CXCR4 and VLA-4 regulate adhesion to fibronectin, stromal cells, and endothelial cells in WM and sensitize WM cells to therapeutic agents. (A) Flow cytometry for VLA-4 expression showing that BCWM.1 and CD19⁺ primary WM cells (N = 10) have high surface expression of VLA-4. (B) Adhesion assay with fibronectin (VLA-4 ligand). BCWM.1 showed significant increase in adhesion to fibronectin compared with BSA used as control. SDF-1 30 nM induced further increase in adhesion. Pretreatment of BCWM.1 for 2 hours with AMD3100 20 μM, PTX 200 ng/mL, and anti–VLA-4 antibody 10 ng/mL significantly inhibit adhesion, even in the presence of SDF-1 30 nM. (C) Adhesion of primary CD19⁺ cells (N = 3) alone or with SDF-1, showing increased adhesion in response to SDF-1 30 nM. Pretreatment of the cells with AMD3100 20 μM, anti–VLA-4 antibody 10 ng/mL, or the combination showed inhibition of adhesion, even in the presence of SDF-1. (D) CXCR4 knockdown cell line showed lower adhesion compared with mock-infected BCWM.1, even in the presence of 30 nM SDF-1. Data show an average of 3 independent experiments (*P = .04, **P = .01). (E) Inhibition of adhesion to stromal cells. Coculture of stromal cells with BCWM.1 resulted in increased adhesion (*P = .008). Pretreatment of BCWM.1 with AMD3100, anti–VLA-4 antibody, PTX, or the combination of AMD3100/anti–VLA-4 antibody, or the combination of PTX/anti–VLA-4 antibody resulted in significant inhibition of adhesion, even in the presence of SDF-1. The combination of AMD3100 and anti–VLA-4 antibody did not induce further decrease in adhesion, indicating that the 2 receptors use the same pathway to regulate adhesion. (F) Coculture of stromal cells with BCWM.1. Bortezomib 2.5 and 5 nM inhibited proliferation in BCWM.1, but less when the cells were cocultured with stromal cells, indicating that stromal cells confer resistance to WM cells. However, when AMD3100 20 μM was added 2 hours before bortezomib, it increased the sensitivity of cells to bortezomib in the coculture experiments at bortezomib 2.5 nM. (G) Adhesion assay to endothelial cells. HUVECs were cultured for 24 hours. BCWM.1 were labeled with calcein AM and cocultured with HUVEC for 4 hours, with or without SDF-1. BCWM.1 showed increased adhesion to endothelial cells, and AMD3100, anti–VLA-4 inhibitor, PTX, and the combination of AMD3100/anti–VLA-4 antibody or anti–VLA-4 antibody/PTX showed decreased adhesion, even in the presence of SDF-1 (*P = .009, **P = .009).