Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 1 Immunization with plasmid-encoded DLL4 results in an immunological response in BALB/c mice. (a) Expression of human DLL4 was verified by transient transfection of HeLa cells with empty vector or the vector encoding DLL4 (pDLL4) and analyzed by western blot. Recombinant human DLL4 was used as a positive control (rhDLL4). (b) Female BALB/c mice were immunized either intramuscularly (IM) or intradermally (ID) according to the depicted protocols. All intradermal injections were given in combination with electroporation (EP). One week after the final immunization animals were challenged by orthotopic injection of D2F2/E2 or TUBO cells in the mammary fat pad. (c) To investigate if the vaccination gave rise to an immunological response, serum was taken 10 days after the final immunization (protocol 1) and analyzed by human DLL4 enzyme-linked immunosorbent assay (ELISA). The figure depicts a representative analysis for one out of four mice analyzed.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Plasmid Preparation, Expressing, Transfection, Western Blot, Recombinant, Positive Control, Electroporation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 2 DNA vaccination against DLL4 results in suppression of tumor growth in BALB/c mice. (a) Expression of DLL4 in D2F2/ E2 tumor endothelial cells was assessed by immunofluorescent staining for the endothelial markers, CD31 (red) and DLL4 (green). Cell nuclei were counterstained by DAPI (blue). Growth of orthotopically implanted D2F2/E2 (b and c) or TUBO (d) mammary carcinomas in BALB/c mice following immunization with empty vector or with DLL4 plasmid DNA according to protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP; n ¼ 8 for all groups). The data shown in (b and d) are representative of three independent experiments. *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Expressing, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 3 Targeting DLL4 by vaccination results in excessive formation of nonfunctional blood vessels. (a) Visual inspection of excised D2F2/E2 tumors revealed overt thrombosis in tumors from DLL4-vaccinated mice. (b) Blood vessel density of D2F2/E2 tumors visualized by immunostaining for the endothelial cell marker CD31 (red; n ¼ 5 for each group). (c) Perfused blood vessels of D2F2/E2 tumors as determined by vascular perfusion using fluorescein-labeled tomato lectin (green; n ¼ 5 for each group). The total area of the vascular bed was visualized by immunostaining for CD31 (red; n ¼ 5 for each group). The fraction perfused area was expressed as perfused area divided by total vascular area for each field. (d) Apoptotic index of D2F2/E2 tumor cells as determined by immunostaining for activated caspase-3 (n ¼ 5 for each group). *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Immunostaining, Marker, Labeling

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 4 T cells are not mediators of the effector functions of the DLL4 vaccine. (a) Mouse DLL4-specific T cell responses following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) were assessed by IFN-g ELISpot by stimulation of splenocytes with mouse DLL4 peptides or recombinant mouse extracellular portion of DLL4 (n ¼ 5 for each group). Recombinant carcinoembryonic antigen protein and carcinoembryonic antigen peptides were used as negative controls. The polyclonal stimulant concanavalin A (conA) was used as a positive control. The experiment was repeated three times. (b) Splenocyte activity in mice following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) at the time of sacrifice after tumor challenge was assessed by IFN-g ELISpot without antigen-specific stimulation (vector, n ¼ 9; protocol 1, n ¼ 8; protocol 2, n ¼ 5). (c) Depletion of CD4 þ or CD8 þ cells was initiated three days before tumor challenge by injection of anti-CD4 or anti-CD8 antibodies and verified by flow cytometric analysis of peripheral blood 5 days after injection of antibodies. Tumor growth curve from vector-immunized mice was duplicated from Figure 2c, which depicts an experiment performed simultaneously to the T cell depletion experiment. (d) Growth of orthotopically implanted D2F2/E2 tumors in control mice and in mice depleted of CD4 þ/CD8 þ cells following vaccination with DLL4 plasmid DNA (n ¼ 6 for all groups). **Po0.01, ***Po0.001; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunospot, Recombinant, Positive Control, Activity Assay, Plasmid Preparation, Injection, Control

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 5 Tumor growth suppression in DLL4-vaccinated mice is mediated by induction of anti-DLL4 antibodies. (a) The presence of anti-mouse DLL4 antibodies in the serum of DLL4-immunized mice was analyzed by ELISA. The figure depicts a representative analysis for one out of four mice analyzed. (b) IsolectinB4 staining of wholemounted P5 mouse retinas from mice treated with immune serum from vector or DLL4 immunized mice according to protocol 1 (3 ID þ EP; n ¼ 4 for each group). Arrowheads indicate endothelial cells morphologically identified as tip cells. (c) Growth of orthotopically implanted D2F2/E2 tumors in mice treated with serum from mice immunized with vector (n ¼ 12) or DLL4 plasmid DNA (n ¼ 16) according to protocol 1 (3 ID þ EP). The experiment was repeated twice with similar results. **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 6 No evidence for liver toxicity or delayed wound healing following immunization with the DLL4 vaccine. (a) Hematoxylin and eosin staining of tissue sections of liver from mice that initiated the immunization procedure with empty vector or the DLL4 vaccine 5 months earlier. (b) Rate of healing expressed as % closure of wounds inflicted to mice immunized with empty vector or the DLL4 vaccine (n ¼ 8 mice per group, two wounds per mouse). *Po0.05, repeated-measures ANOVA.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: DLL4 and PDGF-BB regulate migration of human iPSC-derived skeletal myogenic progenitors

doi: 10.1101/2021.02.28.431778

Figure Lengend Snippet: (A) Principal Component Analysis (PCA) showing mMuSC-derived myoblasts (left), human myoblasts (centre) and hiMPs (right). 4 cell lines were analysed with RNAseq in treated and untreated conditions for each cell population. Each point on the PCA represents a cell population. Additional information in Table S1,2. (B) Volcano plots visualising differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05. (C) Heatmaps showing changes in expression of key myogenic ( MYOD, MYOGENIN ), perivascular ( PDGFRB, NG2, CD146, ALPL ) and NOTCH target ( HEY1, HES1 ) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled. (D) Validation of RNAseq data of panel (C) by real-time PCR analysis of the same myogenic, perivascular and NOTCH target transcripts in treated and untreated hiMPs (N=3; error bars; S.E.M.). Statistical analysis (paired t test) performed on ΔCt values whilst graphs were produced as fold change relative to untreated controls. (E) Curated dot plot Gene Ontology (GO; left), Kyoto Encyclopaedia of Genes and Genomes (KEGG; centre) and Reactome (right) enrichment analyses showing shared gene functions amongst the cell groups; numbers in brackets: genes analysed with a p value threshold set at 0.05; full lists in a dedicated spreadsheet available in Supplemental Information.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506-D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma-Aldrich; A9418-10G) as a carrier protein.

Techniques: Derivative Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Produced

Journal: bioRxiv

Article Title: DLL4 and PDGF-BB regulate migration of human iPSC-derived skeletal myogenic progenitors

doi: 10.1101/2021.02.28.431778

Figure Lengend Snippet: (A) Top cellular and molecular functions associated with DLL4 & PDGFBB modulation generated via ingenuity pathway analysis (IPA). Genes upregulated in the DLL4 & PDGFBB-treated hiMPs relative to the untreated control were subjected to IPA to reveal the predicted most significant associated functions. (B) Fluorescence microscopy images depicting Hoechst-positive nuclei of each cell at sequential time points. Coloured tails represent the locations of the nuclei at previous time points. (C) Unsupervised hierarchical clustering (Ward’s method) visualised with a t-SNE plot showing two distinct clusters (Silhouette Si = 0.19) (n = 408) (perplexity = 35). Cells pooled from 3 independent experimental replicates for each condition were used (untreated and DLL4 & PDGF-BB-treated). (D) Bar chart demonstrating normalised values for comparison of motility phenotypes between cells within the two clusters (mean ±SEM). Statistical significance based on Bonferroni-corrected t -test: all parameters except “hurst_RS” and “autocorr” are statistically significant between the two groups p<0.01 (data points: single cells pooled together from 3 independent experiments). (E) Bar graph displaying the proportions of control and DLL4 & PDGF-BB-treated cells within each cluster. (F) Functional protein association network analysis ( https://string-db.org ). The network view summarises predicted associations for proteins positively regulating cell migration common to all three datasets. The nodes are proteins and the edges represent the predicted functional associations. Red line: fusion evidence; Green line: neighbourhood evidence; Blue line: co-occurrence evidence; Purple line: experimental evidence; Yellow line: text mining evidence; Light blue line: database evidence; Black line: co-expression evidence. Blue nodes: GO:0030335 positive regulation of cell migration, Count in gene set: 8 of 452, false discovery rate: 0.0156. (G) P value-adjusted hierarchical clustering heatmap displaying hierarchical clustering of genes associated with leukocyte trans-endothelial migration (KEGG pathway: hsa04670; P set at 0.05). (H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm 2 , that have migrated through the endothelial layer in each considered condition. (N = 3). A minimum of 10 (1.5 mm 2 ) fields per condition was quantified (mean ±SEM). Scale bar: 250 μm. (I) Bar graph showing fold-change in trans-endothelial migration (mean ±SEM). Statistical significance based on one-way ANOVA with Bonferroni’s multiple comparison.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506-D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma-Aldrich; A9418-10G) as a carrier protein.

Techniques: Generated, Control, Fluorescence, Microscopy, Comparison, Functional Assay, Migration, Expressing, Membrane, Staining