Dmxaa

Recombinant murine IL-12 was overexpressed and purified, as described previously [32 (link)]. IL-12 was formulated in 1.5 w/v% chitosan acetate (CS) (Heppe Medical Chitosan, Halle, Germany) dissolved in DPBS, as described previously [33 (link)]. Unless otherwise noted, the dose of IL-12 was 1 µg and CS/IL-12 was injected intratumorally within an hour of CA. R848 (Resiquimod) (Invivogen, San Diego, CA, USA) was formulated in PBS and delivered intratumorally at 10 µg/mouse. DMXAA (Invivogen) was formulated in DMSO and delivered intratumorally at 500 µg/mouse. For the anti-PD-1 and isotype antibody (BioXCell, Lebanon, NH, USA, clone: RMP1.14) treatments, 300 µg was injected i.p. every 3 days, starting on the day of CA for a total of 6 doses.

All STING and TLR agonists were supplied by InvivoGen: 5,6,-dimethylxanthenone-4-acetic acid (DMXAA, vadimezan), 2’3’-cyclic guanosine monophosphate-adenosine monophosphate (2’3’-cGAMP), 3’3’-cyclic di-adenosine monophosphate (3’3’-c-di-AMP), the Rp,Sp-isomers of the bisphosphorothioate analog of the mammalian cyclic dinucleotide 2’3’-cGAMP (2’3’-cGAM(PS)₂ (Rp/Sp)), 3’3’-cyclic adenosine monophosphate-inosine monophosphate [77 (link)], imiquimod, ODN1826, and ODN2216. STING agonists were diluted in PBS (with 10% DMSO for DMXAA) and administered by local application or i.p. injections. STING agonists were added to the cell medium. In some experiments, the cells were pretreated with digitonin to permeabilize the cell membrane. In brief, medium was removed and cells were permeabilized with digitonin (5 μg/mL) and buffer (0.2% BSA, 50 mM HEPES, 100 mM KCl, 3 mM MgCl₂, 0.1 mM DTT, 85 mM sucrose, 1 mM ATP, 0.1 mM GTP, 2.2 mM NaOH) together with the respective drug for 10 minutes. New antibiotics-free medium was then supplied. TLR agonists were diluted in PBS. Mock treatment was performed with PBS in an appropriated volume.

Menadione, hydrogen peroxide solution, diamide and N-Acetyl-L-Cysteine were purchased from Sigma-Aldrich. Iodoacetamide (IAM), N-Ethylmaleimide (NEM), 5-iodoacetamido-fluorescein (5-IAF) were purchased from Sigma-Aldrich to label free thiols on protein. cGAMP, ISD, DMXAA, poly I:C, poly dA:dT were purchased from Invivogen to induce interferon signaling.

Lung tissue, control or infiltrated by tumors, were harvested after PBS lung circulatory perfusion. The whole tissue was mechanically cut into small pieces and digested with Collagenase type 2 (265 U/mL; Worthington) and DNase (250 U/mL; Thermo scientific) at 37 °C for 30’. Collagenase was then stopped by EDTA 10 mM and the cell suspension was filtered using 70 μm cell strainer (Corning).
CD11c⁺ lung cells were separated by immunomagnetic sorting using CD11c microbeads (Miltenyi Biotec) following manufacturer’s instructions. Dendritic cells were purified by FACS sorting with FACS Aria II (BD Biosciences), using the gating strategy shown in Supplementary Fig. 1c, after CD11c⁺ cells staining in PBS + 2% FBS with the following antibodies: CD11c-A647 (N418), CD103-PE (2E7) purchased from Biolegend and SiglecF-BB515 (E50-2440), CD11b-BV421 (M1/70) and MHCII-BV711 (M5/114.15.2) from BD Biosciences. For gene expression analysis up to 30,000 cells were sorted directly into 350 μL RLT buffer (Qiagen). For ex-vivo stimulation, 10,000 cDC1 were plated in U-bottom 96-well, stimulated with CpGB (1 µg/mL, Invivogen) or DMXAA (25 µg/mL, Invivogen) for 3 h and then lysed in RLT buffer for gene expression analysis.