Most malignant tumors evidence infiltration of many macrophages. In this study, we investigated an anti‐inflammatory macrophage phenotype (M2) in clear cell renal cell carcinoma (RCC) using CD163 and CD204 as markers. Immunostaining showed a correlation between the number of CD163+ cells and age, sex, nuclear grade, and TNM classification. High infiltration of CD163+ cells was significantly associated with poor clinical prognosis in univariate analysis but not in multivariate analysis. We also carried out in vitro studies to examine cell–cell interactions between macrophages and cancer cells. Culture supernatants from RCC cell lines induced polarization of macrophages toward the M2 phenotype. Coculture of macrophages with cancer cells significantly induced activation of signal transducers and activators of transcription‐3 (Stat3) in the cancer cells. Direct coculture of RCC cells with macrophages led to stronger activation of Stat3 in the cancer cells than did indirect coculture using Transwell chamber dishes. Because RCC cells expressed membrane‐type macrophage colony‐stimulating factor (mM‐CSF) on the cell surface, we suggested that this mM‐CSF plays an important role in direct cell–cell interactions. Stat3 activation in cancer cells that was induced by coculture with macrophages was suppressed by downregulation of the M‐CSF receptor (M‐CSFR) in macrophages and by an inhibitor of M‐CSFR. In conclusion, investigation of CD163+ tumor‐associated macrophages would be useful for assessment of the clinical prognosis of patients with ccRCC. Cell–cell interactions mediated by mM‐CSF and M‐CSFR binding could contribute to cancer cell activation. (Cancer Sci 2011; 102: 1424–1431)

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)‐β, 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. (Cancer Sci)

In several malignant tumors including lymphoma, macrophages that infiltrate tumor tissues are called tumor-associated macrophages (TAMs). We discovered that TAMs, especially the CD163 + alternatively activated phenotype (M2), were closely involved with progression of adult T-cell leukemia ⁄ lymphoma (ATLL). We used CD68 (a pan-macrophage marker) and CD163 (an M2 marker) to immunostain 58 ATLL samples. Statistical analyses showed that a high number of CD68 + TAMs and an increased percentage of CD163 + cells among the TAMs were associated with a worse clinical prognosis; multivariate analysis indicated that the percentage of CD163+ cells was an independent prognostic factor. We also carried out in vitro coculture experiments with ATLL cell lines (ATN-1 and TL-Mor) and monocyte-derived macrophages and found that direct coculture with M2 macrophages significantly increased BrdU incorporation into ATLL cell lines. A cytokine array analysis showed that macrophage-derived soluble factors including C5a, tumor necrosis factor-a, growth-related oncogene-a, CCL1 ⁄ I-309, and interleukin-6 stimulated ATLL cell lines. CD163 expression in macrophages was strongly induced by direct contact with ATN-1 cells, and downregulation of CD163 in macrophages significantly suppressed growth of cocultured ATN-1 cells. These results suggest that interaction between M2 macrophages and lymphoma cells may be an appropriate target in treatment of patients with ATLL. (Cancer Sci 2013; 104: 945-951)

We previously showed tumor-associated macrophages/microglia (TAMs) polarized to the M2 phenotype were significantly involved in tumor cell proliferation and poor clinical prognosis in patients with high grade gliomas. However, the detailed molecular mechanisms involved in the interaction between TAMs and tumor cells have been unclear. Current results reveal that, in coculture with human macrophages, BrdU incorporation was significantly elevated in glioma cells, and signal transducer and activator of transcription-3 (Stat3) activation was found in both cell types. Direct mixed coculture led to stronger Stat3 activation in tumor cells than did indirect separate coculture in Transwell chamber dishes. Screening with an array kit for phospho-receptor tyrosine kinases revealed that phosphorylation of macrophagecolony stimulating factor receptor (M-CSFR, CD115, or c-fms) is possibly involved in this cell-cell interaction; M-CSFR activation was detected in both cell types. Coculture-induced tumor cell activation was suppressed by siRNA-mediated downregulation of the M-CSFR in macrophages and by an inhibitor of M-CSFR (GW2580). Immunohistochemical analysis of phosphorylated (p) M-CSFR, pStat3, M-CSF, M2 ratio, and MIB-1(%) in high grade gliomas revealed that higher staining of pM-CSFR in tumor cells was significantly associated with higher M-CSF expression and higher MIB-1(%). Higher staining of pStat3 was associated with higher MIB-1(%). High M2 ratios were closely correlated with high MIB-1(%) and poor clinical prognosis. Targeting these molecules or deactivating M2 macrophages might be useful therapeutic strategies for high grade glioma patients. (Cancer Sci 2012; 103: 2165-2172