Effects of natalizumab, Plerixafor, pertussis toxin, and CS-1 peptide on the migration of MCL cells beneath stromal cells (pseudoemperipolesis). To demonstrate the effect of blocking VLA-4 integrins on pseudoemperipolesis, a confluent layer of M2-10B4 marrow stromal cells was established (A), and untreated (B) or pretreated (with natalizumab in panel C, with CS-1 peptide in panel D) MCL cells were seeded onto the stromal cell layer. After 6 hours, nonmigrated cells were removed by vigorous washing. Migrated cells are characterized by a dark appearance, whereas nonmigrated cells remain bright. The stromal cell layer containing the migrated MCL cells was photographed (100× magnification). These photomicrographs illustrate that pseudoemperipolesis of MCL cells after treatment with natalizumab (C) and CS-1 peptide inhibitor (D) was greatly reduced compared with untreated controls (B). Cells were imaged using a phase contrast microscope (Model ELWD 0.3; Nikon, Garden City, NY) with a 10×/0.25 NA objective lens. Images were captured with a Nikon D40 digital camera (Nikon, Tokyo, Japan) using Camera Control Pro software (Nikon) and processed with Adobe Photoshop 9.0 software (Adobe Systems, San Jose, CA). The MCL cell lines SP-53 (E) and MINO (F) were left untreated (controls) or pretreated with the agents displayed on the horizontal axis and then incubated on confluent MSC layers. After incubation, the nonmigrated cells were vigorously washed off, and the MCL cells that had migrated into the MSC layer were quantified by FACS. The mean ± SEM relative pseudoemperipolesis is shown for 3 independent experiments for each cell line. *Significant inhibition of pseudoemperipolesis compared with control sample (P < .05).