Journal: Molecular Oncology

Article Title: Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

doi: 10.1002/1878-0261.13095

Figure Lengend Snippet: Increased frequencies of Th17 cells after aCRT and pCRT in the blood of cervical cancer patients. PBMCs of 70 cervical cancer patients and 70 female age‐matched healthy controls were analyzed for Th17 frequencies by flow cytometry. (A) Shown is one representative dot blot of the analyzed groups. Numbers indicate percentages of CD4 + T cells or CD4 + IL‐17 + cells, respectively. (B) Frequencies of CD4 + T cells and (D) CD4 + IL‐17 + cells out of lymphocytes. (C, E) Absolute numbers/µL of CD4 + T cells and Th17 cells and (F) proportions of Th17 per total CD4 + T cells were determined. Healthy controls ( n = 70), patients treated with surgery alone ( n = 35; gray dots), aCRT (adjuvant chemoradiotherapy; n = 20; blue dots), and pCRT (primary chemoradiotherapy; n = 15; red dots). Gray line: median value of the respective groups. (G, H) PBMCs of cervical cancer patients which received aCRT ((G); n = 20) or pCRT ((H); n = 15) were analyzed for Th17 frequencies and (I, J) proportions of Th17 per total CD4 + T cells during therapy. P ‐value according to the nonparametric Mann–Whitney U ‐test (H, J) or Kruskal–Wallis test (B, C, D, E, F, G, I). Asterisks represent statistical significances: n.s.: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To reduce nonspecific staining, each section was treated with 2.5% normal horse serum for 30 min. Rabbit anti‐pThr308‐AKT antibody (1 : 250, Abcam; AB_722678), rabbit anti‐pSer473‐AKT antibody (1 : 100, Cell Signaling; 2315049), mouse anti‐CD4 monoclonal antibody 4B12 (1 : 1000, Leica Biosystems; AB_563560), and rabbit anti‐IL‐17 polyclonal antibody (1 : 500, Abcam; AB_1603584) were used.

Techniques: Flow Cytometry, Dot Blot, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

doi: 10.1002/1878-0261.13095

Figure Lengend Snippet: Th17 cells induce resistance of cervical cancer cells toward chemotherapeutic drug cisplatin, irradiation, and combined treatment in an IL‐17‐dependent manner. (A) HeLa cells were cocultured with in vitro generated Th17 cells (proportion 1 : 20; blue line) or medium (dotted line). After 24 h, Th17 cells were removed, HeLa cells (both approaches) were washed with PBS and challenged with serial dilutions of cisplatin. After 18 h, cell viability was assessed by the neutral red uptake method. Shown are the results mean ± SD from two independent experiments performed in triplicates. (B) Timeline of the experimental procedures. (C) SiHa (left), HeLa (middle), and SW756 cells (right panel) were stimulated with medium (dotted lines) or rhIL‐17 (blue lines). After 24 h, cells were washed with PBS and challenged with serial dilutions of cisplatin. After 18 h, cell viability was assessed by the neutral red uptake method. Bars represent summary of three independent experiments of the highest cisplatin dose applied. (D, E) SiHa (left), HeLa (middle), and SW756 cells (right) were stimulated with medium (white, dotted, or striped bars) or rhIL‐17 (blue bars) and (D) irradiated with 6 Gy. (E) Cells were treated with 1.56 µg·mL −1 cisplatin for 2 h and irradiated with 6 Gy. (F) HeLa (left) and SW756 cells (right) were stimulated with medium (white or striped bars) or CM of in vitro generated Th17 cells (blue bars). In neutralization experiments, CM were prestimulated with neutralizing anti‐IL‐17 or respective isotype control antibodies for 2 h (light blue bars). Cells were treated with 1.56 µg·mL −1 cisplatin for 2 h and irradiated with 6 Gy. After 48 h, cell viability was assessed by the neutral red uptake method. Shown are the results mean ± SD from three independent experiments performed in triplicates. P ‐value according to the nonparametric Mann–Whitney U‐test (A, C) or Kruskal–Wallis test (D, E, F). Asterisks represent statistical significances: ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: To reduce nonspecific staining, each section was treated with 2.5% normal horse serum for 30 min. Rabbit anti‐pThr308‐AKT antibody (1 : 250, Abcam; AB_722678), rabbit anti‐pSer473‐AKT antibody (1 : 100, Cell Signaling; 2315049), mouse anti‐CD4 monoclonal antibody 4B12 (1 : 1000, Leica Biosystems; AB_563560), and rabbit anti‐IL‐17 polyclonal antibody (1 : 500, Abcam; AB_1603584) were used.

Techniques: Irradiation, In Vitro, Generated, Neutralization, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

doi: 10.1002/1878-0261.13095

Figure Lengend Snippet: Th17 cells correlated with pThr308‐ and pSer473‐AKT expression in cervical cancer biopsies in situ and are associated with response to primary chemoradiotherapy in cervical cancer patients. (A) Sections of human SCCs of 70 cervical cancer patients were stained for pThr308‐ and pSer473‐AKT expression by IHC or costained for CD4 (green), and IL‐17 (red) by immunofluorescence. Magnification 100× (Scale bars 100 µm), magnification 400× (Scale bars 25 µm). (B, D) The percentage of CD4 + IL‐17 + cells per CD4 + cells correlated with (B) the IRS of pThr308‐AKT or (D) the IRS of pSer473‐AKT. (C, E) The percentage of CD4 + IL‐17 + cells per IL‐17 + cells correlated with (C) the IRS of pThr308‐AKT or (E) the IRS of pSer473‐AKT. (F, G, H, I) The percentage of CD4 + IL‐17 + cells per CD4 + cells, the percentage of CD4 + IL‐17 + cells per IL‐17 + cells, IRS of pThr308‐ and pSer473‐AKT expression were determined in pretherapeutic biopsies of n = 20 cervical cancers and compared with the individual patient's response (non (red circles), partial (orange circles), or complete response (blue circles)) to primary chemoradiotherapy. P ‐value according to the nonparametric Kruskal–Wallis test (F, G, H, I) or Spearman rank correlation with linear regression (B, C, D, E). Asterisks represent statistical significances: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To reduce nonspecific staining, each section was treated with 2.5% normal horse serum for 30 min. Rabbit anti‐pThr308‐AKT antibody (1 : 250, Abcam; AB_722678), rabbit anti‐pSer473‐AKT antibody (1 : 100, Cell Signaling; 2315049), mouse anti‐CD4 monoclonal antibody 4B12 (1 : 1000, Leica Biosystems; AB_563560), and rabbit anti‐IL‐17 polyclonal antibody (1 : 500, Abcam; AB_1603584) were used.

Techniques: Expressing, In Situ, Staining, Immunofluorescence

Journal: Molecular Oncology

Article Title: Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse

doi: 10.1002/1878-0261.13095

Figure Lengend Snippet: Increased Th17 frequencies after chemoradiotherapy in the blood of cervical cancer patients were associated with recurrent cervical cancers. (A) The percentage of CD4 + IL‐17 + cells per CD4 + T cells of 32 patients which received aCRT (circles, n = 18) or pCRT (triangles, n = 14) was evaluated. The percentage of CD4 + IL‐17 + cells per CD4 + T cells was depicted for patients with recurrent cervical cancers ( n = 18; red circles and triangles) in comparison with patients without relapse ( n = 14; gray circles and triangles). P ‐value according to the nonparametric Mann–Whitney U ‐test. Asterisks represent statistical significances: *** P < 0.001. (B) Recurrence‐free survival of 32 patients ( n = 18 after aCRT, n = 14 after pCRT) was determined for a cohort with CD4 + IL‐17 + cells per CD4 + T cells < 8% after therapy (blue line) in comparison with a cohort of post‐therapeutic CD4 + IL‐17 + cells per CD4 + T cells > 8% (red line). Median recurrence‐free survival was 9 months for the cohort of CD4 + IL‐17 + cells per CD4 + T cells > 8%. Comparison of survival analysis was performed using log‐rank (Mantel–Cox) test; chi‐square: 15.22, P < 0.0001.

Article Snippet: To reduce nonspecific staining, each section was treated with 2.5% normal horse serum for 30 min. Rabbit anti‐pThr308‐AKT antibody (1 : 250, Abcam; AB_722678), rabbit anti‐pSer473‐AKT antibody (1 : 100, Cell Signaling; 2315049), mouse anti‐CD4 monoclonal antibody 4B12 (1 : 1000, Leica Biosystems; AB_563560), and rabbit anti‐IL‐17 polyclonal antibody (1 : 500, Abcam; AB_1603584) were used.

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Interleukin-19 Acts as a Negative Autocrine Regulator of Activated Microglia

doi: 10.1371/journal.pone.0118640

Figure Lengend Snippet: (A) Western blotting data for receptors of IL-19. Only microglia express both the IL-20Rα and IL-20Rβ subunits. Neu, neurons; Ast, astrocytes; Mi, microglia. (B) Western blotting data for receptors of IL-19. LPS stimulation did not change the expression levels of IL-19 receptor subunits. Skin was used as the positive control. NT, untreated; LPS, LPS-treated. (C) Western-blotting analysis of STAT3 phosphorylation in microglia treated with 100 ng/ml IL-19. IL-19 induced STAT3 phosphorylation, the main downstream signal of the IL-19 receptor. All quantitative data are expressed as means ± SD (n = 3). *, p < 0.01 vs . 0 min. **, p < 0.001 vs . 0 min.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature, and then incubated overnight at 4°C with rabbit anti-IL-20Rα polyclonal antibodies (Merck Millipore, Billerica, MA, USA), rat anti-IL-20Rβ monoclonal antibody (eBioscience, San Diego, CA, USA), mouse anti-STAT3 monoclonal antibody (BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-pSTAT3 polyclonal antibodies (Cell Signaling Technology, Danvers, MA, USA), or mouse anti-β-actin monoclonal antibody (Sigma-Aldrich), followed by incubation with horseradish peroxidase—conjugated secondary antibodies (GE Healthcare, Buckingham, UK) for 1 h at room temperature.

Techniques: Western Blot, Expressing, Positive Control, Phospho-proteomics

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: ( A ) Relative IL-8 mRNA expression in M2 macrophages treated with leptin with or without the inhibitors. M2 macrophages were pretreated with JAK inhibitor AG490 (50 μmol/L), PI3K inhibitor LY294002 (10 μmol/L), ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative IL-8 mRNA levels were analyzed by qRT-PCR. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01. ( B ) A representative image of Western blot for IL-8 protein expression in M2 macrophages treated in the same manner as (A). ( C ) A representative image of Western blot showing the time course of leptin-induced p38 and ERK 1/2 phosphorylation. M2 macrophages were treated with leptin (100 ng/mL) for 0–24 hours. ( D ) A representative image of Western blot showing leptin-induced ObR-dependent phosphorylation of ERK 1/2 and p38 and production of IL-8. M2 macrophages were pretreated with a polyclonal anti-human ObR antibody (4 μg/mL) for 16 hours and then treated with PBS or leptin (100 ng/mL). ( E ) Luciferase reporter assay to measure leptin-mediated IL-8 promoter activation. M2 macrophages were pretreated with ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Luciferase, Reporter Assay, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: The mouse xenograft model and leptin injection were conducted as in Figure . In the leptin-treated group, polyclonal rabbit anti-mouse IL-8 neutralization antibody (Abbexa Ltd, Cambridge, UK) was injected intraperitoneal in the mice at an initial dose of 0.2 mL per mouse and then 0.1 mL per mouse twice per week for 2 weeks. Polyclonal rabbit IgG (Bioss Ltd, Beijing, China) was used as the control. Mice received the injection of leptin and the antibodies for 2 weeks. ( A ) Tumor volume in the PBS, leptin, leptin + anti-IgG, and leptin + anti-IL8 groups. * P < 0.05. ( B ) Photos of tumor xenografts at day 21 after transplantation of human breast cancer cells. ( C ) Tumor wet weight at day 21 after the cell transplantation. ** P < 0.01. ( D ) Mouse survival. * P < 0.05. ( E ) Representative images of H & E staining of the lung tissue and the figure showing the number of metastatic nodules in the lung. ** P < 0.01. ( F ) Representative images of H & E staining of the liver tissue. ( G ) Representative images of IHC staining of CD68, Ki-67, and IL-8 in the tumor tissue and the figure showing quantitative analysis of the staining. ** P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Injection, Neutralization, Control, Transplantation Assay, Staining, Immunohistochemistry