Rewiring Pyroptosis to Potentiate Cancer Immunotherapy via a Gasdermin D Agonist Bypassing Caspase‐3

Figures

• 

  SCHEME 1. Schematic illustration of DIBDO‐mediated pyroptosis for stimulated antitumor immunotherapy by bypassing Casp‐3 activation.

• 

  FIGURE 1. GSH activated deiodination and catalytic performance of DIBDO. (a) The 1H NMR spectra of a mixture of DIBDO (1 mM) and GSH (2 mM) in D2O before and after reaction. (b) Remaining GSH levels after incubated with or without DIBDO (2 mM) for different times (n = 3, t‐test). (c) Photographs of 1 mL starch solution mixed with H2O, I2 (20 µL, 5 mM), NaI (50 µL, 4 mM), NaI (50 µL, 4 mM) with H2O2 (NaI + H2O2), DIBDO (50 µL, 2 mM), DIBDO (50 µL, 2 mM) with GSH (DIBDO + GSH), DIBDO (50 µL, 2 mM) with GSH and H2O2 (DIBDO + GSH + H2O2), and DIBDO (50 µL, 2 mM) with H2O2 (DIBDO + H2O2). GSH concentration:10 mM; H2O2 concentration: 500 mM. (d) Mechanism of the DIBDO/GSH system in catalyzing the conversion of H2O2 into 1O2. (e) Absorption spectra of TMB in the presence of NaI + H2O2, DIBDO + H2O2, and DIBDO + GSH + H2O2. NaI concentration: 200 µM; H2O2 concentration: 500 mM; DIBDO final concentration: 100 µM. (f) EPR spectra of DIBDO + GSH by using DMPO (100 mM, upper) and TEMP (100 mM, below) as a probe of •OH and 1O2, respectively. (g) Catalytic activity of DIBDO (2 mM) treated with or without GSH (10 mM) for different durations (n = 3). The test conditions: TMB: 50 µg mL−1; DIBDO: 50 µM;…

• 

  FIGURE 2. In vitro antitumor activity of DIBDO. (a) The GSH/GSSG ratio in HeLa cells after 4 h of treatment with DIBDO (200 µM) at various concentration (n = 3). (b) Remaining H2O2 level in HeLa cells after 4 h of treatment with DIBDO (200 µM) (n = 3, t‐test). (c) Cell viability of HeLa cells treated with DBDO, iohexol, and a mixture of DBDO with twice the equivalent of NaI at various concentrations (n = 3). (d) Cell viability of HeLa cells and HUVECs treated with DIBDO at different concentrations (n = 3). (e, f) PI staining (e) and quantitative analysis (f) of HeLa cells after treatment with NaI (400 µM), iohexol (200 µM), and DIBDO (200 µM) (n = 3, ANOVA) for 12 h. Scale bar: 50 µm. (g, h) Flow cytometry analysis (g) and corresponding quantitative analysis (h) of HeLa cells receiving various treatment (400 µM of NaI, 200 µM of iohexol, and 200 µM of DIBDO, respectively) and co‐stained with Annexin V‐FITC and PI. Data = mean ± SD. n = the number of dots as independent replicates in each graph. Statistical analysis was performed using one‐way ANOVA or t‐test.

• 

  FIGURE 3. Characterization of DIBDO‐induced tumor cell death pathway. (a) Fluorescence images of HeLa cells treated with NaI (400 µM), iohexol (200 µM), and DIBDO (200 µM), respectively, and stained with DCFH‐DA (n = 3 independent experiments). (b) Total protein concentration of cells treated with or without DIBDO (200 µM) for 4 h (n = 3). (c) Fluorescence images of Fluo‐4 stained HeLa cells treated with or without DIBDO (200 µM) for 4 h. Scale bar: 50 µm. (d) JC‐1 staining of HeLa cells after treated with NaI (400 µM), iohexol (200 µM), and DIBDO (200 µM) for 4 h. (e, f) Optical micrographs (e) and scanning electron microscopy (SEM) images (f) of HeLa cells after treated with NaI (400 µM), iohexol (200 µM), and DIBDO (200 µM) for 12 h. (g) Western blotting analysis of Cl‐Casp‐1 and GSDMD‐N expression in different treatment groups. The treatment condition consistent with those of (e, f). (h) Western blotting analysis of Cl‐casp‐3 and GSDMD‐N in 4T1, MCF‐7, CaCO2, and HepG 2 cells receiving DIBDO (200 µM, 12 h) and Dox (20 µM, 12 h) treatments. (i) IL‐1β release in cell culture supernatants after the cells incubated with NaI (400 µM), iohexol (200 µM), and DIBDO (200 µM), respectively, for…

• 

  FIGURE 4. Mechanism of DIBDO‐mediated cell death. (a–c) Western blotting analysis of Cyt C (a), Casp‐9 (b), and Cl‐Casp‐8 (c) in HeLa cells. (d) Western blotting analysis of Cl‐Casp‐3 and GSDMD‐N in cells treated with DIBDO (200 µM), I2 (200 µM), RB (32 µM) under 640 nm LED light for 5 min, BSO (100 µM), and Fe3O4 (100 µg mL−1) for 12 h. (e) PCA of blank and DIBDO groups. (f) The volcano plot showing downregulated and upregulated genes in the DIBDO group compared to the blank group. (g) DEGs between the DIBDO and blank groups. (h) KEGG enrichment analysis of DEGs in DIBDO versus blank groups. (i) Enriched chord diagram of KEGG pathways. (j, k) Relative mRNA expression levels of APAF1 (j) and RIPK1 (k) in HeLa cells after treated with DIBDO (200 µM) for 4 h measured by quantitative real‐time polymerase chain reaction (n = 3). (l) Proposed mechanism of Casp‐3 activation downregulation. Data = mean ± SD. n = the number of dots as independent replicates in each graph.

• 

  FIGURE 5. DIBDO‐induced tumor killing and immune activation in vivo. (a) Schematic timeline of in vivo antitumor efficacy experimental time schedule. (b) GSH content in tumor tissue of mice in control, NaI, iohexol, and DIBDO group after treatment (n = 3, ANOVA). Dose: 40 mg/kg. (c) Western blotting analysis of Cl‐Casp‐1 and GSDMD‐N in tumor tissue after varied treatments for 24 h. (d) Time‐dependent tumor volume changing curves for mice in each group (n = 4, ANOVA). (e) Photographs of excised tumors from each mouse in the control, NaI, iohexol, and DIBDO groups after the monitoring period. (f–h) Mature dendritic cells (DCs) (f), CD8+ T cells (g), and M1/M2 macrophage ratio (h) in the spleen of mice (n = 3, ANOVA). (i) The concentration of white blood cell (WBC), red blood cell (RBC), blood platelet (PLT), and hemoglobin (HGB) in the blood of mice (n = 5). (j) The concentration of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (BUN), and creatinine (CREA) in the serum of mice. Blank: untreated; Day 1, 7, 14: Mice treated with DIBDO (1 mg per mouse) for respective time before sacrifice (n = 5) (k) Schematic timeline of DIBDO and…

• 

  FIGURE 6. Construction and evaluation of exogenous tumor vaccines based on DIBDO. (a) Scheme of experimental time schedule. (b, c) Time‐dependent body weight changes (b) and tumor volume growth (c) curves for mice in different groups (n = 10, ANOVA). (d) Relative survival curves of mice across treatment groups (n = 10, ANOVA). (e) Representative photograph (upper) and H&E staining (below) of lung tissue from different groups (n = 5). (f) Counting analysis of metastasis nodules in various groups (n = 5, ANOVA). (g–l) Relative quantifications of mDCs (g), CD8+ T cells (h), Tumor‐associated M1 macrophages (M1) (i), and Tumor‐associated M2 macrophages (M2) (j), Regulatory T cells (Tregs) (k), effector memory T cells (T EMs) (l) in the spleen of mice (n = 5, ANOVA). (m, n) Cytokine levels of TNF‐α (m), and IFN‐γ (n) were isolated from the plasma of the mice in varied treatment groups (n = 3, ANOVA). Data = mean ± SD. n = the number of dots as independent replicates in each graph. Statistical analysis was performed using one‐way ANOVA.