Fig 1: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.
Fig 2: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.
Fig 3: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.
Supplier Page from Abcam for Human CCL16 ELISA Kit