Fig. 2.

HCC-TCM promotes M2 polarization via Wnt2b/β-catenin signalling. a THP-1 derived macrophages (THP-1-M) were incubated with 50% HCC-TCM for 48 h to obtain the HCC-educated macrophages (HCC-TAMs). Transcript expression levels of Wnt2b were determined in HCC-TAMs by qPCR. b The expression levels of Wnt2b (red) in CD68⁺ macrophages (green) were determined by immunofluorescence using TMA containing pairs of tumors and matched para-carcinoma tissues of HCC patients. c, d THP-1-M were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry and qPCR, respectively. THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vector were acquired as described in the Materials and Methods, and then incubated with 50% HCC-TCM for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry (e, i) and qPCR (f, j), respectively. The expression levels of β-catenin in HCC-TAMs that were infected with control vectors or sh-Wnt2b vectors were determined by western blotting and immunofluorescence respectively (g, h). One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721; TMA, Tissue microarray. Data are presented as mean ± SEM from at least three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001)