![Figure 3. Ibrutinib inhibits migration of MCL cells beneath stromal cells (pseudo-emperipoiesis) and the formation of CXCL12-stimulated cortical actin. (A) Phase contrast (left panel) and 4,6 diamidino-2-phenylindole (DAPI) staining (right panel) of Mino and BM stromal cell M2-10B4 coculture 24 hours after Mino cells were pretreated with vehicle (dimethylsulfoxide [DMSO]) (panels a and b) or ibrutinib (1000 nM) (panels c and d). Highlighted yellow outlines show typical cobblestone appearance of migrated Mino cells beneath stromal cells. Black arrow points to a cell that is adhered on top of stromal cells but not migrated underneath. Yellow arrow points between two migrated Mino cells. (B) Mino cell and stromal cell coculture of Mino cells loaded with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (red), and M2 cells loaded with 5-chloromethylfluorescein diacetate (green). (A) Stromal cells alone and (B) in coculture with Mino cells. (C) Mino cells pretreated with G protein coupled receptor inhibitor pertussis toxin at 200 ng/mL or (D) ibrutinib at 1000 nM. (C) Mino cells were stimulated with CXCL12 at 100 ng/mL and stained with rhodamine-phalloidin to determine actin polymerization and were counterstained with DAPI to identify nuclei of cells. Phalloidin staining of Mino cells (A) before and (B) after CXCL12 stimulation and after treatment with (C) pertussis toxin at 200 ng/mL or (D) ibrutinib at 100 nM. Magnification, ×200. (D) Mino cells were pretreated with ibrutinib, pertussis toxin, or vehicle for 30 minutes and then placed on a stromal cell–populated plate. After 4 hours, coculture was washed several times and migrated, and adhered Mino cells were counted in a flow cytometer with calibrated beads after staining with hCD19. Both pertussis toxin and ibrutinib dose-dependently inhibited migration and adhesion of Mino cells (left panel). Mino cells stimulated with CXCL12 and treated with vehicle or drug were stained with phalloidin, and its intensity was determined by using flow cytometry (right panel). (E) Ibrutinib (100 nM) inhibited pseudo-emperipoiesis of primary MCL (hCD19+ cells) in coculture with M2-10B4 stromal cells (left panel). Actin polymerization as assessed by phalloidin staining was significantly reduced by ibrutinib treatment in primary MCL cells. **P < .01; ***P < .001. One-way analysis of variance (ANOVA) compared with vehicle control.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/122/14/10.1182_blood-2013-02-482125/4/2412f3.jpeg?Expires=1758089224&Signature=R6iPtN1ep9xkcw1kasjnRGc3cHBtzBR11nI-wSMNZ40vj7rKdi4vCRcimHwkoLw~6FQmPVWtpaMQaOiiy5GNAUKL58t0AIYuehiZFXmXFgVWiyTZf7UxHLrRahUva6JiHrAWHzcNoe7AozVcODSK~v-4KO8CKj1J8kXqAL7h2eSIndHG14NIcLDmL4PLeZOkmtpp5~g45pIaI~IqsFKKL5uLJWy0kYOxM2KxJcTHMRr6TL7ME1ZxyIfAVVisxii9Jnlx1yjVelpJ~SFRQJwpOXaIIC7lTvh91-4FBTBXfFzEXXoKfm9LxXa2ktdiug2xX7pWeRvuuWqoUpuGSnqxXg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ibrutinib inhibits migration of MCL cells beneath stromal cells (pseudo-emperipoiesis) and the formation of CXCL12-stimulated cortical actin. (A) Phase contrast (left panel) and 4,6 diamidino-2-phenylindole (DAPI) staining (right panel) of Mino and BM stromal cell M2-10B4 coculture 24 hours after Mino cells were pretreated with vehicle (dimethylsulfoxide [DMSO]) (panels a and b) or ibrutinib (1000 nM) (panels c and d). Highlighted yellow outlines show typical cobblestone appearance of migrated Mino cells beneath stromal cells. Black arrow points to a cell that is adhered on top of stromal cells but not migrated underneath. Yellow arrow points between two migrated Mino cells. (B) Mino cell and stromal cell coculture of Mino cells loaded with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (red), and M2 cells loaded with 5-chloromethylfluorescein diacetate (green). (A) Stromal cells alone and (B) in coculture with Mino cells. (C) Mino cells pretreated with G protein coupled receptor inhibitor pertussis toxin at 200 ng/mL or (D) ibrutinib at 1000 nM. (C) Mino cells were stimulated with CXCL12 at 100 ng/mL and stained with rhodamine-phalloidin to determine actin polymerization and were counterstained with DAPI to identify nuclei of cells. Phalloidin staining of Mino cells (A) before and (B) after CXCL12 stimulation and after treatment with (C) pertussis toxin at 200 ng/mL or (D) ibrutinib at 100 nM. Magnification, ×200. (D) Mino cells were pretreated with ibrutinib, pertussis toxin, or vehicle for 30 minutes and then placed on a stromal cell–populated plate. After 4 hours, coculture was washed several times and migrated, and adhered Mino cells were counted in a flow cytometer with calibrated beads after staining with hCD19. Both pertussis toxin and ibrutinib dose-dependently inhibited migration and adhesion of Mino cells (left panel). Mino cells stimulated with CXCL12 and treated with vehicle or drug were stained with phalloidin, and its intensity was determined by using flow cytometry (right panel). (E) Ibrutinib (100 nM) inhibited pseudo-emperipoiesis of primary MCL (hCD19+ cells) in coculture with M2-10B4 stromal cells (left panel). Actin polymerization as assessed by phalloidin staining was significantly reduced by ibrutinib treatment in primary MCL cells. **P < .01; ***P < .001. One-way analysis of variance (ANOVA) compared with vehicle control.
