Bp0117

All animals were maintained in a specific pathogen–free facility. Whsc1-floxed mice were generated by the Beijing Biocytogen Company as previously described (25 (link)). C57BL/6-Tg (TcraTcrb) 1100Mjb/J (OT-I) mice, Apc^min/+ mice, and Villin^Cre/+ mice were purchased from The Jackson Laboratory. All mice were backcrossed with C57BL/6 mice for at least 7 generations. BALB/c, C57BL/6, Rag1^–/–, or NSG male mice aged 4 to 8 weeks were injected subcutaneously with 1 × 10⁶ CT26, MC38, CRC610301, or CRC541051 cells. For cecal injections, C57BL/6 and Rag1^–/– male mice aged 4 to 8 weeks were injected with 5 × 10⁵ KAP cells (derived from Villin^Cre/+Kras^G12DApc^min/+Trp53^fl/fl mice). An isotype or anti-CD8 antibody (10 mg/kg, Bio X Cell, BP0117) was intravenously administered every 3 days. For anti–PD-1 treatment, 10 mg/kg anti–PD-1 (Bio X Cell, BE0273) or an isotype antibody was intravenously administered every 3 days after 8 to 10 days of tumor cell implantations. For IFN-γ treatment, 25 μg/kg IFN-γ (GenScript, Z02915) was intravenously administered over 3 consecutive days after 30 days of tumor cell implantations.

Mice were subcutaneously inoculated with MC38 cells (1 × 10⁶) or TC-1 cells (1 × 10⁵) into the right flank. Tumour size was measured every 2 d or 3 d using digital callipers, and tumour volume was calculated as 0.5 × length × width². On reaching sizes of ~50 mm³, tumours were injected with different STING agonists (50 μl of 5% glucose, 50 μg PC7A polymer, 2.5 μg or 50 μg cGAMP, or 2.5 μg cGAMP in 50 μg PC7A NPs), and some of the groups were intraperitoneally injected with 200 µg depletion antibodies (anti-mCD8α, BioXcell, BP0117 or anti-mNK1.1, BioXcell, BP0036) or 200 µg checkpoint inhibitors (anti-mPD-1, BioXcell, BE0146) every 3 d for comparison or synergy evaluation. For systemic DC depletion, CD11c-DTR transgenic mice were injected intraperitoneally with 100 ng diphtheria toxin (Sigma-Aldrich) every 3 d after tumour inoculation. Mice were injected three times in the MC38 model and four times in the TC-1 model with STING agonist treatments spaced 3 d apart. Mice were euthanized at a tumour burden endpoint of 2,000 mm³. Statistical analysis was performed using GraphPad Prism 7.