Fig. 4

Identification of M1-specific secreted proteins as candidate paracrine signals for pSTAT3 downregulation in GBM using quantitative proteomics and bioinformatic analysis. (A) Venn diagram showing the overlap and number of unique proteins identified in UC-M, CM-M0, and CM-M1 media, including their respective proportions relative to the total proteins detected. (B) Gene Ontology analysis of Biological Process pathway enrichment for the 47 proteins specifically identified in the secretome of pro-inflammatory macrophages. Each dot represents a significantly enriched GO term, grouped by functional similarity. Dot size indicates the number of associated proteins, while the x-axis reflects the enrichment score. Color intensity represents the false discovery rate (FDR), ranging from dark blue (lowest FDR) to grey and yellow (higher FDR). (C) Proteins with potential cytotoxic effects via apoptosis induction and enhancement of TMZ efficacy, resulting from increased STAT1 activation and pSTAT3 inhibition in GBM. A protein–protein interaction (PPI) network was constructed from M1-specific secreted proteins using the STRING database. Nodes represent proteins; edges represent known or predicted interactions. Line thickness reflects interaction confidence. Proteins are colored according to four functional clusters: two red subnetworks representing cytokines/chemokines and proteasome-related proteins; one green network comprising transcription/mRNA-binding proteins; and a core cluster involved in protein synthesis and metabolism. (D) Heatmap of proteins shared between M0 and M1 secretomes. It displays z-score normalized expression levels of 87 overlapping proteins across medium samples. Each row corresponds to a protein, and each column to a replicate of CM-M0 or CM-M1. Color intensity reflects relative abundance, with red tones indicating increased secretion and cooler tones indicating lower secretion. (E) Volcano plot showing differentially secreted proteins between CM-M0 and CM-M1. Each dot represents a protein, positioned by log2 fold change (x-axis) and –log10 adjusted p-value (y-axis). Dashed vertical lines indicate fold change thresholds (log2FC ± 1), and the horizontal dotted line marks a –log10(p-value) of 1.3 (p < 0.05). Proteins with significant changes are highlighted in red (increased secretion in CM-M1, n = 39) or blue (decreased secretion in CM-M1, n = 3). (F) Molecular function enrichment analysis of significantly upregulated proteins in M1-conditioned medium.