Journal: Journal of the National Cancer Institute

Article Title: Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis.

doi: 10.1093/jnci/djs009

Figure Lengend Snippet: Figure 2. Effect of Ang2 expression on lung metastasis. A) Representative biolumines cent images of inguinal lymph nodes 3 weeks after subcutaneous implantation of LNM35 tumors. B) Representative ex vivo bioluminescent images of lungs at 7 weeks after implantation of luciferase-positive LNM35 cells into the abdominal subcutis (4 weeks after the excision of the primary tumor). C) Representative images of hema toxylin- and eosin-stained lung sections from AdAng2- and AdLacZ-treated mice killed 3 weeks after systemic LNM35 inocu lation. D) analysis of lymph node volumes, represented in (A), AdAng2 (n = 10) vs AdLacZ (n = 9) mice. E) Analysis of LNM35 metastatic foci per grid in histological sec tions from the experiment in (C), n = 4 in both groups. F) Quantification of lung weights 7 weeks after s.c. tumor cell implan tation of LNM35 cells and 3 weeks after i.v. of the tumor cells, AdAng2 (n = 3) vs AdLacZ (n = 6) mice and n = 4 in both groups, respec tively. G) Representative lungs with B16-F10 metastases in AdAng2- vs AdLacZ-treated mice at 2 weeks after intravenous adminis tration of tumor cells. H) Bioluminescent images of lungs with B16-F10 metastatic foci. I and J) Analysis of B16-F10 lung metastasis foci per grid (AdAng2 vs AdLacZ) and lung weights, n = 4 in both groups. K) Quantification of B16-F10 melanoma tumor burden in conditionally transgenic Ang2 mice (VEC-tTA/Tet-OS-Ang2 mice) and con trols, VEC-tTA/Tet-OS-Ang2 mice [n = 5], and control [n = 4] mice. All statistical tests were two-sided Student’s t tests. Asterisks indicate statistically significant differences. Error bars = 95% confidence intervals. Scale bar in (A) and (G), 2 mm; in (B), 5 mm; in (C), 1 mm (left panel); 100 µm (right panel). Ang2 = Angiopoietin-2; LacZ = b-galactosidase; i.v. = intravenous; s.c. = subcutaneous; p/s/cm2/sr = photons/s/cm2/steradian; VEC- tTA/Tet-OS-Ang2 = VE-cadherin-tTA/Tet-OS- Ang2 transgenic mouse.

Article Snippet: Sixty percent confluent overnight cultures of BECs expressing the previously characterized Tie2-GFP retrovirus vector (24) were transfected with siRNA targeted against human Ang2 (Santa Cruz, CA; sc-39305) or with control siRNA (Santa Cruz; sc-37007) using Oligofectamine (Invitrogen) and analyzed 48 hours later.

Techniques: Expressing, Ex Vivo, Luciferase, Staining, Transgenic Assay, Control

Journal: Journal of the National Cancer Institute

Article Title: Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis.

doi: 10.1093/jnci/djs009

Figure Lengend Snippet: Figure 3. Ang2-blocking antibodies inhibit primary tumor growth, an giogenesis, and lymphangiogenesis. A) Growth curves of LNM35 primary tumors in nu/nu mice treated with the Ang2-blocking antibodies or hIgG control, n = 8 in both groups. One-way analysis of variance. B) Tumor weights at excision 16 days after implantation, P = .002. Student’s t test. C) Representative immunohistochemical images of LYVE-1- and CD31- stained tumor sections. D) Quantification of densities and area fractions of Lyve-1-positive lymphatic vessels and of CD31-positive blood vessels from at least five histological sections, P = .013 and .019, respectively. Student’s t test. All statistical tests were two-sided. *P < .05. Error bars = 95% CI. Anti-Ang2 = angiopoietin-2-blocking antibody; CD31 = cluster of differenti ation 31; hIgG = human immunoglobulin G; LYVE-1 = lymphatic vessel endothelial hyaluronan receptor-1. Scale bar, 120 µm.

Article Snippet: Sixty percent confluent overnight cultures of BECs expressing the previously characterized Tie2-GFP retrovirus vector (24) were transfected with siRNA targeted against human Ang2 (Santa Cruz, CA; sc-39305) or with control siRNA (Santa Cruz; sc-37007) using Oligofectamine (Invitrogen) and analyzed 48 hours later.

Techniques: Blocking Assay, Control, Immunohistochemical staining, Staining

Journal: Journal of the National Cancer Institute

Article Title: Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis.

doi: 10.1093/jnci/djs009

Figure Lengend Snippet: Figure 6. Effect of Ang2-blocking antibodies on endothelial cell–cell junctions in lung metastases and Ang2 overexpression on vascular integ rity. Transmission electron micrographs (TEM) of capillaries adjacent to metastatic B16-F10 melanoma cell (MC) colonies of Ang2-overexpressing (A and B) and control C57Bl/6J mice (C and D). TEM micrographs of Ang2-blocking antibody (E and F) and HSA-treated (G and H) metastases of NSG mice, samples from at least three mice were evaluated. (B), (D), (F), and (H) show the boxed areas at a higher magnification. Note that in the Ang2-overexpressing mice, the capillaries show more structural abnormalities in endothelial cell (EC), basement membrane (BM) adhe sions and less extensive endothelial cell–cell junctions (arrows in B and D) than in the control mice. Arrowheads in (B) and (D) indicate the interface between the ECs and the BM. Note also that the Ang2-blocking antibody treatment has a normalizing effect; the endothelium in the HSA-treated lung metastasis has less prominent (stars) junctional complexes (arrows) and uneven EC layering. Scale bar in (A), (C), (E), and (G), 1 µm; in (B), (D), (F), and (H), 500 nm. Anti-Ang2 = angiopoietin- 2-blocking antibody; HSA = human serum albumin; NSG = NOD SCID gamma; RBC = red blood cell; VEC-tTA/Tet-OS-Ang2 = VE-cadherin-tTA/ Tet-OS-Ang2 transgenic mouse.

Article Snippet: Sixty percent confluent overnight cultures of BECs expressing the previously characterized Tie2-GFP retrovirus vector (24) were transfected with siRNA targeted against human Ang2 (Santa Cruz, CA; sc-39305) or with control siRNA (Santa Cruz; sc-37007) using Oligofectamine (Invitrogen) and analyzed 48 hours later.

Techniques: Blocking Assay, Over Expression, Transmission Assay, Control, Membrane, Transgenic Assay