Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 1. STING activation elevates pH in Golgi-derived vesicles, leading to LC3 lipidation through V-ATPase and ATG16L1. (A) HeLa-STING(WT), HeLa-STING- ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h, or 100 nM Torin1 for 5 h, or untreated. The cell lysates were analyzed by immunoblotting as indicated. (B) HEK293T FIP200-KO cells stably expressing STING and GFP-LC3B were stimulated with indicated compounds (30 µM MSA2, 100 nM bafilomycin A1) for 2 h, or untreated. Cells were analyzed by confocal microscopy. (Scale bar, 10 µm.) (C) HeLa-STING-ATG16L1 and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 1.5 h or untreated. Protein complexes were immunoprecipitated with a Flag antibody and analyzed by immunoblotting. (D) HeLa cells stably expressing STING, miRFP670-LC3B, and Super Ecliptic pHluorin (SEP)-mKate2-TGN46 were treated with 1 µM diABZI, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min, 18 min, and 27 min are shown. The Left panels show the SEP signal of SEP-mKate2-TGN46 fusion protein. (Scale bar, 10 µm) for images on the Upper panels and 1 µm for Zoom-in images on the Bottom panels.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Derivative Assay, Western Blot, Stable Transfection, Expressing, Confocal Microscopy, Immunoprecipitation, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 2. Noncanonical autophagy is required for STING-induced activation of MiT/TFE transcription factors. (A) HeLa-STING (ctrl) and HeLa-STING-KO cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h or untreated, followed by analyses of cell lysates by immunoblotting with the indicated antibodies. (B) HeLa- STING cells stably expressing TFEB-miRFP670 were treated with 50 nM diABZI for 2 h or untreated, and live cells were imaged by microscopy. (Scale bar, 10 µm.) (C) Quantification of 77 and 80 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING, HeLa-STING-ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h or 100 nM Torin1 for 5 h or untreated. The cell lysates were analyzed by immunoblotting as indicated. (E) HeLa cells stably expressing STING (ctrl) or both STING and SopF (SopF) were treated with 30 µM MSA2 for 2 h or untreated. The cell lysates were analyzed by immunoblotting. (F) HeLa-STING, HeLa-STING-SopF, and HeLa-STING-ATG16L1KO cells stably expressing SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. The cells were stained with an antibody against endogenous TFEB followed by confocal microscopy analyses. (Scale bar, 10 µm.) (G) Quantification of 88 to 228 cells per group as represented by F. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Microscopy, Staining, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 3. TFEB activation by STING does not require TBK1. (A) HeLa-STING and HeLa-STING (1 to 370) cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (B) HeLa-STING (1 to 370) cells were stimulated with 30 µM MSA2 for 2 h or untreated; then, cells were imaged by confocal microscopy using an antibody against TFEB. NucBlue stains nuclear DNA. (Scale bar, 10 µm.) (C) Quantification of 67 and 77 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING (1 to 370), and HeLa-STING (1 to 370)-SopF cells were treated with 10 µM MSA2 for 24 h or untreated. RNA was isolated from these cells for analysis by quantitative RT-PCR to measure the expression of indicated TFEB target genes. CTSD: cathepsin D; CTSB: cathepsin B; GPNMB: Glycoprotein Nonmetastatic Melanoma Protein B. The mRNA expression levels of the MSA-treated cells were normalized to untreated cells (ctrl). Error bars represent SD. The data are representative of 2 independent experiments. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) wild-type (WT) and TBK1/IKKε double knockout (DKO) THP1 cells were treated with 30 µM MSA2 for 2 h or untreated. Then cell lysates were analyzed by immunoblotting.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Confocal Microscopy, Isolation, Quantitative RT-PCR, Expressing, Double Knockout

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 4. STING agonist C53 activates TBK1 and IRF3, but not TFEB, through a mechanism independent of membrane trafficking. (A) HeLa-STING cells were treated with 30 µM MSA2, 50 nM diABZI, or 10 µM C53 for 2 h or untreated, then cell lysates were analyzed by immunoblotting. (B) HeLa STING-KO cells stably expressing STING-mGFP and mCherry-Sec61B were treated with 1.25 µM C53, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min (Ctrl) and 10 min (C53) are shown. (Scale bar, 10 µm) for images on the Left panels and 2 µm for Zoom-in images on the Right panels. (C) HeLa-STING cells were treated with 1 mM BFA or 10 mM GCA for 20 min before stimulation with 50 nM diABZI or 10 µM C53 for 2 h. Cell lysates were analyzed by immunoblotting with indicated antibodies. (D) HeLa-STING cells were stimulated with 1.25 µM C53 for 20 min or untreated, then cells were imaged by confocal microscopy using antibodies against STING and p-TBK1. NucBlue stains nuclear DNA. (Scale bar, 10 µm.)

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Membrane, Western Blot, Stable Transfection, Expressing, Microscopy, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 5. FNIP1/FNIP2–GABARAP interactions mediate the activation of TFEB by STING. (A) HeLa-STING (Ctrl), HeLa-STING-FNIP1/2DKO, HeLa-STING-FNIP1WT, and HeLa-STING-FNIP1MUT cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting as indicated. (B) HeLa-STING (WT), HeLa-STING-LC3sTKO, and HeLa-STING-GABARAPsTKO cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (C) Similar to (B) except that cells were analyzed by immunofluorescent microscopy using an antibody against TFEB. TFEB+NucBlue: merging of TFEB staining with nuclear DNA staining by NucBlue. (Scale bar, 10 µm.) (D) Quantification of 46 to 76 cells per group as represented by C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) HeLa-STING cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h, or 100 nM Torin1 for 5 h, or untreated. Cell lysates were analyzed by immunoblotting. (F) Diagram depicting the mechanism by which STING-induced noncanonical autophagy promotes endolysosomal biogenesis through inhibiting the phosphorylation of TFEB, TFE3, and MITF by mTORC1.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Microscopy, Staining, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 7. STING-induced autophagy recruits the ALIX–ESCRT to mitigate endolysosomal perturbation. (A) HeLa-STING, HeLa-STING-SopF, and HeLa-STING- ATG16L1KO cells that stably express SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. Cells were imaged by microscopy using an antibody against ALIX and other markers as indicated. (Scale bar, 10 µm.) Arrows indicate ALIX puncta. (B) Quantification of 32 to 70 cells per group represented by A. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (C) HeLa-STING cells transfected with siRNA against ALIX or control siRNA were treated with 50 nM diABZI for 24 h, and the morphology of the cells was analyzed by bright-field microscopy. (Scale bar, 25 µm.) (D) Quantification of 228 and 214 cells per group shown in C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ***P < 0.001. (E) HeLa-STING-ATG16L1 and HeLa-STING- ATG16ΔWD40 cells treated with 50 nM diABZI for 24 h or untreated, then cells were analyzed by bright-field microscopy. (Scale bar, 25 µm.) (F) Quantification of more than 300 cells per group shown in E. Error bars represent SD. Tested by unpaired Student’s t test. ***P < 0.001. (G) A Diagram showing that GABARAPs lipidation facilitates ALIX-mediated ESCRT recruitment which mitigates endolysosomal perturbation.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Stable Transfection, Microscopy, Transfection, Control

Journal: Journal of Hematology & Oncology

Article Title: Combination of oral STING agonist MSA-2 and anti-TGF-β/PD-L1 bispecific antibody YM101: a novel immune cocktail therapy for non-inflamed tumors

doi: 10.1186/s13045-022-01363-8

Figure Lengend Snippet: MSA-2 enhanced dendritic cell (DC) maturation. a–c STING pathway-associated cytokine detection. Immature bone marrow-derived DCs (BMDCs) were cultured with MSA-2 for one day, and supernatants were collected for cytokine detection. IFN-β was measured by ELISA assays; TNF-α and IL-6 were determined by multiplex fluorescence-encoded beads; and cell viability was assessed by AO/PI staining. d–g FACS for DC maturation markers. Immature BMDCs were cultured with MSA-2 for one day and collected for flow cytometry. MSA-2 increased CD80, CD86, H-2Kd (MHC-I), I-A/I-E (MHC-II) dose-dependent. h Multiplex fluorescence-encoded beads for proinflammatory chemokine detection. Immature BMDCs were cultured with MSA-2 for one day, and supernatants were collected for cytokine detection. i OVA peptide-plus to evaluate antigen presentation capability. Immature BMDCs were treated LPS or MSA-2 for one day. Then, cells were pulsed with OVA peptide SIINFEKL for six hours and collected for flow cytometry. j One-way mixed lymphocyte reaction (MLR). Stimulating cells were BMDCs derived from BALB/c mice, while responding cells were spleen cells from C57BL/6 in the MLR assays. The mixed cells (the ratio of stimulator to responder = 1:2) were cultured for four days. On day 5, the supernatants and mixed cells were collected for CFSE dilution assay. k–m RNA-seq assay revealing the effect of MSA-2 on BMDC. The heatmap presenting the level of genes encoding cytokines and chemokines. GO and KEGG enrichment analysis showing the pathways or biological processes significantly enriched in MSA-2-treated BMDC. * p < 0.05 means the significant difference compared to the vehicle

Article Snippet: The stimulating cells were activated with 200 ng/ml LPS or 0.01 mg/ml MSA-2 for one day, and then pretreated with 50 μg/ml Mitomycin-C (S8146, Selleck) for 20 min.

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Fluorescence, Staining, Flow Cytometry, Immunopeptidomics, Dilution Assay, RNA Sequencing

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist. Nu, nucleophile. Panels a and b created with BioRender.com .

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques:

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a , Structures of reactive MSA2 analogues. b , HPLC traces of the dimerization reactions. Each reacting pair (50 μM) was allowed to react for 2 h under physiological conditions (37 °C, 10 mM NaPi pH 7, 150 mM NaCl). Reactions were quenched with iodoacetamide (500 μM) to block the remaining free thiol. A260, absorbance at 260 nm; a.u., arbitrary unit; abs, absorbance. See Supplementary Fig. for a detailed analysis of all identified species. c , Structures of electrophiles used as non-specific competitors. d , A colorimetric assay for rate constant measurement. e , A summary of calculated rate constants (mean ± s.d.). Data were averaged from three independent experiments performed on freshly prepared compound dilutions. See Extended Data Fig. for the original plots. f , Top: HPLC traces of the N1–E4 dimerization reaction (50 μM each, 37 °C, 10 mM NaPi pH 7, 150 mM NaCl, 50 µM TCEP) in the presence of competitors. Reactions were quenched with iodoacetamide after 2 h. Right: yields calculated by comparing the integration value of the dimer peak using a calibration curve constructed with pure standards. See Supplementary Figs. and for details. g , Left: non-covalent dimerization between the non-reactive E4 analogue (E4-ctrl) and N1. Right: dose-dependent H NMR chemical shift perturbations of E4-ctrl by N1. [E4-ctrl] = 2 mM; 10 mM NaPi pH 7, 150 mM NaCl, 100 μM TCEP in D 2 O; 25 °C.

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Analogues, Blocking Assay, Colorimetric Assay, Construct

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a , The structure of SC2S dimer formed from N1 and E4 under mild conditions. b , Left: dose–response curves of purified SC2S (red) and MSA2 (blue) on THP-1 Lucia ISG reporter cells. RLU, relative luminescence unit, reported as folds compared with DMSO-treated controls. Curves shown are averaged from three biological replicates, with cells that were split and passaged at least once. EC 50 values are reported as mean ± s.d. Right: representative ITC data for binding constant determination of SC2S and purified LBD of wild-type human STING (hSTINGwt, Supplementary Fig. ). c , Phosphorylation of major STING pathway effectors in THP-1 Lucia ISG cells treated with purified SC2S dimer (5 μM). The experiment was repeated once with similar results. d , Structures of thioether-containing dimers D1–7 with systematically explored linker lengths. e , Left: dose–response curves of dimers D1–7 on THP-1 Lucia ISG reporter cells. Right: THP-1 EC 50 values and binding constants with hSTINGwt of D1–7. Cellular EC 50 values were calculated from three biological replicates and reported as mean ± s.d. Binding constants were averaged from duplicate measurements. See Extended Data Fig. for all ITC data. f , Binding mode of D5 with STING LBD. Left: crystal structure of D5-bound STING (PDB: 9QVT ; dark and light green) superimposed on 2′,3′-cGAMP-bound STING (PDB: 4KSY ; dark and light grey). Right: key interacting residues with D5. All structural representations were generated using PyMOL. See Extended Data Fig. for more structural analysis. g , Top: workflow to test the in situ formation of SC2S on THP-1 cells. Analogues N1 and E4 were serially diluted in two separate 96-well plates and mixed to generate an 8 × 8 matrix of various concentration combinations. The crude mixtures were then seeded with THP-1 cells. IFN-β concentrations in the supernatants were then measured by ELISA. Bottom: IFN-β secretion levels of THP-1 cells treated with N1 and E4 combined at different concentrations. The response from cells treated with 25 μM of purified SC2S dimer was defined as 100%. Panel g created with BioRender.com .

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Purification, Binding Assay, Phospho-proteomics, Generated, In Situ, Analogues, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a Cartoon representation of the dimeric crystal structure of STING LBD bound to compound D5 at 2.31 Å resolution, followed by a 90° rotated view. The displayed distance was measured between the α-carbon (Cα) of Y186 in both chains (as spheres). Superposition of apo (PDB: 4EMU) and D5-bound (PDB: 9QVT) STING structures, aligned by chain A and shown in cartoon representation. The distances displayed were calculated between the Cα atoms of residue Y186 in chains A and B (represented as spheres). b Electron density map (2Fo-Fc, contoured at 1.0 σ) of the ligand, overlaid with its stick representation. c Superposition of D5- and 2’,3’ cGAMP-bound STING LBD crystal structures, emphasizing the angle formed by the C–S–C bonds in the linker region of D5. In both views, it is apparent that D5 penetrates deeper into the binding pocket and is also narrower at the base of the site compared to 2’,3’-cGAMP. d Superposition of D5- (PDB: 9QVT) and MSA2-bound (PDB: 6UKM) STING LBD crystal structures, highlighting conformational differences in chain B when chains A are aligned.

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Residue, Binding Assay

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a MTC of β-GlcA-N1/E4 treatments on non-tumor zebrafish larvae. Groups of 30 noninjected zebrafish larvae were exposed to different concentrations of the compounds (MSA2, SC2S, β-GlcA-N1/E4 and β-GlcA-N1/E4 + β-glucuronidase) for 3 consecutive days, with daily renewal of the E2 media containing the drugs. The tested concentrations of the compounds, in different combinations, were the following: MSA2: 15 μM, 75 μM and 150 μM; SC2S: 0.62 μM, 3.1 μM and 6.2 μM; β-GlcA-N1/E4: 5 μM + 5 μM, 10 μM + 10 μM, 20 μM + 20 μM and 100 μM + 100 μM; β-GlcA-N1/E4 + β-glucuronidase: 20 μM + 20 μM + 166 U/mL, 20 μM + 20 μM + 250 U/mL and 20 μM + 20 μM + 500 U/mL. Toxicity was assessed daily by counting the total number of dead larvae and checking for the presence of morphologic changes such cardiac edemas or curved tails. Control groups were treated with the highest matched DMSO concentration (0.04%, v/v). b Left: representative image of phagocytosis in a xenograft treated with β-GlcA-N1/E4 and β-glucuronidase at 2dpi in Tg(mpeg1:mcherry-F). Each yellow arrow indicates a phagocytic event. Right: n° of phagocytic events at 2dpi. Data are presented as mean ± SEM. Statistical analysis follows that of Fig. .

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Control, Concentration Assay

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a , Experimental design. Hs578T cells were injected into the PVS of 2 dpf zebrafish embryos. At 1 dpi, xenografts were randomly distributed into five treatment groups: DMSO, MSA2 (15 μM), SC2S (0.62 μM), β-GlcA-N1/E4 (20 μM each) and β-GlcA-N1/E4 + enzyme (20 μM each + 250 units ml −1 of β-glucuronidase) with daily renewal of the compounds. At 4 dpi (3 dpt), xenografts were fixed and analysed for apoptosis, tumour size, phagocytosis and macrophage infiltration. Macrophage polarization analysis was performed at 2 dpi (1 dpt) and 4 dpi (3 dpt) by live imaging. All compounds were administrated at concentrations below their MTCs (Extended Data Fig. ). mac, macrophages; enz, enzyme (β-glucuronidase). b , Treated xenografts at 4 dpi. Blue, DAPI; white, activated Caspase-3. c , Quantification of Caspase-3 activation. Act., activated. d , Quantification of tumour sizes (number of tumour cells). For c and d , the results of two independent experiments were averaged and are presented as mean ± s.e.m. Each dot represents one xenograft. e , Representative confocal projection images of macrophages (red) in Hs578T xenografts in Tg(mpeg1:mcherry-F) at 4 dpi. f , Fold induction of TAM (defined by the ratio of the total number of macrophages and that of tumour cells) normalized to control treatments at 4 dpi. Data were averaged from two independent experiments and are presented as mean ± s.e.m. g , Representative confocal images of Hs578T xenografts injected in Tg(mpeg1:mcherry-F, tnfa:GFP-F) at 2 and 4 dpi. Red, macrophages; green, tnfa-positive cells; yellow, macrophages expressing TNF (that is, M1-like macrophages). h , Ratios of M1- to M2-like macrophages in the TME at 2 dpi. i , Ratios of M1-to M2-like macrophages at 4 dpi. For h and i , data are presented as mean ± s.e.m. Images are maximum intensity projections. All images are anterior to the left, posterior to right, dorsal up and ventral down. Scale bars, 50 μm. Dashed lines delineate the tumours. All datasets were challenged by D’Agostino and Pearson and Shapiro–Wilk normality tests. Those with a Gaussian distribution were analysed by parametric unpaired t -test, and those that did not pass the normality tests were analysed by non-parametric unpaired Mann–Whitney test. All tests were two-sided.

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Injection, Imaging, Activation Assay, Control, Expressing, MANN-WHITNEY

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a Experimental design. MC-38 cells were inoculated into the right flank of C57BL/6 mice and treated once tumors reached about 90 mm 3 . MSA2: 5 mg/kg. β-GlcA-N1: 12.5 mg/kg; E4: 5 mg/kg; N1 + E4: N1 12.5 mg/kg and E4 5 mg/kg; β-GlcA-N1/E4 + enz: 12.5 mg/kg of β-GlcA-N1 and 5 mg/kg of E4, preincubated with 500 units of E. coli β-glucuronidase at 37 °C for one hour. N1 was administered intraperitoneally (IP); all other compounds were administered intratumorally (IT). b Tumor growth curves. Data were analyzed by one-way ANOVA, followed by multiple comparisons adjusted with the Sidák-Holm correction. c Survival curves. Differences between the compound-treated groups and the vehicle-treated control group were analyzed by the Log-Rank test. d Body weight monitoring. e Cytokine profiling of sera obtained on Day 9.

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Control

Journal: Nature Chemistry

Article Title: Tumour-specific STING agonist synthesis via a two-component prodrug system

doi: 10.1038/s41557-025-01930-9

Figure Lengend Snippet: a , Experimental design. CT26 cells overexpressing the murine β-glucuronidase (CT26mβGUS) were inoculated into the right flank of BALB/c mice. Overexpression of β-glucuronidase was confirmed by western blotting (Extended Data Fig. ). Treatments began once the tumour volume reached about 90 mm 3 . MSA2: 5 mg kg −1 (IT); β-GlcA-N1: 12.5 mg kg −1 (IT or IP); E4: 5 mg kg −1 (IT); SC2S dimer: 8.6 mg kg −1 (IT, matching molar concentration of the β-GlcA-N1/E4 treatment group). n = 6 for each treatment group. IP, intraperitoneal; IT, intratumoral. b , Tumour volume curves. Data were analysed by one-way ANOVA, followed by multiple comparisons adjusted with the Šidák–Holm correction. c , Body weight monitoring. d , Survival curves. Differences between the compound-treated groups and the vehicle-treated control group were analysed by the log-rank test. e , Tissue distribution of SC2S dimer formed after the β-GlcA-N1 (IP) + E4 (IT) treatment. Here, 0.1 ng g −1 indicates that [SC2S] was below the detection limit. All data points are presented as mean ± s.e.m. n = 3 for each timepoint. Panel a created with BioRender.com .

Article Snippet: Fig. 1 Activation of a two-component prodrug system based on the unique mechanism of MSA2. a , The unique predimerization mechanism of STING agonist MSA2 discovered by Merck. b , Our two-component prodrug strategy with enhanced tumour specificity for on-site synthesis of a potent STING agonist.

Techniques: Over Expression, Western Blot, Concentration Assay, Control