Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 1 Cancer-conditioned myeloid cells chronicle the evolution of pancreatic ductal adenocarcinoma (PDA) in KPC mice. (A) The number of pancreatic Treg (CD45+CD4+FoxP3+), macrophages (CD45+CD11b+F4/80+), myeloid-derived suppressor cells (MDSC) (CD45+CD11b+RB6-8C5 [Ly6G/ Ly6C]+) and NK cells (CD45+NK1.1+) for each pancreas were quantified from normal pancreas (nl), 6–8-week-old KPC pancreata with confirmed preinvasive disease (Pre) and invasive tumours (PDA). Significant differences were detected in the number of Treg, tumour-associated macrophages and MDSC during disease progression. (B) Evolving profiles of three distinct populations of myeloid cells (gated on CD45+CD11b+) were seen in various organs based on expression patterns of Gr-1 and Ly6C. BM, bone marrow; LN, lymph node. (C) The percentages (top panel) and absolute numbers (bottom panel) of CD45+CD11b+ myeloid populations in the spleen and pancreas in normal (black filled circles) and preinvasive (grey filled circles) and invasive (open circles) disease settings. Data are plotted as mean±SEM and each data point represents an individual mouse. Granulocytic MDSC (Gr-MDSC)=CD45+CD11b+Gr-1highLy6Cint; monocytic MDSC (Mo-MDSC)=CD45+CD11b+Gr-1intLy6Chigh and macrophage (Mac) =CD45+CD11b+Gr1intLy6Cint. (D) Specific immunofluorescence reveals rare Ly6G/Ly6C+ (RB6-8C5) cells in normal pancreas, focal accumulation in pancreata with preinvasive disease and diffuse infiltration in invasive PDA. Specific Ly6G immunofluorescence demonstrates that Gr-MDSC are absent from normal pancreas, rare in preinvasive disease and abundant in invasive PDA. The majority of myeloid cells in normal pancreas and surrounding preinvasive lesions appear to be macrophages. Arrowheads, epithelial cells; arrows, myeloid cells; asterisk, Gr-MDSC. Scale bars, 50 mm. (E) Ly6G/Ly6C (RB6-8C5) staining in normal pancreas and KPC salivary gland. Arrowheads, epithelial cells; arrows, myeloid cells. Scale bars, 10 mm. *p<0.05; **p<0.005; ***p<0.0005.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, Biomarker Discovery, Expressing, Staining

Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 4 Systemic administration of 1A8 (αLy6G) specifically depletes Gr-myeloid-derived suppressor cells (Gr-MDSC) in autochthonous pancreatic ductal adenocarcinoma (PDA). (A) Representative myeloid cell profiles in peripheral blood mononuclear cells from a normal mouse and untreated (KPC) and 1A8-treated KPC (KPC + 1A8) mice. Numbers indicate the percentage of each subset gated on CD45 mononuclear cells. We note that the gates for the discrete subpopulations defined in the blood were then applied to the tissue-specific analyses. (B) Percentage of Gr-MDSC (squares) and monocytic MDSC (Mo-MDSC; circles) in blood after 1A8 treatment. Data represent mean±SD from three independently treated animals. (C) Representative fluorescence activated cell sorting (FACS) profiles of CD45+ CD11b+ splenocytes from control (−) and 1A8-treated (+) KPC mice (4–6 animals per group). (D) Both the percentage and number of splenic Gr-MDSC at endpoint of 1A8 treatment (day 12) are significantly decreased (**, p=0.005). (E) Representative FACS profiles of intratumoral myeloid cells in control (−) and 1A8-treated (+) KPC mice at day 12 of Gr-MDSC depletion. (F) The percentage and number of Gr-MDSC in PDA at the endpoint are significantly decreased in 1A8-treated mice compared with control KPC mice.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, FACS, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Glucocorticoids Disrupt Skeletal Angiogenesis Through Transrepression of NF-κB–Mediated Preosteoclast Pdgfb Transcription in Young Mice

doi: 10.1002/jbmr.3987

Figure Lengend Snippet: Glucocorticoids inhibit preosteoclast PDGF-BB through GR/NF-κB–mediated transrepression. (a) Schematic illustration of NF-κB binding site in mouse Pdgfb promoter binding sites for p65 NF-κB and PCR primer design strategy for the analysis of ChIP. (b,c) PDGF-BB concentrations of mouse bone marrow monocytes and RAW246.7 cells cultured for 3 days in M-CSF and RANKL to form preosteoclasts, followed by stimulation with TNFα at denoted concentrations (b) or time after TNFα 20 ng/mL (c). (d) Immunoblot of GR after transfection of preosteoclasts with siRNA control or siRNA GR. GAPDH serves as control to ensure appropriate loading. (e,f) Immunoblot (e) and quantification (f) of ChIP assay detecting binding activity between p65 subunit of NF-κB and Pdgfb promoter using a NF-κB-p65 antibody in preosteoclasts culture plus addition of prednisolone 10−7M, 10−6M alone, or 10−6M plus GR siRNA, RU486 (progesterone antagonist that disrupts the GR/NF-κB interaction), or TNFα. IgG served as negative control, input as positive control. (g) Western blot and quantification of PDGF-BB from cultured conditions utilized in CHIP assay. (h,i) Quantification of Pdgfb mRNA (h) and PDGF-BB protein (i) concentration by real-time PCR and ELISA, respectively. mRNA normalized to GAPDH. (j,k) Representative images (j) and tube length (k) from Matrigel endothelial tube formation assay with addition of preosteoclast culture media under denoted conditions. Assays performed in triplicate. Data are shown as mean ± SD. ChIP = chromatin immunoprecipitation; GR = glucocorticoid receptor; PDGF-BB = platelet-derived growth factor type BB.

Article Snippet: POCs were transfected with either GR siRNA or control siRNA duplex diluted in siRNA transfection medium (Santa Cruz Biotechnology; sc-36868) and mixed with siRNA transfection reagent (Santa Cruz Biotechnology; sc-29528) into siRNA transfection medium after incubation for 30 min at room temperature.

Techniques: Binding Assay, Cell Culture, Western Blot, Transfection, Control, Activity Assay, Negative Control, Positive Control, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Endothelial Tube Formation Assay, Chromatin Immunoprecipitation, Derivative Assay

Journal: PLoS ONE

Article Title: Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

doi: 10.1371/journal.pone.0119415

Figure Lengend Snippet: ( A ) MKN-7 and MKN-74 cells were cultured alone (Mono) or co-cultured with Hs738 (Cocul) for 3 days in the presence of MEK inhibitor I or U0126. Hs738 cells were cultured with the inhibitors for 2 days and CM was prepared. Both gastric cancer lines were cultured in the CM for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without test compounds in each culture condition. ( B ) Hs738 cell extracts were incubated with b-MEK inh-pretreated streptavidin resin in the presence or absence of MEK inhibitor I (M) or U0126 (U) and the bound proteins were analyzed by Western blotting. L, 1/50 of loaded cell extracts. ( C ) Hs738 cells were cultured with MEK inhibitor I for 2 days and the cell lysates were analyzed by Western blotting. ( D ) Hs738 cells were cultured with MEK inhibitor I for 2 days and the concentrations of IL-6 and CXCL1 in the CM were determined. The values are means ± s.d. (n = 3). ( E ) Hs738 cells were treated with siRNA specific for RPL-18A (siRPL) or negative control (siCont) for 2 days and then re-inoculated followed by further culture for 2 days. The cell lysates were analyzed by Western blotting and the amounts of IL-6 in the CM were determined. The values are means ± s.d. (n = 3).

Article Snippet: Anti-human IL-6 neutralizing antibody (MAB206), recombinant human IL-6 (206-IL), and recombinant human CXCL1 (275-CR/CF) were purchased from R&D Systems.

Techniques: Cell Culture, Fluorescence, Incubation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

doi: 10.1371/journal.pone.0119415

Figure Lengend Snippet: ( A ) MKN-7 and MKN-74 cells were cultured for 15 min with or without 50 μg/mL anti-IL-6 neutralizing antibody in Hs738 CM prepared by 2 days of culture. The activation of STAT3 was analyzed by Western blotting. ( B ) MKN-7 and MKN-74 cells were cultured for 15 min with IL-6. The activation of STAT3 was analyzed by Western blotting. ( C ) MKN-7 and MKN-74 cells were cultured with IL-6 or CXCL1 for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). ( D ) MKN-7 and MKN-74 cells were cultured alone (Mono) or co-cultured with Hs738 cells (Cocul) with the indicated antibodies for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without antibodies in each culture condition.

Article Snippet: Anti-human IL-6 neutralizing antibody (MAB206), recombinant human IL-6 (206-IL), and recombinant human CXCL1 (275-CR/CF) were purchased from R&D Systems.

Techniques: Cell Culture, Activation Assay, Western Blot, Fluorescence