Panoply™ Human CCR1 Knockdown Stable Cell Line

Heterogeneity in the tumor microenvironment significantly impacts the prognosis of patients with hepatocellular carcinoma (HCC), with cellular communication through ligand-receptor complexes playing a central role. Here, researchers performed single-cell transcriptome analysis of 10 HCC tissues using CellChat to identify ligand-receptor genes involved in malignant HCC cell communication. Single-cell analysis revealed significant interactions between malignant HCC cells and macrophages, identifying 76 relevant ligand-receptor genes. Patients were stratified into three subtypes based on the expression patterns of eight prognostic-associated ligand-receptor genes. The subtype with the worst prognosis exhibited decreased T cell-related immune cell infiltration, downregulation of immune checkpoint genes, and an increased score of M2-like tumor-associated macrophages. In vitro experiments confirmed the critical role of the CCL16-CCR1 axis in the recruitment and M2 polarization of tumor-associated macrophages. Clinical samples showed that the expression level of CCL16 protein was significantly correlated with advanced stage, lymph node metastasis, and distant metastasis. Immunohistochemistry and immunofluorescence staining further confirmed the correlation between CCL16 and CCR1, CD68, CD206, and CD68+CCR1+ macrophage infiltration.

Studies have reported that CCL16 binds to known receptors, including CCR1, CCR2, CCR5, and CCR8. Here, the researchers added a synthetic Flag-CCL16 protein to cultured THP1 cells and performed coimmunoprecipitation experiments. The results showed that CCL16 primarily interacted with the CCR1 receptor on macrophages, with less affinity for other receptors (Figure 1A). Immunofluorescence analysis further confirmed this interaction, showing colocalization of CCL16 and CCR1 on the THP1 cell membrane (Figure 1B). To further investigate the role of CCR1, the researchers performed CCR1 knockdown in THP1 cells (Figure 1C) and cocultured them with HEPG2 cells to assess macrophage migration. The results showed that CCR1 knockdown in THP1 cells significantly inhibited macrophage recruitment by HEPG2 cells (Figure 1D). Interestingly, after treatment with the CCR1 inhibitor BX471, the overexpression of CCL16 no longer promoted THP1 cell recruitment (Figure 1E). Similarly, co-culture of CCR1-knockdown THP1 cells with CCL16-overexpressing tumor cells no longer promoted macrophage recruitment (Figure 1F). These results suggest that CCL16 secreted by HCC cells promotes macrophage recruitment by binding to the CCR1 receptor on macrophages.

Figure 1. The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (Dai Z, et al., 2024)