Fig 1: CA12 induces CCL8 production in tumor-associated monocytes and macrophages.(A–C) CD14+ cells were purified from paired nontumor tissues (N-MO) and tumor tissues (T-MO) from 3 patients with HCC. Expression levels of metastasis-related genes were quantified by qPCR (A). Levels of CCL8 production by these cells were determined by ELISA (B) and confocal microscopy (C) (CCL8: green; DAPI: blue). Scale bar: 20 µm. (D–H) CD14+ cells were purified from the peripheral blood of healthy donors. (D) Cells were left untreated or treated with HepG2 TSN in the presence or absence of 2DG for the indicated times, and CCL8 production was measured by ELISA (n = 3). (E) Cells were left untreated or treated with HepG2 TSN in the presence or absence of CAi (100 µM) for 72 hours, and CCL8 production was measured by ELISA (n = 4). (F and G) Cells were transfected with siNC or siCA12 and then left untreated or treated with HepG2 TSN for 72 hours. Levels of CCL8 production were measured by ELISA (F) (n = 4). Expression levels of p-p38, p38, p-p65, p65, p-I?Ba, I?Ba, p-JNK, JNK, p-Erk, and Erk were determined by immunoblotting (G) (n = 5). (H) Cells were left untreated or treated with HepG2 TSN in the presence or absence of SB202190 (SB) (25 µM) for 72 hours, and CCL8 production was measured by ELISA (n = 3). (I and J) CD14+ cells were purified from tumor tissues from 36 patients with HCC. Correlations between the mRNA levels of CCL8 and GLUT1 or CA12 in the cells were analyzed by qPCR. Results shown in B–F and H are represented as mean ± SEM. P values were obtained by 2-tailed Student’s t test (B and C), 2-way ANOVA (D–F and H), or Pearson’s correlation and linear regression analysis (I and J). *P < 0.05; **P < 0.01.
Fig 2: CCL8 released by tumor-associated monocytes and macrophages induces EMT and migration of hepatoma cells.(A–D) HepG2 cells were left untreated or treated with CCL8 (20 ng/ml) for 20 hours. Cell migration was analyzed (A and B) (n = 4). Expression levels of vimentin, E-cadherin, N-cadherin, SNAI1, SNAI2, TWIST1, and TWIST2 in the cells were determined by immunoblotting (C and D) (n = 5). One out of five representative graphs is shown in A. Scale bar: 200 µm. (E–G) CD14+ cells were purified from the peripheral blood of healthy donors. Cells were left untreated or treated with HepG2 TSN for 2 hours, washed, and cultured for another 48 hours before their supernatants were collected. HepG2 cells were then treated with CCM or TCM in the presence or absence of control IgG or anti-CCL8 neutralizing antibody (2 µg/ml) for 20 hours. HepG2 cell migration was analyzed (E and F) (n = 4), and the expression of vimentin, E-cadherin, and SNAI1 was determined by immunoblotting (G) (n = 5). One out of five representative graphs is shown in E. Scale bar: 200 µm. (H and I) CD14+ cells and cancer cells were purified from tumor tissues from 13 patients with HCC. Expression levels of CCL8 in CD14+ cells and VIM and CDH1 expression in cancer cells were determined by qPCR. Correlations between the mRNA levels of CCL8 and those of VIM or CDH1 were analyzed. Results shown in B and F are represented as mean ± SEM. P values were obtained by 2-tailed Student’s t test (B), 2-way ANOVA (F), or Pearson’s correlation and linear regression analysis (H and I). *P < 0.05.
Fig 3: Blockade of CA12 reduces macrophage infiltration and induces tumor regression in mice in vivo.(A–C) C57BL/6J mice were s.c. injected with Hepa1-6 cells, DMSO or CAi was then i.p. administered. Tumors were excised and photographed (A), tumor volumes (B) and weights (C) were measured. (D) An orthotopic tumor model was established by intrahepatic injection of Hepa1-6 cells, DMSO or CAi was then i.p. administered. Lungs were excised from mice, and tumor lung metastases were counted. (E–G) Mice bearing with subcutaneous tumors were i.p. injected with GdCl3 and DMSO or Cai. Tumors were excised and photographed (E), tumor volumes (F) and weights (G) were measured. (H–J) Mice bearing orthotopic Hepa1-6 tumors were i.p. injected with (H) or without (I and J) GdCl3, and treated with DMSO or CAi. Lungs were excised from mice, and tumor lung metastases were counted (H). Paraffin-embedded sections of orthotopic hepatic tumors were stained with anti-mouse F4/80 antibody, and infiltration of F4/80+ cells was analyzed, scale bar: 500 µm (I). CD11b+Ly6G– cells were isolated from orthotopic hepatic tumors by FACS, and Ccl8 expression in these cells were quantified by qPCR (J). (K and L) Mice bearing with subcutaneous tumors were i.p. injected with DMSO together with control IgG (designated as control), CAi, anti-mouse PD-1 antibody, or CAi in combination with the anti-mouse PD-1 antibody. Tumor volumes (K) and weights (L) were measured. There were 5 representatives for each group in A–L. Results shown in B–D and F–L are represented as mean ± SEM. P values were obtained by 2-way ANOVA with Tukey’s correction t test (B, F, and K), 2-tailed Student’s t test (C, D, and G–J), or 1-way ANOVA (L). *P < 0.05; **P < 0.01; ***P < 0.001.
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