The scavenger receptor, CD163, is a macrophage-specific marker. Recent studies have shown that CD163 expression in breast and rectal cancer cells is associated with poor prognosis. This study was conducted to evaluate the relationship between CD163 expression as a macrophage trait in cancer cells, and macrophage infiltration and its clinical significance in colorectal cancer. Immunostaining of CD163 and macrophage infiltration were evaluated in paraffin-embedded specimens, earlier analyzed for CD31, D2-40 and S-phase fraction, from primary tumors and normal colorectal mucosa of 75 patients with colorectal carcinoma. The outcomes were analyzed in relation to clinical-pathological data. CD163 expression was positive in cancer cells in 20 % of colorectal cancer patients and was related to advanced tumor stages (P = 0.008) and unfavorable prognosis (p = 0.001). High macrophage infiltration was related to shorter survival and positive CD163 expression in tumor cells. The prognostic impact of macrophage infiltration was independent of tumor stage and CD163 expression in cancer cells (p = 0.034). The expression of macrophage phenotype in colorectal cancer cells is associated with macrophage density in tumor stroma and lower survival rates. Macrophage infiltration has an independent prognostic impact on mortality in colorectal cancer. In accordance with previous experimental studies, these findings provide new insights into the role of macrophages in colorectal cancer.

Recent advances in tumor biology led to the realization that, in order to understand the mechanisms involved in proliferation and invasion of tumor cells, an analysis of the complex interactions that tumor cells establish with host cells of tumor microenvironment is required. The bidirectional interactions between tumor cells and components of tumor microenvironment, in particular endothelial cells, cells of monocyte/macrophage lineage and fibroblasts/myofibroblasts, play a critical role in most of the events that characterize tumor progression and metastasis. Interactions between these "reactive" normal cells and the genetically altered tumor cells, by either cell-to-cell contacts or soluble mediators, control the most aspects of tumor formation and progression. This review addresses some of the experimental evidences documenting that tumor cells may influence host cells of their own microenvironment by triggering changes that facilitate their local as well as distant dissemination. Therefore, it focuses on macrophages and fibroblasts that, upon stimulation by tumor cells, change their state towards a tumor-promoting-like phenotype.

The aim of this study was to investigate whether tumor cells as well as tumor-associated macrophages (TAMs) contribute to the generation of protease activities essential to tumor cell invasiveness, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9), and the urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). We found that the enhanced invasiveness through Matrigel-coated filters of B16 murine melanoma cells stimulated with IFNgamma was associated with an higher expression of uPAR and MMP-9 in these cells. Moreover, treatment with anti-MMP-9 or anti-uPAR monoclonal antibodies abrogated the increase of invasiveness in IFNgamma-stimulated melanoma cells, suggesting a cooperation of uPA system and MMP-9 in cytokine-stimulated invasiveness. Invasiveness through Matrigel was also enhanced in B16 melanoma cells exposed to a medium conditioned by TAMs, represented in our experimental model by thioglycollate-elicited macrophages co-cultivated with melanoma cells. Macrophages isolated from these co-cultures were found to express higher levels of uPAR and MMP-9 compared to macrophage cultures alone, and the pro-invasive activity of the co-culture-conditioned medium was abrogated by anti-MMP-9 monoclonal antibodies, but not anti-uPAR monoclonal antibodies. Furthermore, the enhanced uPAR and MMP-9 expression in macrophages co-cultivated with tumor cells seems a rather specific phenomenon, generated through a cell-to-cell contact mechanism. On the whole, our data point to a cooperation between tumor cells and macrophages elicited by tumor cells themselves in generating key enzymes essential in the promotion of tumor invasiveness, such as uPAR and MMP-9.

Tumor-associated macrophages (TAMs) may elicit contrasting effects on tumor growth, depending on their biological activities. Macrophages use arginine either to synthesize nitric oxide (NO) through the inducible NO synthase (iNOS) or to produce ornithine through arginase activity. Although the effects of NO are primarily cytotoxic, production of ornithine may promote tumor cell proliferation. Thus, iNOS/arginase balance in TAMs may be crucial in tumor progression. The aim of this study was (a) to explore iNOS and arginase expression in TAMs associated with human melanoma at different stages of tumor progression and (b) to explore whether melanoma cells influence iNOS and/or arginase expression in TAMs under basal condition and in the presence of interferon gamma and/or lipopolysaccharide. Immunohistochemical analyses performed on tissue sections from in situ melanoma, invasive melanoma of different pT categories, and metastatic melanoma revealed that (a) the percentage of iNOS-positive TAMs was significantly higher in in situ and thin melanomas in comparison with more advanced, thicker tumors; (b) the percentage of arginase-positive TAMs did not change among the pT categories analyzed; and (c) the percentage of iNOS-positive TAMs was greater than that of arginase-positive TAMs in peritumoral and intratumoral locations of thin melanomas (pT1). Moreover, by the use of an in vitro experimental protocol represented by B16 murine melanoma cells cocultivated with inflammatory macrophages, we found that melanoma cells stimulate iNOS expression and NO production in macrophages. In conclusion, our in vivo and in vitro results suggest that, mainly in early melanoma lesions, iNOS prevails over arginase in TAMs, a phenomenon possibly stimulated by contact with tumor cells. However, macrophages stimulated by murine melanoma cells secreted a level of NO compatible with an antitumor activity only in the presence of interferon gamma.

The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.

In previous studies, we found that IFNgamma and TNFalpha generated by activated macrophages stimulate the metastatic potential in F10-M3 cells, a clone isolated from B16-F10 murine melanoma line. In this phenomenon, TNFalpha promoted the expression of a metastatic phenotype in tumor cells previously primed with IFNgamma. Here, we demonstrate that IFNalpha or IFNbeta may replace IFNgamma in priming tumor cells. We also noticed that an enhancement of the expression of p55TNFalpha receptor was associated with the preconditioning of tumor cells with IFNgamma and IFNbeta. By the use of an appropriate inhibitor, we observed that JAK1 signal transduction pathway was involved in the expression of a metastatic phenotype and of p55TNFalpha receptor shown in IFNgamma- and IFNbeta-primed melanoma cells stimulated with TNFalpha. Furthermore, the activity of the protein kinase C (PKC) was required for IFNgamma-primed melanoma cells to express a metastatic phenotype after stimulation with TNFalpha. In conclusion, our study shows that a metastatic phenotype was expressed in B16 murine melanoma cells stimulated with TNFalpha regardless of whether the cells were primed with IFNgamma IFNalpha or IFNbeta. The molecular events leading to the expression of a metastatic phenotype in F10-M3 melanoma cells are represented by: (a) an enhanced expression of p55TNFalpha receptor in IFNs-primed tumor cells dependent on JAK1 signal transduction pathway; and (b) an intact PKC activity during TNFalpha stimulation.

In the present study we investigated whether synthesis of nitric oxide (NO) by macrophages is affected by contact with tumor cells. Although it is well known that NO generated by macrophages influences different activities related to tumor progression, there is limited information on the modulatory role of tumor cells on NO release by macrophages. The experimental protocol used in our study consisted in the determination of NO secreted by macrophages, either resident or inflammatory, co-cultivated with tumor cells (B16 melanoma and L929 fibrosarcoma cells) at different cell densities and macrophage:tumor cell ratios. This experimental in vitro protocol simulates the different interactions between macrophages and tumor cells that occur during the development of a tumor mass. We found that the co-cultivation with tumor cells induced an increased secretion of NO in macrophages provided that they express an inflammatory phenotype, and they were challenged with LPS or IFNgamma/LPS. Two more variables were found to be critical in the increase of NO generation in inflammatory macrophages cultivated with tumor cells: a high cell density and a prevalence of tumor cells over macrophages. The enhancement of NO secreted in inflammatory macrophages stimulated by tumor cells was not observed in normal murine fibroblasts co-cultivated with tumor cells.

Although there is a great deal of interest in the role played by tumor-associated macrophages in tumor progression, the knowledge of the biological mediators involved in the interplay between macrophages and tumor cells is still limited. In the present study, we investigated whether the lipoxygenase pathway in resident murine peritoneal macrophages is affected by contact with tumor cells of a different origin, e.g. murine B16 melanoma and L929 fibrosarcoma cells, and human Hs294T melanoma and HT1080 fibrosarcoma cells. Our experiments have been carried out by using macrophages co-cultivated with tumor cells at different ratios, in order to simulate the relative proportions between macrophages and tumor cells during the in vivo development of a tumor. Reverse phase HPLC analyses of the lipoxygenase products of resident peritoneal macrophages revealed a rather complex profile characterized by a high level of 12(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid followed by leukotriene B(4), 5(S)-hydroxyeicosatetraenoic acid, and lipoxins. Macrophages co-cultivated with tumor cells, both murine and human, showed a marked reduction of lipoxygenase products, mainly in the co-cultures where tumor cells prevailed over macrophages. The characteristic profile of macrophage lipoxygenase products was re-established after removal of tumor cells from the co-cultures. The inhibitory effect on lipoxygenase pathways exerted by tumor cells, was not seen when macrophages were co-cultivated with normal primary murine and human fibroblasts.

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parnum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors.