α4β1 integrin and CD44 mediate soluble proMMP-9 binding to B-CLL cells and adhesion of these cells to immobilized proMMP-9. (A) B-CLL cells, with or without previous incubation with the indicated Abs or peptides, were incubated for 30 minutes with or without proMMP-9 (10 μg/mL) and analyzed by flow cytometry using anti–MMP-9 pAbs. Const MMP-9 indicates constitutive MMP-9. (B) Identical binding experiments were performed with B-CLL cells transfected with the indicated siRNAs. HLA expression was also analyzed as a control for membrane integrity. A representative sample of the 3 studied with identical results is shown. (C) BRO168 cells were incubated with proMMP-9 or VCAM-1 and analyzed by flow cytometry. The binding of VCAM-1 was also measured after cell preincubation with the CS1 peptide. (D) BCECF-AM-labeled B-CLL cells (5 × 10⁵; 3 patients), with or without previous incubation with the indicated Abs or peptides, or transfected with the indicated siRNAs, were added to wells coated with 10 μg/mL proMMP-9 or VCAM-1. After 90 minutes at 37°C, attached cells were quantitated using a fluorescence analyzer. Average values, each with duplicate determinations, represent the percentage of the total number of cells added. (E) The indicated BCECF-AM–labeled B cells were added to proMMP-9– or VCAM-1–coated wells and attached cells measured as explained. All determinations were done in triplicate and values represent the average of 3 (BRO168, HUT112, PBL-BL) or 2 (tonsillar and mantle lymphoma BL) independent experiments. Bar codes are as in panel D. *P ≤ .05; **P ≤ .01.