Reduction of spontaneous migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis) by PIK-90 and PI-103. CLL B cells were preincubated with PIK-90 or PI-103 and seeded onto confluent marrow stromal cell layers. After overnight incubation, the CLL cells that had not migrated into the stromal cell layer were vigorously washed off. Then, CLL B cells that migrated beneath the stromal cells were microphotographed (A) and quantified by flow cytometry (B). In phase-contrast, pseudoemperipolesis is characterized by the dark appearance of CLL cells that have migrated into the same focal plane as the stromal cells. Cells were imaged in medium 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 Japan); when necessary, Adobe Photoshop 9.0 (Adobe Systems, San Jose, CA) was used for image processing. (A) Representative phase-contrast photomicrographs of pseudoemperipolesis of untreated CLL cells, labeled “control,” and, in comparison, reduced pseudoemperipolesis of the same CLL sample pretreated with 10 μM PIK-90 (bottom row). White bars in the left panels represent 100 μM (100× magnification); bars in the right panels, 50 μM (400× magnification); white filled arrow, a nonmigrated CLL cell; gray filled arrow, a migrated CLL cell; black filled arrow, a marrow stromal cell (framed). In panel B, each bar represents the mean (± SEM) relative number of migrated CLL cells from 4 different patients after pretreatment with the agents displayed on the horizontal axis, compared with the untreated controls. *Significant inhibition with P values less than .05.