Inhibition of CXCL12-induced responses in CLL cells by T140 and AMD3100. (A) Actin polymerization of CLL cells in response to CXCL12 preincubated with different concentrations of T140. Intracellular F-actin in cells was measured using FITC-labeled phalloidin 15, 60, 120, and 300 seconds after the addition of 200 mg/mL CXCL12. Results are shown as percentages of intracellular F-actin relative to the values before the addition of CXCL12 and are mean ± SEM of 9 experiments. (B) Inhibition of actin polymerization of CLL cells in response to CXCL12 by the CXCR4 antagonists T140 and AMD3100. Cells were preincubated with 4 μM or 40 μM AMD3100 before stimulation with CXCL12. Inhibition of the response by 10 μg/mL T140 (4 μM) is shown for comparison. Results are displayed as percentages of intracellular F-actin relative to the values before the addition of CXCL12 and are the mean ± SEM of 4 CLL samples from different patients. (C) CXCL12-induced chemotaxis is inhibited by T140. Results indicate relative migration compared with control samples migrating to 200 ng/mL CXCL12 (100%) and samples preincubated with different concentrations of T140, representing the mean ± SEM values of 12 experiments with CLL cells from different patients (^*P < .05). (D) In vitro migration of CLL cells beneath stromal cells (pseudoemperipolesis) is inhibited to T140. Cells were seeded onto M2-10B4 cells and allowed to migrate to the stromal cell layer. After vigorous washings, the remaining CLL cells were quantified using flow cytometry. Results are represented relative to untreated controls (100%). T140 was used at different concentrations, as indicated in the figure. Data shown are mean ± SEM values of 10 experiments with CLL cells from different patients (^*P < .05).