Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: EGFL6 is highly expressed during osteoblast differentiation and co-localizes with blood vessels in bone. (A) EGFL6 expression profiled from an array of normal tissues, organs, and cell lines in mice. Data is adapted from BioGPS ( http://biogps.org/ ). Red box indicates the EGFL6 signal intensity in osteoblasts. (B) Alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining showing the osteogenic differentiation of neonatal calvarial osteoblasts on day 4 and day 10 respectively. (C) qPCR analysis of osteogenic gene Bglap as well as EGFL6 during osteoblast differentiation (n = 3 per group). (D) Representative confocal images of the immunostaining of EGFL6 and endomucin (EMCN) in 12-week-old male mice tibiae and bone defect area. Growth plate (GP) is indicated with white dashed line. All data are presented as mean ± SD. **P < 0.01 relative to the control group. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Expressing, Staining, Immunostaining

Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: EGFL6 mediates osteoblast differentiation through BMP-Smad and MAPK signaling pathways. (A) Alizarin Red S (ARS) staining of mesenchymal stem cells (MSCs) transduced with lentiviral EGFL6 shRNA or a vector control and induced osteogenic differentiation for 14 days. (B) Quantification of ARS-stained area of (A) (n = 3 per group). (C) qPCR analysis of mRNA expressions of EGFL6 , Bglap (encoding osteocalcin), Sp7 (encoding osterix), Runx2 , and Vegfa (n = 3 per group). (D) ARS staining of MSCs transduced with lentiviral EGFL6 overexpression vector or a vector control and induced osteogenic differentiation for 14 days. (E) Quantification of ARS-stained area of (D) (n = 3 per group). (F) qPCR analysis of mRNA expressions of EGFL6, Bglap, Sp7, Runx2 , and Vegfa (n = 3 per group). (G) ARS staining of MSCs induced into mineralization in the absence or presence of BMP2 and EGFL6 protein. (H) ARS staining of the mineralization of MC3T3-E1 cells stably transduced with lentiviral EGFL6 overexpressing vector or a control vector (n = 3 per group). (I) Gel electrophoresis showing EGFL6 expression in MC3T3-E1 cells following osteogenic induction for 21 days. (J) Western Blot assay showing the proteins level of basal canonical (Smad) and non-canonical (MAPK) BMP signaling pathways including P-Smad 1/5/8, P-ERK, and P-P38 in MC3T3-E1 cells induced by BMP-2. (K) Quantifications of the band intensities of (J) (n = 3 per group). All bar graphs are presented as mean ± SD. *P < 0.05, **P < 0.01 relative to the control group. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Staining, Transduction, shRNA, Plasmid Preparation, Over Expression, Stable Transfection, Nucleic Acid Electrophoresis, Expressing, Western Blot

Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: Osteoblast-specific deletion of EGFL6 has no significant effect on bone phenotype. (A) Schematic illustration of the strategy used to generate the osteoblast - specific EGFL6 conditional knockout (cKO) mice. (B) Photographs of a 12-week-old male EGF6 conditional knockout mouse (EGFL6 OCN ) and its littermate control (EGFL6 fl/Y ). (C) Weight and body length of 12-week-old male EGFL6 OCN mice (n = 5) and EGFL6 fl/Y mice (n = 7). (D) Representative three-dimensional reconstructed micro-CT images showing the femurs 12-week-old male EGFL6 OCN and EGFL6 fl/Y mice. (E) Quantification of the trabecular bone parameters including bone volume per tissue volume (BV/TV) and trabecular number (Tb.N) (n = 14 per group). (F) Representative micro-CT images of cortical bone, and quantification of cross-sectional thickness (Cs.Th) of the cortical bone (n = 9 per group). (G) Representative hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining of femurs. (H) Representative images of bone growth rates as determined by calcein and alizarin red labelling, and quantification of mineral apposition rate (MAR) (n = 4 per group). All bar graphs are presented as mean ± SD. ns, no significance. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Knock-Out, Micro-CT, Staining

Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: EGFL6 global knockout (gKO) mice display normal bone phenotype. (A) Schematic illustration of the strategy used to generate the EGFL6 global knockout (gKO) mice. (B) Representative three-dimensional reconstructed micro-CT images showing the femurs of 24-week-old male EGFL6 WT and EGFL6 gKO mice. (C) Quantification of the trabecular bone parameters including bone volume per tissue volume (BV/TV) and trabecular number (Tb.N) (n = 10 per group). (D) Representative micro-CT images of lumbar 1 (L1) of EGFL6 WT and EGFL6 gKO mice, and (E) quantification of BV/TV and Tb.N of the trabecular bone (n = 10 per group). (F) Representative micro-CT images of cortical bone of EGFL6 WT and EGFL6 gKO mice, and (G) quantification of bone marrow area (B.Ar) and cross-sectional thickness (Cs.Th) of the cortical bone (n = 10 per group). (H) Representative hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining of femurs of EGFL6 WT and EGFL6 gKO mice. Black arrows indicate osteoblasts. (I) Quantifications of number of osteoblast per bone perimeter (N.Ob/Pm) and number of osteoclast per bone perimeter (N.Oc/Pm). (J) Representative confocal images of type H vessel stained for CD31 (red) and EMCN (green) in tibia of EGFL6 WT and EGFL6 gKO mice. (K) EGFL6 gene expression in differentiating bone marrow mesenchymal stem cells (MSCs) derived from EGFL6 WT and EGFL6 gKO mice. (L) Alizarin red S (ARS) staining of mineralization of MSCs from EGFL6 WT and EGFL6 gKO mice. Growth plate is indicated with white dashed line. All bar graphs are presented as mean ± SD. ns, no significance. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Knock-Out, Micro-CT, Staining, Expressing, Derivative Assay

Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: Deletion of EGFL6 in osteoblasts leads to impaired bone repair characterized by reduced angiogenesis. (A) Schematic illustration of mono-cortical bone defect model. (B) Representative three-dimensional reconstructed micro-CT images of bone repair in EGFL6 OCN and EGFL6 fl/Y mice 1 week after surgical procedure, and bone histomorphometric analysis including hematoxylin-eosin (HE), picrosirius red (PSR), Masson trichrome staining of bone defect region. CB, cortical bone. Scale bar = 200 μm (C) Quantification of newly formed bone in defect region by micro-CT scanning and Masson trichrome staining (n = 5 per group). BV/TV, bone volume per tissue volume; BS/TS, bone surface and tissue surface. (D) Representative micro-CT and bone histomorphometric images of bone defect at week 2. Scale bar = 200 μm. (E) Quantification of the newly formed bone in defect region at week 2 by micro-CT scaning (WT, n = 11; cKO, n = 10) and Masson trichrome staining (n = 5 per group). (F) Representative confocal images of type H vessels stained for CD31 (red) and EMCN (green) in bone defect region of EGFL6 OCN and EGFL6 fl/Y mice at week 2. (G) Quantification of EMCN + and CD31 + area in (F) (n = 5 per group). White dashed lines indicate the edge of bone tissue. All bar graphs are presented as mean ± SD. *P < 0.05, **P < 0.01 relative to the WT group. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Micro-CT, Staining

Journal: Theranostics

Article Title: Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

doi: 10.7150/thno.60902

Figure Lengend Snippet: EGFL6 deficiency reduced osteogenesis during bone repair. (A) Representative confocal images of immunofluorescence staining for Runx2 (green) and CD31 (red) in bone defect region of EGFL6 OCN and EGFL6 fl/Y mice at week 2. (B) Quantification of Runx2-positive area in (A) (n = 5 per group). (C) Representative confocal images of immunofluorescence staining for P-Smad1/5/8 (orange) and CD31 (red) in bone defect region. (D) Quantification of P-Smad1/5/8-positive area in (C) (n = 5 per group). (E) Schematic illustration of osteoblast derived EGFL6 which contributes to the coupling of osteogenesis and angiogenesis in bone repair. White dashed lines indicate the edge of bone tissue. *P < 0.05, **P < 0.01 relative to the WT group. Differences are analyzed using Student's t-test.

Article Snippet: Full-length EGFL6 protein was purified from HEK293 using EGFL6 (NM_015507) human tagged ORF clone (RC207729) by OriGene Technologies Inc. (Rockville, US).

Techniques: Immunofluorescence, Staining, Derivative Assay

Journal: In Vivo

Article Title: Cytoplasmic Expression of the EGFL6 Protein Is an Independent Prognostic Factor for Shortened Patient Survival in Human Hepatocellular Carcinoma

doi: 10.21873/invivo.13715

Figure Lengend Snippet: EGFL6 (–): low-expression; EGFL6 (+): high-expression. p-Value was measured using Fisher Extract Test. *p<0.05; **p<0.01.

Article Snippet: The relationship between EGFL6 protein expression levels and the clinicopathological parameters of HCC was assessed using the Chi-square test or Fisher exact test.

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 ) 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