Gas therapy potentiates aggregation-induced emission luminogen-based photoimmunotherapy of poorly immunogenic tumors through cGAS-STING pathway activation

Abstract

The immunologically "cold" microenvironment of triple negative breast cancer results in resistance to current immunotherapy. Here, we reveal the immunoadjuvant property of gas therapy with cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway activation to augment aggregation-induced emission (AIE)-active luminogen (AIEgen)-based photoimmunotherapy. A virus-mimicking hollow mesoporous tetrasulfide-doped organosilica is developed for co-encapsulation of AIEgen and manganese carbonyl to fabricate gas nanoadjuvant. As tetra-sulfide bonds are responsive to intratumoral glutathione, the gas nanoadjuvant achieves tumor-specific drug release, promotes photodynamic therapy, and produces hydrogen sulfide (H₂S). Upon near-infrared laser irradiation, the AIEgen-mediated phototherapy triggers the burst of carbon monoxide (CO)/Mn²⁺. Both H₂S and CO can destroy mitochondrial integrity to induce leakage of mitochondrial DNA into the cytoplasm, serving as gas immunoadjuvants to activate cGAS-STING pathway. Meanwhile, Mn²⁺ can sensitize cGAS to augment STING-mediated type I interferon production. Consequently, the gas nanoadjuvant potentiates photoimmunotherapy of poorly immunogenic breast tumors in female mice.

Fig. 1. The fabrication and biological functions of gas nanoadjuvant.

a Schematic illustrating preparation routes for gas nanoadjuvant. b Schematic diagram of gas nanoadjuvant-based cGAS-STING pathway-dependent antitumor immune responses. Following tumor accumulation, the virus-like surface helps gas nanoadjuvant effectively invade cancer cell through spike surface-assisted adhesion. After entering cancer cell, the overexpressed GSH could break tetrasulfide bond that enables H₂S generation and drug release. Upon NIR laser irradiation, the AIEgen-based phototherapy could activate MnCO to produce CO and Mn²⁺. Both H₂S and CO stimulate the intracellular release of mtDNA to exert immunoadjuvant property by cGAS-STING pathway activation. Meanwhile, Mn²⁺ is a powerful cGAS activator to enhance STING-mediated type I IFN response. The gas nanoadjuvant can engage cGAS-STING pathway in both tumor cells and DCs, leading to DC maturation and potent antitumor immune responses.

Fig. 2. Preparation and characterization of MTHMS.

a Synthesis routes of MTHMS. b Representative EDS element mapping of tvHMS (n = 3 independent experiments). Scale bar, 100 nm. c Representative transmission electron microscope (TEM) images of (i) solid silica, (ii) solid silica@tMS, (iii) tvHMS, and (iv) MTHMS (n = 3 independent experiments). Scale bar, 100 nm. d Diameter distribution and zeta potential of tvHMS and MTHMS through DLS (n = 3 independent experiments). Data represent the mean ± SD. e Representative UV-vis spectra of tvHMS, MnCO, TSSI, and MTHMS (n = 3 independent experiments). f Representative PL spectra of MTHMS (1 × 10⁻⁵M TSSI) (n = 3 independent experiments). g Representative photoacoustic images of MTHMS (n = 3 independent experiments). h Representative UV-vis spectra of DTNB for the detection of GSH depleting ability of tvHMS (n = 3 independent experiments). i H₂S production from tvHMS dispersed in GSH solution (n = 3 independent experiments). j Representative detection results of H₂S production from tvHMS in GSH solution by Pb(NO₃)₂ soaked circular paper (n = 3 independent experiments). k Time-dependent TSSI release from MTHMS in GSH solution (n = 3 independent experiments). l Representative TEM images of MTHMS under GSH stimulus (n = 3 independent experiments). Scale bar, 100 nm. m Representative measurement of ROS production of MTHMS (1 × 10⁻⁶M TSSI) under NIR irradiation (n = 3 independent experiments). n Representative thermographic images and (o) temperature change curves of H₂O, MHMS, THMS, and MTHMS (1 × 10⁻⁴M TSSI) after NIR exposure (660 nm, 0.3 W cm⁻²) (n = 3 independent experiments). p Representative measurement of CO generation at 37 °C after various treatments (n = 3 independent experiments). Source data underlying d–f, h, i, k, m, o, p are provided as a Source Data file.

Fig. 3. Evaluation of MTHMS in vitro.

a Schematic diagram of fabrication and b TEM and scanning electron microscope (SEM) imaging of virus-like and sphere-like MTHMS (scale bar = 50 nm). CLSM observation and flow cytometric analysis of 4T1 cells incubated with virus-like and sphere-like MTHMS for 1 h c and 4 h d (scale bar = 15 μm). e WSP-1 (H₂S), f FL-CO-1 (CO), g JC-1 (red for aggregate and green for monomer), and h DCFH-DA (ROS) fluorescence imaging with various treatments (scale bar = 15 μm). For b–h, experiment was repeated three times independently with similar results.

Fig. 4. Evaluation of gas nanoadjuvant-induced mtDNA release and antitumor immune response through cGAS-STING activation.

a RT-PCR detection of relative mtDNA copy number in cytosol and supernatant of 4T1 cancer cells after various treatments (n = 3 independent experiments). The detection of cytokines (IFN-β, CXCL10, and IL-6) in culture supernatants of b 4T1 cells following the indicated treatments and c BMDCs incubated with pretreated cancer cells (n = 3 independent experiments). b the p values of MTHMS + L to THMS + L in the detection of IFN-β, CXCL10, IL-6 are 0.0052, 0.0143, and <0.0001, respectively. And the p values of THMS + L to TSSI + L in the detection of IFN-β, CXCL10, IL-6 are 0.0133, 0.0307, and 0.0015, respectively. d Flow cytometric assessment images and e relative quantification of DC maturation (CD11c⁺CD80⁺CD86⁺⁾ triggered by cancer cells with different treatments (n = 3 independent experiments). f Inhibitory effects of formulations on proliferation of 4T1 cancer cells investigated via CCK-8 assay (n = 3 independent experiments). h Flow cytometric assessment images and g relative quantification of apoptosis of 4T1 cancer cells after receiving the indicated treatments, cells were stained with Annexin V-FITC/PI (n = 3 independent experiments). g The p values of MTHMS + L to THMS + L and MTHMS + L to MnCO+TSSI + L are 0.0002 and <0.0001, respectively. i Calcein-AM/PI staining of 4T1 cells with various treatments (scale bar = 40 μm). Experiment was repeated three times independently with similar results. Data represent the mean ± SD. Statistical significance was calculated through one-way ANOVA using a Tukey post hoc test. Source data underlying a–c, e–g are provided as a Source Data file.

Fig. 5. Assessment of therapeutic efficiency of MTHMS.

a Schematic depicting 4T1 tumor model, 4T1 cells were intravenously (i.v.) administrated into the tumor-bearing mice on day 10 to simulate the hematogenous metastasis (WB: western blotting assay; FC: flow cytometry analysis; IF: immunofluorescence; ELISA: enzyme-linked immunosorbent assay; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; H&E: hematoxylin and eosin). b Tumor growth curve (n = 5 mice), (c) tumor weight variations (n = 5 mice), (d) tumor photographs (n = 5 mice), and e survival curve (n = 6 mice) following different treatments. For (c), the p value of MTHMS + L to THMS + L is <0.0001. f Tumor growth curve and (g) survival curve of mice rechallenged with 4T1 cancer cells (n = 5 mice). h Representative images of H&E, TUNEL, and Ki67 staining of tumor slices collected from mice receiving various treatments (n = 3 mice). i Representative photos of lung stained with Bouin’s fluid and representative images of H&E staining of lung and liver following different treatments (n = 3 mice). Dashed outlines indicate lung and liver metastases in H&E staining. Scale bar = 100 μm. Data represent the mean ± SD. Statistical significance was calculated through one-way ANOVA using a Tukey post-hoc test (b), log-rank (Mantel–Cox) test (e, g), or two-tailed student’s t test (f). Source data underlying b, c, e–g are provided as a Source Data file.

Fig. 6. The cGAS-STING pathway activation and antitumor immunity after treatments.

G1:Saline, G2:Saline+L, G3: MHMS, G4: MnCO+TSSI + L, G5: THMS + L, G6: MTHMS + L. a Schematic illustration of gas nanoadjuvant-mediated cGAS-STING promotion. b Western blotting assay of STING, TBK1, IRF-3, and phosphorylation of proteins in tumor. Samples derived from the same experiment and gels/blots were processed in parallel. Experiment was repeated twice independently with similar results. c Flow cytometric assay of mature DC (CD11c⁺CD80⁺CD86⁺) in TDLNs (n = 4 mice). The p values of G6 to G5 and G6 to G4 are 0.0003 and <0.0001. d Flow cytometric assay of tumor-infiltrating CD8⁺ in CD3⁺ T cells (n = 4 mice). The p values of G6 to G5 and G6 to G4 are 0.0001 and <0.0001. e Flow cytometric assay of tumor-infiltrating CD4⁺Foxp3⁺ Tregs (n = 4 mice). The p values of G6 to G5 and G6 to G4 are both <0.0001. Flow cytometric assay of tumor-infiltrating f M2-like macrophages (CD206^hiCD11b⁺F4/80⁺) and g M1-like macrophages (CD80^hiCD11b⁺F4/80⁺) (n = 4 mice). f p values of G6 to G5 and G6 to G4 are 0.0006 and 0.0001. g p values of G6 to G5 and G6 to G4 are 0.0002 and <0.0001. h Flow cytometric assay of CD3⁺CD8⁺CD62L^lowCD44^hi T_EM in spleen (n = 4 mice).The p values of G6 to G5 and G6 to G4 are 0.0001 and <0.0001. Data represent mean ± SD. Data were compared through one-way ANOVA using Tukey post-hoc test. Source data underlying b–h are provided as a Source Data file.

Fig. 7. Gas nanoadjuvant-induced systemic antitumor immunity.

a Schematic illustration of bilateral tumor model. Tumor on the right side represents “primary tumor” with laser irradiation, whereas tumor on the left side was defined as “distant tumor” without any treatment (FC: flow cytometry analysis; ELISA: enzyme-linked immunosorbent assay). b Tumor photographs, c tumor growth profiles, d tumor weight variations after the indicated treatments (n = 5 mice). e Flow cytometric assay and relative quantification of tumor-infiltrating CD8⁺ in CD3⁺ T cells (n = 4 mice). f Flow cytometric assay and relative quantification of tumor-infiltrating CD4⁺Foxp3⁺ Tregs (n = 4 mice). c–f The p values of MTHMS + L (right) to Saline+L (right) and MTHMS + L (left) to Saline+L (left) are all <0.0001. g Flow cytometric assay and relative quantification of CD3⁺CD8⁺CD62L^lowCD44^hi T_EM in spleen (n = 4 mice). g The p value of MTHMS + L to Saline+L is <0.0001. h The secretion of cytokines (IL-6, TNF-α, and IFN-γ) in serum (n = 4 mice). h The p values of MTHMS + L to Saline+L in IL-6, TNF-α, IFN-γ secretion are <0.0001, 0.0019, <0.0001, respectively. Data represent the mean ± SD. Statistical significance was calculated through one-way ANOVA using a Tukey post-hoc test (c–f) or two-tailed student’s t test (g, h). Source data underlying c–h are provided as a Source Data file.