Fig 2: TEER and the percentage of blood monocytes that migrated across the BBB in different models. The cell-induced as well as co-culture TEER values were evaluated by subtracting the TEER of the blank insert. (A) Percentage of spontaneous migration vs. TEER values. (B) Percentage of specific migration toward CCL19 vs. TEER values. For the last condition, monocytes were treated for 24 h with PGE2 before the migration assay through the BBB model. Data represent the mean of four independent experiments, ±SD. Statistical analysis was performed using Student t-test; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: Unmutated chronic lymphocytic leukemia (U-CLL) COBLL1-high cells show deregulated chemotaxis and motility. Migratory properties of 10 mutated CLL (M-CLL), 10 U-CLL COBLL1-low, and 6 U-CLL COBLL1-high samples were assessed using transwell plates. (A) Chemotaxis towards chemokine CCL19 expressed as migration index (MI). (B) Chemotaxis towards chemokine CXCL12 expressed as MI. (C) Basal migration. MI was calculated as the number of cells migrated towards chemokine divided by the number of cells migrated in chemokine-free media. Basal migration was calculated as the percentage of migrated cells from all seeded cells. Each measurement was performed in a technical triplicate. Bars represent mean+Standard Deviation (S.D.) (A and B) Individual dots represent individual patients (C). *P≤0.05, **P≤0.01. (Mann-Whitney test).

Fig 4: Cells migration under the agarose spot can be selectively modulated by antagonists. (A) Migration of PC-3 cells under agarose spot containing 100 nM CXCL12 (ligand for CXCR4), is modulated in a dose-dependent manner by CXCR4 selective antagonist, AMD3100. (B and C) However, migration of PC-3 cells under agarose spot is not abrogated by CXCR4 selective antagonist, AMD3100 for the spots containing 100 nM CCL21 and 100 nM CCL19 (ligands for CCR7). (D) Migration of PC-3 cells under agarose spot containing 100 nM chemokines in the absence (grey bars) or presence (purple bars) of CCR7 antagonist ICT13069 (10 µM) shows that migration against CXCL12 is not modulated but that migration is reduced against CCL19 and CCL21. (E) Comparison of the inhibition of CXCL12 induced migration of PC-3 cells by AMD3100 in a Boyden assay.

Fig 5: Cell surface dynamics of ROR1 in migrating primary CLL cells is attenuated by Lyn inhibition. (A) Scheme of the transwell assay indicating the upper and lower compartment that was used for the separate analysis of the surface markers. (B,C) Surface expression of ROR1 (B) and CCR7 (C) in the upper (○) and lower (●) compartments of transwell chamber in the panel of primary CLL cells was analyzed by flow-cytometry. Paired t-test (B) n = 15, (C) n = 13. (D) Correlation of the change in the surface expression of ROR1 during migration represented as the ratio of receptor levels in the lower:upper compartment with the chemotactic properties of cells (expressed as the number of cells in the lower chamber) in the panel of primary CLL cells. Spearman's rank correlation coefficient correlation coefficient (n = 15). (E) The primary CLL cell chemotaxis towards CCL19 (expressed as the number of cells in the lower chamber) is reduced by 25 nM Dasatinib (Paired t-test). (F) Dynamics in the ROR1 surface levels during migration of primary CLL cells. Cells were allowed to migrate to CCL19 in the presence and absence of Dasatinib (25 nM) and ROR1 surface levels were analyzed by flow cytometry as in (B). ROR1 dynamics is expressed as a ratio of ROR1 level on cells in the lower and upper transwell compartment (n = 15). Smaller graphs show separately CLL cells with high and low ROR1 surface dynamics. Paired t-test.