Figure 1. GDF-15 protects transformed cells against macrophages and promotes tumor development in vivo.

(A) p65^–/–Ras MEFs were cocultured with peritoneal macrophages (MΦ) with normal or conditioned media from p65^+/+Ras MEFs. Graph represents cell survival scored by trypan blue exclusion, normalized to untreated p65^–/–Ras MEFs. n = 6. Data are shown as mean ± SEM. *P ≤ 0.05, 1-way ANOVA. (B) Gdf-15 analyzed by qRT-PCR in Ras MEFs normalized to Gapdh ± SEM. *P ≤ 0.05, Student’s t test. n = 3. (C) GDF-15 ELISA from Ras MEF–conditioned media. n = 3. Data are shown as mean ± SEM. *P ≤ 0.05, Student’s t test. (D) Ras MEFs were cocultured with macrophages and GDF-15–neutralizing antibody (GDF-15 Ab) at concentrations of 0, 25, 625, and 2,500 ng/ml. Graph represents cell survival similar to that shown in A. Data are shown as mean ± SEM from 2 independent experiments, each performed in triplicate. *P ≤ 0.05, 1-way ANOVA. (E) p65^–/–Ras MEFs were cocultured with macrophages and recombinant GDF-15 (rGDF-15) at concentrations of 0, 5, and 10 ng/ml. Graph represents cell survival similar to that shown in A. Data represent mean ± SEM derived from 2 independent experiments, each performed in triplicate. *P ≤ 0.05, 1-way ANOVA. (F) p65^+/+Ras MEFs (1 × 10⁶) were injected subcutaneously into SCID mice. Cohorts of mice (n = 5) were intravenously injected with GDF-15 antibody (20 μg/mouse) or IgG control (20 μg/mouse) and tumor sizes measured. Arrowheads indicate time points for injections. Data are shown as mean ± SEM. *P ≤ 0.05, SPSS repeated measures, general linear model. (G) Single clones from Ras MEFs expressing Gdf-15 shRNA or scrambled control (sh control) were subcutaneously injected into SCID mice and tumor sizes measured. Data represent mean tumor diameter from 2 single clones (Scr-1 and Scr-2 for scrambled controls and C2 and D2 for Gdf-15–knockdown) injected into 5 mice each. Data are shown as mean ± SEM. *P ≤ 0.05, SPSS repeated measures, general linear model.