recombinant egfl6 (regfl6  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Graphs represent the percentage of B, CD4 + , CD8 + , and CD11b + cells in BM ( A ) and spleen ( B ) of WT and Egfl6 mice. ( C ) Gating and quantification of Ly6G and Ly6C subsets of CD11b + BM and splenic cells from healthy C57BL/6J (WT) and Egfl6 mice. ( D ) Volcano plot showing differentially expressed genes (DEGs) between BM CD11b + cells of Egfl6 mice versus C57BL/6J (WT). P values determined via t test are plotted on the y axis. DEGs are colored in red. ( E ) Gating and quantification of BM-derived CD11b + Ly6G + Ly6C – cells stimulated with rGM-CSF ± rEgfl6. ( F ) qPCR analyses of indicated genes in sorted BM CD11b + cells stimulated with rGM-CSF + rEgfl6. Stimulation with rGM-CSF alone was used as control. ( G and H ) ELISA of Granzyme B (GZMB) in IL-2 + CD3/CD28 activated CD8 + T cells and cultured directly with rEgfl6-stimulated BM-derived MDSC cells or MDSC control at different ratio ( G ) or with the conditioned media (CM) of rEgfl6-stimulated BM-derived MDSC cells or MDSC control ( H ). Unstimulated CD8 + T cells were used as negative control. Results were analyzed using unpaired 2-tailed t test or 2-way ANOVA. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Tumor volume changes (mm 3 ) and images of 2F8c and 2F8c-Egfl6 subcutaneous tumors resected and measured 3 weeks after tumor cell inoculation ( n = 6 mice per group). ( B ) Time-dependent body weight gain in mice i.p. injected with ID8-CV and ID8-Egfl6 tumors ( n = 8 mice per group). ( C ) Evaluation of peritoneal metastases of ID8-CV and ID8-Egfl6 that had a weight increase of over 35% of their original weight on the day of tumor cell injections ( n = 6 mice per group). ( D and E ) Kaplan-Meier overall survival analysis for 2F8c+/–Egfl6 ( D ) and ID8+/–Egfl6 ( E ). Survival statistics were calculated using log-rank analysis from Kaplan-Meier survival plots. ( F and G ) Flow cytometric evaluation and summary of PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( F , top panel), M-MDSC (CD11b + Ly6G – Ly6C + ) ( F , bottom panel), and TAM (CD11b + F4/80 + CD206 + ) ( G ) in ID8+/–Egfl6 tumors. ( H ) Flow cytometric evaluation and quantification of CD8 T (CD45 + Thy1.2 + ) cells and their expression of IFN-γ in ID8+/–Egfl6 tumors. ( I and J ) ELISA of Granzyme B (GZMB) ( I ) and IFN ( J ) in IL-2 + CD3/CD28 activated CD8 T cells (Pos Control) and cultured directly with F4/80 + or Ly6G + cells isolated from ID8 and ID8-Egfl6 ascites at ratio of 1:1. ( K and L ) Time-dependent volume changes (mm 3 ) of 2F8c and 2F8c-Egfl6 tumor cells ( K ) or body-weight gain in mice i.p. injected with ID8 and ID8-Egfl6 tumor cells ( L ) and treated with anti-Ly6G/Ly6C Ab or IgG isotype control ( n = 6 mice per group). P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes (DEGs) between CD11b + cells infiltrating 2F8c-Egfl6 versus 2F8c tumors. Negative Log 10 P values determined via t test are plotted on the y axis. ( B ) IPA protein analysis of Egfl6 treatment associated DEG pathways identified as significantly ( P < 0.05) upregulated (left panel) or downregulated (right panel). ( C and D ) Summary of PD-L1 expression determined by flow cytometry in infiltrating TAMs ( C ) and by qPCR in BM-derived macrophages polarized with different stimuli as indicated D . ( E ) Western blotting analysis of IL-10 and Cxcl2 in TAMs and PMN-MDSCs isolated from ID8+/–Egfl6 ascites. Actin was used as loading control. ( F ) ELISA of IFN-γ in CD8 + T cells cultured with the Ly6G + cells isolated from ID8+/–Egfl6 ascites in the absence/presence of IL-10 or Cxcl2 NAbs. ( G ) Western blotting showing the indicated protein expression in BM-isolated CD11b + cells treated with GM-CSF and rEgfl6 for 0, 7.5, and 15 minutes. β-Actin was used as loading control. Results are representative of at least 3 independent experiments. ( H and I ) ELISA showing IL-10 and Cxcl2 protein secretion in GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or Syk inhibitor (R406) ( H ), and GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or the integrin inhibitor Cyclo-RGD (c-RGD) ( I ). ( J ) Graph represents a ChIP assay performed with anti-Jun Ab followed by qPCR to measure IL-10 promoter in ID8+/–Egfl6 ascites. Data are presented as mean ± SEM. P values were calculated using unpaired 2-tailed t test or 1-way ANOVA with Tukey’s post test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All results are representative of 3 independent experiments.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Expressing, Flow Cytometry, Derivative Assay, Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) 2F8c and 2F8c-Egfl6 tumor growth in mice treated with anti-PD-L1 Ab or IgG isotype control Ab ( n = 8 mice per group). * P < 0.05, 2F8c + IgG versus 2F8c-Egfl6 + IgG; *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. ( B ) Kaplan-Meier survival analysis for the indicated treatment groups. *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. Survival statistics were calculated using log-rank (Mantel-Cox) analysis from Kaplan-Meier survival plots. ( C ) Flow cytometry quantification of intratumoral PMN-MDSCs (CD11b + Ly6G + Ly6C – ), M-MDSCs (CD11b + Ly6G – Ly6C + ), CD206 + TAMs, and CD8 + T cells in the indicated tumors. ( D – F ) qPCR analysis of mRNA expression of S100A9 , IL-10 , and Cxcl2 gene expression in ( D ) 2F8c-Egfl6 versus 2F8c, ( E ) anti-PD-L1–treated 2F8c versus IgG-treated 2F8c, ( F ) anti-PD-L1–treated 2F8c-Egfl6 versus IgG-treated 2F8c-Egfl6 tumor samples. ( G ) Representative images of IHC staining showing Cxcl2-expressing cells in control and a-PD-L1–treated tumor tissue sections. Graph represents the number of Cxcl2 + cells in the indicated tumors. Scale bars: 20 μm. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Control, Flow Cytometry, Expressing, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volume changes (mm 3 ) and representative images of 2F8c-Egfl6 subcutaneous tumors treated with IgG isotype Ab (Control), a-PD-L1 Ab, and a-Egfl6 Ab, alone or in combination, were resected and measured 2 days after the last treatment ( n = 8 mice per group). ** P < 0.01, IgG Ab versus a-Egfl6 Ab; *** P < 0.001, anti-PD-L1 Ab versus a-Egfl6 Ab and IgG Ab versus anti-PD-L1+ a-Egfl6 Abs. ( B and C ) Kaplan-Meier overall survival analysis for 2F8c-Egfl6 ( B ) and ID8 p53–/– Brca2–/— -Egfl6 ( C ) mice receiving the indicated treatment. Survival statistics were calculated using the Log-rank (Mantel-Cox) test analysis. ( D – G ) Flow cytometric gating and quantification of CD206 + TAMs ( D ), PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( E ), MHCII + TAMs ( F ), and CD8 + T (CD45 + Thy1.2 + ) ( G ) cells in 2F8c-Egfl6 and ID8 p53–/– Brca2–/— -Egfl6 tumors. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) qPCR analyses of IL-10 and Cxcl2 in the indicated treated Egfl6 + 2F8c tumors. ( B ) IF images and quantification of IL-10 expression in the indicated treated Egfl6 + 2F8c tumors. P values were calculated using unpaired 2-tailed t test. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 , **** P < 0.0001. All results are representative of 3 independent experiments. Scale bar: 30 μm.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Gating and quantification of human CD11b + CD66b + ( A ) and CD11b + CD163 + CD64 + ( B ) cells in CD33 + cells isolated from ascites of patients with HGSOC and stimulated with rEGFL6 +/– c-RGD. ( C ) Cytokine array and densitometry of the CM of CD33 + ascites from patients with HGSOC stimulated with GM-CSF +/– rEGFL6. Spot intensities were calculated using ImageJ software. ( D ) Representative immunofluorescence images showing EGFL6 expression (red) and CD68 cell (green) localization in HGSOC tumor tissue sections ( n = 6 per group). DAPI stained nuclei. Graph represents the number of CD68-positive cells in tissues expressing high or low levels of EGFL6. Scale bar: 100 μm. ( E ) Spatial feature plots of EGFL6 and CD163 markers and spatial autocorrelation of selected genes. Moran’s I test, implemented in the Seurat FindSpatiallyVariableFeatures function, was applied to compute spatial autocorrelation of the expression of each gene. Data are from a previously published dataset . ( F – H ) Sorted correlation plots between mRNA expression of EGFL6 in CD45 – cells and mRNA expression of cytokines and surface proteins in the indicated immune cells. Correlation was computed using the Spearman’s correlation with the sample-wise averaged gene expression. Each dot represents the Spearman’s correlation coefficients of a gene, and the dots were sorted in ascending order. P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Isolation, Software, Immunofluorescence, Expressing, Staining