Journal: iScience

Article Title: A tumor endothelial cell-specific microRNA replacement therapy for hepatocellular carcinoma

doi: 10.1016/j.isci.2024.108797

Figure Lengend Snippet:

Article Snippet: Anti-PCNA antibody , Santa Cruz , #sc-7907.

Techniques: Angiogenesis Assay, Sequencing, Control, Positive Control, Software, Functional Assay, Microarray

Journal: Molecular Oncology

Article Title: REST promotes ETS1‐dependent vascular growth in medulloblastoma

doi: 10.1002/1878-0261.12903

Figure Lengend Snippet: REST promotes vasculature in REST TG cerebella and Ptch +/− /REST TG tumors. H&E staining and IHC for CD31 were performed on (A) cerebellar sections from WT and REST TG mice and (B) tumor sections from Ptch +/− and Ptch +/− /REST TG transgenic mice, to demonstrate the vasculature changes (left panels). Arrowheads show the blood vessels. Quantitation of blood vessels in sections ( n = 3; three fields /section), and average blood vessel diameter is shown in the right panels for A & B. Scale bars in A, B; H&E = 20 μm; CD31 = 10 μm. Data show individual variability and means ± SD. P ‐values were obtained using Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Slides were placed in distilled water, washed with 0.1% Tween‐20 in PBS (PBS‐T), blocked in 1% FBS in PBS for 1 h, and then coincubated with goat anti‐luciferase (1 : 150, Cat# NB100‐1677SS; Novus Biologicals, Centennial, CO, USA) and mouse anti‐CD31 (1 : 250, Cat# Dia‐310; DIANOVA GmbH).

Techniques: Staining, Transgenic Assay, Quantitation Assay

Journal: Molecular Oncology

Article Title: REST promotes ETS1‐dependent vascular growth in medulloblastoma

doi: 10.1002/1878-0261.12903

Figure Lengend Snippet: Human MBs with REST elevation have increased vasculature. (A, B) Relative REST mRNA and protein levels in DAOY/DAOY‐R, UW228/UW228‐R, and UW426/UW426‐R cells were measured by RT‐PCR and western blot analysis, respectively. Right panel in B shows quantification data. Tubulin served as a loading control. Data show individual variability and means ± SD. P ‐values were obtained using Student's t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) Growth of DAOY‐ffluc and DAOY‐R‐ffluc cells implanted in the cerebella of immunodeficient mice ( n = 5) was monitored by BLI. Color scale bar indicates luminescence intensity across the images. (D) Brain sections from the above mice were studied by H&E (top panel) to locate tumors, and IHC for CD31 (bottom panel) to identify tumor vasculature. Arrowheads show the blood vessels. Quantitation of blood vessels and average blood vessel diameter is shown in the right panel ( n = 3; three fields/section). Scale bars: H&E = 20 μm; CD31 = 10 μm. (E) IHC for CD31 in tumor‐bearing brain sections of PDOX mice ( n = 3., three fields/section) to demonstrate vasculature changes in tumors (left panels). Arrowheads show the blood vessels. Scale bars: top (40×) = 10 μm, bottom (10×) = 20 μm. (F) CD31 mRNA expression profile in four subtypes of SHH‐MB patient samples measured by microarray (α; n = 65, β; n = 35, γ; n = 47, δ; n = 76) from GSE85217 data set . Each dot corresponds to one individual patient. Data show individual variability and means ± SD. P ‐values were obtained using the unpaired t ‐test with Welch's correction. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G) Scatter plot of correlation of REST mRNA expression and CD31 mRNA expression. Figure shows the plot across all 223 SHH‐MB patients ( r = 0.33, P < 0.0001). (H) CD31 mRNA expression profile in SHH‐MB patient samples. Hierarchical clustering based on expression of neuronal differentiation markers divided the SHH‐MB patient samples into six distinct clusters (Cluster 1; n = 39, Cluster 2; n = 31, Cluster 3; n = 32, Cluster 4; n = 61, Cluster 5; n = 39, Cluster 6; n = 21) . Each dot corresponds to an individual patient. Data show individual variability and means ± SD. ns, not significant. P ‐values were obtained using the unpaired t ‐test with Welch's correction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Slides were placed in distilled water, washed with 0.1% Tween‐20 in PBS (PBS‐T), blocked in 1% FBS in PBS for 1 h, and then coincubated with goat anti‐luciferase (1 : 150, Cat# NB100‐1677SS; Novus Biologicals, Centennial, CO, USA) and mouse anti‐CD31 (1 : 250, Cat# Dia‐310; DIANOVA GmbH).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitation Assay, Expressing, Microarray

Journal: Molecular Oncology

Article Title: REST promotes ETS1‐dependent vascular growth in medulloblastoma

doi: 10.1002/1878-0261.12903

Figure Lengend Snippet: REST elevation promotes secretion of proangiogenic factors. (A) Conditioned culture medium from UW228, UW426, and DAOY cells was used to probe a commercially available human angiogenesis array for a panel of known angiogenesis‐related secreted proteins (left panel). The boxes and box numbers represent pro‐ or antiangiogenic proteins and correspond to molecules listed in Table . Densitometric quantitation is shown on the right (also see Table ). (B) Volcano plots show angiogenesis‐related genes DE between SHH‐MB samples ( GSE85217 ) with high‐ and low‐REST expression . Samples with HR expression ( n = 117); α; n = 42/65 (64.6%), β; n = 28/35 (80.0%), γ; n = 15/47 (31.9%), δ; n = 32/76 (42.1%). Samples with low‐REST expression ( n = 106); α; n = 23/65 (35.4%), β; n = 7/35 (20.0%), γ; n = 32/47 (68.1%), δ; n = 44/76 (57.9%). The gene expression difference between HR/LR samples is shown on the x ‐axis, the P ‐value (in –log 10 scale) on the y ‐axis. The horizontal line indicates a P ‐value of 0.05. Circles represent genes encoding proteins analyzed in A (also see Tables and ). Black dots identify expression levels for CD31 , VEGFR1, and ETS1 . (C) In vitro angiogenesis assay to show relative tube formation by HUVECs cultivated in endothelial culture (EC) alone or with conditioned media from DAOY/DAOY‐REST(R), UW228/UW228‐REST(R), and UW426/UW426‐REST(R) cell cultures. EC medium and conditioned medium were used in a 1 : 1 ratio. Tubes formed in matrigel were photographed after 16 h (left panel) and quantitated (right panel). Data shown are mean ± SD. P ‐values were obtained using Student's t ‐test. * P < 0.05, ** P < 0.01, **** P < 0.0001 , n = 3. Scale bar; 100 μm.

Article Snippet: Slides were placed in distilled water, washed with 0.1% Tween‐20 in PBS (PBS‐T), blocked in 1% FBS in PBS for 1 h, and then coincubated with goat anti‐luciferase (1 : 150, Cat# NB100‐1677SS; Novus Biologicals, Centennial, CO, USA) and mouse anti‐CD31 (1 : 250, Cat# Dia‐310; DIANOVA GmbH).

Techniques: Quantitation Assay, Expressing, In Vitro, Angiogenesis Assay

Journal: Molecular Oncology

Article Title: REST promotes ETS1‐dependent vascular growth in medulloblastoma

doi: 10.1002/1878-0261.12903

Figure Lengend Snippet: MBs with elevated REST expression upregulate VEGFR1 and colocalize with endothelial cells. (A‐B) IHC was performed on cerebellar sections ( n = 3) from Ptch +/− and Ptch +/− /REST TG mice and PDOX animals ( n = 3) to demonstrate VEGFR1 expression. Arrowheads show blood vessels; Scale bars: top (40×) = 10 μm, bottom (10×) = 20 μm. (C‐D) Western blot analysis to measure VEGFR1 levels in human DAOY/DAOY‐R and UW426/UW426‐R cells, and VEGFR1 and REST protein levels in mouse C17.2/ST2 cells, and in CGNPs from WT/ REST TG mice. Tubulin served as a loading control. (E) HUVECs were cocultured with DAOY or DAOY‐R cells on matrigel, and tube formation was assessed after 16 h. DAOY/DAOY‐R are in red, while HUVECs are shown in green color. Quantitation of the relative numbers of DAOY or DAOY‐R cells colocalized with HUVECs is shown on the right. (F) Immunofluorescence assay to show colocalization in yellow of CD31‐positive endothelial cells (red) and luciferase‐positive tumor cells (green) in tumor sections from DAOY or DAOY‐R xenografts. Quantitative data ( n = 3, three fields/section) is shown on the right. P ‐values were obtained using Student's t ‐test. *** P < 0.001. (G) Profile of VEGFR1 mRNA expression in microarray data of four subtypes of human SHH‐MB samples from GSE85217 data set . Each dot corresponds to one individual patient. Data show individual variability and means ± SD. P ‐values were obtained using the unpaired t ‐test with Welch's correction. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (H) Scatter plot of correlation of REST mRNA expression and VEGFR1 mRNA expression. Figure shows the plot across all 223 SHH‐MB patients ( r = 0.29, P < 0.0001). (I) VEGFR1 mRNA expression profile in SHH‐MB patient samples. Hierarchical clustering based on expression of neuronal differentiation markers divided the SHH‐MB patient samples into six distinct clusters . Each dot represents an individual patient. Data show individual variability and means ± SD. P ‐values were obtained using the unpaired t ‐test with Welch's correction. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Slides were placed in distilled water, washed with 0.1% Tween‐20 in PBS (PBS‐T), blocked in 1% FBS in PBS for 1 h, and then coincubated with goat anti‐luciferase (1 : 150, Cat# NB100‐1677SS; Novus Biologicals, Centennial, CO, USA) and mouse anti‐CD31 (1 : 250, Cat# Dia‐310; DIANOVA GmbH).

Techniques: Expressing, Western Blot, Quantitation Assay, Immunofluorescence, Luciferase, Microarray

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 2. CCL27 and CCL28 protein localization in tumor-associated lymphangiogenesis. A, Apex-293EBNA, VEGFD-293EBNA, and FEMX-I xenograft tumors were stained via IHC with LYVE1, CCL27, or CCL28 antibodies. Top insets show the position of the main image (black box) in a lower magnification overview of the tumor section. Lower insets show a serial field of the main image at the same magnification stained with a corresponding isotype-matched negative control antibody. Scale bar, 100 mm in main; 200 mm in overview. B, Immunofluorescence staining of VEGFD-293EBNA tumors with LYVE1 and CCL27 antibodies. Scale bar, 100 mm. C, CCL28 staining on a lymphatic vessel in a FEMX-I tumor section. Scale bar, 100 mm. D, Relative expression of CCR10 and CCL27 mRNA in samples of human melanoma (n ¼ 45), benign nevi (n ¼ 18), and normal skin (n ¼ 7) from the publicly available microarray expression dataset GSE3189 (20). Data are presented as individual points with mean SEM (, P < 0.05; , P < 0.001; , P < 0.0001 by Mann–Whitney test).

Article Snippet: Cryosections of Matrigel plugs were stained using antibodies for rabbit anti-mouse LYVE1 (Fitzgerald Industries International catalog no. 70R-LR003, RRID: AB_1287923), hamster anti-mouse podoplanin (Fitzgerald catalog no. 10R-P155a, RRID:AB_1288912), or rat anti-mouse PECAM1/CD31 (BD Pharmingen catalog no. 553370, RRID: AB_394816) to assess the extent of lymphangiogenesis and angiogenesis, as described previously (17).

Techniques: Staining, Negative Control, Expressing, Microarray, MANN-WHITNEY

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 3. Proinflammatory cytokines stimulate CCR10 expression in LECs. A, qRT-PCR analysis of CCR10 mRNA levels in LECs after stimulation with 10 ng/mL TNFa. Data represent mean SEM of three experiments. , P < 0.05 by ANOVA with Dunnett posttest compared with unstimulated (Unstim). B and C, Immunofluorescence staining with antibodies to the macrophage marker F4/80 (red) and the LEC marker LYVE1 (green) demonstrates the infiltration of macrophages into VEGFD– expressing tumors (C), with empty vector (Apex)-293EBNA tumors (not expressing VEGFD) showing fewer macrophages and lymphatic vessels (B). Filled arrows, macrophages; open arrows, lymphatics. Scale bars, 100 mm. D and E, Quantification of the F4/80–stained area (D) or LYVE1-stained area (E) in VEGFD- 293EBNA tumors versus Apex-293EBNA tumors. Data represent mean SEM of 5 fields each from 10 tumors. , P < 0.05 by Student t test. F, Quantification showing minimal costaining of F4/80 with LYVE1 in VEGFD-293EBNA tumors. Data represent mean SEM, n ¼ 5 tumor-bearing mice, 2 sections per tumor.

Article Snippet: Cryosections of Matrigel plugs were stained using antibodies for rabbit anti-mouse LYVE1 (Fitzgerald Industries International catalog no. 70R-LR003, RRID: AB_1287923), hamster anti-mouse podoplanin (Fitzgerald catalog no. 10R-P155a, RRID:AB_1288912), or rat anti-mouse PECAM1/CD31 (BD Pharmingen catalog no. 553370, RRID: AB_394816) to assess the extent of lymphangiogenesis and angiogenesis, as described previously (17).

Techniques: Expressing, Quantitative RT-PCR, Staining, Marker, Plasmid Preparation

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 5. CCL28 and CCL27 cooperate with VEGFD to attract LECs in vivo. A–E, FVB mice were injected subcutaneously with 200 mL Matrigel containing PBS (A) or 2 mg/mL of recombinant human CCL27 (B), 4 mg/mL VEGFD (C), 2 mg/mL CCL27 and 4 mg/mL VEGFD (D), or 4 mg/mL VEGFA (E). Matrigel plugs were harvested after one week and sections thereof stained with antibodies to LYVE1 (green) and PECAM1 (red) and nuclei counterstained with DAPI (blue). Scale bars, 100 mm. F and G, Quantification of LYVE1-positive vessels (F) or PECAM1-positive vessels (G) in Matrigel plugs. Data points represent average % stained area of <15 fields per mouse with mean SEM of n ¼ 6 mice per group (5 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test. , P < 0.05; , P < 0.01; , P < 0.001. H and I, A similar experiment was conducted in which 2.5 mg CCL28 and VEGFD, alone or in combination as shown, were injected subcutaneously into FVB mice in 500 mL of Matrigel. LYVE1 staining was quantified in H and PECAM1 staining in I. Data points represent mean average % stained area of <15 fields per mouse with mean SEM of n ¼ 4 mice (3 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test; , P < 0.05; , P < 0.01. J, CCL28 Matrigel plug costained with antibodies to LYVE1 (green) and podoplanin (red). Right, magnified view of the boxed area. Scale bars, 50 mm. K, CCL28 Matrigel plug costained with LYVE1 (green) and the macrophage marker F4/80 (red) showing infiltration of macrophages into the plug. Right, a magnified view of the boxed area. Open arrows, LYVE1þ LECs; closed arrows, F4/80þ macrophages. Scale bars, 50 mm.

Article Snippet: Cryosections of Matrigel plugs were stained using antibodies for rabbit anti-mouse LYVE1 (Fitzgerald Industries International catalog no. 70R-LR003, RRID: AB_1287923), hamster anti-mouse podoplanin (Fitzgerald catalog no. 10R-P155a, RRID:AB_1288912), or rat anti-mouse PECAM1/CD31 (BD Pharmingen catalog no. 553370, RRID: AB_394816) to assess the extent of lymphangiogenesis and angiogenesis, as described previously (17).

Techniques: In Vivo, Injection, Recombinant, Staining, Marker

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 6. CCL27 promotes tumor-associated lymphangiogenesis. A, Western blot (WB) for immunoprecipitated (IP) CCL27 from conditioned medium of tumor cells. Positions of molecular weight markers (kDa) are shown on the left. B–D, Mouse xenograft tumors produced from subcutaneous flank injection of pVITRO3- 293EBNA (control; B), CCL27-293EBNA (C), or VEGFD-293EBNA (D) cells. E–G, LYVE1 (green) and PECAM1 (red) immunofluorescence of pVITRO3-293EBNA (E), CCL27-293EBNA (F), or VEGFD-293EBNA (G) tumors. Right, magnified view of the boxed area. Scale bars, 50 mm. H and I, Quantification of LYVE1-positive vessels (H) or PECAM1-positive vessels (I) in xenograft tumors. Data points represent the average % stained area of <15 fields with mean SEM of n ¼ 5 (pVITRO3), n ¼ 10 (CCL27), or n ¼ 4 (VEGFD) tumors. , P < 0.05; , P < 0.001 by Kruskal–Wallis test with uncorrected Dunn test. J–L, Collecting lymphatic vessels filled with Patent Blue V from mice with pVITRO3-293EBNA (J), CCL27-293EBNA (K), or VEGFD-293EBNA (L) tumors. Arrowheads demarcate the vessel. M, Collecting lymphatic vessel diameter in mice with subcutaneous tumors. Data represent mean SEM of 5 mice. , P < 0.01 by Student t test. N, Volumes of CCL27-293EBNA, VEGFD-293EBNA, or pVITRO3-293EBNA tumors plotted over time. Data represents mean tumor volume SEM of two independent experiments, each with 5–10 mice per group.

Article Snippet: Cryosections of Matrigel plugs were stained using antibodies for rabbit anti-mouse LYVE1 (Fitzgerald Industries International catalog no. 70R-LR003, RRID: AB_1287923), hamster anti-mouse podoplanin (Fitzgerald catalog no. 10R-P155a, RRID:AB_1288912), or rat anti-mouse PECAM1/CD31 (BD Pharmingen catalog no. 553370, RRID: AB_394816) to assess the extent of lymphangiogenesis and angiogenesis, as described previously (17).

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Produced, Injection, Control, Staining