Role of MMP-9 in B-CLL migration through Matrigel and HUVECs. (A) B-CLL cells (5 × 105) from 3 different patients, with or without previous incubation with the indicated Abs (10 μg/mL), TIMP-1 (1.5 nM), or transfected with control siRNA or 3 different MMP-9 siRNAs (MMP9.1, MMP9.2, and MMP9.3), were added to the upper chamber of Transwell filters coated with Matrigel. CXCL12 (150 ng/mL) was added to the medium in the bottom chamber, except for the control. After 24 hours, invasive cells were counted by flow cytometry. Values represent the average of the 3 samples studied and are expressed as the percentage of total cells added. (B) B-CLL cells from 3 patients, with or without previous incubation with the indicated Abs, or transfected with the same siRNAs shown in panel A, were added to Transwell filters previously coated with inactivated (control) or TNF-α–activated HUVECs. CXCL12 was added to the bottom chamber except for the control. After 24 hours, transmigrated cells in the bottom chamber were counted by flow cytometry. Values represent the average of the 3 samples studied. (C) Transfected B-CLL cells from 2 patients were incubated for 24 hours, and the conditioned medium was concentrated and analyzed by gelatin zymography. Values are the average of the 2 samples studied. (D) B-CLL cells (5 × 105) from 2 different patients were incubated for 1 hour with the indicated inhibitor or with medium (None) and added to Transwell filters coated with TNF-α–activated HUVECs. Control cells were added to inactivated HUVECs. After 24 hours, cells in the bottom chamber were counted by flow cytometry. Values represent the average of the 2 samples studied. * P < .05; ** P < .01; *** P < .001.
