Fig 2: cSCC development is reduced in OSM-deficient mice(A) OSM-KO mice and their WT littermates were subcutaneously injected with 1.106 PDVC57 cells and tumor volume was measured each week using a digital caliper. Data are presented as the mean ± SEM tumor volume (mm3) from five independent experiments, Two-way ANOVA test with Bonferroni post-tests, **p < 0.01, ***p < 0.001. (B) The size of draining lymph nodes were measured under the microscope using a ruler and the volume calculated according to the formula: length x width x thickness. (C) Relative mRNA expression was measured by real-time qPCR in tumors (T) (n = 25) and normal skin (N) (n = 25) of OSM-KO mice and WT littermates. Data are presented as min to max box plots of relative mRNA expression (A.U.). Kruskall-Wallis test with Dunn's Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: OSM increases the migration and proliferation o PDVC57 cells(A) In vitro wounds were made in cultures of mitomycin-treated PDVC57 cells and the cells cultured for a further 48 h, with or without OSM, (10 ng/mL). Cell migration into the wounded area was assessed as described in Materials & Methods. Scale bar: 100 µm. Data are presented as the mean ± SEM of the migration area (mm2) from three independent experiments. Mann-Whitney test, *p < 0.05. (B) PDVC57 cells were cultured, with or without OSM (10 ng/mL), for 48,h. Cells were fixed and stained with an anti-Ki67 antibody (green) and the cell nuclei stained with DAPI (blue). Scale bar: 100 µm. Data are represented as the mean ± SEM of the percentage of Ki67+ cells from three independent experiments. Mann-Whitney test, *p < 0.05, **p < 0.01.

Fig 4: PDVC57 cells express functional OSMR type II involved in ERK1/2 and STAT3 signaling and modulation of gene expression(A) Relative mRNA expression of normal murine epidermal keratinocytes (NMEK) and PDVC57 cells cultured under normal conditions for 6 h was measured by real-time qPCR. Data are presented as the mean ± SEM of relative mRNA expression (A.U.). Two-way ANOVA test with Bonferroni post-tests, *p < 0.05, **p < 0.01. (B) PDVC57 cells were cultured with or without OSM (10 ng/mL) for 5 min, 15 min, 30 min, 1 h, or 6 h. Protein levels of P-STAT3, total STAT3, P-ERK 1/2 and total ERK were measured by western blotting. Data are presented as the mean ± SEM of phosphorylated to-total protein ratio for STAT3 or ERK from three independent experiments. Kruskall-Wallis test with Dunn's Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Scatter-plot graphs representing the relative mRNA expression of PDVC57 cells stimulated, or not, with OSM (10 ng/mL) for 6 or 24 h. The X axis indicates the relative expression in the control and the Y axis the relative expression of OSM stimulated PDVC57 cells. The red dots reflect up-regulated genes and the green dots, down-regulated genes. Total RNA was isolated and labelled according to the Affymetrix protocol. Transcriptional profiles were obtained using Affymetrix GeneChip™ Mouse Gene 2.1 ST Array Plate.

Fig 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.