Figure 6.

GM-CSF signalling blockade reduces monopoiesis, viability and alternative polarisation of macrophages in the tumour microenvironment. (A) Graph compares CFU-GM in bone marrow mononuclear cell and splenocyte suspensions isolated from mice bearing established orthotopic URCCA4.3 tumours after 17 days of treatment with IgG control (n=5) or anti-GM-CSF (n=5). (B) Graph shows mean number of viable cells per well after culturing bone marrow-derived macrophages in URCCA4.3 conditioned media containing IgG control (n=6) or anti-GM-CSF neutralising antibody (n=6) for 72 hours. (C) Representative flow cytometry histogram of phosphorylated-Signal Transducer and Activator of Transcription 5 (pSTAT5) staining in F4/80⁺ bone marrow-derived macrophages cultured in URCCA4.3 conditioned media with IgG control (n=6) or anti-GM-CSF neutralising antibody (n=6) for 72 hours. (D) Graph compares the MFI of pSTAT5 staining in F4/80⁺ bone marrow-derived macrophages cultured in URCCA4.3 conditioned media with IgG control (n=6) versus anti-GM-CSF neutralising antibody (n=6) for 72 hours. (E) Heatmap shows expression of the top 25 and bottom 25 most differentially expressed protein-coding genes (DEGs) from RNA-seq analysis of bone marrow-derived macrophages cultured in URCCA4.3 conditioned media with IgG control (n=6) or anti-GM-CSF neutralising antibody (n=6) for 72 hours. Results were previously filtered to include DEGs based on p value <0.05 and log₂ fold change<−1.0 or >1.0. (F) Graph shows quantitative real-time PCR (qRT-PCR) analysis of select downregulated DEGs determined by RNA-seq that are involved in M2 polarisation and immune modulation in bone marrow-derived macrophages cultured in URCCA4.3-conditioned media with IgG control (n=6) or anti-GM-CSF neutralising antibody (n=6) for 72 hours. (G) Table shows the top 20 downregulated Gene Ontology (GO) biological processes in bone marrow-derived macrophages cultured in URCCA4.3 conditioned media and in orthotopic URCCA4.3 tumours after GM-CSF neutralisation. Highlighted GO terms denote convergent pathways in bone marrow-derived macrophages and URCCA4.3 tumours. All graph bars depict means±SEM, and p values were determined by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. MFI, median fluorescence intensity; CFU-GM, granulocyte–macrophage colony-forming unit; GM-CSF, granulocyte–macrophage colony-stimulating factor.