SPP1⁺ macrophages promote colorectal cancer progression by activating JAK2/STAT3 signaling pathway

Tumor metastasis is an important risk factor for death in patients with colorectal cancer (CRC). This study aims to explore the effect of CXCL9 and SPP1 (CS) polarity alteration of tumor-associated macrophages (TAMs) on CRC progression and its associated molecular mechanisms. The heterogeneity of cellular subsets between the CRC and Normal groups was analyzed using single-cell RNA sequencing (scRNA-seq) technology. Developmental trajectories of the cellular subsets were plotted using pseudotime analysis, and the differences in enrichment scores among the cellular subsets were analyzed by combining gene set variation analysis (GSVA). Mouse CRC models were constructed, and SPP1^- TAMs or SPP1⁺ TAMs were isolated from mouse tumor tissues by flow cytometry sorting. MC38 cells were treated with the JAK/STAT3 inhibitor WP1066 and co-cultured with SPP1⁺ TAMs. MC38 cell viability was detected by the cell counting kit-8 (CCK-8) assay. The apoptosis rate of MC38 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. The expression of JAK/STAT3 pathway proteins was detected using western blot (WB), and the expression of epithelial-mesenchymal transition (EMT)-related proteins was detected using immunofluorescence. There were significant differences in SPP1⁺ TAMs between the CRC and Normal groups by scRNA-seq, and JAK/STAT3 signaling pathway had significant scores in each cell subset. In vitro assays showed that compared with the Negative group, MC38 cells in the Positive group exhibited higher viability and lower apoptosis rate, protein levels of p-JAK2 and p-STAT3 were significantly up-regulated, and the EMT levels were increased. In contrast, after the cells were co-treated with WP1066 on the basis of co-culture with SPP1⁺ TAMs, MC38 cell viability was decreased, and apoptosis was increased; the levels of JAK2, STAT3, and their phosphorylation were decreased, and the EMT process was inhibited. Therefore, SPP1⁺ TAMs promote CRC cell proliferation and EMT by activating JAK2/STAT3 signaling pathway.