Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma - PubMed (original) (raw)

Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma

Horlad Hasita et al. Cancer Sci. 2010 Aug.

Abstract

Many studies have shown that tumor-associated macrophages (TAMs) contribute to tumor development and poor prognosis in various cancers. In this study, we investigated the macrophage populations and phenotypes, and their correlation to angiogenesis, immunosuppression, and clinical prognosis in intrahepatic cholangiocarcinoma (ICC). CD68 (+) and CD163 (+) macrophage infiltration was analyzed in paraffin-embedded tissue samples from 39 patients. CD163 is used as a marker of M2 macrophages. Neovascularization and infiltration of forkhead box P3 (FOXP3) (+) regulatory T cells were also evaluated. The number of CD68 (+) and CD163 (+) macrophages was positively correlated with the numbers of vessels and regulatory T cells. The number of CD163 (+) cells was more closely associated with them. Intrahepatic cholangiocarcinoma (ICC) patients with high counts of CD163 (+) macrophages showed poor disease-free survival (P = 0.0426). The macrophage density was not correlated with overall survival. In an in vitro study using ICC cell lines (HuCCT1, RBE, and MEC) and human macrophages, tumor cell supernatant (TCS) from cell lines induced an activation of signal transducers and activators of transcription-3 (Stat3) and macrophage polarization toward the M2 phenotype. Tumor cell supernatant (TCS) from HuCCT1 most strongly induced Stat3 activation and production of cytokines and other bioactive molecules such as interleukin (IL)-10, vascular endothelial growth factor (VEGF)-A, transforming growth factor (TGF)-beta, and matrix metalloproteinase (MMP)-2. Down-regulation of Stat3 by siRNA significantly suppressed the production of IL-10 and VEGF-A. These results provide suggestive evidence that TAMs contribute to cancer progression via Stat3 activation, and CD163 is useful for evaluating M2 TAMs and predicting the clinical prognosis of ICC patients.

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Figures

Figure 1

Immunohistochemical analysis of CD68‐ and CD163‐positive cells in intrahepatic cholangiocarcinoma. (a) In patient no. 3 (Pt.3), CD68‐positive tumor‐associated macrophages (TAMs) highly expressed CD163. However, CD163 was not detected in CD68‐positive TAMs in patient no. 25 (Pt.25). The density of macrophages was classified into two groups (high density or low density). (b) Double‐immunostaining of CD68 and CD163 antigens were performed. CD68 (brown) and CD163 (blue) antigens were mainly localized in cytoplasm and surface cell membrane respectively. Double‐positive cells are indicated by arrows.

Figure 2

Immunohistochemical analysis of FOXP3‐positive regulatory T cells. (a) Double‐immunostaining of FOXP3 and CD4 antigens. FOXP3 (blue) and CD4 (brown) were stained in the nucleus and cytoplasm of infiltrating leukocytes respectively. Double positive cells are indicated by arrows. Scale bar: 20 μm. (b) FOXP3‐positive cells were counted and classified into two groups (high density or low density).

Figure 3

Correlation between the numbers of CD68(+) or CD163(+) macrophages and the number of vessels and regulatory T cells. Significant association was observed between the numbers of CD68(+) or CD163(+) macrophages, and the numbers of regulatory T cells or vessels. FOXP3.

Figure 4

Correlation between macrophages and clinical prognosis. (a) Disease‐free survival (DFS) and macrophage density. (b) Overall survival (OS) and macrophage density.

Figure 5

Macrophage differentiation into the M2 phenotype by tumor‐cell supernatant (TCS). (a) Cultured macrophages were stimulated by TCS for 2 days, and then CD163 expression, a marker of M2 status, was examined by cell‐ELISA assay. Macrophages cultured with TCS were stimulated with LPS and interleukin (IL)‐10 (M2‐related cytokine) production was evaluated by ELISA. (b) Signal transducers and activators of transcription‐3 (Stat3) activation by TCS was examined by using the monocytic cell line THP‐1 and cultured macrophages. The following cells were cultured with TCS for 1 h, and pStat3 was evaluated by immunostaining. (c) Cultured macrophages were stimulated by TCS for 15 h, and the mRNA expression of molecules and cytokines which related to the M2 phenotype was examined by quantitative PCR (Q‐PCR). *P < 0.05. MMP2, matrix metalloproteinase 2; TGF‐β, transforming growth factor‐β. VEGF‐A, vascular endothelial growth factor A. (d) Signal transducers and activators of transcription‐3 (Stat3) protein in macrophages was silenced by siRNA. Western blot analysis revealed down‐regulation of Stat3 protein by siRNA. (e) Following the inhibition of Stat3 expression, macrophages were stimulated by TCS from HuCCT1. The mRNA expression of CD163 and cytokines was evaluated by Q‐PCR. *P < 0.05 (compared with control siRNA).

Figure 6

(a) Interleukin (IL)‐6 expression in intrahepatic cholangio‐carcinoma (ICC) cancer cells. Strong immunostaing of IL‐6 was observed in patient (Pt.) 14 (right panel), whereas no IL‐6 expression in cancer cells was seen in Pt. 25 (left panel). Plasma cells (arrowheads) were positively stained as an internal control (right panel). (b) Significant correlation between the numbers of CD68(+) macrophages and IL‐6 production in cancer cells.

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Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma - PubMed (original) (raw)