Fig 2: Multiple mechanisms are involved in inhibition of cGAS-mediated pathways in mitotic cells.a, b CDK1 inhibition partially restores cGAMP production. Asynchronized (Asyn) or synchronized (Mitotic) HT1080 cells were left untreated or treated with RO-3306 (10 μM) for 30 min, and then cell extracts containing cGAMP were delivered to digitonin-permeabilized Raw264.7 cells for 4 h before qPCR analysis for mRNA levels of the indicated genes (a), or were directly analyzed by mass spectrometry (b). **P < 0.01, ***P < 0.001 (Student’s t-test, unpaired, two-tailed). Data shown are mean ± S.D. of one representative experiment performed in triplet (a). c Effects of CDK1 inhibition on activation of downstream components of cGAS in mitotic cells. Synchronized (Mitotic) HT1080 cells were left untreated or treated with RO-3306 (10 μM) for the indicated times before immunoblotting analysis with the indicated antibodies. Asynchronized (Asyn) HT1080 cells transfected with HT-DNA were used as a positive control. d Inhibition of downstream components of cGAS in mitosis. HT1080 cells asynchronized (Asyn) or synchronized by paclitaxel (Mitotic) were left mock-transfected or transfected with cGAMP for the indicated times before immunoblotting analysis with the indicated antibodies. e cGAMP-induced recruitment of TBK1 to MITA is barely affected in mitotic cells. Asynchronized (Asyn) or synchronized (Mitotic) HT1080 cells were left untreated or treated with cGAMP for 2 h followed by co-immunoprecipitation and immunoblotting analysis. f CA but not OA restores mcGas phosphorylation at S291. Raw264.7 cells were synchronized by nocodazole, pretreated with the phosphatase inhibitors (100 nM) for 30 min, followed by RO-3306 treatment (10 μM) for 30 min or release into fresh medium for 2 h before immunoblotting analysis. g CDK1 and PP1 coordinately regulate cGAS phosphorylation during mitosis. Synchronized (Mitotic) Raw264.7 cells were pretreated with RO-3306 (10 μM) for 30 min then treated with CA or OA (100 nM) for 30 min, or were pretreated with CA or OA (100 nM) for 30 min and then treated with RO-3306 (10 μM) for 30 min. The cells were then analyzed by immunoblots with the indicated antibodies. Data shown are representative of three (a) or two (d–g) biological repeats.

Fig 3: BSA-Man@Mn2+-Ft@Lap nanoassembly stimulates cGAS-STING-mediated dsDNA sensing machineries in DCs.a Schematical illustration of the nanoassembly-stimulated cGAS/STING signaling of DCs in the TME. b Western blot analysis of the expression levels of STING, p-STING, TBK1, p-TBK1, IRF-3, and p-IRF-3 in BMDCs after the conditioning with supernatants from 4T1 cells treated with (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn2+-Ft, and (V) BSA-Man@Mn2+-Ft@Lap, respectively. c, d Flow cytometric analysis of dsDNA uptake and p-TBK1 levels in BMDCs incubated with 4T1 cells after different treatments. e Western blot analysis of the expression level of STING, p-STING, IRF-3, p-IRF-3, TBK1, and p-TBK1 in BMDCs after treatment with supernatants from 4T1 cells treated with BSA-Man@Mn2+-Ft@Lap, BSA-Man@Mn2+-Ft@Lap+DNase, and BSA-Man@Mn2+-Ft@Lap+HMGB1 antibody, respectively. f, g Flow cytometric analysis of dsDNA uptake and p-TBK1 levels in splenic cells co-cultured with 4T1 cells after different treatments. h cGAMP production in BMDCs after treatment with (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn2+-Ft, (V) BSA-Man@Mn2+-Ft@Lap, (VI) BSA-Man@Mn2+-Ft@Lap+DNase, and (VI) BSA-Man@Mn2+-Ft@Lap+HMGB1 antibody, measured by ELISA. Flow cytometry and western blot experiments in panels b–g were repeated three times independently with similar results. Data are presented as mean values ± SEM (n = 5 biologically independent samples for panel h). Statistical analysis was carried out via two-way ANOVA method. * Indicates significance at p < 0.05, *** indicates significance at p < 0.001, **** indicates significance at p < 0.0001. Source data are provided as a Source data file.

Fig 4: On-demand activation of the protein-based nanoassembly in TME enhances the immune response of poorly immunogenic solid tumors.a Preparation procedure of the protein nanoassembly and its activation in TME. b Schematic illustration of nanoassembly-mediated enhanced immunotherapy in poorly immunogenic solid tumors. The systemically administered BSA-Man@Mn2+-Ft@Lap nanoassembly rapidly disintegrated in the perivascular region of solid tumors into highly diffusive BSA-Man@Mn2+ and Ft@Lap components, thus enabling enhanced penetration into the tumor interior. Ft@Lap molecules would bind to the transferrin receptors on tumor cells for targeted Lap delivery to elicit efficient immunogenic apoptosis, leading to the release of abundant DAMPs including HMGB1 and dsDNA. Meanwhile, BSA-Man@Mn2+ molecules were selectively internalized by immature DCs to enhance the dsDNA sensitivity of the cGAS-STING axis. These activities would act in a cooperative manner to promote DC maturation and cross-priming, eventually enhancing the infiltration and effector function of tumor-specific T cells for efficient immunotherapy.

Fig 5: F. nucleatum enhanced the therapeutic effect of PD-L1 blockade by activating STING signaling. a–d The indicated proteins were detected by Western blotting. Actin was used as a loading control. a DLD1 cells were treated with Fn (1:1000) for different time course. b DLD1 cells were pre-treated with H151 for 2 h, and then treated with Fn (1:1000) for 12 h. c CT26.WT cells were treated with Fn (1:1000) for different time course. d CT26.WT cells were pre-treated with C176 for 2 h, and then treated with Fn (1:1000) for different time course. e–h CT26.WT cells were subcutaneously injected into BALB/c mice (n = 7 for each group). Tumor-bearing mice were pre-treated with C176 or vehicle, then intratumorally injected with F. nucleatum or PBS and intraperitoneally injected treated with an anti-PD-L1 mAb or an isotype control mAb every three days until the end of the experiment. Tumor volumes were measured. e An image of tumors collected at the end of the experiment is shown. f, g Tumor volumes and relative tumor volumes at various time points are shown. One-way ANOVA and Bonferroni’s multiple comparison test. h Flow cytometry was used to detect the proportion of IFN-γ+ cells in CD8+ TILs from mice. One-way ANOVA and Bonferroni’s multiple comparison test. *P < 0.05; **P < 0.01. Fn, F. nucleatum