Figure 5. Mouse tumors are moderately differentiated cSCC associated with OSM overexpression by infiltrating neutrophils.

(A) Mouse cSCC sections were fixed, embedded in paraffin, and stained with H&E or an anti-CD3, anti-CD68, or anti-MPO antibody. Scale bar: 100 μm. (B) Relative OSM mRNA expression was measured by real-time qPCR in tumors (T) (n = 25) and normal skin (N) (n =2 5). Data are presents as min to max box plots of relative mRNA expression (A.U.). Mann-Whitney test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. (C) 8-μm frozen sections of tumors from WT mice were fixed in 4% paraformaldehyde and stained with an anti-OSM antibody (R&D system, MAB4951) at 1:50 followed by staining with a goat anti-rat-IgG-AF555 secondary antibody (Thermofisher, A-21434) 1:100. Cell nuclei were stained with TOPRO (Invitrogen). Image acquisition was performed with an Olympus FV1000 confocal microscope using FlowView software (ImageUP platform, University of Poitiers). Scale bar: 20 μm. (D) Tumor cells and infiltrating tumor cells were isolated from WT mice. Cells were stained with anti-CD45-V500, anti-CD3-BV421, anti-Ly6G/C-FITC, anti-F4/80-PE, anti-CD68-PerCP-Cy5.5, or antiNK1.1-PE-Cy7. Data are presented as the mean ± SEM of total counts of PN: polynuclear neutrophils, MDSC: myeloid-derived suppressor cells, macrophages, T cells, and NK cells per tumor from three independent experiments.