Tumour-specific STING agonist synthesis via a two-component prodrug system

General material and instrumentation

Most chemicals and solvents were purchased from Acros, Alfa Aesar, Sigma-Aldrich, MedChemExpress, Fluorochem, Biosynth Carbosynth and Cayman Chemical. Milli-Q ultrapure water was used in all relevant experiments. Thin-layer chromatography was performed with Supelco silica gel 60 F254 plates. Flash column chromatography was performed with Millipore silica gel 60 (0.040–0.063 mm, 230–400 mesh). NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer equipped with a BBO smart probe or on a Bruker Avance 700 MHz spectrometer equipped with a TXO cryoprobe. Spectra were processed with MestreNova. High-resolution mass spectra of small molecules were recorded on a Waters LCT Premier mass analyser equipped with an Agilent 1100 high-performance liquid chromatography (HPLC) system or on a Waters Xevo G2-S bench top QTOF. Liquid chromatography–mass spectrometry (LC–MS) analysis of reaction mixtures was performed on a Waters SQD2 mass analyser coupled with a Waters H-class UPLC system (Waters Acquity UPLC BEH C18 reversed-phase column). Preparative HPLC was performed on an Agilent 1260 Infinity II chromatography system (ZORBAX 5 Eclipse Plus C8 21.2 × 150 mm reversed-phase column).

Alkylation rate constant measurements

Electrophiles C1–C5 and E1, E2 and E4 were prepared as 50 mM dimethyl sulfoxide (DMSO) stock solutions, diluted in the reaction buffer (10 mM NaPi pH 7, 150 mM NaCl) to 500 µM, and dispensed (100 μl each well) into an ultraviolet-transparent 96-well plate (Corning 3635). Before the start of each assay, thiol N1 (or Ellman’s reagent) was freshly weighed and prepared as a 50 mM DMSO stock solution. N1 was diluted in the reaction buffer containing 200 µM Tris(2-carboxyethyl)phosphine) (TCEP) (400 μM for Ellman’s reagent) to prepare a 200 µM working solution, which was then added (100 μl each well) to the electrophiles, resulting in the following initial condition: 250 μM of the electrophile, 100 μM of the nucleophile, 10 mM NaPi pH 7, 150 mM NaCl and 100 µM TCEP (assuming complete reduction of the Ellman’s reagent). The absorbance at 410 nm (412 nm for Ellman’s reagent) was monitored at 25 °C with a plate reader (Molecular Devices SpectraMax i3x). The concentration of the remaining nucleophile at each timepoint was determined by comparing the background-subtracted absorbance with a calibration curve obtained by serial dilutions of the 200 µM working solution. The concentration of the remaining electrophile at each timepoint was determined by calculating the amount of nucleophile consumed. Rate constants were obtained by fitting the data with the standard second-order rate law (GraphPad Prism).

Michaelis–Menten kinetics of β-GlcA-N1 cleavage by human β-glucuronidase

Human β-glucuronidase (AcroBiosystems BEB-H52H3) was reconstituted in deionized water to a concentration of 1 mg ml⁻¹. The enzyme stock solution was further diluted in assay buffer (0.2 M acetate pH 4.5) to 10 μg ml⁻¹ (134 nM). β-GlcA-N1 (50 mM DMSO stock) was twofold serially diluted in assay buffer from 5 to 0.078 mM. The enzyme solution and diluted β-GlcA-N1 were incubated in a transparent 96-well plate and incubated at 37 °C until thermo equilibrium was reached. Then, 100 µl of β-GlcA-N1 at various concentrations was mixed with 100 µl of β-glucuronidase using a multichannel pipette, resulting in the following initial condition: [β-GlcA-N1] = 2.5, 1.25, 0.62, 0.31, 0.16, 0.08 and 0.04 mM; [β-glucuronidase] = 67 nM; 5% DMSO (v/v). The reactions were incubated at 37 °C. At 30-min and 60-min timepoints, a 50-µl portion was withdrawn from each reaction mixture and mixed with 50 µl of quenching buffer (1 M sodium carbonate, 1 mM TCEP). The concentration of liberated N1 was determined by comparing the absorbance of each quenched reaction at 410 nm to a calibration curve of purified N1. Reaction velocities were calculated from the slopes of [N1] versus time plots. The velocity versus [β-GlcA-N1] plot was then fitted with the standard Michaelis–Menten kinetic model (GraphPad Prism).

Purification of human STING wild-type 138-379

Escherichia coli BL21 (DE3) (Thermo Scientific EC0114) was transformed with a pET28a plasmid (Genscript) harbouring the codon-optimized sequence of SUMO-hSTINGwt (aa138-379). Transformed BL21 cells were inoculated into LB medium (50 µg ml⁻¹ kanamycin) and incubated at 180 rpm, 37 °C overnight. The overnight culture was then diluted (1:100 dilution) in 2× YT medium (50 µg ml⁻¹ kanamycin) and incubated at 180 rpm, 37 °C until optical density at 600 nm (OD₆₀₀) reached 0.6 (approximately 3 h). The bacterial culture was then cooled to 16 °C, induced with 0.5 mM isopropyl 1-thio-d-galactopyranoside and incubated overnight at 180 rpm, 16 °C. The bacterial cells were pelleted at 4,000g for 30 min, resuspended in 20 ml of lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 20 mM imidazole and 2 mM TCEP supplied with 2 tablets of cOmplete mini EDTA-free protease inhibitors) and disrupted by sonication on ice. The total lysate was clarified by centrifugation at 22,000g for 30 min. The supernatant was collected and filtered through a 0.45-µm filter (Millipore SLHAR33SS). The clarified lysate was then incubated on a roller with 10 ml bed volume of Ni-NTA resin (Thermo Scientific 88223) at 4 °C for 1 h. The Ni-NTA resin was then packed in a column, flushed with 20 ml of wash buffer (25 mM HEPES pH 7.5, 150 mM NaCl and 20 mM imidazole) and eluted with 40 ml of elution buffer (25 mM HEPES pH 8.0, 150 mM NaCl and 250 mM imidazole). The eluted protein was concentrated to approximately 5 ml (Millipore Amicon 10 kDa molecular weight cut-off (MWCO)) and buffer-exchanged into storage buffer (25 mM HEPES pH 7.5 and 150 mM NaCl). TCEP was then added to a final concentration of 2 mM, and 250 units of SUMO protease (Merck SAE0067) were added. The cleavage reaction mixture was left to stand at 4 °C overnight until LC–MS analysis indicated the completion of the cleavage reaction. The cleavage mixture was incubated with 10 ml bed volume of Ni-NTA resin on a roller at 4 °C for 1 h and packed in a disposable column. The flowthrough was collected, and the resin was further flushed with 20 ml of wash buffer. The eluted fractions were combined, concentrated and further purified by size-exclusion chromatography (Cytiva, Superdex 200 Increase 10/300 GL). The purified protein in storage buffer was concentrated to 330 µM (dimer concentration) as determined by bicinchoninic acid (BCA) assay, aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C for future use.

ITC

The binding constants of purified dimers were measured by ITC (Malvern Panalytical AutoITC200) in a reverse-titration format. Stock DMSO solutions of each dimer were dissolved in titration buffer (25 mM HEPES pH 7.5 and 150 mM NaCl) to a final concentration of 25 µM and added to the titration cell. The syringe was filled with 250 µM of purified STING in a matching buffer. Titration was performed at 25 °C with the following parameters: 750 rpm stirring, 0.2 µl initial injection followed by 2-µl injections of the dimer solution every 120 s. Titration of STING protein into blank buffer was separately performed and subtracted from the raw data before the analysis. The background-subtracted titration curves were fitted with the built-in Origin software using the standard one-site model.

MD simulations

The crystal structure of MSA2 in complex with STING (PDB entry: 6UKM)¹⁵ was used as initial structure for all simulations. MD simulations were performed with the AMBER 22 package⁵³, implemented with ff14SB⁵⁴ and gaff2⁵⁵ to correctly reproduce the conformational behaviour of the protein and the ligands, respectively. The LEaP module of AMBER 22 was used to generate the topology and coordinate files for the MD simulations, which were carried out using the CUDA version of the PMEMD module of the AMBER simulation package. Each complex was immersed in a water box with a 10 Å buffer of TIP3P water molecules⁵⁶, and the system was neutralized by adding explicit counter ions (Na⁺). A two-stage geometry optimization approach was performed with the PMEMD module. The first stage minimizes only the positions of solvent molecules and ions, using a 50 kcal mol⁻¹ Å⁻² harmonic potential, and the second stage is an unrestrained minimization of all the atoms in the simulation cell. In both stages, 2,500 steps of steepest descent minimization were followed by 2,500 steps of conjugate gradient minimization. The systems were then heated by incrementing the temperature from 0 to 300 K under constant pressure of 1 atm and periodic boundary conditions for 2 ns. Harmonic restraints of 10 kcal mol⁻¹ were applied to the solute, and the Andersen temperature coupling scheme⁵⁷ was used to control and equalize the temperature. The time step was kept at 1 fs during the heating stages. The SHAKE algorithm was applied to constrain all bonds involving hydrogen atoms⁵⁸. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method⁵⁹. A sharp cut-off of 8 Å was applied to Lennard–Jones interactions. Each system was equilibrated for 2 ns with a 2-fs time step at a constant volume and temperature of 300 K. Production trajectories were then run for an additional microsecond under the same simulation conditions. Binding free energy calculations were performed using the MM-GBSA method⁶⁰. This approach combines molecular mechanics energies with solvation terms estimated using the generalized Born implicit solvent model. Representative snapshots were extracted from the equilibrated MD trajectories for postprocessing. Entropic contributions were not considered in this study.

Potency test of purified dimers on THP-1 cells

THP-1 Lucia ISG cells (InvivoGen thpl-isg) were maintained in the growth medium (RPMI 1640, 2 mM GlutaMAX, 25 mM HEPES, 10% FBS and Pen-Strep/Normocin) at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were pelleted (150g, 10 min) and resuspended in the growth medium (40% conditioned) at a density of 1 × 10⁶ cells ml⁻¹. The cell suspension was dispensed (50 µl each well) into a lidded white 96-well plate (BrandTech 781665). Test compounds or DMSO control were diluted in the growth medium and sterilized by filtering through 0.22-µm polyethersulfone (PES) membranes (Merck SLGP033RS). Compounds serially diluted in the growth medium were then added to (50 µl each well) the cell suspension, resulting in a final density of 5 × 10⁵ cells ml⁻¹ with 20% conditioned medium. In all cases, the DMSO content did not exceed 0.5%. Treated cells were incubated at 37 °C for 24 h. Upon cooling down to room temperature, 50 µl of reconstituted luciferase substrate (InvivoGen QUANTI-Luc) was added to each well via the injector of the plate reader (Molecular Devices SpectraMax i3x), and the luminescence output was recorded (4 s delay, 0.1 s integration). Raw signals were divided by the averaged signal of the DMSO-treated control wells and reported in relative luminescence unit (RLU). The dose–response curve of each replicate was fitted individually using the standard 4PL model (GraphPad Prism). The EC₅₀ value of each compound was averaged from three independent experiments and reported as mean ± s.e.m.

Testing the dilution limits of N1/E4 combination to activate THP-1 cells

THP-1 cells (Cell Line Service) were maintained in the growth medium (RPMI 1640, 2 mM GlutaMAX, 1 mM sodium pyruvate, 1× non-essential amino acids, 10% FBS and Pen-Strep) at 37 °C in a humidified atmosphere containing 5% CO₂. On the day before the experiment, the cells were resuspended in fresh growth medium at a density of 5 × 10⁵ cells ml⁻¹, dispensed (75 µl per well) to a round-bottom 96-well plate (Sarstedt 83.3925.500), and incubated at 37 °C for 24 h. DMSO stocks of Compound N1 and E4 were serially diluted in the growth medium in two separate 96-well plates (Sarstedt 83.3925.500). The contents of the two plates were mixed and incubated briefly (5 min) at 37 °C before being transferred to the cells (75 µl per well), resulting in a final density of 2.5 × 10⁵ cells ml⁻¹ per well. Cells treated with purified SC2S dimers (25 µM) served as the positive control. All test compounds diluted in the growth medium were sterilized by filtering through 0.22 µm PES membranes (Merck SLGP033RS). In all cases, the final concentration of DMSO did not exceed 0.5% (v/v). Treated cells were incubated at 37 °C for 6 h and pelleted by centrifugation (120g, 10 min). The supernatants were collected and immediately analysed for interferon-β concentrations by enzyme-linked immunosorbent assay (ELISA; PBL assay science 41435) following the manufacturer’s instructions. The signal strength of cells treated with 25 µM of purified SC2S dimer was defined as 100% response. All other wells were reported as percentage activation and plotted on a heatmap (GraphPad Prism) accordingly.

β-Glucuronidase-specific STING activation by the β-GlcA-N1/E4 pair

THP-1 Lucia ISG cells were maintained as described above. On the day of the experiment, cells were pelleted (150g, 10 min) and resuspended in the growth medium (40% conditioned) at a density of 1 × 10⁶ cells ml⁻¹. Compound β-GlcA-N1, E4 and E4-ctrl were prepared as DMSO stock solutions. β-Glucuronidase (Sigma-Aldrich G8295) was reconstituted in PBS (25,000 units ml⁻¹). Various test groups (group A: 50 µM β-GlcA-N1, 50 µM E4, 500 units ml⁻¹ β-glucuronidase; group B: 50 µM β-GlcA-N1, 50 µM E4-control, 500 units ml⁻¹ β-glucuronidase; group C: 50 µM β-GlcA-N1, 50 µM E4-ctrl; group D: 500 units ml⁻¹ β-glucuronidase) were prepared, incubated at 37 °C for 1 h, sterile-filtered through 0.22-µm PES membranes (Merck SLGP033RS) and combined with the cell suspension at an equal volume (1 + 1 ml) on a 48-well plate (Greiner 677102). The final DMSO content of each well did not exceed 0.5%. Treated cells were incubated at 37 °C for 24 h and pelleted by centrifugation (300g, 10 min). The supernatants were collected, aliquoted and frozen at −20 °C for future analysis. The concentrations of cytokines from three biological replicates were quantified by ELISA (CXCL-10: Bio-Techne QK266; IL-6: Bio-Techne D6050B; IFN-β: PBL assay science 41435). Data were analysed by two-tailed Welch’s t-test (GraphPad Prism).

Western blots of STING pathway activation in THP-1 Lucia ISG cells

THP-1 Lucia ISG cells were maintained as described above. On the day of the experiment, cells were collected by centrifugation (150g, 10 min) and resuspended in fresh growth medium at a density of 2 × 10⁶ cells ml⁻¹. Cells were treated with cycloheximide (50 µg ml⁻¹) and incubated for 50 min at 37 °C. Treated cells were then combined with the growth medium containing SC2S dimer (5 µM final concentration) to 1 × 10⁶ cells ml⁻¹ and incubated at 37 °C. At each timepoint, a portion (3 ml) of the cell suspension was withdrawn. Withdrawn cells were pelleted (300g, 3 min), washed with PBS (1 ml), pelleted again (300g, 3 min) and resuspended to a density of 1 × 10⁷ cells ml⁻¹ in ice-cold lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) supplied with protease/phosphatase inhibitor tablets (Pierce A32961) and freshly prepared phenylmethylsulfonyl fluoride (1 mM). Lysates were further disrupted by sonication at low power (5 s × 3 cycles). Sonicated lysates were clarified by centrifugation at 4 °C (13,000g, 5 min). The supernatants were collected, and the total protein concentrations were determined by BCA assay (Pierce 23225). Samples were reduced and denatured at 95 °C for 5 min before being separated on a precast polyacrylamide gel (Invitrogen NuPAGE 4–12% Bis-Tris, 20 µg total protein per lane). Protein bands were then transferred to a polyvinylidene difluoride membrane (Invitrogen iBlot2). The membrane was incubated in blocking buffer (5% bovine serum albumin (BSA) in TBST pH 7.4) on a shaker at room temperature for 1 h and probed by the respective primary antibody (anti-pSTING: Cell Signaling Technology E9A9K, 1:1,000 dilution; anti-pIRF3: Cell Signaling Technology E7J8G, 1:1,000 dilution; anti-pTBK1: Cell Signaling Technology D52C2, 1:1,000 dilution; anti-β-actin: Cell Signaling Technology 13E5, 1:1,000 dilution) at 4 °C for 15 h. The membrane was washed three times with TBST and incubated with HRP-conjugated secondary antibody (anti-rabbit IgG, Cell Signaling Technology, 1:1,000 dilution) for 1 h at room temperature. After being washed three times with TBST, the membrane was treated with ECL reagent (Pierce 32106) and imaged using automatic exposure (Bio-Rad ChemiDoc MP).

Western blots of β-glucuronidase expression in CT26 mouse tumour models

Tumour samples isolated from the mice were homogenized and lysed in RIPA buffer containing protease inhibitor (Roche), phosphatase inhibitors (Sigma) and 0.1% Benzonase (Sigma) on ice for 30 min. The lysates were centrifuged at 21,000g for 15 min at 4 °C. The supernatant was collected, and the total protein concentrations were determined using BCA assay (Sigma). The supernatants were separated on 12% SDS–PAGE gels (30 µg total protein per lane) and transferred to polyvinylidene difluoride membranes (GE Healthcare). Membranes were then blocked with 5% BSA in TBST for 1 h at room temperature and then probed with the primary antibodies (anti-GUSB: Proteintech 16332-1-AP, 1:1,000 dilution; anti-Vinculin, Cell Signaling Technology #18799, 1:1,000 dilution) at 4 °C overnight. The membranes were then washed three times with TBST and incubated with the HRP-conjugated secondary antibody (goat anti-mouse IgG, Abcam ab205719, 1:10,000 dilution) for 1 h at room temperature. The membranes were then washed three times and imaged using ECL substrate (BioRad #170-5060) and Amersham 800 Imaging System (Cytiva).

Zebrafish care and handling

The zebrafish (Danio rerio) model was handled and maintained according to the standard protocols of the European Animal Welfare Legislation, Directive 2010/63/EU (European Commission, 2016) and Champalimaud Fish Platform. All protocols were approved by the Champalimaud Animal Ethical Committee and Portuguese institutional organizations—ORBEA (Órgão de Bem-Estar e Ética Animal/Animal Welfare and Ethics Body) and DGAV (Direção Geral de Alimentação e Veterinária/Directorate General for Food and Veterinary).

Zebrafish transgenic and mutant lines

Depending on the purpose of each experiment, different genetically modified zebrafish lines were used in this study: Tg(mpeg1:mcherry-F; tnfa:GFP-F)⁴¹, Tg(mpeg1:mcherry-F)⁴¹ and mutants casper and nacre⁶¹.

Hs578T cell labelling

TNBC cell line Hs578T was cultured and expanded in Dulbecco’s modified Eagle medium (DMEM) high glucose (Biowest) supplemented with 10% FBS (Sigma-Aldrich), 100 U ml⁻¹ of penicillin–streptomycin (Hyclone) and supplemented with insulin at 10 μg ml⁻¹ (Sigma-Aldrich). Hs578T cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C. Hs578T were authenticated through short tandem repeat profile analysis and tested routinely for mycoplasma contamination. Hs578T cells were grown to 70% confluence, washed once with Dulbecco’s PBS (Biowest) and detached with EDTA (1 mM) by scrapping. Cell suspension was collected to a microcentrifuge tube, mixed with lipophilic dye Deep Red Cell Tracker (1 μl ml⁻¹ of cell suspension, 10 mM stock) (Life Technologies) for 10 min at 37 °C in darkness and washed with Dulbecco’s PBS. Cells were centrifuged at 250g, for 4 min at 4 °C, and resuspended in DMEM. Cell viability was assessed by the trypan blue exclusion method, and cell number was determined by haemocytometer counting. Cells were resuspended in growth medium to a final density of 5 × 10⁵ cells μl⁻¹. Fluorescently labelled Hs578T cells were injected using borosilicate glass microcapillaries under a fluorescence scope (Zeiss Axio Zoom. V16) equipped with a mechanical micropipette (World Precision Instruments, Pneumatic Pico pump PV820). Cells were injected into the PVS of 2 dpf zebrafish embryo previously anaesthetized with 1× Tricaine (Sigma-Aldrich). After the injection, zebrafish xenografts were left for 10 min in 1× Tricaine, transferred to E3 medium and kept at 34 °C. At 1 dpi, zebrafish xenografts were screened for the presence of tumoural mass. Xenografts with cells in the yolk sac or cellular debris were discarded, while the successful ones were grouped according to the sizes of their tumours using the size of their eyes as a scale. The xenografts were then exposed to E2 media containing DMSO or the test compounds. Xenografts were checked daily; dead ones were removed, and the E2 medium with DMSO or compounds was refreshed. Four days later, the zebrafish xenografts were euthanized, fixed overnight with 4% (v/v) formaldehyde (Thermo Scientific) at 4 °C and preserved at −20 °C in pure methanol. For transgenic zebrafish line Tg(mpeg1:mcherry-F; tnfa:GFP-F), fixation was performed with PIPES for optimal fluorescence signal preservation.

Zebrafish xenograft live imaging

At 2 dpi (1 dpt), controls and treated Tg(mpeg1:mcherry-F; tnfa:GFP-F) xenografts were carefully selected under a fluorescent scope to ensure that only the double-positive transgenics were imaged. Selected xenografts were anaesthetized and mounted onto a coverslip with 0.8% low-melting agarose with 1× Tricaine in E2 medium. The mounted xenografts were imaged using a LSM 980 Upright confocal laser scanning microscope, with a 25× water objective lens. Then, z-stack images of the tumours were obtained within a 3-µm interval. At the end of the acquisition, xenografts were carefully recovered and returned to the initial conditions (DMSO, MSA2, SC2S, N1/E4 and β-GlcA-N1/E4). At 3 dpt, the same xenografts were imaged once again. Image analysis was performed using FIJI software (ImageJ 2.14).

Zebrafish xenograft imaging and analysis

Fixed zebrafish xenograft images were acquired using a LSM 980 Upright confocal laser scanning microscope, with a 5-μm interval. Generated images were processed using the FIJI/ImageJ software. The number of cells was quantified using the Cell Counter plugin in ImageJ software. To assess tumour size, three representative slices of the tumour, from the top (Z_first), middle (Z_middle) and bottom (Z_last), per z stack per xenograft were analysed, and a proxy of total cell number of the entire tumour (4′,6-diamidino-2-phenylindole (DAPI) nuclei) was estimated as follows: tumour size = (total number of slices/1.5) × (Z_first + Z_middle + Z_last)/3. The correction factor of 1.5 was estimated based on human cells with nuclei averaging 10–12 μm in diameter. The number of activated caspase-3-positive cells, macrophages, M1 and M2-like TNF-positive macrophages were counted in every slide along the tumour. The transgenic xenografts negative for TNF were used to quantify TAMs and phagocytosis. To get the percentage of each population of M1 and M2-like, the obtained number was divided by its corresponding tumour size.

Whole-mount immunofluorescence

Xenografts stored in pure MeOH were rehydrated by a series of decreasing MeOH concentrations (75%, 50%, 25%, v/v MeOH diluted in PBS/0.1% Triton, w/v). Xenografts were then permeabilized with 0.1% (w/v) Triton in PBS and blocked in a mixture of 1× PBS, BSA, DMSO, Triton 1% (w/v) and goat serum for 1 h at room temperature. The xenografts were then probed with primary antibodies (rabbit anti-cleaved Caspase-3, Cell Signaling Technologies, 1:100 dilution; rabbit anti-mCherry, Abcam, 1:100 dilution; mouse anti-GFP, Roche, 1:100 dilution) overnight and incubated with secondary antibodies (Alexa goat anti-rabbit 594, Thermo Scientific, 1:400 dilution; Alexa goat anti-mouse 488, Thermo Scientific, 1:400 dilution) and DAPI (50 μg ml⁻¹) at 4 °C overnight. The xenografts were washed, fixed and mounted between two coverslips, allowing for double side acquisition using Mowiol mounting media (Sigma).

Zebrafish experiment statistical analysis

Statistical analysis was performed using the GraphPad Prism 9.0 software. All datasets were challenged by D’Agostino and Pearson and Shapiro–Wilk normality tests. In general, datasets with a Gaussian distribution were analysed by parametric unpaired t-test, and datasets that did not pass the normality tests were analysed by nonparametric unpaired Mann–Whitney test. Clearance datasets were analysed using Fisher’s exact test. All tests were two-sided with a confidence interval of 95%.

Maintenance of cell lines for mouse experiments

Mouse colon adenocarcinoma MC38 cells were purchased from Kerafast (ENH204-FP). The cells were cultured in DMEM (Gibco, Thermo Scientific #21969035), supplemented with 10% heat-inactivated FBS (Gibco, Thermo Scientific), 1× GlutaMAX (Gibco, Thermo Scientific) and 1× penicillin–streptomycin solution (Gibco, Thermo Scientific). Murine colon adenocarcinoma cell line CT26-overexpressing mouse β-glucuronidase enzyme (CT26mβGUS) was a gift from Dr Steve R. Roffler from the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The cells were cultured in RPMI 1640 medium (Gibco, Thermo Scientific) supplemented with 10% heat-inactivated FBS (Gibco, Thermo Scientific), 1× GlutaMAX (Gibco, Thermo Scientific) and 1× penicillin–streptomycin solution (Gibco, Thermo Scientific). Both cell lines were cultured at 37 °C under a humidified atmosphere containing 5% CO₂.

Mouse tumour models

All animal experiments were conducted at the Gulbenkian Institute for Molecular Medicine (GIMM, Lisbon). Animal work was performed with strict adherence to the Portuguese Law (Portaria 1005/92) and the European Guideline 86/609/EEC. The Federation of European Laboratory Animal Science Associations guidelines and recommendations concerning laboratory animal welfare were followed. All animal experiments were approved by the Portuguese official veterinary department for welfare licensing – Direção Geral de Alimentação e Veterinária (DGAV) and the IMM Animal Ethics Committee (authorization AWB_2021_03_GB_TargCancerDrugs). Eight-week-old male and female C57BL/6J or BALB/c mice (purchased from Charles River) were subcutaneously inoculated in the right flank with 100 μl of 1 × 10⁶ MC38 cells or 5 × 10⁶ CT26mβGUS, respectively, in a 1:1 mixture of Dulbecco’s modified Eagle medium (Gibco) with Matrigel (Corning). Tumour growth was monitored over time by performing daily bilateral vernier caliper measurements. Tumour volumes were estimated with the formula: (length × width²)/2. Treatments were initiated when tumours reached 90–100 mm³. Treatments were randomly assigned to mice according to tumour volumes. Treatments were administered intratumourally (or intraperitoneally for component β-GlcA-N1). Animals were observed every 1–2 days and euthanized when either the tumour volume reached ~1,000 mm³, the body weight loss exceeded 20% or ulceration started to appear. For immune characterizations, inflammatory cells were isolated from the tumours, spleens and inguinal lymph nodes 24 h after the second treatment. Blood was collected and sera was obtained for cytokine profiling. Collected data were analysed with GraphPad Prism 9.

Multiplexed analysis of cytokines

This study used Luminex xMAP technology for multiplexed quantification of 44 mouse cytokines, chemokines and growth factors. The multiplexing analysis was performed using the Luminex 200 system (Luminex) by Eve Technologies Corp. Forty-five markers were simultaneously measured in the samples using Eve Technologies’ Mouse Cytokine 44-Plex Discovery Assay, which consists of two separate kits, one 32-plex and one 12-plex (MilliporeSigma), following the manufacturer’s protocol. The 32-plex consisted of eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF and VEGF. The 12-plex consisted of 6Ckine/Exodus2, erythropoietin, fractalkine, IFNβ-1, IL-11, IL-16, IL-20, MCP-5, MDC, MIP-3α, MIP-3β and TARC. Assay sensitivities of these markers range from 0.3 to 30.6 pg ml⁻¹ for the 45-plex. The sensitivities of individual analytes are available in the MilliporeSigma MILLIPLEX MAP protocol.

LC–MS/MS analysis of in situ SC2S formation

Tumour-bearing mice treated with β-GlcA-N1(IP) + E4 (IT) were euthanized at 1, 2, 4, 8, 24 and 72 h posttreatment (n = 3 for each timepoint). The blood, tumour, lung, kidney, liver and spleen were collected for each mouse. Three mice treated with vehicle were included as control samples. Whole blood samples were centrifuged at 2,000g, 4 °C for 10 min. Supernatant plasma was aspirated into an Eppendorf tube and frozen at −80 °C. Tumour and organ tissues were snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. The plasma samples (10-µl aliquots) were prepared by protein precipitation extraction. The organic layer was evaporated to dryness and reconstituted in a solution equivalent to the initial chromatographic starting conditions. The tissue samples were analysed by homogenization to give a 200 ng ml⁻¹ homogenate concentration. The homogenised tissue samples (10-µl aliquots) were then taken through the same preparation procedure as the plasma samples. All samples were analysed using the PKB Core Facility’s 6500 LC–MS/MS system, in negative ion mode. The chromatography was carried out using a gradient method on a Kinetex EVO C18 column (50 × 2.1 mm). Analysis was conducted in four batches, consisting of calibration standards, quality control samples, blank control samples, internal standard-only samples and study samples. Calibration curves were constructed using linear regression with a 1/x² weighting. A calibration range of 1–500 ng ml⁻¹ (5–2,500 ng g⁻¹ in tissue) was used. Precision and accuracy were well within the PK/B core facility’s acceptance criteria of ±20%. The LC–MS/MS method was developed and qualified ahead of the sample analysis. The performance of the method met normal Core Facility acceptance criteria for analysis.

X-ray crystallography

The 8xHis-tagged R232-STING_138–379 construct was expressed using vector pEXP-MBP (Addgene 112568). E. coli BL21(DE3) cells, harbouring the recombinant plasmid, were cultivated in 2xYT medium (supplemented with 0.1 mg ml⁻¹ ampicillin) at 37 °C until OD₆₀₀ reached ~0.8 and then cooled to 18 °C for 30 min. Isopropyl 1-thio-d-galactopyranoside was added to 0.4 mM, and growth continued at 18 °C for 16 h. Cells were pelleted by centrifugation and resuspended in lysis buffer (30 mM HEPES buffer, pH 7.5, 0.5 M NaCl,10 mM imidazole, 0.5 mM TCEP, 5% glycerol and 100 mM phenylmethylsulfonyl fluoride) before flash-freezing in liquid nitrogen. After 1 day, cells were thawed and lysed by sonication on ice (5 s ON, 10 s OFF, 5 min total ON). Proteins were purified using Ni-NTA resin (Qiagen) and eluted stepwise in binding buffer with 300 mM imidazole. Removal of histidine tag was performed at 4 °C overnight using recombinant TEV protease (at 1:20 molar ratio) while dialysing (SnakeSkin Dialysis Tubing, 10 kDa cut-off weight, Thermo Fisher Scientific) against gel filtration buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP and 5% glycerol). Proteins were further purified by reverse affinity in Ni-NTA followed by gel filtration (Superdex 200 16/60, GE Healthcare). Protein in gel filtration buffer was concentrated to 6.7 mg ml⁻¹ using 10 kDa MWCO centrifugal concentrators (Millipore) at 5 °C. Compounds in 100% DMSO were added to protein (247 µM final solution) at 2.2 mM final concentration (~2.2% DMSO) and incubated on ice for approximately 1 h. This mixture was centrifuged at maximum speed for 10 min at 5 °C before setting up 200-nl sitting drops with protein–inhibitor complex and reservoir solution at ratios of 1:1 and 1:2. Crystallization experiments were performed at 20 °C. Crystals were flash-cooled in liquid nitrogen for data collection. Diffraction data were collected at the Diamond Light Source at the I04 beamline. The best-diffracting crystals grew under the conditions described in the table below. Diffraction data were integrated using XDS (within autoPROC)⁶² and scaled using AIMLESS from the CCP4 software suite⁶³. Molecular replacement and initial refinement were performed using Dimple (PDB ID 4KSY was used as the molecular replacement search model for phasing). Follow-up refinements and manual model adjustments were conducted using BUSTER⁶⁴ and Coot⁶⁵, respectively. Structure validation was performed using MolProbity⁶⁶. The final model and structure factors were deposited in the PDB (PDB ID: 9QVT).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.