Secondary Logo

Journal Logo

Tumour-associated macrophage infiltration, neovascularization and aggressiveness in malignant melanoma: role of monocyte chemotactic protein-1 and vascular endothelial growth factor-A

Varney, Michelle L.; Johansson, Sonny L.; Singh, Rakesh K.

ORIGINAL ARTICLES
Free

The objective of this study was to evaluate the role of tumour-associated macrophages (TAMs) in malignant melanoma progression, invasion and angiogenesis. We examined the levels of macrophage infiltration and monocyte chemotactic protein-1 (MCP-1), neovascularization and vascular endothelial growth factor-A (VEGF-A) in different Clark's level melanomas with varying thicknesses and metastases. The level of TAM density was significantly higher in thick (>0.75 mm) than thin (≤0.75 mm) melanomas, and positively correlated with melanoma invasiveness and metastasis. In contrast, MCP-1 expression was significantly lower in thick (>0.75 mm) than thin (≤0.75 mm) melanomas and negatively correlated with melanoma aggressiveness and invasion. We did not observe any significant difference in the levels of neovascularization between thin and thick melanomas, and no correlation with VEGF-A expression, TAM density or melanoma aggressiveness and invasion. Interestingly, levels of VEGF-A were significantly higher in metastatic melanoma than in thick melanoma. In addition, we observed lower levels of MCP-1 messenger RNA (mRNA) expression in more aggressive melanomas and in cell lines with higher metastatic potential. In summary, our data suggest a distinct pattern of TAM infiltration, MCP-1 expression, neovascularization and VEGF-A expression during human melanoma progression, and a complex interaction between TAMs and melanoma cells in the regulation of melanoma progression, angiogenesis and metastasis.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA

Sponsorship: This work was supported in part by grant CA72781 (R.K.S.) from the National Cancer Institute, National Institutes of Health and the Nebraska Research Initiative Molecular Therapeutics Program (R.K.S.).

Correspondence and requests for reprints to Rakesh K. Singh, Department of Pathology and Microbiology, The University of Nebraska Medical Center, 985845 Nebraska Medical Center, Omaha, NE 68198-5845, USA

Tel: 402 559 9949; fax: 402 559 5551;

e-mail: rsingh@unmc.edu

Received 12 October 2004 Accepted (after revision) 9 May 2005

Back to Top | Article Outline

Introduction

Human malignant melanoma is a highly aggressive form of skin cancer, the incidence of which is increasing at a rate greater than any other type of cancer, and for which there is no effective treatment for advanced disease [1–5]. The pathogenesis and progression of melanoma depend on interactions between tumour cells and stromal cells in the host [6,7]. Tumour-associated macrophages (TAMs) derived from the host are major components of the tumour microenvironment and have been shown to be associated with tumour progression, angiogenesis and metastasis [8–15].

Tumour cells attract circulating monocytes through the production of chemotactic factors, such as monocyte chemotactic protein-1 (MCP-1), monocyte-colony-stimulating factor (M-CSF) and vascular endothelial growth factor-A (VEGF-A) [16,17]. Indeed, infiltration of TAMs has been shown to be involved in the course of progression of melanoma [13]. In the tumour microenvironment, TAMs have been shown to secrete factors, including cytokines and enzymes, implicated in angiogenesis as well as in the modulation of the extracellular matrix [18–28].

In malignant melanoma, the significance of angiogenesis as a prognostic indicator remains controversial [8,9,29–31] and its role in melanoma metastasis remains unclear. In this study, we hypothesize that melanoma cells recruit circulating monocytes by the production of MCP-1, and TAM–melanoma cell interaction leads to increased production of VEGF-A and angiogenesis. To test this hypothesis, we selected melanoma specimens with different Clark's levels, which provides a well-defined clinical pathological determinant of melanoma progression and metastasis [3,4,32], and examined TAM infiltration, the production of MCP-1 and VEGF-A and neovascularization in relation to Clark's level and invasion. Our data suggest a distinct pattern of TAM infiltration, MCP-1 expression, angiogenesis and VEGF-A expression in malignant melanomas with different aggressiveness and a complex interaction between melanoma cells and monocytes in the regulation of melanoma progression and metastasis.

Back to Top | Article Outline

Materials and methods

Tumour specimens

Paraffin-embedded cutaneous malignant melanoma tumour specimens from the tissue archives of the Department of Pathology and Microbiology, University of Nebraska Medical Center (UNMC), Omaha, Nebraska, USA were studied. All primary melanoma samples were classified by a pathologist (S.L.J.) according to thickness and depth of invasion using previously defined parameters [3,4,33] (Table 1). Similarly, metastatic lesions were categorized according to organ site (Table 2). After an initial review of the available haematoxylin and eosin-stained slides of the tumours, representative paraffin blocks were selected and consecutive sections, 6 μm thick, were re-cut from the tumours to include adjacent uninvolved tissue and processed for immunohistochemistry.

Table 1

Table 1

Table 2

Table 2

Back to Top | Article Outline

Immunohistochemical analysis

TAMs, neovascularization, MCP-1 and VEGF-A expression in primary tumours and metastases were analysed by immunohistochemistry [34]. Specifically, sections (6 μm thick) on glass slides were deparaffinized by incubation in EZ-Dewax (Biogenex, San Roman, California, USA). Slides were then rinsed and processed for antigen retrieval by microwave citrate buffer treatment. After rinsing in Dulbecco's phosphate-buffered saline (D-PBS) (Mediatech, Herndon, Virginia, USA), endogenous peroxide activity was blocked by incubation in hydrogen peroxide. After washing, non-specific binding was blocked and sections were then incubated with primary antibody [anti-CD68 (Dako, Carpinteria, California, USA), anti-CD34 (Biogenex), anti-MCP-1 (Santa Cruz Biotechnology, Santa Cruz, California, USA) or anti-VEGF-A (R&D Systems, Minneapolis, Minnesota, USA)] in a humidified chamber overnight at 4°C. The slides were rinsed and biotinylated secondary antibody [anti-mouse/anti-rabbit IgG (Vector Laboratories, Burlingame, California, USA) for CD68 and CD34; anti-goat IgG (Sigma Chemical, St Louis, Missouri, USA) for MCP-1 and VEGF] was added. Immunoreactivity was detected using the ABC Elite and DAB substrate kits (Vector Laboratories) according to the manufacturer's instructions. A reddish brown precipitate in the cytoplasm indicated a positive reaction. Negative controls used all reagents except the primary antibody.

The intensity of staining for CD68 (TAMs), CD34 (neovascularization), MCP-1 and VEGF-A expression was graded on a scale of 0 to 3+, with 0 representing no detectable stain and 3+ representing the strongest stain. Two independent observers (R.K.S. and M.L.V.) examined each slide using a Nikon E400 microscope (Nikon, Melville, New York, USA); their observations were positively correlated with each other (P<0.05). If the two observers differed in their scoring, a third observer examined the slide. In addition, the macrophage content and vessel density were quantified microscopically with a 5×5 reticle grid (Klarmann Rulings, Litchfield, New Hampshire, USA) at 400×magnification (total area, 250 μm).

Back to Top | Article Outline

Melanoma cell lines with different metastatic potential

A375P (tumorigenic, low-metastatic), A375SM (tumorigenic, high-metastatic) and SBC-2 (non-tumorigenic, non-metastatic) human melanoma cell lines were used [35,36]. A375P and A375SM cell lines were maintained in culture as an adherent monolayer in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1×non-essential amino acids, 2 mM L-glutamine, 1×vitamin solution and 40 μg/ml gentamicin (Mediatech). SBC-2 was maintained in culture as an adherent monolayer in RPMI-1640 with 10% FBS, 2 mM L-glutamine and 40 μg/ml gentamicin.

Back to Top | Article Outline

Messenger RNA (mRNA) analysis

Total cellular RNA was isolated using Trizol reagent according to the manufacturer's protocol. RNA was quantified spectrophotometrically with a Pharmacia LKB spectrophotometer (Uppsala, Sweden). Complementary DNA (cDNA) was prepared by reverse transcription of 2.0 μg of total RNA. Two microlitres of first-strand cDNA (1 : 10 dilution) was amplified by polymerase chain reaction (PCR) cycle sets using a denaturing temperature of 94°C, annealing temperatures optimized for each primer and extension at 72°C. The primers were synthesized by Sigma-Genosys (The Woodlands, Texas, USA) or the UNMC Molecular Biology Core Laboratory (Omaha, Nebraska, USA). The primers used were: MCP-1 forward, 5′-CCC CAG TCA CCT GCT GTT AT-3′; MCP-1 reverse, 5′-GAG TTT GGG TTT GCT TGT CC-3′; M-CSF forward, 5′-CTC TGT CTC CCC TCA TCA GC-3′; M-CSF reverse, 5′-TCC TTG ACA ACT GGG GTC TC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-ACG CAT TTG GTC GTA TTG GG-3′; GAPDH reverse, 5′-TGA TTT TGG AGG GAT CTC GC-3′. PCR fragments were separated on 2% agarose 3 : 1 (Midwest Scientific, St Louis, Missouri, USA) gels containing ethidium bromide (EtBr) (0.5 μg/ml). EtBr-stained cDNA amplicons were visualized and analysed using a gel documentation system (AlphaInnotech, San Leandro, California, USA). For quantitative studies, amplified sequences were analysed and the relative mRNA transcript levels were obtained using an equal number of cells with simultaneous amplification within the linear range and ImageQuant software (Molecular Dynamics, Sunnyvale, California, USA). MCP-1 and M-CSF signals are presented as an expression index: the ratio of each gene-specific transcript to the signal from the housekeeping gene, GAPDH.

Back to Top | Article Outline

Statistical analysis

SPSS for Windows (SPSS Inc., Chicago, Illinois, USA) was used for all statistical calculations. P<0.05 was deemed to be significant. Bivariate correlation analysis was performed using Spearman's rho correlation coefficient for non-parametric distributions. Correlation coefficients range from −1 (a perfect negative relationship) to +1 (a perfect positive relationship). When interpreting these results, no cause–effect conclusions are made as a result of a significant correlation. To assess significance between groups, the Mann–Whitney U-test was used.

Back to Top | Article Outline

Results

TAM infiltration in malignant melanoma

We examined the relationship between the pattern and extent of macrophage infiltration and tumour progression and metastasis by immunostaining melanoma specimens with different Clark's levels and tumours of varying thickness. Consecutive sections stained with all reagents except primary antibody were used as controls to confirm specific antibody staining (Fig. 1a–1c). We observed a differential pattern of immunostaining for TAMs (Fig. 1g–1i). In all the tumours examined, irrespective of grade, the intensity of TAM staining was higher in the periphery of the tumour when compared with the centre. We observed a positive correlation between macrophage staining intensity and Clark's level (r=0.561, P<0.01). In addition, when the tumours were grouped according to thin (≤0.75 mm) or thick (>0.75 mm), an even stronger correlation (r=0.625, P<0.01) was observed between macrophage infiltration and melanoma aggressiveness. We observed a significant difference in overall TAM staining between thin and thick lesions (P<0.001) and thin and metastatic specimens (P<0.001), but observed no difference between thick and metastatic samples.

Fig. 1

Fig. 1

Macrophages in three hot spots (areas of intense staining) were counted using a reticle with a 5×5 mm Chalkley grid under 400×magnification. Correlation analysis revealed a significant positive correlation between TAM count and Clark's level (r=0.548, P<0.001). Similarly, when the tumours were stratified according to thickness, a significant difference (P<0.01) between macrophage density in thin (≤0.75 mm) and thick (>0.75 mm) tumours was observed (Fig. 2a). Furthermore, macrophage infiltration in metastatic lesions was significantly increased when compared with thin but not thick tumours (Fig. 2a).

Fig. 2

Fig. 2

Back to Top | Article Outline

MCP-1 expression in archival malignant melanoma specimens with different aggressiveness

Macrophage infiltration into a tumour is regulated by a number of cytokines and chemokines, in particular MCP-1. We examined the production of MCP-1 in melanoma specimens by immunohistochemistry of serial sections using an anti-MCP-1 antibody and determined the relative staining intensity. MCP-1 staining was observed in the proliferative area of the tumours (Fig. 1d–1f). No immunoreactivity was observed in antibody control sections. A significant negative correlation (r=−0.307, P<0.01) was observed between Clark's level and MCP-1 expression. A negative correlation was observed (r=−0.340, P<0.01) after stratification of the samples according to thin (≤0.75 mm) versus thick (>0.75 mm) melanoma. We did not observe any correlation between TAM intensity and MCP-1 expression.

In addition, Mann–Whitney analysis of the samples indicated a significant difference in MCP-1 expression between thin and thick lesions (P<0.05). Similarly a significant difference was observed between thin and metastatic lesions (P<0.05) (Fig. 2c). However, no significant difference was seen between thick and metastatic lesions (Fig. 2c).

Back to Top | Article Outline

Neovascularization in malignant melanoma specimens

The extent of neovascularization in the melanoma specimens was examined by immunostaining with an anti-CD34 antibody, a marker for vascular endothelial cells (Fig. 3a–3c). Overall blood vessel staining was evaluated and analysis revealed no correlation between neovascularization and Clark's level (r=0.191, P=0.193) or thickness (r=0.155, P=0.294). Similar to macrophage hot spot counting, vessel numbers were counted. There was no correlation between blood vessel density and either Clark's level (r=−0.181, P=0.223) or thickness of the tumour (r=−0.110, P=0.461). In addition, no significant difference was seen between different tumour stages or tumour thicknesses (Fig. 2b). Statistical analysis revealed a negative correlation between the extent of neovascularization in tumours and MCP-1 (r=−0.515, P<0.05) and VEGF (r=−0.415, P<0.05).

Fig. 3

Fig. 3

Back to Top | Article Outline

VEGF-A staining in malignant melanoma

We used consecutive tissue sections stained for TAMs to look for VEGF-A production (Fig. 3d–3f). Statistical analysis of VEGF-A staining revealed no correlation with either Clark's level (r=−0.129, P=0.289) or depth of invasion (r=−0.179, P=0.138). Interestingly, VEGF levels showed a positive correlation with MCP-1 levels (r=0.415, P<0.001). We observed no significant difference in VEGF-A expression between different tumour thicknesses (Fig. 2d).

Back to Top | Article Outline

Expression of MCP-1 and M-CSF mRNA by melanoma cell lines with different metastatic potential and tumour tissue with different Clark's levels

Our immunohistochemical analyses demonstrated a negative correlation between MCP-1 expression and Clark's level. However, we did not observe any correlation between MCP-1 expression and TAM density. M-CSF has been demonstrated to be involved in TAM recruitment, activation and differentiation. The expression of MCP-1 and M-CSF mRNA was examined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in human melanoma specimens and in cell lines with different metastatic potential. All the melanoma cell lines and patient tumour samples analysed expressed MCP-1 and M-CSF mRNA. The expression level of MCP-1 mRNA was highest in Clark's level I tumours (Fig. 4). MCP-1 levels decreased as the Clark level increased. A similar trend was found in MCP-1 expression in melanoma cell lines. SBC-2 cells, which are non-tumorigenic in nude mice, showed the highest levels of MCP-1 mRNA expression (Fig. 4). A375SM, which is an aggressive, metastatic cell line, expressed the lowest levels; A375P cells expressed intermediate levels of MCP-1 mRNA. In contrast, Clark's level IV and A375SM cells expressed higher levels of M-CSF than Clark's level I or SBC-2 cells. Interestingly, when the ratio of MCP-1 to M-CSF was calculated, it was higher in Clark's level I than in Clark's level II, which was higher than Clark's level IV. Similarly, in the human cell lines, the ratio of MCP-1 to M-CSF mRNA for SBC-2 (non-metastatic) was higher than that for A375P (low-metastatic), which was higher than that for A375SM (high-metastatic) (Fig. 4).

Fig. 4

Fig. 4

Back to Top | Article Outline

Discussion

In this study, we have demonstrated that TAM levels in malignant melanoma positively correlate with aggressiveness (i.e. Clark's level or depth of invasion and metastasis). In melanoma, there is an established correlation between thickness and aggressiveness of the tumour [33]. Our results indicate a direct correlation between TAMs and both Clark's level and thickness of the melanoma. We also observed a higher overall TAM density in high Clark's level tumours when compared with low Clark's level tumours. These results are similar to those of earlier reports, suggesting the significance of TAMs in melanoma progression and metastasis [27,37]. The regulation of TAM infiltration is controlled by a number of cytokines and chemokines, including MCP-1 [38,39], M-CSF [40] and VEGF [17]. Both MCP-1 and M-CSF are also important in the activation and differentiation of peripheral blood monocytes into TAMs [41–43]. As MCP-1 induces TAM infiltration, and we detected a direct correlation with melanoma progression, we examined MCP-1 expression and expected it to be upregulated in advanced melanoma.

We observed an inverse correlation between MCP-1 production and melanoma aggressiveness. A significant difference in the level of MCP-1 expression was observed between thin and thick melanoma and metastases. We observed higher levels of MCP-1 in early melanoma (Clark's level I–III) compared with aggressive melanoma (Clark's level IV–V). High levels of MCP-1 have been shown to be associated with the inhibition of tumour growth in multiple tumour systems [44–46]. In contrast, reports examining MCP-1 expression in human mammary carcinomas demonstrated that higher MCP-1 expression was associated with early relapse and poor prognosis [47]. The role of chemokines in melanoma progression and metastasis is complex and not clearly established [48]. Nesbit et al. [49] demonstrated that the biological effects of tumour-derived MCP-1 were biphasic, depending on the level of secretion; specifically, higher levels of MCP-1 were associated with tumour destruction, whereas lower levels of MCP-1 were associated with enhanced neovascularization, tumour growth and metastasis. The presence of a macrophage infiltrate may be important in promoting an anti-tumour response during the early stages of tumour progression when MCP-1 levels are higher due to monocyte recruitment and activation.

Previous reports have suggested a significant role for M-CSF in the regulation of TAMs in more aggressive tumours as they strive to continue the symbiotic host–tumour relationship [50–52]. In the present study, we observed higher levels of M-CSF expression in high-level tumours. In a mouse sarcoma model, Walter et al. [53] found an increase in TAM proliferation, together with a correlation between high levels of M-CSF transcripts and the associated TAMs, which contained high levels of c-fms mRNA, the receptor for M-CSF, suggesting a paracrine interaction between TAMs and sarcomas. A recent study demonstrated that local M-CSF production by primary breast tumours in M-CSF-deficient mice led to the accumulation of TAMs and promoted growth and metastasis [51]. These data suggest that M-CSF expression is positively associated with disease and monocyte recruitment may depend on complex interactions between chemokines and growth factors. Whether these results may be due in part to the recruitment of new monocytes, survival and proliferation of TAMs or production of other macrophage chemoattractants remains to be determined, and a more precise role for M-CSF in TAM accumulation and progression and metastasis in melanoma is currently under investigation.

In malignant melanoma, the significance of angiogenesis as a prognostic indicator remains controversial [8,9,29–31]. Similar to these reports, we observed no correlation between the extent of neovascularization and aggressiveness. As discussed earlier, our data demonstrate a correlation between TAM density and aggressiveness; however, we did not observe a correlation between TAM density and neovascularization. This is in contrast with an earlier report which suggested an increase in the numbers of macrophages and microvessel density with increasing depth of the tumour and with tumour angiogenesis [27]. The observed differences between the two studies may be due to the different classification of melanoma specimens used (Clark's classification in this study versus radial and vertical growth phase in Torisu et al. [27]).

Macrophages have been shown to produce angiogenic factors [18] and tumour-derived factors have been implicated in the induction of VEGF-A and interleukin-8 (IL-8) in macrophages [27,28]. We did not observe any correlation between TAM density and VEGF-A expression. In a report by Goede et al. [39], using a rat corneal assay, it was shown that MCP-1 could induce inflammatory angiogenesis at a level similar to VEGF-A, although the induction was more rapid. These results suggest that MCP-1 may act as an indirect inducer of angiogenesis by recruiting macrophages. Our results suggest higher levels of MCP-1 in the early stages of melanoma progression and a corresponding infiltration of macrophages that continues in advanced disease; therefore, we investigated whether this resulted in a differential expression of angiogenic factors. Our analysis did not show a correlation between VEGF-A expression and Clark's level or tumour thickness. We did not observe any correlation between vascular density or VEGF-A expression and the metastatic potential of melanoma specimens. We did not find a relationship between TAMs and VEGF-A expression. This implies that macrophages may play a multifunctional role by promoting both neovascularization, through the production of angiogenic cytokines, and tumour growth, by the production of paracrine growth factors, such as IL-8, which has been shown to be a macrophage-derived angiogenic factor [18] and a paracrine growth factor for melanoma [7,54]. Therefore, it is possible that another cytokine may facilitate tumour growth and tumour angiogenesis in this model.

We observed a differential TAM immunostaining pattern between tumours of different levels and TAM staining was higher in the periphery than in the centre of the tumour. Several reports have demonstrated that tumour-derived cytokines differentially regulate macrophages in different microenvironments [20,55–57]. For example, cytokine production from proximal and distal macrophages is distinct, similar to the differences seen between M1 (classically activated) and M2 (alternatively activated) macrophages [58–60]. It has been reported that macrophages infiltrating tumours driven by tumour- and T-cell-derived cytokines express a predominantly M2 phenotype [61].

Although no correlation was detected between VEGF-A and tumour stage or depth of invasion, our data indicate that, perhaps like macrophages, VEGF-A may play a multifunctional role in recruiting circulating monocytes and simultaneously promoting angiogenesis through increased vessel survival, vessel co-option or vasculogenic mimicry [62–64]. Our data suggest that perhaps another mechanism of vascularization is taking place in melanoma. In addition, this can be explained by the fact that there are a number of redundant pro-angiogenic factors produced in malignant melanoma.

In summary, our results imply an association between macrophage infiltration and progression in malignant melanoma, and suggest an interaction between MCP-1 and M-CSF in macrophage recruitment. We observed an inverse correlation between MCP-1 production and disease aggressiveness, but no correlation between vascular density or VEGF expression and aggressiveness in melanoma specimens.

Back to Top | Article Outline

Acknowledgement

We thank Mitsuru Futakuchi for reviewing the manuscript.

Back to Top | Article Outline

References

1. Lee JA. Trends in melanoma incidence and mortality. Clin Dermatol 1992; 10:9–13.
2. Lee JE. Factors associated with melanoma incidence and prognosis. Semin Surg Oncol 1996; 12:379–385.
3. Clark WH, Elder DE, Guerry D, Epstein MN, Greene MH, Van Horn M. A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma. Hum Pathol 1984; 15:1147–1165.
4. Clark WH. Tumour progression and the nature of cancer. Br J Cancer 1991; 64:631–644.
5. Franceschi S, La Vecchia C, Lucchini F, Cristofolini M. The epidemiology of cutaneous malignant melanoma: aetiology and European data. Eur J Cancer Prev 1991; 1:9–22.
6. Fidler IJ, Radinsky R. Search for genes that suppress cancer metastasis. J Natl Cancer Inst 1996; 88:1700–1703.
7. Singh RK, Varney ML. IL-8 expression in malignant melanoma: implications in growth and metastasis. Histol Histopathol 2000; 15:843–849.
8. Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, Cascinelli N. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 1996; 77:1303–1310.
9. Mihm MC Jr, Clemente CG, Cascinelli N. Tumor infiltrating lymphocytes in lymph node melanoma metastases: a histopathologic prognostic indicator and an expression of local immune response. Lab Invest 1996; 74:43–47.
10. Menard S, Tomasic G, Casalini P, Balsari A, Pilotti S, Cascinelli N, et al. Lymphoid infiltration as a prognostic variable for early-onset breast carcinomas. Clin Cancer Res 1997; 3:817–819.
11. Setala LP, Kosma VM, Marin S, Lipponen PK, Eskelinen MJ, Syrjanen KJ, et al. Prognostic factors in gastric cancer: the value of vascular invasion, mitotic rate and lymphoplasmacytic infiltration. Br J Cancer 1996; 74:766–772.
12. Ropponen KM, Eskelinen MJ, Lipponen PK, Alhava E, Kosma VM. Prognostic value of tumour-infiltrating lymphocytes (TILs) in colorectal cancer. J Pathol 1997; 182:318–324.
13. Brocker EB, Zwadlo G, Holzmann B, Macher E, Sorg C. Inflammatory cell infiltrates in human melanoma at different stages of tumor progression. Int J Cancer 1988; 41:562–567.
14. Lin EY, Pollard JW. Role of infiltrated leucocytes in tumour growth and spread. Br J Cancer 2004; 90:2053–2058.
15. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004; 4:71–78.
16. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13:265–270.
17. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996; 87:3336–3343.
18. Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992; 258:1798–1801.
19. Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, Nuseir N. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha. Nature 1987; 329:630–632.
20. Mantovani A. Tumor-associated macrophages in neoplastic progression: a paradigm for the in vivo function of chemokines. Lab Invest 1994; 71:5–16.
21. Polverini PJ, Leibovich SJ. Induction of neovascularization in vivo and endothelial proliferation in vitro by tumor-associated macrophages. Lab Invest 1984; 51:635–642.
22. Salvesen HB, Akslen LA. Significance of tumour-associated macrophages, vascular endothelial growth factor and thrombospondin-1 expression for tumour angiogenesis and prognosis in endometrial carcinomas. Int J Cancer 1999; 84:538–543.
23. Shapiro SD. Diverse roles of macrophage matrix metalloproteinases in tissue destruction and tumor growth. Thromb Haemost 1999; 82:846–849.
24. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leuk Biol 1994; 55:410–422.
25. Sunderkotter C, Goebeler M, Schulze-Osthoff K, Bhardwaj R, Sorg C. Macrophage-derived angiogenesis factors. Pharmacol Ther 1991; 51:195–216.
26. Baskic D, Acimovic L, Samardzic G, Vujanovic NL, Arsenijevic NN. Blood monocytes and tumor-associated macrophages in human cancer: differences in activation levels. Neoplasma 2001; 48:169–174.
27. Torisu H, Ono M, Kiryu H, Furue M, Ohmoto Y, Nakayama J, et al. Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and IL-1alpha. Int J Cancer 2000; 85:182–188.
28. Varney ML, Olsen KJ, Mosley RL, Bucana CD, Talmadge JE, Singh RK. Monocyte/macrophage recruitment, activation and differentiation modulate interleukin-8 production: a paracrine role of tumor-associated macrophages in tumor angiogenesis. In Vivo 2002; 16:471–477.
29. Bayer-Garner IB, Hough AJ Jr, Smoller BR. Vascular endothelial growth factor expression in malignant melanoma: prognostic versus diagnostic usefulness. Mod Pathol 1999; 12:770–774.
30. Graham CH, Rivers J, Kerbel RS, Stankiewicz KS, White WL. Extent of vascularization as a prognostic indicator in thin (<0.76 mm) malignant melanomas. Am J Pathol 1994; 145:510–514.
31. Massi D, Franchi A, Borgognoni L, Paglierani M, Reali UM, Santucci M. Tumor angiogenesis as a prognostic factor in thick cutaneous malignant melanoma. A quantitative morphologic analysis. Virchows Arch 2002; 440:22–28.
32. Herlyn M. Human melanoma: development and progression. Cancer Metastasis 1990; 9:101–109.
33. Breslow A. Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg 1970; 172:902–908.
34. Singh RK, Varney ML, Bucana CD, Johansson SL. Expression of interleukin-8 in primary and metastatic malignant melanoma of the skin. Melanoma Res 1999; 9:383–387.
35. Singh RK, Gutman M, Radinsky R, Bucana CD, Fidler IJ. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 1994; 54:3242–3247.
36. Kozlowski JM, Hart IR, Fidler IJ, Hanna N. A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice. J Natl Cancer Inst 1984; 72:913–917.
37. Ono M, Torisu H, Fukushi J, Nishie A, Kuwano M. Biological implications of macrophage infiltration in human tumor angiogenesis. Cancer Chemother Pharmacol 1999; 43(Suppl.):S69–S71.
38. Graves DT, Barnhill R, Galanopoulos T, Antoniades HN. Expression of monocyte chemotactic protein-1 in human melanoma in vivo. Am J Pathol 1992; 140:9–14.
39. Goede V, Brogelli L, Ziche M, Augustin HG. Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer 1999; 82:765–770.
40. Elgert KD, Alleva DG, Mullins DW. Tumor-induced immune dysfunction: the macrophage connection. J Leuk Biol 1998; 64:275–290.
41. Dorsch M, Hock H, Kunzendorf U, Diamantstein T, Blankenstein T. Macrophage colony-stimulating factor gene transfer into tumor cells induces macrophage infiltration but not tumor suppression. Eur J Immunol 1993; 23:186–190.
42. Finnin M, Hamilton JA, Moss ST. Characterization of a CSF-induced proliferating subpopulation of human peripheral blood monocytes by surface marker expression and cytokine production. J Leuk Biol 1999; 66:953–960.
43. Munn DH, Armstrong E. Cytokine regulation of human monocyte differentiation in vitro: the tumor-cytotoxic phenotype induced by macrophage colony-stimulating factor is developmentally regulated by gamma-interferon. Cancer Res 1993; 53:2603–2613.
44. Zhang L, Khayat A, Cheng H, Graves DT. The pattern of monocyte recruitment in tumors is modulated by MCP-1 expression and influences the rate of tumor growth. Lab Invest 1997; 76:579–590.
45. Zhang L, Yoshimura T, Graves DT. Antibody to Mac-1 or monocyte chemoattractant protein-1 inhibits monocyte recruitment and promotes tumor growth. J Immunol 1997; 158:4855–4861.
46. Huang S, Singh RK, Xie K, Gutman M, Berry KK, Bucana CD, et al. Expression of the JE/MCP-1 gene suppresses metastatic potential in murine colon carcinoma cells. Cancer Immunol Immunother 1994; 39:231–238.
47. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, et al. Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 2000; 6:3282–3289.
48. Payne AS, Cornelius LA. The role of chemokines in melanoma tumor growth and metastasis. J Invest Dermatol 2002; 118:915–922.
49. Nesbit M, Schaider H, Miller TH, Herlyn M. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells. J Immunol 2001; 166:6483–6490.
50. Janowska-Wieczorek A, Belch AR, Jacobs A, Bowen D, Padua RA, Paietta E, et al. Increased circulating colony-stimulating factor-1 in patients with preleukemia, leukemia, and lymphoid malignancies. Blood 1991; 77:1796–1803.
51. Lin EY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 2001; 193:727–740.
52. McDermott RS, Deneux L, Mosseri V, Vedrenne J, Clough K, Fourquet A, et al. Circulating macrophage colony stimulating factor as a marker of tumour progression. Eur Cytokine Netw 2002; 13:121–127.
53. Walter S, Govoni D, Bottazzi B, Mantovani A. The role of macrophages in the regulation of primary tumor growth. Pathobiology 1991; 59:239–242.
54. Schadendorf D, Moller A, Algermissen B, Worm M, Sticherling M, Czarnetzki BM. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol 1994; 153:3360.
55. Crowther M, Brown NJ, Bishop ET, Lewis CE. Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leuk Biol 2001; 70:478–490.
56. Elgert KD, Alleva DG, Mullins DW. Tumor-induced immune dysfunction: the macrophage connection. J Leuk Biol 1998; 64:275–290.
57. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13:265–270.
58. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002; 23:549–555.
59. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 2000; 164:6166–6173.
60. Goerdt S, Politz O, Schledzewski K, Birk R, Gratchev A, Guillot P, et al. Alternative versus classical activation of macrophages. Pathobiology 1999; 67:222–226.
61. Mantovani A, Schioppa T, Biswas SK, Marchesi F, Allavena P, Sica A. Tumor-associated macrophages and dendritic cells as prototypic type II polarized myeloid populations. Tumori 2003; 89:459–468.
62. Folberg R, Hendrix MJ, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol 2000; 156:361–381.
63. Hendrix MJ, Seftor EA, Meltzer PS, Gardner LM, Hess AR, Kirschmann DA, et al. Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry. Proc Natl Acad Sci USA 2001; 98:8018–8023.
64. Maniotis AJ, Folberg R, Hess A, Seftor EA, Gardner LM, Pe'er J, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol 1999; 155:739–752.
Keywords:

Angiogenesis; monocyte chemotactic protein-1 (MCP-1); melanoma; monocytes; tumour-associated macrophages; vascular endothelial growth factor

© 2005 Lippincott Williams & Wilkins, Inc.