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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

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: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Isolation, Software, Immunofluorescence, Expressing, Staining

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Human normal ovary, ovarian tumor, and healing wound tissues were dissociated, and isolated endothelial cells and samples were processed for microarray. b, Gene microarray of endothelial cells from normal ovary, healing-wound tissue, and ovarian tumor–associated endothelial cells. c, Expression of EGFL6 in human normal ovary, wound, and ovarian tumor samples. Representative images were taken from different samples. Scale bar =100 µm d, Validation of gene microarray data using q-PCR.(Normal ovary; n = 5, Ovarian tumor; n = 10, Wound; n = 7). Validation Error bars indicates SEM. *p<0.05 vs. Normal. See also Figure S1.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Isolation, Microarray, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Graph of wound area on mice treated with either control IgG antibody or DC101 (anti-VEGFR2) quantified on days 0 through 15 after a skin excision wound. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control IgG. b, One day after SKOV3ip1 tumor cell injection, a wound was created on the dorsal side of the mice. Animals were treated with either Control siRNA-CH or mEGFL6 siRNA-CH. Graphical depiction of wound areas quantified on days 0 through 15 after skin excision wound. c, Effect of mEGFL6 silencing on tumor growth; representative images of tumor burden. Tumor weights are shown in d and numbers of tumor nodules in e. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control siRNA. f, Hind limb ischemia. After arterial ligation, mice were separated into 3 groups (n = 5 mice per group): normal, ischemia-24 h, and 96 h. Blood flow was monitored before and after femoral artery ligation with use of serial color doppler. At each time point, tissue was harvested and frozen so that immunofluorescence staining could be performed. g, EGFL6 expression was increased in endothelial cells in the ischemic (hypoxic) condition compared with the normal conditions. Error bars indicate SEM. *p<0.05 vs. Normal. See also Figure S2.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Injection, Ligation, Immunofluorescence, Staining, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, EGFL6 promoter reporter analysis under normoxia and hypoxic conditions. b, TWIST1 ectopic expression increases EGFL6 transcription activity. c, Increase in TWIST1 and EGFL6 expression in hypoxia and CoCl2 treatment. d, Ectopic expression of TWIST1 increases EGFL6 expression in RF24 cells. e, ChIP analysis of TWIST1 binding to EGFL6 promoter region in hypoxia compared with normoxia. Cross-linked chromatin from RF24 cells incubated in hypoxia chamber for 48 h and immunoprecipitated with TWIST1 or IgG control antibodies. The input and immunoprecipitated DNA was subjected to PCR using primers corresponding to the base pairs upstream of EGFL6 transcription start site. f, EGFL6 gene silencing using siRNA leads to increased cell death in hypoxia condition. (n = 3) **p<0.005, *p<0.05 See also Figure S3.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation, Immunoprecipitation

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Expression level change in selected proteins after normalization by RPPA analysis. b and c, Western blotting of EGFL6-mediated activation of PI3K/AKT signaling, Tie2 and EGFR signaling. d, RF24 cells treated with Control (PBS) or EGFL6. Extracts were subjected to anti-Tie2 immunoprecipitation (IP) and integrin α5, αV, β1, and β3 detected by immunoblotting. e, Expression level of pTie2 and pAKT in cytosol and membrane fractions with Control (PBS) and EGFL6 treatment. αβ-tubulin was used as internal control of cytosolic fraction; NaK ATPase was used as membrane marker. f, Silencing of Integrin β1 (ITGB1) and Tie2 using specific siRNAs decreases Tie2 and AKT signaling. g–h, Silencing of Integrin β1 (ITGB1) and Tie2 decreases EGFL6-mediated tube formation (g) and migration (h) in endothelial cells. *p<0.05 vs. Control siRNA. In i–k, RGD blocking peptide decreases Tie2/AKT activation (i), tube formation (j) and migration (k). (n=3) *p<0.05 vs. Control. See also Figure S4.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Expressing, Western Blot, Activation Assay, Immunoprecipitation, Marker, Migration, Blocking Assay

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, The binding affinity of recombinant EGFL6 to monoclonal antibody #93 and #135 was measured by Biacore. The dissociation constant (Kd) value of the monoclonal antibodies were calculated to be 1.89 nM (#93) and 2.19 nM (#135). b, Effect of EGFL6 blocking antibodies on Tie2/AKT activation in RF24 cells (n=3). c, Effect of EGFL6 blocking antibodies on wound healing in vivo (n=5 mice/group, #135; 5 mg/kg). d and e, EGFL6 antibodies decreased tube formation and migration of RF24 cells. f, Effect of EGFL6 blocking antibodies on tumor weight and tumor nodules in SKOV3ip1 tumor-bearing mice. Seven days after tumor cell injection, mice were randomly divided into three groups (10 mice/group) to receive therapy: (1) Control IgG Ab, (2) EGFL6 #93, and (3) EGFL6 Ab #135 (5 mg/kg). Antibody treatment was given once a week. g, Effect of targeted EGFL6 on proliferation (Ki67) and microvessel density (CD31). Scale bar = 100µm. The bars in the graphs correspond sequentially to the labeled columns of images on the left. Error bars indicates SEM. *p<0.05 vs. Control IgG. See also Figure S5.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Binding Assay, Recombinant, Blocking Assay, Activation Assay, In Vivo, Migration, Injection, Labeling

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Tumor growth in Tie2;EGFL6 KO mice (KO) and WT mice. ID8 murine ovarian cancer cells were injected into KO and WT mice (n=10 mice per group). b, Double immunofluorescence staining of CD31 and EGFL6 in ID8 tumor from WT and KO mice. Scale bar = 100 µm. c, Bar graph represents tumor weight. d and e, Proliferation (Ki67) and microvessel density (CD31) counted in WT and KO mice tumor sections. Error bars indicate SEM. Scale bar = 100 µm. *p<0.05 vs. WT mice. f, Representative images of human ovarian cancer vasculature with low or high immunohistochemical staining for EGFL6. Scale bar =200 µm. Representative images were taken from different samples. g, Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EGFL6. See also Figure S6 and Table S1.

Article Snippet: Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Injection, Double Immunofluorescence Staining, Immunohistochemical staining, Staining