Identification of CCL4-positive macrophages and their associated immune microenvironment in thymic epithelial tumors

Thymic epithelial tumors (TETs) are one of the primary tumor types in the mediastinum, originating from thymic epithelial cells. Recent studies have highlighted the close association between specific macrophage subpopulations and the tumor immunosuppressive microenvironment. However, the characteristics and immunological functions of macrophages within TETs are still poorly understood. Single-cell transcriptome sequencing and the TCGA-THYM data were collected for integrated analysis of macrophage characteristics. The target cells were verified using multiplex immunofluorescence (mIF) staining. Functional validation was performed using Western blot and Transwell migration assays. Drug sensitivity was evaluated by predicting the half-maximal inhibitory concentration through oncoPredict. A group of CCL4⁺ macrophage subpopulations was clustered from the TETs myeloid single-cell subpopulation. By cell communication analysis, CCL4⁺ macrophages were positively correlated with exhausted T cells and regulatory T cells (Tregs). mIF staining confirmed a significant positive association between CCL4⁺ CD163⁺ cells and FOXP3⁺ CD4⁺ cells in terms of their proportions (r = 0.679, p < 0.001). Furthermore, in vitro experiments demonstrated that macrophages promoted Treg migration, which was significantly reduced by CCL4 blockade (p < 0.05). Additionally, there are significant differences in drug sensitivity predictions and survival between TET patients with high and low CCL4 expression. These findings suggest that CCL4⁺ macrophages are specifically present in TETs and are related to the immune microenvironment of Treg infiltration.The expression of CCL4 in other immune cell subpopulations and cohorts still needs further verification to clarify its unique role in TET macrophages.