Relationship between preBCR signaling and CXCR4- and integrin-mediated preB cell motility. (A) FAK geometric mean fluorescence intensity (GMFI) in preB cells from C57BL/6 mice treated with BCR signaling inhibitors (blue, fostamatinib and ibrutinib) or vehicle (red), or with PI3K inhibitor wortmannin (green) or vehicle (red) for 6 h in vitro. (B) α4 integrin expression in preB cells of C57BL/6 mice treated as in A. Numbers indicate geometric mean fluorescence intensity ± SD (n = 3 mice per condition). (C and D) FAK geometric mean fluorescence intensity (C) and α4 integrin expression (D) in preB cells treated with stimulatory CD79b antibody for 6 h in vitro. Con., control. (A and C) Bars indicate mean; circles depict individual mice/assays. (D) Numbers inside histogram indicate geometric mean fluorescence intensity ± SD (n = 3 individual mice/assays). (E) Transwell migration toward CXCL12 of preB cells treated with stimulatory CD79b antibody for 6 h in vitro. Y axis indicates frequency of migrated cells; x axis indicates CXCL12 concentration (nanograms per milliliter). (F) PreB cell migration across VCAM-1–coated transwells (5 µm) toward CXCL12 gradient (300 ng/ml). Y axis indicates percent reduction in migrated cells; x axis indicates VCAM-1 concentration (micrograms per milliliter) used for coating transwells. (E and F) Circles represent average of three independent experiments. Error bars indicate SD. (G) Distribution of EV-transduced GFP+ preB-ALL cells (green, top left) and of FAK-transduced GFP+ preB-ALL cells (green, bottom left) in BM calvaria of Cxcl12dsRed/+ mice at day 14 after transplantation. Movement of GFP+ B cells tracked for 30 min by IVM. Colored lines represent cell trajectories (right panels). Scale bar, 50 µm. (H) PreB-ALL cell velocity (micrometers per minute). Each circle represents a single cell analyzed. (I) Mean motility coefficient of EV- and FAK-transduced preB-ALL cells. Cell displacement from starting coordinates is plotted against time (minutes). Lines depict the mean motility coefficient from three videos imaged/mouse. (J) Time (minutes) spent in direct contact with CXCL12+ cells over a 30-min period. (K) Average time (minutes) that EV- and FAK-transduced preB-ALL cells spent in direct contact with individual CXCL12+ cells. (J and K) Circles represent single cells. Data in panels G–K are from Video 3. (L) Model of the preB/IL-7+ cell circuit. **, P < 0.005; ***, P< 0.0005; ****, P < 0.00005 by unpaired Student’s t test. Data in all panels are representative of two to four independent experiments.
