Cell culture

UMUC3 (human bladder carcinoma) and A549 (human lung adenocarcinoma), HCT116 (human colon carcinoma), MP41 (human uveal melanoma) were purchased from ATCC. B16F10 (mouse melanoma), 4T1 (mouse malignant neoplasms of mammary gland), LL2 (mouse Lewis lung carcinoma), Renca (mouse kidney carcinoma) and CT26 (mouse colon adenocarcinoma) were gifts from Prof. Miguel López Lázaro, Department of Pharmacology, University of Seville, Spain. NTUB1 (human bladder carcinoma) was a gift from Prof. Te-Chang Lee, IBMS, Academia Sinica, Taipei Tiwan. A549 and B16F10 cells were cultured in DMEM with GlutaMax. NTUB1, 4T1, LL2, Renca, CT26 and MP41 cells were cultured in RPMI1640 with GlutaMax, UMUC3 cells were cultured in Minimum Essential Medium with GlutaMax and HCT116 cells were cultured in McCoy’s 5 A Medium with GlutaMax. Media were supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. All cells were maintained at 37 °C, 5% CO2 and a humid incubator.

Compounds and blockades

TH1579 was prepared according to published methods (WO2015187088). Cisplatin was purchased from Sigma-Aldrich. Atezolizumab (Tecentriq, 1200 mg) was from Roche.

Cell viability

TH1579 was dissolved in dimethyl sulfoxide (DMSO) at 10 mM and dispensed to final concentration using D300e digital dispenser (Tecan). Cells were seeded in the described complete media, and plates were incubated for 96 h. Cell viability was determined by adding 10 μg/mL resazurin (Sigma Aldrich) and measured after 4–6 h. Fluorescence at 595 nm was measured by Hidex Sense reader. Half-inhibition concentration (IC50) was calculated in GraphPad Prism v.9.4.1

Mice and treatment

All animal experiments were approved and conducted as per the European directive, ethical guideline, and regulations of the Institutional Review Committee, that is, Regional Animal Ethical Committee Stockholm (approval Dnr: 5718-2019). C57BL6/N female mice were purchased from Charles River. All mice (6–8 weeks old) were housed in 3–5 mice / cage with a 12-h light cycle. Temperature and humidity set according to laboratory animal guidelines and regulation.

0.3 million (or 0.15 million) B16F10 cells were injected subcutaneously at the right flank to generate a syngeneic model. Animals were randomised into treatment groups when tumours reached 50 mm3. Animals were euthanized when human endpoints were reached.

TH1579 (90 mg/kg, twice daily, per oral, p.o.) was formulated in a vehicle solution of 22.5% Hydroxypropyl-β-cyclodextrin with sterile water. Atezolizumab (diluted to 0.5 mg/mL) was formulated in saline.

Tumour dissociation and flow cytometry

Mice were sacrificed at the indicated days. Tumours were extracted, finely minced and digested with the MACS Miltenyi Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Dissociated tumour cells were washed with RPMI-1640 medium and lysed with ACK Lysing Buffer (Gibco). Cells were resuspended in staining buffer (DPBS with 5% FBS and 2 mM EDTA). LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen) was applied to cells in combination with Rat anti-mouse CD16/CD32 Fc Block (BD Biosciences, #553142) for 10 min at room temperature, prior to incubation with antibodies for 45 min at 4 °C. For immune cell staining penal, cells were fixed with 2% PFA for 30 min and washed, resuspended in staining buffer. For tumour cell penal, as gp-100 is an intracellular marker, cells were fixed and permeabilized with Foxp3 Transcription factor staining buffer kit (Thermo Fisher) according to the manufacturer’s instructions, followed by incubation with antibodies for 60 min at 4 °C. Compensation was performed using UltraComp eBeads™ Plus Compensation Beads (Invitrogen) incubated with antibodies and ArC™ Amine Reactive Compensation Bead Kit (Invitrogen) incubated with LIVE/DEAD staining. Signal threshold definition was defined using all-stain, unstained, and FMO controls. Gating strategies are provided in Supplementary Fig. S6. Samples were analysed on NovoCyte Flow Cytometer (Aglient) and data was analysed by FlowJo v.10.8.1.

Following antibodies were used: BV786 Rat Anti-Mouse CD45 (BD Bioscience, clone 30-F11, 1:100), BV711 Hamster Anti-Mouse CD3e (BD Bioscience, clone 145-2C11, 1:100), Pacific Blue™ Rat Anti-Mouse CD8a (BD Bioscience, clone 53-6.7, 1:100), PE Anti-Melanoma gp100 (Abcam, ab246731, 1:5000).

Western blot

Cell pellets were incubated on ice in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 1x protease inhibitor cocktail (Sigma-Aldrich), and 1x Halts phosphatase inhibitor cocktail (Thermo Fisher Scientific)) and sonicated. Lysate was centrifuged at 13,000 rpm, 20 min to collect supernatant. Protein concentration was determined by BCA (Bicinchoninic Acid) Protein Assay (Thermo Fisher Scientific). Samples were prepared in NuPAGE™ LDS sample buffer (Invitrogen) with NuPAGE™ Sample Reducing Agent (Invitrogen). Samples were denatured at 70 °C for 10 min. Samples were loaded on 4–15% SDS-PAGE gel (Criterion™ TGX™ Precast Midi Protein Gel, Bio-Rad) and the proteins were transferred to nitrocellulose membranes using the Trans-Blot Turbo instrument (Bio-Rad) according to the standard protocol. Membranes were stained with Ponceau S and blocked in 5% milk powder or 1% BSA in tris-buffered saline with Tween and then probed with primary antibodies over night at 4 °C. Secondary antibodies were probed for 2 h at room temperature. Images of blots were obtained using the LI-COR Odyssey Fc Imaging system (LI-COR) and analysed by ImageStudioLite v.5.2 (LI-COR).

Antibodies

Following antibodies were used: mouse anti beta-Actin (Abcam, ab6276, 1:1000), mouse anti-H2A.X phospho S139 (Millipore, 05-636, 1:1000), mouse anti-Histone H3 phospho-S10 (H3-pS10; Abcam, ab5176, 1:1000), rabbit anti-cleaved PARP (Cell Signaling, #9541, 1:1000), rabbit anti-PDL1 (Cell Signaling, #13684, 1:1000), rabbit anti-pTBK1 (Cell Signaling, #5483, 1:500), rabbit anti-TBK1 (Cell Signaling, #3504, 1:1000), rabbit anti-pSTING (Cell Signaling, #50907, 1:500), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-cGAS (Cell Signaling, #15102, 1:1000), rabbit anti-MTH1 (Novus Biologicals, NB100-109, 1:1000). Secondary antibodies were: Peroxidase AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, 711-035-152, 1:5000), Peroxidase AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch, 715-035-150, 1:5000), IRDye 680RD Goat Anti-Mouse IgG (LI-COR, 926-68072, 1:5000) and IRDye 800CW Donkey Anti-Rabbit IgG (LI-COR, 926-32213, 1:5000).

Transfection of siRNA

To established cGAS knock down cells, CGAS siNRA (SMARTpool) was purchased from Horizon Discovery. 20 nM (for NTUB1) or 10 nM (for UMUC3) of siRNA was transfected by using INTERFERin transfection reagent according to manufacturer’s instruction. The same concentration of All-Stars negative control (Qiagen) was used as non-targeting control. After transfection, cells stilled at least 48 h (for NTUB1) or 24 h (for UMUC3) and then add compounds for treatment.

PD-L1 expression analysis by flow cytometry

Cells were cultured with different compounds. After treatment, cells were collected, washed and resuspended in DPBS with 5% FBS and 2 mM EDTA. Cells were stained with APC conjugated rat anti-mouse CD274 (BD Biosciences, clone MIH5, 1:100) for murine cells or APC conjugated mouse anti-human CD274 (BioLegend, clone 29E.2A3, 1:40) for human cells, or isotype controls including APC Rat IgG2a, λ Isotype Control for anti-mouse CD274 (BD Biosciences, clone B39-4, 1:100), APC Mouse IgG2b, κ Isotype Ctrl (Biolegend, clone MPC-11, 1:40) in dark. Cells were washed and resuspended in DPBS with 5% FBS and 2 mM EDTA. Representative gating strategies are provided in Supplementary Fig. S1D. Samples were analysed on Navios flow cytometer (Beckman Coulter) and data was analysed by FlowJo v.10.8.1.

RT-qPCR

Cells were collected by scraping in TRI Reagent (Zymo Research). Total RNA was prepared with the Direct-zol RNA miniprep kit (Zymo Research) and cDNA was prepared with QuantiTect Reverse Transcriptase kit (Qiagen) according to manufacturer’s instructions. Each reaction contained 40 ng of cDNA, 1 µM forward and reverse primers and 1x iTaq universal SYBR green supermix (Bio-Rad). The qPCR reactions were performed in a Rotor-Gene Q instrument (Qiagen). Each qPCR reaction was made in triplicates and expression of target genes were normalised to the control geneactin beta.

Following primers for human were used:

PDL1_Forward: 5′-CCTCCAAATGAAAGGACTCAC-3′

PDL1_Reverse: 5′-TTTTCACATCCATCATTCTCCC-3′

CCL5_Forward: 5′-TGCCACTGGTGTAGAAATACTC-3′

CCL5_Reverse: 5′-GCTGTCATCCTCATTGCTACT-3′

CXCL10_Forward: 5′-GACATATTCTGAGCCTACAGCA-3′

CXCL10_Reverse: 5′-CAGTTCTAGAGAGAGGTACTCCT-3′

IFNB_Forward: 5′-AACTTGCTTGGATTCCTACAAAG-3′

IFNB_Reverse: 5′-TATTCAAGCCTCCCATTCAATTG-3′

ACTB_Forward: 5′-CATTGCTGACAGGATGCAGAAGG-3′

ACTB_Reverse: 5′-TGCTGGAAGGTGGACAGTGAGG-3′

Following primers for mouse were used:

Pdl1_Forward: 5′-CCACATTTCTCCACATCTAGCA-3′

Pdl1_Reverse: 5′-TCCATCCTGTTGTTCCTCATTG-3′

Ccl5_Forward: 5′-CCTCTATCCTAGCTCATCTCCA-3′

Ccl5_Reverse: 5′-GCTCCAATCTTGCAGTCGT-3′

Cxcl10_Forward: 5′-ATTTTCTGCCTCATCCTGCT-3′

Cxcl10_Reverse: 5′-TGATTTCAAGCTTCCCTATGGC-3′

Ifnb_Forward: 5′-CCAGCTCCAAGAAAGGACGA-3′

Ifnb_Reverse: 5′-CGCCCTGTAGGTGAGGTTGAT-3′

Actb_Forward: 5′-CATTGCTGACAGGATGCAGAAGG-3′

Actb_Reverse: 5′-TGCTGGAAGGTGGACAGTGAGG-3′

Modified comet assay

Cells were seeded in 6-well plates at a density of 300,000–400,000 cells/well and the next day treated with TH1579 or DMSO for 24 h. Cells were harvested and washed once with DPBS and finally resuspended in DPBS at a concentration of 1 million/mL. 100 μL of cell suspension was mixed with 500 μL 1.2% low melting point agarose at 37 °C and the mixture were added to agarose coated slides and a coverslip was added on top. The slides were lysed overnight at 4 °C in Lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X100). Slides were washed three times in enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 g/L BSA, pH 8.0) and treated with hOGG1 enzyme (2 µg/mL) or buffer alone for 45 min at 37 °C. Slides were transferred to alkaline electrophoresis buffer (300 mM NaOH, 10 mM EDTA) for 20 min and electrophoresis was performed at 25 V, 300 mA for 30 min at 4 °C. Slides were washed in Neutralization buffer (400 mM Tris, pH 7.5) for 45 min. DNA was stained with SYBR gold dye (Thermo Fisher Scientific), and comets were imaged and quantified with Comet Assay IV software.

Quantification and statistical analysis

All data were plotted and statistical analysis was carried out in GraphPad Prism v.9.4.1. Data were plotted as means ± standard error mean (SEM).