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) Metastasis-free survival Kaplan-Meier plots of athymic mice intracardially inoculated with 1×105 H2030-BrM or H2087-LCC cells. One set of mice inoculated with H2087-LCC cells was treated with anti-asialo-GM1 antibody to deplete NK cells 30 days after cell inoculation. n=9 (H2030-BrM) or 10 (H2087-LCC) mice per group, log-rank test. (b) Flow cytometry analysis of Lineage−CD45+NKP46+ NK cells in blood from athymic mice that were treated with IgG control or anti-asialo-GM1 antibody for 4 days. Lineage− : TCRβ−CD3−CD19−B220−CD11c−Ly6G−F4/80−. n=3 mice per group. Mean ± s.e.m., two-sided unpaired t-test. (c) Petal charts of metastasis incidence in specific organs of 10 athymic mice intracardially inoculated with 1×105 H2087-LCC cells, treated with or without anti-asialo-GM1 antibody. Metastasis progression was monitored by BLI for 160 days after cell inoculation. (d) Petal charts of metastasis incidence in specific organs after intracardiac inoculation of 2×104 KPad1 cells into 8–9 B6-albino mice or NSG mice. Metastasis progression was monitored by BLI for 60 days (B6-albino mice) or 28 days (NSG mice) after cell inoculation. (e) Flow cytometry analysis of Lineage−CD45+NKP46+ NK cells, CD45+CD3+CD4+, and CD45+CD3+CD8+ T cells in blood from B6-albino mice that were treated for 3 days with IgG control, anti-NK1.1, anti-CD4, or anti-CD8 antibodies. n=3 mice per group. Mean ± s.e.m., two-sided unpaired t-test. (f) Metastasis-free survival Kaplan-Meier plots of NSG mice inoculated intracardially with KPad1 cells, or B6-albino mice inoculated intracardially with KPad1 cells or KP-482T1 cells, and treated with the indicated antibodies to deplete specific types of immune cells. 1×105 cells were inoculated per mouse. Antibody treatments started 12 days after inoculation. n=8 (KPad1/control, KPad1/αCD4), 7 (KPad1/αNK1.1, KPad1/αCD8, KP-482T1), or 9 (NSG) mice per group, log-rank test. *, p=0.03 (KPad1/αNK1.1 vs. KPad1/control), *, p=0.0146 (KPad1/αCD8 vs. KPad1/control), **, p=0.0094 (KPad1/αCD4 vs. KPad1/control), ***, p=0.0002 (KP-482T1 vs. KPad1/control), ****, p<0.0001 (KPad1/NSG vs. KPad1/control in B6-albino). (g) Gene set enrichment analysis (GSEA) showing pathways enriched in dormant H2087-LCC cells that were induced to proliferate in culture compared to cells from spontaneous metastatic outbreaks, analyzed from previous scRNA-seq data sets. (h-i) Metastasis-free survival of athymic mice (h) or B6-albino mice (i) intracardially inoculated with 2.5×105 H2087-LCC (h) or KPad1 cells (i), expressing a scrambled RNA control or various sgRNA pools. n=9 (Scrambled), 10 (NK ligands and MHC class I), 12 (STING pathway), 10 (LPS and RNA-sensing pathways), or 11 (Interferon response, Complement pathway) mice per group (h); n=7 (Scrambled, LPS and RNA-sensing pathways), 8 (Complement pathway), or 9 (other groups) mice per group (i). (j) BLI quantification of lung metastatic colonies after intravenous inoculation of 2.5×105 KPad1 cells expressing a scrambled control or different sgRNA pools in B6-albino mice. Tissues above the dotted line were harvested 57 days after cell inoculation, then subjected to sgRNA recovery and analysis. n=9 (Additional MHC class I) or 10 (other groups) mice per group. (k-l) Gene rank based on the average fold enrichment of sgRNAs in multi-organ metastases from H2087-LCC (k) or KPad1 (l) cells. (m) Gene rank based on average fold change of sgRNAs enriched in the lung colonies formed after tail-vein inoculation of 2.5×105 KPad1 cells into B6-albino mice. For k-m, genes with average log2FC >0 are plotted and genes targeted by the top 10 enriched sgRNAs are listed.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: In Vivo, CRISPR, Flow Cytometry, Control, 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) Schematic of flow cytometry or immunofluorescence analysis of immune cells in bone metastasis. (b) Flow cytometry analysis and quantification of CD45+ leukocytes, CD45+CD19−TCRβ−XCR1+CD11c+ cDC1 cells, CD45+NK1.1+TCRβ−CD49b+NKp46+ NK cells, CD45+TCRβ+NK1.1−CD8+ T cells, and CD45+TCRβ+NK1.1−CD4+ T cells in KP-482T1 metastasis-bearing femurs after Dox-induced expression of STING for 2 days or 7 days. Cell numbers per gram of femur tissue were counted and normalized to control (-Dox). Metastasis-bearing femurs were collected 2 weeks after intracardiac inoculation of 2×104 KP-482T1 cells in B6-albino mice. n=7 mice per group. Mean ± s.e.m., two-sided unpaired t-test. p values comparing each Dox treatment group with control (-Dox). (c-f) Percentage of NK cells degranulating (CD107a+) (c) or producing IFNγ (d), and percentage of CD44+CD8+ T cells producing IFNγ (e) or TNF (f), isolated from KP-482T1 metastasis-bearing femurs after Dox-induced expression of STING for 2 days or 7 days, and then cultured ex vivo with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4h. n=5 (+Dox 7 days) or 6 (other groups) mice per group. Mean ± s.e.m. (g) Schematic of the NK cell-mediated killing assay, and the percentage of WT or STING KO H2087-LCC cells killed by incubation with naïve NK cells for 4h at the indicated effector:target ratios. n=3 per group. Mean ± s.e.m. (h) Schematic of the trans-well migration assay, and the number of NK cells migrated into cell culture media conditioned by WT or STING knockout H2087-LCC cells. n=3 per group. Mean ± s.e.m., two-sided unpaired t-test.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: Flow Cytometry, Immunofluorescence, Expressing, Control, Isolation, Cell Culture, Ex Vivo, Incubation, Migration, Knock-Out

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) Rationale for experimental design. (b-c) qRT-PCR analysis of CXCL10 and CCL5 mRNA levels in WT or STING knockout KPad1 (b) or H2087-LCC (c) cells treated with 33 μM MSA-2 for 4h. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (d-e) Treatment was performed as in (b, c) but with 10 μM (KPad1) or 50 μM (H2087-LCC) ADU-S100. Mean ± s.e.m., representative of 2 independent experiments. Each dot represents a technical replicate of the assay. (f-j) Metastasis-free survival plots of B6-albino mice intracardially inoculated with 2.5×105 KPad1 cells, treated with both MSA-2 and IgG control (f, n=10 mice) or individual antibodies to deplete NK cells (g, n=10 mice for vehicle or 8 mice for MSA-2), CD4+ T cells (h, n=10 mice), CD8+ T cells (i, n=10 mice), or a combination of these antibodies (j, n=10 mice for vehicle or 9 mice for MSA-2). Mice were administered antibodies (200 μg/mouse) once weekly for 3 weeks starting 6 days after cell inoculation, and vehicle or MSA-2 (50 mg/kg of body weight) once weekly for 2 weeks starting 9 days after cell inoculation. Log-rank test. (k) Metastasis-free survival plots of B6-albino mice intracardially inoculated with 2.5×105 KPad1 cells pre-treated with 33 μM MSA-2 for 24h before inoculation. n=10 mice per group. (l) Metastasis-free survival plots of C57BL/6J mice intravenously inoculated with 2.5×105 WT or Sting knockout KPad1 cells and treated with vehicle or ADU-S100 (1.25 mg/kg of body weight) once weekly by intratracheal delivery until the study endpoint. ADU-S100 treatment started 5 days after cell inoculation. n=15 mice (control) or 8 mice (ADU-S100). Log-rank test. (m) Quantification of WT or STING knockout H2087-LCC cell numbers in lungs from athymic mice treated with vehicle or ADU-S100 (6.25 mg/kg of body weight) once weekly for 4 weeks by intratracheal delivery. ADU-S100 treatment started 1 week after intravenous inoculation of 1×105 cells. Lungs were harvested 5 weeks after inoculation. n=5 mice per group. Mean ± s.e.m., two-sided unpaired t-test.

Article Snippet: Single-cell suspensions were then incubated with rat antibodies against mouse Ly6G, CD19, CD3ε, CD8α, CD4, and Ter-119 antibodies (Bio X Cell, clones 1A8, 1D3, 17A2, 2.43, GK1.5 and TER-119, respectively, 1:100), washed twice in PBS supplemented with 2% FBS, then incubated with BioMag goat anti-rat IgG beads (Qiagen, Cat# 310107, 5mg per spleen) to remove antibody-bound cells.

Techniques: Quantitative RT-PCR, Knock-Out, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Efficacy of WWQ-131, a highly selective JAK2 inhibitor, in mouse models of myeloproliferative neoplasms.

doi: 10.1016/j.biopha.2022.113884

Figure Lengend Snippet: Fig. 5. WWQ-131 suppressed rhEPO-mediated polycythemia and splenomegaly in BALB/c mice. (A) Reticulocyte count. (B) Hematocrit. (C) Ter119/CD71 eryth roblast population in spleen (top) and bone (down). (D) Spleen weight. (E) Representative photographs of the spleen. (F) p-STAT5 levels in spleen samples. All graphs show mean ± SEM. ###P < 0.001 versus control. *P < 0.05; **P < 0.01; ***P < 0.001 versus model. ^P < 0.05; ^^P < 0.01 versus 120 mg/kg of fedratinib group.

Article Snippet: Anti-JAK2 (Cat No. E-AB-70193), anti-β-ACTIN (Cat No. E-AB-20034), anti-Mouse CD71 (Cat No. E-AB-F1093D) and Ter119 (Cat No. E-AB-F1125E) antibodies were purchased from Elabscience (China).

Techniques: Control

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 3. Tumour-derived EV-transported Sema3A enhances vascular permeability. (a–c) EVs were purified from conditioned medium of GSC#4 that received either non-silencing RNA (siC) or Sema3A-targeting (siS3A), and applied to the left hemisphere of mouse brain. Vehicle (PBS) served as a control. (a) Evans blue extravasation is shown in OCT-included brains. Arrow indicated the site of EV dropping (left hemisphere). (b) Intensity of Evans blue extravasation was quantified in left and right hemispheres, including all brain areas (whole), only cerebrum (crbum) or cerebellum (crbellum). *Po0.05; **Po0.01; ***Po0.001. (c) Confocal analysis of the red blood cell marker Ter119 (red), Claudin5 (Cldn5, green) and Evans Blue (grey) in frozen brain sections. Nucleic acid staining is shown in blue (DAPI). Scale bar: 40 μm. Graph shows the quantification of Ter119 signal intensity from confocal analysis. **Po0.01. (d) Frozen tissue sections from subcutaneous ectopic and intracranial orthotopic GSC#9 xenografts were stained with Pecam (green) and Ter119 (red). Nucleic acid staining is shown in blue (DAPI). Scale bar: 20 μm. (e) Circulating blood EVs were purified from non-injected (non-tum EV, blue) and GSC-xenografted (xeno EV, red) mice and were analysed by flow cytometry for mouse (m) and human (h) CD63. Respective isotype controls (IgG) are shown in grey. Percentage represents the number of positive events for hCD63. (f) Human Sema3A (hSema3A) concentrations were measured by ELISA in circulating EVs purified from sera of non-injected ( −, light orange) and ectopically (ecto.) or orthotopically (ortho.) GSC xenografted (GSC, orange) mice. *Po0.05; **Po0.01. Each panel is representative of at least three independent experiments.

Article Snippet: Antibodies The following antibodies were used: Sema3A (ab23393, Abcam, Paris, France), Alix and Claudin5 (Santa Cruz, Heidelberg, Germany), mouse and human CD63, PECAM (BD Biosciences, Le Pont de Claix, France), Ter119 (R&D Systems, Lille, France), CD9, Hsp70, Rab4, EEA1 (System Biosciences, Ozyme, Saint-Quentin-en-Yvelines, France), Tom20, Kinectin (Cell Signaling, Ozyme), Annexin V, SytoRNA Select and Choleratoxin (Life Technologies).

Techniques: Derivative Assay, Permeability, Control, Marker, Staining, Injection, Cytometry, Enzyme-linked Immunosorbent Assay