Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: a Heat map to summarise secretion of cytokines and chemokines from a panel of BTC cell lines. BTC cell lines were grown to 70–80% confluency, at which point supernatants were harvested and analysed using a multiplex platform. Data in the heat map represent the mean pg/mL values from at least n = 2 individual experiments. b PBMCs from healthy adult donors were incubated with 10 ng/mL of IL-6/GM-CSF (positive control) or with 10% of culture media supplemented with supernatants from individual human BTC cell lines for 7 days. These cells were then harvested, stained for MDSC phenotypic markers, and analysed via flow cytometry. Error bars represent standard deviation across donors. Asterisk (*) denotes statistical significance as compared to paired DMSO-treated PBMCs. For each condition, a minimum of n = 8 donors were analysed; Supplementary Table indicates an exact n for each condition. c The gating schema for flow cytometric analysis of cells with an MDSC phenotype. Gates and voltage were set using appropriate fluorochrome-conjugated isotype control antibodies.

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Multiplex Assay, Incubation, Positive Control, Staining, Flow Cytometry, Standard Deviation, Control

Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: a PBMCs were stimulated with 10 ng/mL of IL-6/GM-CSF (positive control) or with 10% of culture media supplemented with supernatants from individual BTC cell lines ± IL-6 or GM-CSF neutralising antibody for 7 days. Cells were then harvested and stained for the MDSC phenotype and analysed via flow cytometry. Error bars represent standard error of measurement. Asterisk (*) denotes significance compared to GM-CSF neutralisation alone. For each condition, a minimum of n = 3 donors were analysed; Supplementary Table indicates an exact n for each condition. b Basal activation of the Jak/STAT pathway in a panel of BTC cell lines. Immunoblot analysis was conducted to assess constitutive expression of phosphorylated STAT3 (Tyr 705 ), STAT5 (Tyr 694 ), and STAT1 (Tyr 701 ). Levels of total STAT proteins and β-actin were included as loading controls. Data shown are representative from at least n = 3 individual experiments. c Healthy donor PBMCs were incubated for 20 min with culture media supplemented with 10% supernatants from individual BTC cell lines. Cells were then lysed and analysed by immunoblot. PBMCs showed increased pSTAT3 (Tyr 705 ) and d pSTAT5 (Tyr 694 ) following a 20-min incubation with BTC supernatants.

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Positive Control, Staining, Flow Cytometry, Activation Assay, Western Blot, Expressing, Incubation

Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: Immunoblot analysis showing increased pSTAT3 and pSTAT5 in PBMCs after culture with BTC supernatants are abrogated by a neutralising Ab against IL-6 or b neutralising Ab to GM-CSF (α-IL-6 or α-GM-CSF). PBMCs stimulated with IL-6 and GM-CSF served as positive controls for pSTAT3 and pSTAT5, respectively. Data shown are representative of n = 3 individual experiments. β-Actin was used as a loading control. (−) = media alone, (+) = stimulation with IL-6 (10 ng/mL, positive control for pSTAT3) or GM-CSF (20 ng/ml, positive control for pSTAT5).

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Western Blot, Control, Positive Control

Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: Analysis of IL-6, GM-CSF, and CD33 + S100a9 + staining of a human BTC tissue microarray revealed significant correlations of these soluble and cellular biomarkers with each other on both a subtype-specific and nonspecific level.

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Staining, Microarray

Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: Association of IL-6 expression, GM-CSF expression, and CD33 + S100a9 + cell infiltration as measured by immunofluorescence with clinicopathologic features of patients within biliary malignancy tissue microarray ( n = 69).

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Expressing, Immunofluorescence, Microarray

Journal: British Journal of Cancer

Article Title: Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease

doi: 10.1038/s41416-020-1018-0

Figure Lengend Snippet: a Images (×40) of biliary tract cancer tissue microarray stained for DAPI (blue), S100A9 (red) and CD33 (green). Dual stained cells (yellow) are shown in merged image. b Images (×20) of patient tissue demonstrating high expression of IL-6 and GM-CSF in biliary tract cancer tissue microarray. Stained for DAPI (blue). GM-CSF (red) and IL-6 (green). c Representative images (×20) from 4 separate patients showing variability in IL-6 and GM-CSF staining, with white arrows indicating localisation of stains to ductal regions of tissue. d Graph demonstrating the correlation between increased staining for IL-6, GM-CSF and MDSC infiltration with the presence of satellite lesions. Significnace is indicated by an asterisk ( p Value < 0.05) e Graph demonstrating the relationship between patients with GM-CSF staining above the median (red line) or below the median (blue line) and recurrence-free survival ( p = 0.051).

Article Snippet: To block the effects of IL-6 and GM-CSF, cultured BTC supernatants, or media containing recombinant IL-6 (10 ng/mL) plus recombinant GM-CSF (10 ng/mL), were pre-incubated with anti-IL-6 antibody (Ab; 5 μg/mL, clone 6708, R&D Systems), anti-GM-CSF Ab (10 μg/mL, clone 3209, R&D Systems), or both for 30 min prior to addition to peripheral blood mononuclear cell (PBMC) cultures as described.

Techniques: Microarray, Staining, Expressing