Fig. 5: MEKi suppress M2-like macrophages and MDSCs.

a Flow cytometric analysis of the impact of MEKi GDC-0623 and/or anti-CD40 Ab treatment on the macrophage infiltrate in MC-38 and PDA30364 tumors, as performed on four mice per treatment group of the experiments shown in Fig. 3 b–c. Representative dot plots are shown. Cells were pre-gated on living CD45+, CD11b+, F4/80+ cells. One-way ANOVA with post hoc Dunnetts’s test (treatment groups vs. control). Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). b Polarization of mouse bone marrow-derived macrophage cultures towards M1- or M2-like phenotype, as assessed by flow cytometry marker analysis. Mean ± s.e.m, n = 6. c Cell viability of polarized macrophages after 3-day treatment with GDC-0623. Left panel: representative dose–response curves. Relative IC50 values were calculated by four-parameter logistic curve fit with a bottom constraint > 0. Mean ± s.e.m, n = 3. Right panel: Ki67/PI-based cell cycle analyses of tumor cells treated with 1 µm GDC-0623 and respective quantification. Mean ± s.e.m, n = 3. Unpaired student t test (medium vs. GDC-0623 for each cell cycle phase; FDR (Q = 1%), two-stage step-up method of Benjamini, Krieger and Yuketieli). d Representative dot plots of myeloid cell populations in MC-38 and PDA30364 tumors. e Representative dot plots of 'Gr1+', Ly6C+Ly6G+, and Ly6C+ Ly6G− myeloid cell populations in PDA30364 tumors. Cells were pre-gated on living CD45+, CD11b+ cells; quantitation normalized to all living cells. Mean ± s.e.m, n = 4. One-way ANOVA with post hoc Dunnett’s test (treatment groups vs. control). F Cell viability of bone marrow-derived MDSC cultures after 3-day treatment with GDC-0623; assay performed as described above. Mean ± s.e.m, n = 3.