Figure 4

Crosstalk of EL4, EL4-v10 with bone marrow- and spleen-stroma. (A,B) CFSE-labeled EL4 and EL4-v10 (1×105) were seeded in 24-well plates on a confluent BM-Str monolayer, cultures containing 10 μg/ml IM7 or K926. After 4 h (37°C) and washing, cells were lysed to determine fluorescence. Adhesion of EL4 and EL4-v10 to BM-Str and spleen-Str and inhibition by antibodies (mean±SD, triplicates); (A) significant differences (p <0.05) in adhesion of EL4 vs. EL4-v10: s; and (B) by antibody: *. (C) CFSE-labeled EL4 and EL4-v10 (1×105) were seeded in 24-well plates on a 90% confluent BM-Str monolayer, cultures containing 10 μg/ml IM7 or K926. Migration of EL4 and EL4-v10 on BM-Str (videomicroscopy, pictures every 20 min for 12 h). Mean±SD of migration (20 EL4 or EL4-v10/well) and representative examples; significant differences (p <0.05) between EL4 vs. EL4-v10: s, and in the presence of antibody: *; (D) EL4 and EL4-v10 (2×105) were seeded in triplicates in 24-well plates coated with BSA or a BM-Str monolayer. After starving (48 h) RPMI1640/10% FCS was added (2 h), medium contained 10 μg/ml IM7 or K926. After washing and fixation (ethanol) EL4/EL4-v10 were stained with PI; Mean values (triplicates) of cell cycle progression and representative examples; significant differences (p <0.05) in dependence on BM-Str: s, and in the presence of antibody: *; (E) CFSE-labeled EL4/EL4-v10 (2×105) were seeded in 24-well plates coated with BSA or BM-Str or spleen-Str. Proliferation of EL4 and EL4-v10 was evaluated by flow cytometry (CFSE dilution) after 24 h and 72 h; mean±SD (triplicates) of EL4/EL4-v10 having progressed through 2 divisions; significant differences (p <0.05) in the presence of stroma: s. Experiments A-E were repeated 3-times. EL4 adhere slightly better than EL4-v10 to BM- and spleen-Str, adhesion is mostly inhibited by IM7. Contact with BM-Str affects EL4 cell cycle progression, which is neutralized by IM7.