Fig. 4: Morphological and functional changes in Frount-deficient monocytes /macrophages.

a Morphology of macrophages isolated from tumor tissue of Flox/flox or FROUNT-cKO mice observed by differential interference-contrast microscopy (left panel) and ratios of lamellipodial to filopodial macrophages (based on analysis of >140 cells) (right panel). b In vivo expression of activation markers on macrophages in LLC-tumor tissue as measured by flow cytometry (Flox/flox: n = 11, cKO: n = 9). c In vitro expression of CD86, MHC class II and CD206 on monocytes from control (Cre⁺) or FROUNT-cKO (Cre⁺/Flox/flox) mice cultured with DsRed⁺ LLC tumor cells as measured by flow cytometry (n = 9). d Direct effect of macrophages on tumor cell growth in vitro. Representative image of DsRed⁺ LLC tumor cell clusters cultured alone or with monocyte-derived macrophages from control or FROUNT-cKO mice (upper panel) and enumeration of the clusters (>10 cells) per well (n = 9) (middle panel). Flow cytometry measurement of the number of DsRed⁺ tumor cells (middle panel), EdU⁺ proliferating tumor cells, and annexin V⁺ tumor cells (lower panel) (n = 9). e Effect of CCR2 or CCR5 deficiency on the upregulation of CD86 on monocytes, and tumor cell cluster formation in tumor-monocyte co-culture experiments (n = 8). f–h Myeloid cell-specific deletion of FROUNT in LysM-Cre mice with Frount-floxed allele (Flox/flox), and the wild-type allele (+/+) as a control. Frount mRNA expression in CD11b⁺ myeloid cells and CD11b^- nonmyeloid cells isolated from tumor tissue (f) (n = 3) and immunoblot analyses of FROUNT and pan-actin as a control in lysate of bone marrow-derived macrophages (g). h Myeloid cell-specific deletion of FROUNT inhibits tumor growth. Combined tumor-growth kinetics in LysM-Cre mice with the wild-type allele (+/+) or Frount-floxed allele (Flox/flox) subcutaneously inoculated with B16 tumors (n = 6 per group; representative of three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed, unpaired Student’s t-test. Error bars indicate s.e.m. Scale bars, 50 μm.