Figure 4.

Bim is required for the death of tumor-associated ECs and inhibition of tumor growth on VEGF neutralization. (A) WT or Bim^−/− mice bearing subcutaneous 3LL-LLCs were treated with anti-VEGF antibody or a control Ig isotype–matched antibody when the tumors reached a volume of ∼50 mm³. CD31 immunohistochemistry revealed tumor vascularization at day 6 of treatment. (B) FACS analysis was used to enumerate viable tumor-associated ECs. Data represent mean ± SEM (n = 5–7 for each treatment and genotype). (C) Bar graph depicts the percentages of tumor-associated ECs with high levels of β-galactosidase (β-gal) activity. Mice were treated with two daily injections of control IgG or anti-VEGF antibody and analyzed 1 d after cessation of treatment. Data represent the mean ± SEM (n = 3–5 for both control Ig and anti-VEGF treatments). (D) Day 6 tumors from saline- or anti-VEGF antibody–treated WT or Bim^−/− mice were subjected to TUNEL and CD31 staining. Inset shows magnification of a boxed region showing an apoptotic EC (yellow). Boxes highlight regions containing apoptotic ECs. Bars: (A) 200 µm; (D) 40 µm. (E) WT, Bim^−/−, Bid^−/−, and Puma^−/− mice bearing 3LL-LLC were treated with anti-VEGF neutralizing antibody as described in Mice, expression constructs, and tumor models. FACS analysis was used to enumerate viable tumor-associated ECs. Data represent mean ± SEM (n = 3–5 for each treatment and genotype of mice). The data for WT and Bim^−/− mice are the same as those shown in B and are reproduced in this panel to facilitate comparison. n.s., not significant. (F) The growth of 3LL-LLC in WT or Bim^−/− mice treated with VEGF neutralizing antibody or a control Ig antibody. Mice were sacrificed when tumors exceeded a diameter of 1 cm on any orthogonal parameter. Data represent mean tumor volume and SEs (n = 5–17 per time point and genotype of mice; *, P = 0.047 control Ig–treated Bim^−/− mice vs. control Ig–treated WT mice; **, P = 0.0002 anti-VEGF antibody–treated WT mice vs. control Ig–treated WT mice; ***, P = 0.0001 anti-VEGF–treated Bim^−/− mice vs. anti-VEGF antibody–treated WT mice). All statistical comparisons were made at day 8 of treatment. Arrows designate the days of treatment. (G) WT CD45.1⁺ (closed bars) or Bim^−/− CD45.2⁺ (open bars) recipient mice were reconstituted with unfractionated bone marrow from Bim^−/− CD45.2⁺ or WT CD45.1⁺ donors, respectively. The hematopoietic compartment was reconstituted for 8 wk before inoculation of 3LL-LLC cells, and mice were subjected to treatment as described in Mice, expression constructs, and tumor models. FACS analysis was used to enumerate viable tumor-associated ECs. Data represent mean ± SEM (n = 4–13 for each treatment and genotype of mice).