−1, mass ratio = 7:3) for the indicated period, washed and imaged using confocal laser scanning microscopy. The fluorescence intensity of internalized NPs was quantified by flow cytometry, and relative uptake among treatment groups was compared. Statistical significance was evaluated using one-way analysis of variance (ANOVA).
Mice
STING−/− and Batf3−/− mice were purchased from the Jackson Laboratory, while C57BL/6 WT and BALB/c mice were obtained from Charles River Laboratories. All mice were kept under specific pathogen-free conditions in a barrier facility with a 12 h light–12 h dark cycle and fed standard chow (2916, Teklad Global). The experimental groups consisted of randomly selected female littermates, approximately 6–8 weeks old, of the same strain. All procedures were conducted in accordance with the ethical guidelines and protocols approved by the AAALAC-accredited Institutional Animal Care and Use Committee at UT Southwestern Medical Center under protocol number 2017-102331.
Tumour models and treatment protocols
To establish the lung metastasis models, C57BL/6 mice were intravenously injected with either 1 × 106 LL/2 lung cancer cells or 1 × 105 B16F10 melanoma cells on day 0. The metastatic breast cancer model was generated in Balb/c mice by injecting 1 × 106 4T1 cancer cells into the mammary fat pad. For the LL/2 lung cancer model, tumour-bearing C57BL/6 mice were treated via i.v. injection of AND logic NPs (100 µl per mouse), MSA-2 or PBS (control) on day 5. Lungs were collected on day 14 and fixed in Bouin’s solution for 24 h. In the B16F10 melanoma model, mice were treated on day 7, and lungs were collected on day 17 following fixation in formalin for 24 h. For the 4T1 breast cancer model, mice were treated on days 9, 11 and 15. Primary tumour volumes were monitored, and lungs were collected on day 24 after fixation in Bouin’s solution for 24 h. In a subset of experiments, aPD1 (200 µg) was administered intraperitoneally 1 day before PHM NP treatment and repeated 3 times during the study. 4T1 tumour volumes were measured using calipers, with length (L) and width (W) recorded. Volumes were calculated using the formula V = L × W × W/2. According to the institutional animal care guidelines, the maximal permitted tumour size was 1,500 mm3, at which point animals were euthanized.
Safety evaluations
Mouse serum was collected 6 h post-injection of PHM NP, MSA-2, PSC7A or PBS for cytokine analysis. The samples were analysed using the BD cytometric bead array (CBA) mouse inflammation kit (catalogue number 552364) and flow cytometry to quantify inflammatory cytokines. For liver and kidney function tests, serum samples were collected 24 h post-treatments. The samples were stored at 4 °C and subsequently sent to the UTSW Metabolic Phenotyping Core for analysis.
Pharmacokinetic analysis
C57BL/6 mice bearing LL/2 lung metastases were intravenously administered dye-labelled PHM NP, formulated as a hybrid NP consisting of PHM NP and PSC7A-ICG at a mass ratio of 7:3. Each PSC7A polymer was conjugated with ~3 ICG molecules, and the total injected NP dose was 20 mg kg−1. At predefined time points post-injection, 50 µl of blood was collected from each 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 using the Pearl Trilogy Small Animal Imaging System (LI-COR). Fluorescent NPs were quantified at 800 nm (for ICG). Data were presented as the percentage of the injected dose (% ID), with plasma collected at 5 min post-injection representing the maximum dose (100% ID). Pharmacokinetic profiles were analysed using nonlinear regression and a two-phase decay model with GraphPad Prism software v10.5.0.
Biodistribution analysis
To assess organ distribution, C57BL/6 mice bearing LL/2 lung metastases and Balb/c mice bearing 4T1 orthotopic solid tumours (~120 mm3) were intravenously injected with dye-labelled STING NPs as described above. Mice were euthanized 24 h post-injection, and tissues, including metastatic lung, TDLNs, liver, lungs, heart, kidney and spleen, were collected and weighed. Tissues were mechanically dissociated and homogenized in lysis buffer (2% Triton X-100, 100 mM HEPES, 5 mM EDTA, pH 7.1) using tissue grinder tubes (Precellys Lysing Kits). Homogenates were centrifuged at 500 × g for 3 min, and the supernatants were transferred to black 96-well plates for fluorescence quantification using the Pearl Trilogy Small Animal Imaging System (LI-COR). Fluorescent NPs were measured at 800 nm (ICG), and NP concentrations were calculated using tissue-specific standard curves generated from untreated mice. Organ uptake was reported as the percentage of injected dose per gram of tissue.
Cell tropism studies
For cell tropism studies, C57BL/6 mice bearing LL/2 lung metastases and Balb/c mice bearing 4T1 orthotopic solid tumours (~120 mm3) were intravenously injected with Cy5-labelled STING NPs. Mice were euthanized 24 h post-injection, and tissues, including lung metastases, TDLNs and spleens, were collected and processed into single-cell suspensions. Cells were stained with fluorochrome-conjugated antibodies, including 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 Dead Cell Stain Kit (Invitrogen, L34966) was used to assess cell viability. Data were acquired using BD LSRFortessa or Beckman CytoFLEX flow cytometers and analysed with CytExpert v2.4 and FlowJo v10.10.0. All gating strategies are shown in Supplementary Fig. 20.
APC activation and T cell priming studies
For APC and T cell analysis, C57BL/6 mice bearing B16F10-OVA lung metastases were intravenously injected with PHM NPs at schematic time. Mice were euthanized 1 or 5 days post-injection, and tissues, including lung metastases, TDLNs and spleens, were collected and processed into single-cell suspensions. Cells were stained with fluorochrome-conjugated antibodies, including 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 Dead Cell Stain Kit (Invitrogen, L34966) was used to assess cell viability. Data were acquired using BD LSRFortessa or Beckman CytoFLEX flow cytometers and analysed with CytExpert v2.4 and FlowJo. All gating strategies are shown in Supplementary Figs. 20–23.
mIHC analysis
Formalin-fixed, paraffin-embedded LL/2 lung tissues were collected for mIHC staining. mIHC was performed using the Opal 7-Color Manual IHC Kit (Akoya Biosciences, NEL811001KT) according to the manufacturer’s protocol. Multispectral images were acquired using the Akoya Biosciences Vectra Polaris system at ×20 magnification. For each slide, ten randomly selected fields were imaged. The resulting multispectral images were processed and analysed using the Halo v3.6.4134.464 image analysis software. Individual cells were identified using a nuclear segmentation algorithm based on DAPI staining, with a cellular mask applied around each nucleus. This allowed for the quantification of surface marker expression at the single-cell level. Staining agents 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 were obtained from Bio-Rad, and primary antibodies included anti-NQO1 (N5288, Sigma, 1:800) and β-actin (A2228, Sigma-Aldrich, mouse monoclonal antibody, 1:5,000). Cells were lysed in SDS sample buffer containing protease and phosphatase inhibitors, followed by heating for protein denaturation. The lysates were centrifuged, and the supernatant was loaded onto a 4–15% Mini-PROTEAN gel (Bio-Rad). Electrophoresis was performed at 50 V for 20 min, followed by 100 V for 60 min. Proteins were transferred to a PVDF membrane using 100 V for 60 min on ice. After transfer, membranes were blocked for 1 h at room temperature in either 5% non-fat milk or BSA (for phosphorylated proteins). Membranes were then incubated overnight at 4 °C with primary antibodies. Secondary antibodies (goat anti-mouse or goat anti-rabbit IgG, HRP-linked; BioLegend, 1:3,000) were applied for 1 h at room temperature. Protein bands were visualized using the GelDoc Go Gel Imaging System (Bio-Rad).
In vivo immune cell depletion experiments
For NK cell depletion, mice were intraperitoneally injected with 500 μg of anti-NK1.1 antibody (clone PK136, BioXcell) on day 4 following LL/2 inoculation. Maintenance doses of 250 μg of anti-NK1.1 antibody were administered every 3 days. For CD8+ or CD4+ T cell depletion, mice received 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. Maintenance doses of the same antibodies (200 μg) were administered every 3 days.
Statistical analyses
Statistical analyses were conducted using Microsoft Excel and GraphPad Prism (version 9.0). Data are presented as the mean ± standard error of the mean (s.e.m.), unless otherwise specified. Appropriate post hoc statistical tests were applied throughout. For normally distributed datasets, one-way ANOVA followed by Tukey’s or Bonferroni’s multiple-comparison tests was used for comparisons among three or more groups, and two-way ANOVA with Bonferroni’s post hoc correction or main-effect-only models was applied for longitudinal tumour growth curves. For pairwise comparisons, two-tailed unpaired Student’s t-tests were used. Survival analysis was conducted using the Mantel–Cox test.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
