cdgmp  (InvivoGen)

Journal: Cancer Research Communications

Article Title: NLRP3 Inflammasome Activation Expands the Immunosuppressive Myeloid Stroma and Antagonizes the Therapeutic Benefit of STING Activation in Glioblastoma

doi: 10.1158/2767-9764.CRC-23-0189

Figure Lengend Snippet: In vivo characterization of STING and NLRP3 inflammasome activation in GL261. A, Mice received subcutaneous injection of 1.0 × 10 6 GL261 cells in 30% Matrigel and then were injected intratumorally with vehicle, STING agonist cdGMP (25 μg), NLRP3 agonist nigericin (25 μg), or combination on days 10, 14, and 18. Tumors were then processed for tumor harvest and flow cytometry as described in methods ( B, C, H–K ) or measured for survival ( D and E ). Data shown represent ( B ) GL261 harvested tumor weights. C, Analyzed CD45 + cell frequency of total single cells. D, GL261 tumor volumes and ( E ) survival data. F–I, Overall composition and fold changes of cell densities vs. vehicle-treated tumors for analyzed CD45 + ( F and G ) myeloid and ( H and I ) lymphoid cell populations. J, Mice received intracranial injection of 5.0 × 10 4 GL261 cells in PBS in the right striatum via a guide bolt system, then were treated intratumorally via the guide bolt on day 7 with vehicle, STING agonist cdGMP (5 μg), NLRP3 agonist nigericin (5 μg), or combination, and survival monitored ( K ). Error bars represent mean ± SEM. Statistical significance was calculated using the Student t test or log-rank test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. DC, dendritic cells; SubQ, sub-cutaneous.

Article Snippet: Cyclic di-GMP (cdGMP), LPS, nigericin, and MCC950 were purchased from InvivoGen and reconstituted as directed.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry

Journal: Cancer Research Communications

Article Title: NLRP3 Inflammasome Activation Expands the Immunosuppressive Myeloid Stroma and Antagonizes the Therapeutic Benefit of STING Activation in Glioblastoma

doi: 10.1158/2767-9764.CRC-23-0189

Figure Lengend Snippet: In vivo analysis of the GL261 tumor immune microenvironment following STING and NLRP3 inflammasome activation. Mice received subcutaneous injection of 1.0 × 10 6 GL261 cells in 30% Matrigel and then were injected intratumorally with vehicle ( n = 6), STING agonist cdGMP (25 μg; n = 10), NLRP3 agonist nigericin (25 μg; n = 9), or combination ( n = 10) on days 10, 14, and 18. Tumors were then processed for tumor harvest and flow cytometry as described in methods. Data shown represent Gr-MDSC ( A ), Mono-MDSC ( B ), and TAM ( C ) frequency as a percent of total analyzed CD45 + cells. D, Arginase, ( E ) CD206, ( F ) Ki67, and ( G ) iNOS expression on Gr-MDSC, Mono-MDSC, and TAM populations as indicated. Ratios of number of ( H ) CD8 T cells/FOXP3 + CD4 Tregs and ( I ) Gr-MDSC/CD8 T cells. Among NK cells, granzyme + NK cells were identified and reported as ( J ) density per mg of tumor tissue and ( K ) frequency as a percent of total analyzed CD45 + cells. L, Granzyme and ( M ) Ki67 expression on CD4, CD8, and NK cell populations as indicated. Error bars represent mean ± SEM. Statistical significance was calculated using the Student t test. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. iNOS, inducible nitric oxide synthase.

Article Snippet: Cyclic di-GMP (cdGMP), LPS, nigericin, and MCC950 were purchased from InvivoGen and reconstituted as directed.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, Expressing

Journal: Cancer Research Communications

Article Title: NLRP3 Inflammasome Activation Expands the Immunosuppressive Myeloid Stroma and Antagonizes the Therapeutic Benefit of STING Activation in Glioblastoma

doi: 10.1158/2767-9764.CRC-23-0189

Figure Lengend Snippet: In vivo analysis of intracranial GL261 tumor immune microenvironment composition following STING and NLRP3 inflammasome activation. A, Mice received intracranial injection of 5.0 × 10 4 GL261 cells, then were treated with 5 μg of cdGMP and/or nigericin, and tumor-bearing hemispheres harvested 48 hours following treatment for spectral flow cytometry analysis. B, Live CD45 + cell densities reported as cells per tumor-bearing hemisphere. C, Live CD45 + cell frequency as a fraction of total single cells. D–G, Overall composition and fold changes of cell densities vs. vehicle-treated tumors for analyzed CD45 + ( D and E ) myeloid and ( F and G ) lymphoid cell populations. Error bars represent mean ± SEM. Statistical significance was calculated using the Student t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. cDC1, type 1 conventional dendritic cells; Neg., negatively.

Article Snippet: Cyclic di-GMP (cdGMP), LPS, nigericin, and MCC950 were purchased from InvivoGen and reconstituted as directed.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry

Journal: Cancer Research Communications

Article Title: NLRP3 Inflammasome Activation Expands the Immunosuppressive Myeloid Stroma and Antagonizes the Therapeutic Benefit of STING Activation in Glioblastoma

doi: 10.1158/2767-9764.CRC-23-0189

Figure Lengend Snippet: In vivo analysis of intracranial GL261 tumor immune microenvironment phenotypes following STING and NLRP3 inflammasome activation. Mice received intracranial injection of 5.0 × 10 4 GL261 cells, then were treated with 5 μg of cdGMP and/or nigericin, and tumor-bearing hemispheres harvested 48 hours following treatment for spectral flow cytometry analysis. A, Gr-MDSC, ( B ) Mono-MDSC, and ( C ) microglia frequency as a percent of total analyzed CD45 + cells. D, CD206, ( E ) arginase, and ( F ) PD-L1 expression on Gr-MDSCs, Mono-MDSCs, and microglia as indicated. Ratios of the number of ( G ) CD8 T cells/FOXP3 + CD4 Tregs and ( H ) Gr-MDSC/CD8 T cells. Expression of ( I ) PD-1 and ( J ) LAG-3 on CD8 T cells. K, Granzyme expression on CD8 Teff, NK cells, and NKT cells as indicated. Among CD8 Teff, NK cells, and NKT cells, granzyme + cells were identified and reported as ( L ) density as cells per tumor-bearing hemisphere and ( M ) frequency as a percent of total analyzed CD45 + cells. Error bars represent mean ± SEM. Statistical significance was calculated using the Student t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. MFI, mean fluorescence intensity.

Article Snippet: Cyclic di-GMP (cdGMP), LPS, nigericin, and MCC950 were purchased from InvivoGen and reconstituted as directed.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Chimeric Exosomes Functionalized with STING Activation for Personalized Glioblastoma Immunotherapy

doi: 10.1002/advs.202306336

Figure Lengend Snippet: Preparation and characterization of DT‐Exo‐STING. a) Flow cytometry histograms of CD63 on T‐Exos, D‐Exos and DT‐Exos. b) TEM images and size distributions of T‐Exos, D‐Exos and DT‐Exos. c) Flow cytometry plots of anti‐CD44–labeled T‐Exos, anti‐CD11c–marked D‐Exos and the double‐antibody–labeled chimeic DT‐Exos. d) SDS‐PAGE protein analysis of T‐Exos, D‐Exos, and DT‐Exos. e) Imaging flow cytometry to determine the encapsulation of cdGMP‐Dy547 within DT‐Exos. cdGMP (green) and CD63 (red) in images. f) CLSM images of cdGMP‐Dy547 distribution within DCs after 4 h incubation with individual cdGMP‐Dy547 and cdGMP‐Dy547–loaded DT‐Exos. Nucleus (blue), lysosome (green), and cdGMP‐Dy547 (red) in confocal images.

Article Snippet: This embarkation behavior was assessed using a fluorescently‐labeled CDN, that is, cdGMP‐Dy547 (Axxora) under the surveillance of an imaging flow cytometer (Amnis ImageStreamX Mk II), and the encapsulation efficiencies into diverse exosomes were further determined by LS55 fluorescence spectrometer (Perkin‐Elmer).

Techniques: Flow Cytometry, Labeling, SDS Page, Imaging, Encapsulation, Incubation

Journal: Advanced Science

Article Title: Chimeric Exosomes Functionalized with STING Activation for Personalized Glioblastoma Immunotherapy

doi: 10.1002/advs.202306336

Figure Lengend Snippet: Targeted delivery of DT‐Exo‐STING in vivo. a) Representative in vivo fluorescence imaging and b) quantification in brain regions of GL261‐bearing mice at the indicated time points after subcutaneous administration with cdGMP‐Dy547–containing formulations ( n = 3; two‐way ANOVA with Tukey's multiple comparisons test). c) Ex vivo fluorescence imaging of the major organs (heart, liver, spleen, lung, kidney, and brain) 12 h after subcutaneous administration. Inserted images exhibited the brain penetration of cdGMP‐Dy547–containing formulations. cdGMP‐Dy547 (red), nucleus (blue), and CD31 (green) in images. d) 3D fluorescence imaging to visualize the distribution of cdGMP‐Dy547–loaded DT‐Exos within the optically transparent brain tissues performed with CLARITY technique. cdGMP‐Dy547 (red) in images. e) Ex vivo fluorescent images of brain tissues and f) cervical LNs of GL261 tumor‐bearing mice following subcutaneous injection with various vesicles containing cdGMP‐Dy547. cdGMP‐Dy547 (red) and nucleus (blue) in images. Data in (b) are represented as means ± SD. **** p < 0.0001.

Article Snippet: This embarkation behavior was assessed using a fluorescently‐labeled CDN, that is, cdGMP‐Dy547 (Axxora) under the surveillance of an imaging flow cytometer (Amnis ImageStreamX Mk II), and the encapsulation efficiencies into diverse exosomes were further determined by LS55 fluorescence spectrometer (Perkin‐Elmer).

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Injection