Journal: Cancer Research

Article Title: Slit2 Inhibits Breast Cancer Metastasis by Activating M1-Like Phagocytic and Antifibrotic Macrophages

doi: 10.1158/0008-5472.can-20-3909

Figure Lengend Snippet: Figure 5. Slit2 reduces the expression of IL6, thereby inhibiting TAMs activity. A, Total RNA of CD11bþ cells sorted from rSlit2-N PBS-treated MMTV-PyMT tumorswas subjected to gene expression analysis using NanoString technology. The heatmap shows differentially expressed top genes. B, Total RNA was isolated from MDA-MB-231 cells overexpressing Slit2 (231-Slit2) or vector control (231-vec) and analyzed for gene expression using microarray technology. The heatmap shows differentially expressed top genes. C, 231-Slit2 or 231-Vec cells CM was subjected to cytokine array analysis. The representative image shows differentially expressed molecules. D, The graph shows levels of IL6 in CM derived from 231-Sli2 or Vec CM detected by ELISA. E, Expression of Robo1 in MDA-MB-231 cells transduced with lentivirus expressing siRNA specific to Robo1 (si-Robo1) or control (si-ctrl) by Western blot. F, b-actin was used as loading control. MDA-MB-231 parental control, si-Robo1, or si-ctrl from E was treated with rSlit2-N or PBS and phosphorylation at p65 of NFkB (p-NFkB) or total NFkB (t-NFkB) was analyzed. G, Graph showing levels of IL6 in si-Robo1 or si-ctrl MDA-MB-231 cells treated with rSlit2-N or PBS detected by ELISA (n ¼ 3 each group). H, BMDMs were pretreated with serum-free CM (CTRL), or 231-Slit2 CM or 231-Vec CM or 231-Vec CM preincubated with IL6 nAb (231-Vec þ IL6nAb) or IL6 nAb. After 2 hours, pH-Rhodo–labeled S. Aureus particles were added to the cells and recombinant IL6 was added to the 231-Slit2 CM cells (231-Slit2-rIL6). The kinetics of S. Aureus phagocytosis was analyzed using a fluorescence plate reader at every 15 minutes up to 4 hours. , P < 0.05; , P < 0.01; , P < 0.001 using Student t test.

Article Snippet: The cells were pretreated with mouse rSlit2-N (100 ng/mL) or IL6 neutralization antibody (BioXCell# BE0046) for 2 hours.

Techniques: Expressing, Activity Assay, Gene Expression, Isolation, Plasmid Preparation, Control, Microarray, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Phospho-proteomics, Labeling, Recombinant

Journal: Cancer Research

Article Title: Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation

doi: 10.1158/0008-5472.can-15-1671

Figure Lengend Snippet: Figure 3. IL6 is crucial for persistent DDR signaling in irradiated salivary glands. A, analysis of serum IL6 protein levels by ELISA. Data, mean SEM; , P < 0.05; , P < 0.01; , P < 0.001 versus na€ve controls using two-tailed Wilcoxon signed rank test; n ¼ 3–4. B, IL6 mRNA in the submandibular salivary glands by real-time PCR in irradiated (13 Gy) mice. Data, mean SEM; , P < 0.05; , P < 0.01; , P < 0.001 versus na€ve controls using two-tailed Mann– Whitney test; n ¼ 3–4. A single-dose irradiation was used in order to mark a specific time of initiation of the DNA damage–inducing events. C, quantification of gH2AX foci in ductal cells of salivary glands in irradiated (5 5.6 Gy) wild-type and IL6/ mice removed 8 weeks after irradiation. Data, means SEM; , P < 0.05 using two-tailed Mann–Whitney test; WT, n ¼ 7; IL6/, n ¼ 6; WTþIR and IL6/þIR, n ¼ 4. D, relative mRNA levels of senescence and SASP markers in salivary glands from na€ve and irradiated (5 5.6 Gy) wild-type and IL6/ mice, 8 weeks after irradiation. Data, means SEM; , P < 0.05; , P < 0.01; , P < 0.001 by two-tailed Mann– Whitney test; WT, n ¼ 5; WTþIR, n ¼ 6; IL6/, n ¼ 4; IL6/þIR, n ¼ 6). E, representative photomicrographs of DcR2 immunostaining (brown) in thin sections of salivary glands from mice in C. Scale bars, 50 mm; d, duct.

Article Snippet: Antibody-mediated inhibition experiments were performed using neutralizing monoclonal anti-mouse IL6 antibody (MAB406; R&D Systems Inc.), and a rat monoclonal IgG1 isotype control antibody (MAB005; R&D Systems Inc.; 100 mg, i.v.) administered 2 hours prior to irradiation.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunostaining

Journal: Cancer Research

Article Title: Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation

doi: 10.1158/0008-5472.can-15-1671

Figure Lengend Snippet: Figure 4. IL6R is expressed on ductal cells of the salivary gland and is activated by IL6 infusion. A, IL6R mRNA expression by RT-PCR analysis in the salivary gland compared with liver and kidney. Control is salivary gland RNA (0 hour) without reverse transcription. B, Western blot analysis of IL6R in submandibular glands in na€ve and irradiated (15 Gy) mice at 3 and 6 hours after irradiation. C, IL6R immunofluorescence staining (red) in submandibular glands counterstained to show nuclei (blue). Scale bars, 50 mm. D, Western blot analysis and quantification (right) of pSTAT3 in salivary glands taken 45 minutes following retrograde infusion of normal saline, IL6 (15 ng), or HIL6 (50 ng). Data represent mean SEM; , P < 0.001 using two- tailed Mann–Whitney test; n ¼ 5. E, representative images of pSTAT3 immunostaining of salivary gland thin sections from mice treated as in D reveal strong STAT3 phosphorylation (red nuclear staining) primarily in ductal cells. Scale bars, 100 mm.

Article Snippet: Antibody-mediated inhibition experiments were performed using neutralizing monoclonal anti-mouse IL6 antibody (MAB406; R&D Systems Inc.), and a rat monoclonal IgG1 isotype control antibody (MAB005; R&D Systems Inc.; 100 mg, i.v.) administered 2 hours prior to irradiation.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Western Blot, Irradiation, Staining, Saline, Two Tailed Test, MANN-WHITNEY, Immunostaining, Phospho-proteomics

Journal: Cancer Research

Article Title: Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation

doi: 10.1158/0008-5472.can-15-1671

Figure Lengend Snippet: Figure 5. Local pretreatment with IL6 or HIL6 reduces irradiation-induced senescence in the salivary gland. A, quantification of nuclear gH2AX foci in ductal cells in submandibular salivary glands from mice treated by retrograde infusion of either IL6 (15 ng) or Hyper- IL6 (50 ng) protein and irradiated (5 5.6 Gy). Data, means SEM; , P < 0.05; , P < 0.01 using two-tailed Mann–Whitney test; na€ve, n ¼ 5; salineþIR, n ¼ 4; IL6þIR and HIL6þIR, n ¼ 6. B, relative mRNA levels by real-time PCR of senescence and SASP markers in na€ve and irradiated (5 5.6 Gy) salivary glands 8 weeks after irradiation from mice treated in A. Data, mean SEM; , P < 0.05; , P < 0.01, using two-tailed Mann– Whitney test; na€ve, n ¼ 3; salineþIR, n ¼ 4; IL6þIR and HIL6þIR, n ¼ 6). C, representative photomicrographs of p21 immunostaining of salivary glands from A (left). Scale bars, 100 mm. Right, quantification of p21þ acinar cells. Data, means SEM; , P < 0.05; , P < 0.01; using two-tailed Mann– Whitney test; na€ve and salineþIR, n ¼ 4; IL6þIR and HIL6þIR, n ¼ 5.

Article Snippet: Antibody-mediated inhibition experiments were performed using neutralizing monoclonal anti-mouse IL6 antibody (MAB406; R&D Systems Inc.), and a rat monoclonal IgG1 isotype control antibody (MAB005; R&D Systems Inc.; 100 mg, i.v.) administered 2 hours prior to irradiation.

Techniques: Irradiation, Two Tailed Test, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Cancer Research

Article Title: Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation

doi: 10.1158/0008-5472.can-15-1671

Figure Lengend Snippet: Figure 6. IL6 pretreatment, but not IL6 deficiency, accelerates DNA damage repair shortly following irradiation. A, quantification of nuclear gH2AX foci in ductal cells in na€ve submandibular salivary glands and glands pretreated by retrograde infusion of IL6 (30 ng) or normal saline 48 and 72 hours after irradiation (5.6 Gy). Data, means SEM; , P < 0.01; , P < 0.001; #, P < 0.01 versus all other groups using two-tailed Mann–Whitney test; na€ve, n ¼ 3; saline (48 hours) and IL6 (72 hours), n ¼ 9; saline (72 hours) and IL6 (48 h), n ¼ 10). B, quantification of nuclear gH2AX foci in ductal cells of submandibular salivary glands from na€ve wild-type mice and wild-type and IL6/ mice removed 48 and 72 hours after irradiation (5.6 Gy). Data, means SEM; #, P < 0.05 versus all other groups using two-tailed Mann–Whitney test; na€ve, n ¼ 3; WT (48 hours) and IL6/ (48 hours), n ¼ 4; WT (72 hours) and IL6/ (72 hours), n ¼ 6.

Article Snippet: Antibody-mediated inhibition experiments were performed using neutralizing monoclonal anti-mouse IL6 antibody (MAB406; R&D Systems Inc.), and a rat monoclonal IgG1 isotype control antibody (MAB005; R&D Systems Inc.; 100 mg, i.v.) administered 2 hours prior to irradiation.

Techniques: Irradiation, Saline, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Research

Article Title: Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation

doi: 10.1158/0008-5472.can-15-1671

Figure Lengend Snippet: Figure 7. IL6 and HIL6 pretreatment, and also IL6 deficiency, ameliorate radiation-induced salivary hypofunction. Salivary gland function analyzed by pilocarpine stimulation in mice 8 weeks following irradiation with single (13 Gy; A, C–E), or fractionated (5 5.6 Gy; B, F) irradiation doses: wild-type and IL6-deficient (IL6/) mice (A and B); anti-IL6 mAb (Anti-IL6)- and control mAb IgG (CNTL Ab)-treated mice (C); wild-type and sgp130Fc transgenic mice (D); and following pretreatment by infusion to the submandibular salivary glands with saline (control), IL6 (15 ng), or HIL6 (50 ng; E and F). Data, means SEM; , P < 0.05; , P < 0.01; , P < 0.001, using one-tailed Mann–Whitney test; n ¼ 4–7.

Article Snippet: Antibody-mediated inhibition experiments were performed using neutralizing monoclonal anti-mouse IL6 antibody (MAB406; R&D Systems Inc.), and a rat monoclonal IgG1 isotype control antibody (MAB005; R&D Systems Inc.; 100 mg, i.v.) administered 2 hours prior to irradiation.

Techniques: Irradiation, Control, Transgenic Assay, Saline, One-tailed Test, MANN-WHITNEY