Fig. 6: OPN activates the PI3K/AKT signaling axis to promote macrophage secretion of CSF1.

A Cytokine array profiling of macrophage secretomes under OPN-modulated co-culture conditions: representative array membranes comparing OPN knockdown (shOPN) vs. overexpression (OE) in CRC-TAM co-cultures. (left) and quantification of CSF1 as the most differentially secreted factor. (right). B Macrophages treated with conditioned medium (CM) from CRC cells for 72 hours show CSF1 expression by ELISA (left) and CSF1R expression by qPCR (right). C Macrophages treated with rhOPN for 72 hours show CSF1 expression by ELISA (left) and CSF1R expression by qPCR (right). D Transwell migration assays of M0 macrophages or PLX3397-treated macrophages co-cultured with CRC cell CM (left), with summarized quantitative results (right). E, F RNA-seq of M0 macrophages co-cultured with SW480-shNC and SW480-shOPN#2 CM for 48 hours. E Heatmap of macrophage polarization-related genes (z-score analysis, upregulated in red). F KEGG analysis of enriched pathways. G Western blot of PI3K/AKT phosphorylation in macrophages cultured with CRC cells CM for 72 hours (left), with quantitative results (right). H–I Pharmacological perturbation of the signaling pathway hierarchy. H Western blot of PI3K(p85/p55)/AKT(Ser473) in macrophages pretreated with LY294002 (PI3K inhibitor, 10 μM) or PLX3397 (CSF1R inhibitor, 10 μM) followed by rhOPN (400 ng/ml) treatment for 48 hours. I CSF1 secretion was quantified by ELISA in macrophage supernatants under corresponding treatments. All data are shown as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant.