Figure 2. Clodronate treatment inhibits cytokine production by endothelial cells and macrophages but has no significant effect on tumor cytokine secretion or cell proliferation. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay shows the response of endothelial cells (RF24) to increasing doses of clodronate at 180 h. Cells showed an IC₅₀ of 3.725 mM (95% CI: 3.540–3.909 mM) determined by interpolation of data. (B) MTT cell viability assay shows the response of response of tumor cells (SKOV3) to increasing doses of clodronate at 72 h. Cells showed an IC₅₀ of 3.941 mM (95% CI: 3.488–4.393 mM) as determined by interpolation of data. (C) Quantification of EGF, FGF2, TGFα, IFNγ, IL6, IL8, IL10, TNFα, and VEGF secretion (pg/mL) by in vitro endothelial (RF24) tumor (SKOV3) cells and activated macrophages (THP-1) treated with media (control) or filtered clodronate salt (2 mM) shows significant decrease in cytokine secretion of endothelial cells in the clodronate treatment group relative to the control group, but shows mixed results in cytokine secretion of activated macrophages and no significant change in cytokine secretion of tumor cells between the clodronate treatment and control groups. Supernatant was collected 48 h after treatment administration for endothelial and tumor cells, and 12 h after treatment administration for activated macrophages. n = 3 samples collected per group, measured in duplicates by ELISA.*P < 0.05, ****P < 0.0001 and NS > 0.05, not significant, as determined by the Student t test. Data are shown as means ± SEM.