Decrease of CNS infiltration of leukemia cells by combination therapy with dasatinib and BTKi. (A) Upper panels, images of mouse brain rostral leptomeninges slices from WT C57/BL6 mice, vehicle-, dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice tissues, stained using the hematoxylin and eosin (H&E) method. Vehicle-treated mice show an increased level of leukemic infiltration (arrow) compared with the dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice. Samples were collected from sacrificed animals with signs of disease from vehicle- (mean = 18 days), dasatinib (mean = 24 days), ibrutinib (mean = 30 days), and dasatinib + ibrutinib (mean = 37 days) treated animals. Objective lens: 40×; scale bar: 50 μm. Lower panels, immunohistochemical staining of CNS sections with anti-GFP antibody showing less GFP⁺ E2A-PBX1⁺/preBCR⁺ leukemic cells infiltration in double- and single-treated mice compared with vehicle-treated mice. Arrows pointing at leukemic CNS infiltration. Unstained CNS sections are showed. Objective lens: 20×; scale bar: 200 μm. (B) GFP positivity from 3 representative mice of each group was estimated after transformation of images to DAB and GFP staining intensity quantified by a single cutoff threshold of 0.20. Statistical analysis was performed by 1-way analysis of variance (ANOVA), Dunnett multiple comparison test. (C) Single-cell suspensions from fresh tissue from both brain and spinal cord after cutting into small pieces by scalpel and resuspended in a cell dissociation solution were used for quantification of GFP⁺ cells in flow cytometry. Panels show the gating strategy for phospho-flow cytometry staining for GFP⁺ murine E2A-PBX1⁺/pre-BCR⁺ leukemia cells from vehicle-, dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice. Gating was performed on lymphocytes, single cells, and GFP⁺ cells. The frequency of GFP⁺ murine E2A-PBX1⁺/preBCR⁺ leukemia cells decrease in the combination treatment. (D-E) Frequency of GFP⁺ murine leukemia infiltrating (D) the brain and (E) the spinal cord of C57BL/6 WT, mice. Each symbol represents an individual mouse. Statistical analysis was performed by 1-way ANOVA, Dunnett multiple comparison test. Scatter dot plots represent mean ± SEM. ∗∗P < .01; ∗∗∗P < .0001. IHC, immunohistochemistry; WT, wild type.