Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: Inhibition of ephrin B2 suppresses multiple myeloma growth in vivo . A, Representative immunofluorescence images of RPMI-8226 multiple myeloma (MM) cells stained for BCMA (green) and EFNB2; merged image with DAPI (blue) nuclear stain at right (red). Scale bar, 100 μm. B, Representative immunofluorescence images of VE-cadherin (Cdh5) expression (white) and Ephb4 colocalization (green) in BM blood vessels in NSG mice. Scale bar, 100 μm. C, Representative images of femur cross sections from NSG mice at 5 weeks posttransplant of RPMI-8226 multiple myeloma cells; sinusoidal blood vessel expressing VE-cadherin (Cdh5; white), multiple myeloma cells expressing BCMA (green), and EFNB2 (red) and merged image shown at right. Scale bar, 100 μm. Arrows, multiple myeloma cells adjacent to sinusoidal vessel. D, Left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 5 weeks posttransplant of 5 × 10 6 RPMI-8226 cells treated with sh-EFNB2 or sh-Control . SSC, side scatter. Right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. n = 8/group. E, Percent survival of NSG mice after transplantation of 5 × 10 6 RPMI-8226 cells transduced with sh-EFNB2 or sh-Control . Log-rank test, P = 0.0012, n = 12/group. F, Mean percentages of Annexin V + RPMI-8226 cells at 24 hours posttreatment with EFNB2 scFv ( n = 6/group). G, Top, schematic representation of experiment. Bottom left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 4 weeks posttransplantation of 3 × 10 6 RPMI-8226 cells and intravenous administration of EFNB2 scFv or IgG on days +4, 6, 8, 10, and 12. Bottom right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. One-way ANOVA was utilized for analyses of three or more groups; Student two-sided t test was utilized otherwise. Error bars, SD. **, P < 0.01; ****, P < 0.0001; # , P < 0.05; ### , P < 0.001. ( G, Created with BioRender.com.)

Article Snippet: Cells were stimulated with either human IgG1 mAb (R&D Systems, MAB9894-SP) or preclustered recombinant mouse EPHB4 Fc Chimera Protein (R&D Systems, 446-B4).

Techniques: Inhibition, In Vivo, Immunofluorescence, Staining, Expressing, Control, Transplantation Assay, Transduction

Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: The effects of ephrin B2 on multiple myeloma are mediated by STAT5. A, Heat map of differentially expressed genes by RNA-seq of sh-EFNB2 versus sh-Control cells. B, Upstream regulator analysis from IPA of RNA-seq data. C, Selected STAT5 target genes in the RNA-seq data. D, Left, representative analysis of phospho-STAT5 in RPMI-8226 cells treated with sh-EFNB2 or sh-Control in presence of EPHB4-Fc. Right, mean fluorescence intensity (MFI) of pSTAT5 in the sh-EFNB2 - and sh-Control –treated multiple myeloma cells. E, Numbers of U266 cells in culture over time when transduced with gRNA1-EFNB2 and gRNA2-EFNB2 . F, Left, representative histograms of BrdU incorporation over 6 hours in U266 cells transduced with the labeled construct. Right, %BrdU + cells in each group. G, Left, representative flow cytometric analysis of Annexin V + 7AAD + (necrotic) and Annexin V + 7AAD − (apoptotic) cells. Right, mean levels of total Annexin V + cells within each treatment group. H, Mean percentages of Annexin V + U266 cells engineered with the indicated construct and exposed to bortezomib (Bor) or dexamethasone (Dex) for 24 hours. n = 6/group. I, Representative histograms of p-STAT5 in U266 cells transduced with gRNA-EFNB2 or gRNA-control , with and without stimulation by EPHB4-Fc. IgG (isotype) was used as a negative control for EPHB4-Fc. Right, mean MFI of pSTAT5 in each treatment population. J, Multiple myeloma cells transduced with gRNA-EFNB2 or gRNA-control were cultured with increasing concentrations of the STAT5 inhibitor 2-[(4-oxo-4H-1-benzopyran-3-yl)methylene]hydrazide 3-pyridinecarboxylic acid (STAT5i) for 24 hours, then BrdU incorporation analysis was performed. n = 6/group. One-way ANOVA used for analyses in which there were three or more groups, Student two-sided t test for the other analyses. Error bars, SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Cells were stimulated with either human IgG1 mAb (R&D Systems, MAB9894-SP) or preclustered recombinant mouse EPHB4 Fc Chimera Protein (R&D Systems, 446-B4).

Techniques: RNA Sequencing, Control, Fluorescence, Transduction, BrdU Incorporation Assay, Labeling, Construct, Negative Control, Cell Culture

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: (A) EPCs were stimulated with 3 μg/ml ephrin-B2–Fc for 30 minutes at 37°C. Cell lysates were prepared, subjected to immunoprecipitation with an anti-human EphB4 antibody, and resolved by SDS-PAGE, and proteins were transferred to nitrocellulose membranes as described in Methods. Membranes were then blotted with the 4G10 anti-phosphotyrosine antibody. To check for equal protein loading, membranes were stripped and reprobed with an anti-EphB4 antibody. pTyr, phosphotyrosine; WB, Western blot. (B) To confirm EphB4 activation, ephrin-B2–Fc–stimulated EPCs were subjected to double immunostaining with EphB4 and anti-phosphotyrosine antibodies. Note the membrane colocalization of the EphB4 and the 4G10 signals corresponding to clusters (yellow color) in ephrin-B2–stimulated cells. Inset: magnification showing clustering and internalization of the phosphorylated EphB4 when cells were stimulated with ephrin-B2–Fc (right) but not with control CD6-Fc (left). Scale bar: 10 μm.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Activation Assay, Double Immunostaining, Membrane, Control

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: (A) Representative photomicrographs and quantitative analysis of microangiography. (B) H&E staining and capillary density. CD31-positive capillaries appear in green. Scale bar: 100 μm. (C) Foot perfusion in mice injected with PBS, nonstimulated EPCs (EPCs), and stimulated EPCs (EPCs + ephrin-B2–Fc, EPC + EphB4-Fc, or EPC + CD6-Fc). Values are expressed as means ± SEM; n = 10 per group. **P < 0.01, ***P < 0.001 versus PBS-injected mice; ##P < 0.01, ###P < 0.001 versus nonstimulated EPC-injected mice. NI, nonischemic; Isch, ischemic.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Staining, Injection

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: Western blot quantification of EphB4 protein demonstrating the efficiency of EphB4 siRNA in transfected cells. The membrane was reprobed for β-actin to check for equal loading. NT, nontransfected cells; control siRNA, luciferase siRNA; transfection reagent, Dharmafect2. n = 3. ***P < 0.001 versus nontransfected cells.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Western Blot, Transfection, Membrane, Control, Luciferase

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: EphB4 siRNA abolishes ephrin-B2–Fc–induced postischemic neovascularization

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques:

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: EPCs were stimulated for 6 hours with 3 μg/ml of either EphB4-Fc, ephrin-B2–Fc, or CD6-Fc or left unstimulated and then allowed to attach to IL-1β preactivated HUVEC monolayer. EPCs transfected with EphB4 siRNA were stimulated with 3 μg/ml ephrin-B2–Fc or left unstimulated and then allowed to attach in a similar manner. Cells transfected with luciferase siRNA were used as control (siRNA control cells). Data are expressed as means ± SEM. n = 3. *P < 0.05 versus nonstimulated EPCs (EPCs). EPCs represented in this figure were nonstimulated.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Transfection, Luciferase, Control

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: (A and B) PSGL-1 expression in EPCs. EPCs were stimulated with 3 μg/ml of either ephrin-B2–Fc, EphB4-Fc, or CD6-Fc and then processed for FACS analysis of PSGL-1 expression. (A) FACS profiles of nontransfected (top panel) and EphB4 siRNA–transfected (bottom panel) EPCs. (B) Expression of PSGL-1 in nontransfected (top panel) and EphB4 siRNA–transfected (bottom panel) EPCs. n = 3. **P < 0.01 versus nonstimulated EPCs. (C) Effect of PSGL-1 siRNA on PSGL-1 protein expression. Control siRNA– and PSGL-1 siRNA–transfected EPCs were stimulated with 3 μg/ml ephrin-B2–Fc or left unstimulated and then processed for immunocytochemistry with an anti–PSGL-1 antibody as described in Methods. PSGL-1–positive staining appears in red. Scale bar: 20 μm. (D) Effect of PSGL-1 siRNA on EPC adhesion to IL-1β prestimulated HUVEC monolayer. Adhesion was quantified by measuring OD at 570 nm. n = 3. *P < 0.05 versus nonstimulated EPCs transfected with control siRNA.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Expressing, Transfection, Control, Immunocytochemistry, Staining

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: (A) Ephrin-B2–Fc treatment increased EPC adhesion to immobilized E selectin and P selectin fusion proteins. EPCs were stimulated with 3 μg/ml of either ephrin-B2–Fc, EphB4-Fc, or CD6-Fc. Then the cells were allowed to adhere to immobilized recombinant E selectin–Fc (black bars), P selectin–Fc (gray bars), or to human IgG Fcγ fragments (white bars). Adhesion was quantified by measuring OD at 570 nm. Results are expressed as percentages of control nonstimulated EPCs (EPC). EPCs represented in this figure were nonstimulated. n = 3. *P < 0.05; **P < 0.01 versus nonstimulated EPCs. (B) Blocking antibodies directed against E selectin and P selectin neutralized ephrin-B2–Fc–induced EPC adhesion to IL-1β–prestimulated HUVEC monolayers. n = 3. *P < 0.05 versus EPCs treated with control IgG1.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Recombinant, Control, Blocking Assay

Journal:

Article Title: PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

doi: 10.1172/JCI28338

Figure Lengend Snippet: E selectin and P selectin were overexpressed by the ischemic endothelium. EphB4 activation enhances PSGL-1 expression at the surface of EPCs. This allows attachment of the circulating ephrin-B2–Fc–stimulated EPCs via interaction between PSGL-1 and E selectin and P selectin. The attached cells can then migrate to the ischemic tissue where they can integrate into the nascent vessels and/or participate in a paracrine fashion in the neoangiogenic process.

Article Snippet: To check for equal protein loading, membranes were probed with an EphB4 antibody (R&D Systems).

Techniques: Activation Assay, Expressing

Journal: Neoplasia

Article Title: Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

doi: 10.1593/neo.03457

Figure Lengend Snippet: Figure 1. Effect of monomeric and dimeric soluble EphB4 on gel sprouting angiogenesis. Concentrations as low as 2 ng/ml VEGF induce a robust sprouting angiogenesis effect originating from collagen gel–embedded HUVEC spheroids (negative control versus positive control) (A and B). Dimeric EphB4-Fc (5 g/ml) significantly enhances VEGF-induced sprouting angiogenesis (*P < .05) (A and C). In contrast, soluble monomeric EphB4 (5 g/ml) does not affect VEGF-induced sprouting angiogenesis. An excess of monomeric sEphB4 (25 g/ml), however, completely abrogates EphB4-Fc induced sprouting angiogenesis (*P < .05) (A and D). Scale bar, 100 m.

Article Snippet: Recombinant mouse EphB4-Fc, anti– murine EphB4 antibody, and human recombinant vascular endothelial growth factor (VEGF) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Negative Control, Positive Control

Journal: Neoplasia

Article Title: Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

doi: 10.1593/neo.03457

Figure Lengend Snippet: Figure 2. Growth of sEphB4-expressing and mock-transfected A375 mela- nomas. Wild-type A375 cells express the receptors EphB1 and EphB2 as well as the EphB2/B3/B4 ligand, ephrinB2. Expression of the receptors EphB3 and EphB4 is not detectable by RT-PCR analysis (A). Constitutively sEphB4- transfected cells abundantly express sEphB4 mRNA (B) (Northern blot) and sEphB4 protein (C) (Western blot analysis of supernatant; SVEC4-10 cells expressing endogenous full-length EphB4 used as control). Subcutaneous injection of A375 cells (106 each) into nude mice leads to rapid tumor growth (D and F). Mock-transfected tumors form reddish tumors indicative of intense vascularization (E). In contrast, sEphB4-expressing A375 melanomas form small subcutaneous nodules (E) with strongly reduced total tumor weight (F) (**P <0.001). The figure shows the mean ± SEM of one of three experiments with similar results analyzing at least 12 mice per data point.

Article Snippet: Recombinant mouse EphB4-Fc, anti– murine EphB4 antibody, and human recombinant vascular endothelial growth factor (VEGF) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Western Blot, Control, Injection

Journal: Neoplasia

Article Title: Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

doi: 10.1593/neo.03457

Figure Lengend Snippet: Figure 4. Analysis of mock-transfected wild-type and sEphB4-expressing A375 melanoma cells in culture and tumors. Expression of sEphB4 has only a minor effect on proliferation of A375 as evidenced by a nonsignificant difference in the tumor cells’ growth rate (A) (n = 3 in duplicates). Likewise, both sEphB4-transfected and mock-transfected cells similarly form colonies in a 14-day soft agar assay (B and C). Yet, colonies of sEphB4-expressing cells remain smaller (B versus C) and the cells have an altered cell cycle distribution pattern with significantly reduced levels of sub-G1 cells (n = 3; P < .05) (D and E). Mock-transfected A375 cells form compact three-dimensional spheroids (F). In contrast, sEphB4-expressing A375 cells do not form compacted three-dimensional spheroids (G). Soluble EphB4- expressing A375 tumors contain significantly less mitotic cells as evidenced by staining with an anti–phospho-H3 antibody (H) and fewer TUNEL+ cells (I) (n = 4; *P < .05).

Article Snippet: Recombinant mouse EphB4-Fc, anti– murine EphB4 antibody, and human recombinant vascular endothelial growth factor (VEGF) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Expressing, Soft Agar Assay, Staining, TUNEL Assay

Journal: Neoplasia

Article Title: Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

doi: 10.1593/neo.03457

Figure Lengend Snippet: Figure 5. Expression of EphB4 and ephrinB2 mRNA in matched pairs of colon carcinomas and adjacent normal tissue. (A) Total RNA isolated from biopsies of tumor (T) and adjacent (A) tissues from 15 patients was isolated and analyzed by Northern blot analysis (upper panel; lower panel: 28S and 18S loading controls). (B) Ratio of tumor versus adjacent normal tissue (T/A) expression levels of EphB4 and ephrinB2 quantitated by comparative densitometric analysis of Northern blot signals (normalized to loading controls). Relative ephrinB2 expression levels do not differ in colon tumors and adjacent normal tissue. In contrast, EphB4 expression is two-fold upregulated in human colon cancers (**P < .001).

Article Snippet: Recombinant mouse EphB4-Fc, anti– murine EphB4 antibody, and human recombinant vascular endothelial growth factor (VEGF) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Isolation, Northern Blot