Chemokine (C-C Motif) Ligand 2/CCR2/Extracellular Signal-Regulated Kinase Signal Induced through Cancer Cell-Macrophage Interaction Contributes to Hepatocellular Carcinoma Progression

Abstract

Tumor-infiltrating macrophages, known as tumor-associated macrophages, play a crucial role in the tumor microenvironment. Herein, immunohistochemistry revealed that intratumoral CD68-positive macrophages are associated with poor prognosis and clinicopathologic factors in patients with hepatocellular carcinoma (HCC). Subsequently, an indirect co-culture system involving HCC cells and peripheral blood-derived macrophages was developed. cDNA microarray analysis revealed that chemokine (C-C motif) ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with macrophages. CCL2 neutralization suppressed proliferation, migration, and phosphorylation of extracellular signal-regulated kinase (Erk) in HCC cells and macrophages enhanced through co-culture. In contrast, recombinant human CCL2 (rhCCL2) addition facilitated these malignant phenotypes and increased Erk phosphorylation levels in HCC cells and macrophages. The primary CCL2 receptor, CCR2, was expressed in HCC cells and macrophages and was up-regulated in co-cultured HCC cells. CCR2 inhibition suppressed malignant phenotypes and reduced phosphorylated levels of Erk enhanced by rhCCL2. Additionally, the inhibition of Erk signal suppressed rhCCL2-enhanced malignant phenotypes. Moreover, serum CCL2 levels were higher in patients with HCC than those in healthy donors. On the basis of immunohistochemistry, CCL2-positive cases with high CCR2 expression and phosphorylated Erk-positive cases exhibited poor survival outcomes. Therefore, CCL2 up-regulation through interactions between HCC cells and macrophages contributed to HCC progression, making the CCL2/CCR2/Erk signal a potential target for HCC treatment.

Figure 1

The number of macrophages, specifically CD68-positive macrophages, infiltrating into the tumor correlates with the poor prognosis of patients with hepatocellular carcinoma (HCC). A: Infiltrating macrophages in 95 human HCC tissues are assessed using hematoxylin and eosin (H&E) and immunohistochemical staining of macrophage markers (CD68, CD163, and CD204). The images are assessed at high-power fields (three images per case). Stained cells are counted in each image, and the samples are stratified into two groups (high or low) using the median staining cell number of averages of three images. Representative figures are illustrated. Case number 70 (top panels) is a 68-year–old patient with poorly differentiated HCC pathologically diagnosed as stage IV, and case number 34 (bottom panels) is a 47-year–old patient with moderately differentiated HCC pathologically diagnosed as stage II. B: Postoperative survival outcomes of 95 patients with HCC with high or low macrophage markers are assessed using the Kaplan-Meier method. P values are determined on the basis of the log-rank test. C: Survival outcomes of macrophage marker expression in HCC according to the University of Alabama at Birmingham CANcer data analysis Portal databases. Scale bars = 50 μm (A).

Figure 2

Co-culturing with macrophages facilitates the malignant phenotypes of hepatocellular carcinoma (HCC) cells. A: Experimental scheme of establishment of peripheral blood monocyte-derived macrophages. Peripheral blood mononuclear cells (PBMCs) are collected from healthy donors. Peripheral blood CD14-positive monocytes (PBMos) are purified from PBMCs using autoMACS pro Separator and incubated with recombinant human macrophage colony-stimulating factor (rhM-CSF) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 6 days, introducing PBMo-derived macrophages (M0). B: Experimental scheme of transwell proliferation assay of HCC cells (Hep G2, HuH-7, and HLE) and M0. HCC cells (4.0 × 10⁴ cells per well) are seeded in lower plates. For the co-culture system, M0 (3.0 × 10⁴ cells per well) are seeded in upper chambers (0.4-μm pore). After 48 hours, dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS) assays are performed to compare the cell proliferation between monocultured (Mono) and co-cultured (Co) HCC cells. C: Experimental scheme of transwell migration assay of HCC cells (Hep G2, HuH-7, and HLE) and M0. HCC cells (1.0 × 10⁵ cells per well) are seeded in upper chambers (8.0-μm pore). For the co-culture system, M0 (8.0 × 10⁴ cells per well) are seeded in lower plates. After 48 hours, HCC cells migrating to the lower surface are counted to compare the cell migration between monocultured and co-cultured HCC cells. D: Representative images of transwell migration assays shown in C. E: Expression levels of Akt, phosphorylated Akt (p-Akt), extracellular signal-regulated kinase (Erk), phosphorylated Erk (p-Erk), NF-κB, and phosphorylated NF-κB (p-NF-κB) in monocultured and co-cultured HCC cells based on Western blot analysis. β-Actin is used as a loading control. F: Quantification of the Western blot analysis bands illustrated in E. The expression levels of p-Akt (Ser473 and Thr308), p-Erk (Thr202/Tyr204), and p-NF-κB in monocultured and co-cultured HCC cells with M0 are normalized to the respective total protein levels. B, C, and E: Experiments are performed in triplicate and repeated at least thrice. B, C, and F: Two-sided t-tests were performed for the statistical analysis of parametric data. Data are expressed as the means ± SEM (B, C, and F). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 100 μm (D). DMEM, Dulbecco’s modified Eagle’s medium; NS, not significant.

Figure 3

Chemokine (C-C motif) ligand 2 (CCL2) up-regulated among the interactions between hepatocellular carcinoma (HCC) cells and macrophages contributes to the malignant phenotypes of HCC cells. A: CCL2 mRNA expression in monocultured (Mono) and co-cultured (Co) HCC cell lines using real-time quantitative PCR (qPCR). B: CCL2 mRNA expression in monocultured and co-cultured macrophages using qPCR. C: Supernatants of peripheral blood CD14-positive monocyte-derived macrophage (M0) monoculture, HCC cell monoculture, and M0/HCC co-culture are analyzed using enzyme-linked immunosorbent assay for the secretion levels of CCL2. D–F: Transwell proliferation assays (D), transwell migration assays (E), and Western blot analysis (F) of extracellular signal-regulated kinase (Erk) and phosphorylated Erk (p-Erk) are performed to explore the effects of neutralizing antibodies against CCL2 (Ab CCL2) on co-cultured HCC cells with M0. Ab CCL2 or hamster control IgG (IgG) is added to both upper chambers and lower plates. G: Quantification of the Western blot analysis bands illustrated in F. The expression levels of p-Erk (Thr202/Tyr204) in HCC cells co-cultured with M0 treated with Ab CCL2 or IgG are normalized to total Erk expression levels. H–J: Transwell proliferation assays (H), transwell migration assays (I), and Western blot analysis (J) of time-dependent transitions in Erk and p-Erk are performed to explore the effects of recombinant human CCL2 (rhCCL2; 25 ng/mL) on HCC cells. K: Quantification of the Western blot analysis bands illustrated in J. The time-dependent transitions of p-Erk in HCC cells treated with rhCCL2 are normalized to total Erk expression levels. A, B, F, and J: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are used as the loading control for qPCR and Western blot analysis, respectively. A–F and H–J: Experiments are performed in triplicate and repeated at least thrice. A–E and G–I: Two-sided t-tests were performed for the statistical analysis of parametric data. Data are expressed as the means ± SEM (A–E, G–I, and K). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4

Chemokine (C-C motif) ligand 2 (CCL2)/CCR2–extracellular signal-regulated kinase (Erk) signaling controls the malignant phenotypes of hepatocellular carcinoma (HCC) cells. A: CCR2 mRNA expression in HCC cell lines based on RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as the loading control. B: The protein levels of CCR2 in HCC cell lines based on Western blot analysis. C: Quantification of the Western blot analysis bands illustrated in B. The expression levels of CCR2 in HCC cells are normalized to β-actin expression levels. D: The protein levels of CCR2 in monocultured (Mono) and co-cultured (Co) HCC cell lines based on Western blot analysis. E: Quantification of the Western blot analysis bands illustrated in D. The expression levels of CCR2 in monocultured and co-cultured HCC cells are normalized to β-actin expression levels. F: The correlation analysis between CCL2 and CCR2 expression based on the differential gene expression analysis in tumor, node, and metastatic tissues (TNM plot). Spearman correlation is used to measure the correlation. The blue line shows the regression line, and the shaded gray area around the blue line shows the 95% CI. G and H: Transwell proliferation assays (G) and transwell migration assays (H) are performed to explore the effects of ab120812 (0.036 or 0.36 μmol/L; CCR2 antagonist) on the enhanced proliferation and migration of HCC cells owing to recombinant human CCL2 (rhCCL2; 25 ng/mL) addition. I: Western blot analysis of time-dependent transitions in Erk and phosphorylated Erk (p-Erk) is performed to explore the effects of ab120812 (0.36 μmol/L) on the enhanced phosphorylated levels of p-Erk owing to the addition of rhCCL2 (25 ng/mL). J: Quantification of the Western blot analysis bands illustrated in I. The time-dependent transitions of the p-Erk (Thr202/Tyr204) in HCC cells treated with rhCCL2 are normalized to total Erk expression levels. The solid and dotted lines indicate the expression levels of p-Erk in HCC cells treated with dimethyl sulfoxide (DMSO) and ab120812, respectively. K and L: Transwell proliferation assays (K) and transwell migration assays (L) are performed to explore the effects of PD98059 (10 μmol/L; mitogen-activated protein kinase kinase inhibitor) on the enhanced proliferation and migration of HCC cells owing to the addition of rhCCL2 (25 ng/mL). B, D, and I: β-Actin is used as the loading control for Western blot analysis. G–I, K, and L: DMSO is added as a negative control. A, B, D, G–I, K, and L: Experiments are performed in triplicate and repeated at least thrice. E, G, H, and J–L: Two-sided t-tests were performed for the statistical analysis of parametric data. Data are expressed as the means ± SEM (C, E, G, H, and J–L). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. NS, not significant.

Figure 5

Chemokine (C-C motif) ligand 2 (CCL2) facilitates the tumor-associated macrophage–like polarization, proliferation, and migration of macrophages. A: Experimental scheme of peripheral blood CD14-positive monocyte-derived macrophage (M0) culture. M0 are incubated with 50% hepatocellular carcinoma (HCC)–condition media (CM) for 2 days. B: The expression levels of CD163, CD204, IL-10, and IL-12 in M0 cultured with or without 50% HCC-CM are analyzed using real-time quantitative PCR (qPCR). C: The protein levels of CD163 and CD204 in M0 cultured with or without 50% HCC-CM are analyzed using Western blot analysis. D: Quantification of the Western blot analysis bands illustrated in C. The expression levels of CD163 and CD204 in M0 treated with 50% HCC-CM are normalized to β-actin expression levels. E and F: Transwell proliferation assays (E) and transwell migration assays (F) are performed to explore the effects of HCC cells on the proliferation and migration of macrophages. G: The correlation analysis between CCL2 and macrophage markers [CD68, CD163, and macrophage scavenger receptor 1 (CD204)] expression based on the differential gene expression analysis in tumor, node, and metastatic tissues (TNM plot). Spearman correlation is used to measure the correlation. The blue lines show the regression line, and the shaded gray area around the blue lines show the 95% CIs. H–J: Transwell proliferation assays (H), transwell migration assays (I), and Western blot analysis (J) of extracellular signal-regulated kinase (Erk) and phosphorylated Erk (p-Erk) are performed to explore the effects of neutralizing antibodies against CCL2 (Ab CCL2) on M0 co-cultured with HCC cells. Ab CCL2 or hamster control IgG (IgG) is added to both upper chambers and lower plates. K: Quantification of the Western blot analysis bands illustrated in J. The expression levels of p-Erk (Thr202/Tyr204) in macrophages co-cultured with HCC cells treated with Ab CCL2 or IgG are normalized to total Erk expression levels. L: The expression levels of CD163, CD204, IL-10, and IL-12 in M0 cultured with recombinant human CCL2 (rhCCL2; 25 ng/mL) are analyzed using qPCR. M: Western blot analysis of Erk, p-Erk, CD163, and CD204 is performed to explore the effects of rhCCL2 (25 ng/mL) on M0. N: Quantification of the Western blot analysis bands illustrated in M. The expression levels of p-Erk in macrophages treated with rhCCL2 are normalized to total Erk expression levels, and those of CD163 and CD204 are normalized to β-actin expression levels. O and P: Transwell proliferation assays (O) and transwell migration assays (P) are performed to explore the effects of rhCCL2 (25 ng/mL) on M0. B, C, J, L, and M: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as the loading control for qPCR (B and L), and β-actin is used as the loading control for Western blot analysis (C, J, and M). B, C, E, F, H–J, L, M, O, and P: Experiments are performed in triplicate and repeated at least thrice. B, D–F, H, I, K, L, and N–P: Two-sided t-tests were performed for the statistical analysis of parametric data. Data are expressed as the means ± SEM (B, D–F, H, I, K, L, and N–P). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. NS, not significant.

Figure 6

Serum chemokine (C-C motif) ligand 2 (CCL2) levels are up-regulated in patients with hepatocellular carcinoma (HCC). The serum samples are obtained from patients with HCC and healthy donors. The serum CCL2 levels are measured using an enzyme-linked immunosorbent assay. A, B, and D–G: The serum CCL2 levels are compared using box plots based on the presence of HCC (healthy donor or patient with HCC; A), tumor, node, and metastasis (TNM) stage (B), number of intrahepatic tumors (solitary or multiple; D), vascular invasion (E), etiology (viral or nonviral; F), and fibrosis number according to the new Inuyama criteria (G). One patient had no available data on fibrosis number. C: The correlation analysis between maximum tumor size and the serum CCL2 levels. Spearman correlation is used to measure the correlation. The blue line shows the regression line, and the shaded gray area around the blue line shows the 95% CI. For statistical analyses, the Kruskal-Wallis test is performed to compare the differences between the groups, and the Wilcoxon signed-rank test is performed to compute significance. Data are expressed as the means ± SEM (A, B, and D–G). n = 87 patients with HCC (A–G); n = 8 healthy donors (A and B). ∗∗P < 0.01, ∗∗∗P < 0.001. NS, not significant.

Figure 7

The expression levels of chemokine (C-C motif) ligand 2 (CCL2), CCR2, and phosphorylated extracellular signal-regulated kinase (p-Erk) correlate with the poor prognosis of patients with hepatocellular carcinoma (HCC). A: The CCL2, CCR2, and p-Erk expression levels in 95 human HCC tissues are analyzed using immunohistochemical analyses. The samples are stratified into two groups (CCL2 and p-Erk: positive or negative; CCR2: high or low). The representative images of hematoxylin and eosin (H&E) staining and intratumoral CCL2, CCR2, and p-Erk expression levels are illustrated. Case number 21 (top panels) is a 63-year–old patient with poorly differentiated HCC pathologically diagnosed as stage IV, and case number 56 (bottom panels) is a 38-year–old patient with moderately differentiated HCC pathologically diagnosed as stage I. B: Postoperative survival outcomes of 95 patients with HCC are analyzed using the Kaplan-Meier method stratified by the CCL2, CCR2, and p-Erk expression levels. C: Postoperative survival outcomes of 95 patients with HCC are analyzed using the Kaplan-Meier method stratified by the CCL2 expression levels in cases with CCR2-high or CCR2-low expression levels. B and C:P values are determined on the basis of the log-rank test. Scale bars = 50 μm (A).

Figure 8

Schematic diagram of the role of chemokine (C-C motif) ligand 2 (CCL2) in the hepatocellular carcinoma (HCC) microenvironment. Erk, extracellular signal-regulated kinase; TAM, tumor-associated macrophage.