FIGURE 8 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

<p>Dabrafenib reduces MDSC accumulation in the tumor microenvironment. Mice were implanted with 3 × 10⁵ YUMM1.7 cells subcutaneously in the right flank. On day 10 after tumor implantation, mice were either treated with 30 mg/kg of dabrafenib or the equivalent volume of DMSO for 7 consecutive days (<i>n</i> = 10 per group). Mice were sacrificed on day 20 after tumor implantation. <b>A,</b> Number of total live cells in bone marrow, isolated from one tibia and femur per mouse after red cells lysis. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>B,</b> Frequency of MDSCs (CD45⁺CD3^negCD19^negCD11b⁺MHCII^neg) from total live cells (left), from immune cells (middle), and absolute counts of MDSCs (right) in bone marrow. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>C,</b> Representative contour plot of Ly6C and Ly6G expression in total MDSCs (CD45⁺CD3^negCD19^negCD11b⁺MHCII^neg) in bone marrow. Gates show median frequency of m-MDSCs (Ly6C⁺Ly6G^neg), PMN-MDSCs (Ly6C⁺Ly6G⁺) and cells with intermediate phenotype (Int, Ly6C^intLy6G^int). <b>D,</b> Frequency of MDSCs subtypes described in C. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>E,</b> Frequency of MDSCs (CD45⁺CD3^negCD19^negCD11b⁺MHCII^neg) from total immune cells. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>F,</b> Number of circulating MDSCs (CD45⁺CD3^negCD19^negCD11b⁺MHCII^neg) per microliter of blood. <b>G,</b> Representative contour plot of Ly6C and Ly6G expression in total MDSCs (CD45⁺CD3^negCD19^negCD11b⁺MHCII^neg) in blood. Gates show simplified nomenclature for all four subpopulations Ly6C^int (A), Ly6C^hi (B), Ly6C^intLy6G^int (C), and Ly6C^intLy6G^hi (D). <b>H,</b> Frequency of MDSC subtypes described in F. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>I,</b> Absolute counts of MDSC subtypes described in F and G per microliter of blood. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>J,</b> Number of intratumoral MDSCs (CD45⁺CD11b⁺MHCII^neg) per milligram of tumor. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>K,</b> Representative contour plot of Ly6C and Ly6G expression in total intratumoral MDSCs (CD45⁺CD3^negCD19^negNK1.1^negCD11c^negCD11b⁺MHCII^neg). Gates show median frequency of m-MDSCs (Ly6C^hi) and PMN-MDSCs (Ly6C^hiLy6G^hi). <b>L,</b> Absolute counts of MDSC subtypes described in J per milligram of tumor. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>M,</b> Mice were implanted with 1 × 10⁶ YUMMER cells subcutaneously in the right flank and 12 days later treated with dabrafenib as described above. Plots are representative contour plots of Ly6C and Ly6G expression in total intratumoral MDSCs (CD45⁺CD3^negCD19^negNK1.1^negCD11c^negCD11b⁺MHCII^neg). Graphs show relative frequency of m-MDSCs (i.e., Ly6C^hi, left graph) and more mature Ly6G⁺Ly6C^neg PMN cells from total MDSCs (right graph). <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. All panels are representative of three independent experiments. DAB = dabrafenib.</p>