tgf β inhibitor sb 505124  (Merck & Co)

Journal: Nature

Article Title: STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma

doi: 10.1038/s41586-023-05880-5

Figure Lengend Snippet: (a) PCR based quantitation of STING promoter (left) or 3’ enhancer (right) reads in methylated DNA immunoprecipitation (MeDIP) samples from parental H2087-LCC, two spontaneous metastasis outbreaks (SO1 and SO2), and the aggressive cell lines A549 and H2030. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (b) qRT-PCR analysis of STING mRNA levels after 3-day treatment of the indicated cell lines with 100nM 5-aza-2’deoxycytidine (5-azadC). Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (c-d) Schematic of the STING locus and the locations of 30 CpG sites analyzed in the STING promoter (c) and 14 sites in the STING 3’ enhancer (d), and percentage of methylation on each CpG site as determined by bisulfite sequencing of H2087-LCC cells freshly sorted from 4 metastatic outbreaks in 2 athymic mice or 2 outbreaks in 2 NSG mice, and H2087-LCC and H2030 cells in culture. Organs were harvested 5–7 weeks after intracardiac inoculation of 1×105 H2087-LCC cells. n=4 (athymic) or 2 (NSG) outbreaks. Mean ± s.e.m., two-way ANOVA analysis. (e) Western immunoblot analysis of STING in H2087-LCC cells treated with TGF-β or CDK4/6 inhibitor palbociclib for the indicated time periods, representative of 2 independent experiments. For gel source data, see Supplemental Figure 1. (f) IF staining of human vimentin (green), DAPI (blue), and STING (red), in lung metastases generated 7 weeks after intravenous inoculation of 1×105 wild-type (WT) or TGFBR2 knockout H2087-LCC cells in NSG mice. The IF staining intensity in vimentin-positive areas was quantified. Scale bar: 50 μm. n=28 (WT) or 34 (KO) lesions from 6 mice. Mean ± s.e.m., two-sided unpaired t-test. (g) Schematic summary of epigenetic mechanisms regulating STING expression during dormancy and metastasis progression. See also Extended Data Figure 9–10.

Article Snippet: TGF-β1 (100 nM, R&D Systems) or TGF-β receptor inhibitor SB-505124 (2.5 μM, Millipore Sigma, Cat# S4696-5MG)) was added in 2% FBS supplemented media for the indicated time.

Techniques: Quantitation Assay, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Quantitative RT-PCR, Methylation Sequencing, Western Blot, Staining, Generated, Knock-Out, Expressing

Journal: Nature

Article Title: STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma

doi: 10.1038/s41586-023-05880-5

Figure Lengend Snippet: (a) Growth rate of H2087-LCC cells treated with TGF-β or CDK4/6 inhibitor palbociclib for the indicated times. n=4. Mean ± s.e.m., two-way ANOVA analysis. (b) qRT-PCR analysis of STING mRNA levels in H2087-LCC cells treated with TGF-β or palbociclib for 7 days. Mean ± s.e.m., representative of 3 independent experiments. Each dot represents a technical replicate of the assay. (c) qRT-PCR analysis of IFNB1 and CCL5 expression in WT or STING knockout H2087-LCC cells treated with TGF-β for 3 days. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (d) qRT-PCR analysis of cGAS mRNA levels in H2087-LCC cells treated with TGF-β for 7 days. Mean ± s.e.m., representative of 3 independent experiments. Each dot represents a technical replicate of the assay. (e) ELISA analysis of cGAMP levels in H2087-LCC cells treated with TGF-β for 5 days. n=3 per group. Mean ± s.e.m. (f) Gene track view of H3K27ac, H3K4me1, and H3K4me3 ChIP-Seq tags at the STING locus in H2087-LCC cells treated with TGF-β for 4 days. (g) PCR based quantitation of methylated STING promoter and 3’ enhancer sequences in MeDIP samples from H2087-LCC cells that were treated with TGF-β for the indicated times. Mean ± s.e.m., representative of 3 independent experiments. Each dot represents a technical replicate of the assay. (h) Schematic of the TGF-β and SMAD-dependent transcriptional reporter. SBE: Smad Binding Element. mCMV: minimal CMV promoter. TRE: Tetracycline-Responsive promoter Element. (i) Representative IF staining images of H2087-LCC cells (human vimentin, green) expressing SMAD-responsive mCherry reporter in the lungs of athymic mice intravenously inoculated with 1×105 cells. Organs were harvested 8 weeks after inoculation. Disseminated cancer cells were present as single quiescent (Ki67low) or proliferative cells (Ki67high, white). Representative of 2 independent experiments. Scale bar: 20 μm. (j) qRT-PCR analysis of SMAD7, SNAI1 and STING expression in H2087-LCC cells treated with TGF-β or no additions for 2 h or 96 h, respectively. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (k) Representative IHC images (left) and quantification (right) of the proportion of H2087-LCC single cells, small clusters, micrometastases, and macrometastases in the lungs of NSG mice 7 weeks after intravenous injection of 1×105 cells. Scale bar: 50 μm. n=102 (WT) or 33 (KO) single cells, 17 (WT) or 12 (KO) small clusters, 31 (WT) or 71 (KO) micrometastases, and 5 (WT) or 9 (KO) macrometastases. (l) IF staining of human vimentin (green), DAPI (blue), and CCL5 (red), in lung metastases generated 7 weeks after intravenous inoculation of 1×105 WT or TGFBR2 knockout H2087-LCC cells in NSG mice. The IF staining intensity in vimentin-positive areas was quantified. Scale bar: 50 μm. n=41 (WT) or 55 (KO) lesions from 5 mice. Mean ± s.e.m., two-sided unpaired t-test.

Article Snippet: TGF-β1 (100 nM, R&D Systems) or TGF-β receptor inhibitor SB-505124 (2.5 μM, Millipore Sigma, Cat# S4696-5MG)) was added in 2% FBS supplemented media for the indicated time.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Quantitation Assay, Methylation, Methylated DNA Immunoprecipitation, Binding Assay, Staining, Injection, Generated

Journal: Advanced Science

Article Title: Genetically Engineered Hematopoietic Stem Cells Deliver TGF‐ β Inhibitor to Enhance Bone Metastases Immunotherapy

doi: 10.1002/advs.202201451

Figure Lengend Snippet: Comparing the immune microenvironment of subcutaneous tumors and bone metastases. A–C) Comparison of the expression level of PD‐L1 in CD45 + (A), CD45 + CD11b + (B), and CD45 + CD11c + (C) cells in subcutaneous tumors and bone metastases. D,E) The proportions of CD4 + (D) and CD8 + (E) T cells in subcutaneous tumors and bone metastases, respectively. F,G) Comparison of the expression level of PD‐1 in CD4 + (F) and CD8 + (G) T cells in subcutaneous tumors and bone metastases. H) Quantification of TGF‐ β levels in subcutaneous tumors and bone metastases. I–L) The proportion of CD4 T H lineages, T H 1 (I), T H 2 (J), T H 17 (K), and T reg (L), in bone metastases and subcutaneous tumors. Data have been represented as mean ± SD. Statistical significance was calculated using a two‐tailed unpaired Student's t ‐test (n = 3), P ‐value: * P <0.05, ** P <0.01, and *** P <0.001; n.s., no significance; a.u., arbitrary units; MFI, mean fluorescence intensity.

Article Snippet: Different concentrations of the TGF‐ β inhibitor SB‐505124 (SB, MedChemExpress, New Jersey, USA) were added to the HSC suspension (5–50 µg mL −1 ), and these mixtures were oscillated gently for 2 h at 4 °C.

Techniques: Comparison, Expressing, Two Tailed Test, Fluorescence

Journal: Advanced Science

Article Title: Genetically Engineered Hematopoietic Stem Cells Deliver TGF‐ β Inhibitor to Enhance Bone Metastases Immunotherapy

doi: 10.1002/advs.202201451

Figure Lengend Snippet: Characterization of HSCs loaded with TGF‐ β inhibitors. A) Scheme of the preparation process and treatment method for SB@HSCs. B) Purity of the HSCs was assessed using flow cytometry. C) Morphology of the HSCs was observed using confocal microscopy. Scale bar: 10 µm. D) Representative UV–vis absorption peaks of SB, HSCs, and SB@HSCs in PBS. E,F) The loading amount and percentage (E) as well as in vitro cumulative release profile (F) of SB from SB@HSCs (n = 3). G) The effect of SB at different concentrations on HSC apoptosis (n = 6). H,I) Expression levels of Sca‐1 (H) and CXCR4 (I) in HSCs and SB@HSCs were analyzed using flow cytometry (n = 3). J) Representative fluorescence images and quantitative analysis of bone isolated 4 h after injection of DiD‐labeled paraformaldehyde‐fixed HSCs, HSCs, and SB@HSCs (n = 4). Data have been represented as mean ± SD. Statistical significance was calculated using Student's t ‐test and one‐way ANOVA followed by Tukey's post‐hoc test; P ‐value: ** P <0.01; n.s., no significance; a.u., arbitrary units; MFI, mean fluorescence intensity.

Article Snippet: Different concentrations of the TGF‐ β inhibitor SB‐505124 (SB, MedChemExpress, New Jersey, USA) were added to the HSC suspension (5–50 µg mL −1 ), and these mixtures were oscillated gently for 2 h at 4 °C.

Techniques: Flow Cytometry, Confocal Microscopy, In Vitro, Expressing, Fluorescence, Isolation, Injection, Labeling

Journal: Advanced Science

Article Title: Genetically Engineered Hematopoietic Stem Cells Deliver TGF‐ β Inhibitor to Enhance Bone Metastases Immunotherapy

doi: 10.1002/advs.202201451

Figure Lengend Snippet: A) Representative immunofluorescence images of CD4 + (green) and CD8 + (red) T cells in bone metastases, after different treatments. Scale bar: 20 µm. B–G) Representative flow cytometry zebra plots and statistical analyses of immune cells in mice, after different treatments. CD4 + T cells (B), CD8 + T cells (C), T H 1 (D), T H 2 (E), T H 17 (F), and T reg (G) cells (n = 4). H) TGF‐ β levels in the bone marrow of mice in each group (n = 4). I–K) PD‐L1 expression levels of CD45 + (I), CD45 + CD11b + (J), and tumor (K) cells in all groups, as analyzed using flow cytometry (n = 4). Data have been represented as mean ± SD. Statistical significance was calculated using a two‐tailed unpaired Student's t ‐test (n = 4); P ‐value: * P <0.05, ** P <0.01, and *** P <0.001; a.u., arbitrary units; MFI, mean fluorescence intensity.

Article Snippet: Different concentrations of the TGF‐ β inhibitor SB‐505124 (SB, MedChemExpress, New Jersey, USA) were added to the HSC suspension (5–50 µg mL −1 ), and these mixtures were oscillated gently for 2 h at 4 °C.

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Two Tailed Test, Fluorescence