Tumor-Associated Macrophages in the Cutaneous SCC Microenvironment Are Heterogeneously Activated

Abstract

Tumor-associated macrophages (TAMs) may have an important role in tumor immunity. We studied the activation state of TAMs in cutaneous SCC, the second most common human cancer. CD163 was identified as a more abundant, sensitive, and accurate marker of TAMs when compared with CD68. CD163+ TAMs produced protumoral factors, matrix metalloproteinases 9 and 11 (MMP9 and MMP11), at the gene and protein levels. Gene set enrichment analysis (GSEA) was used to evaluate M1 and M2 macrophage gene sets in the SCC genes and to identify candidate genes in order to phenotypically characterize TAMs. There was coexpression of CD163 and alternatively activated “M2” markers, CD209 and CCL18 (chemokine (C-C motif) ligand 18). There was enrichment for classically activated “M1” genes in SCC, which was confirmed in situ by colocalization of CD163 and phosphorylated STAT1 (signal transducer and activator of transcription 1), IL-23p19, IL-12/IL-23p40, and CD127. Also, a subset of TAMs in SCC was bi-activated as CD163+ cells expressed markers for both M1 and M2, shown by triple-label immunofluorescence. These data support heterogeneous activation states of TAMs in SCC, and suggest that a dynamic model of macrophage activation would be more useful to characterize TAMs. Tumor-associated macrophages (TAMs) may have an important role in tumor immunity. We studied the activation state of TAMs in cutaneous SCC, the second most common human cancer. CD163 was identified as a more abundant, sensitive, and accurate marker of TAMs when compared with CD68. CD163+ TAMs produced protumoral factors, matrix metalloproteinases 9 and 11 (MMP9 and MMP11), at the gene and protein levels. Gene set enrichment analysis (GSEA) was used to evaluate M1 and M2 macrophage gene sets in the SCC genes and to identify candidate genes in order to phenotypically characterize TAMs. There was coexpression of CD163 and alternatively activated “M2” markers, CD209 and CCL18 (chemokine (C-C motif) ligand 18). There was enrichment for classically activated “M1” genes in SCC, which was confirmed in situ by colocalization of CD163 and phosphorylated STAT1 (signal transducer and activator of transcription 1), IL-23p19, IL-12/IL-23p40, and CD127. Also, a subset of TAMs in SCC was bi-activated as CD163+ cells expressed markers for both M1 and M2, shown by triple-label immunofluorescence. These data support heterogeneous activation states of TAMs in SCC, and suggest that a dynamic model of macrophage activation would be more useful to characterize TAMs. chemokine (C-C motif) ligand 18 dendritic cell gene set enrichment analysis matrix metalloproteinase reverse transcriptase-PCR squamous cell carcinoma signal transducer and activator of transcription tumor-associated macrophage Cutaneous squamous cell carcinoma (SCC) is the second most common human cancer, affecting >300,000 individuals in the United States annually (Weinberg et al., 2007Weinberg A.S. Ogle C.A. Shim E.K. Metastatic cutaneous squamous cell carcinoma: an update.Dermatol Surg. 2007; 33: 885-899Crossref PubMed Scopus (138) Google Scholar; Brantsch et al., 2008Brantsch K.D. Meisner C. Schonfisch B. et al.Analysis of risk factors determining prognosis of cutaneous squamous-cell carcinoma: a prospective study.Lancet Oncol. 2008; 9: 713-720Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Although most cases can be treated successfully by surgical removal, certain aggressive cases can cause extensive tissue destruction and metastasize to local lymph nodes and distant organs. These aggressive cases are responsible for ∼10,000 non-melanoma skin cancer deaths in the United States each year. Aggressive behavior by SCC is observed in solid organ-transplant recipients (Carucci, 2004Carucci J.A. Cutaneous oncology in organ transplant recipients: meeting the challenge of squamous cell carcinoma.J Invest Dermatol. 2004; 123: 809-816Crossref PubMed Scopus (19) Google Scholar). Based on the potential for the host immunity to regulate tumor behavior in SCC, it is important to characterize the tumor-associated immune microenvironment. Macrophages are one of the major populations of tumor-infiltrating leukocytes associated with solid tumors (Gordon and Taylor, 2005Gordon S. Taylor P.R. Monocyte and macrophage heterogeneity.Nat Rev Immunol. 2005; 5: 953-964Crossref PubMed Scopus (3527) Google Scholar). Macrophages that infiltrate and surround tumor nodules are defined as tumor-associated macrophages (TAMs) (Wang et al., 2010Wang Y.C. He F. Feng F. et al.Notch signaling determines the M1 versus M2 polarization of macrophages in antitumor immune responses.Cancer Res. 2010; 70: 4840-4849Crossref PubMed Scopus (278) Google Scholar), and different studies have shown that macrophages may either inhibit or stimulate tumor growth. Initially, TAMs were shown to participate in the early eradication of tumor cells in vitro (Romieu-Mourez et al., 2006Romieu-Mourez R. Solis M. Nardin A. et al.Distinct roles for IFN regulatory factor (IRF)-3 and IRF-7 in the activation of antitumor properties of human macrophages.Cancer Res. 2006; 66: 10576-10585Crossref PubMed Scopus (61) Google Scholar). However, other studies have suggested that TAMs may contribute to carcinogenesis, as there is a positive correlation between increased numbers of TAMs and poor prognosis in some human cancers (Leek et al., 1996Leek R.D. Lewis C.E. Whitehouse R. et al.Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.Cancer Res. 1996; 56: 4625-4629PubMed Google Scholar; Bingle et al., 2002Bingle L. Brown N.J. Lewis C.E. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.J Pathol. 2002; 196: 254-265Crossref PubMed Scopus (1442) Google Scholar; Sica et al., 2006Sica A. Schioppa T. Mantovani A. et al.Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy.Eur J Cancer. 2006; 42: 717-727Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar; Lin and Pollard, 2007Lin E.Y. Pollard J.W. Tumor-associated macrophages press the angiogenic switch in breast cancer.Cancer Res. 2007; 67: 5064-5066Crossref PubMed Scopus (308) Google Scholar; Shabo et al., 2008Shabo I. Stal O. Olsson H. et al.Breast cancer expression of CD163, a macrophage scavenger receptor, is related to early distant recurrence and reduced patient survival.Int J Cancer. 2008; 123: 780-786Crossref PubMed Scopus (151) Google Scholar; El-Rouby, 2010El-Rouby D.H. Association of macrophages with angiogenesis in oral verrucous and squamous cell carcinomas.J Oral Pathol Med. 2010; 39: 559-564Crossref PubMed Scopus (40) Google Scholar; Nonomura et al., 2010Nonomura N. Takayama H. Kawashima A. et al.Decreased infiltration of macrophage scavenger receptor-positive cells in initial negative biopsy specimens is correlated with positive repeat biopsies of the prostate.Cancer Sci. 2010; 101: 1570-1573Crossref PubMed Scopus (18) Google Scholar; Steidl et al., 2010Steidl C. Lee T. Shah S.P. et al.Tumor-associated macrophages and survival in classic Hodgkin's lymphoma.N Engl J Med. 2010; 362: 875-885Crossref PubMed Scopus (904) Google Scholar). TAMs can fail to recognize tumor antigens (Fadok et al., 1998Fadok V.A. Bratton D.L. Konowal A. et al.Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.J Clin Invest. 1998; 101: 890-898Crossref PubMed Scopus (2405) Google Scholar) and may release factors that directly stimulate tumor growth and angiogenesis (Lin et al., 2006Lin E.Y. Li J.F. Gnatovskiy L. et al.Macrophages regulate the angiogenic switch in a mouse model of breast cancer.Cancer Res. 2006; 66: 11238-11246Crossref PubMed Scopus (737) Google Scholar; Lin and Pollard, 2007Lin E.Y. Pollard J.W. Tumor-associated macrophages press the angiogenic switch in breast cancer.Cancer Res. 2007; 67: 5064-5066Crossref PubMed Scopus (308) Google Scholar). Furthermore, the tumor itself can create a dynamic microenvironment that can transform TAMs (Gocheva et al., 2010Gocheva V. Wang H.W. Gadea B.B. et al.IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion.Genes Dev. 2010; 24: 241-255Crossref PubMed Scopus (455) Google Scholar). Thus, TAMs in the SCC microenvironment may be associated with tumor growth. Currently, the general classification of macrophage activation parallels the Th1/Th2 paradigm, defining classically activated (M1) and alternatively activated (M2) cells (Mantovani et al., 2004Mantovani A. Sica A. Sozzani S. et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol. 2004; 25: 677-686Abstract Full Text Full Text PDF PubMed Scopus (3923) Google Scholar; Mosser and Edwards, 2008Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat Rev Immunol. 2008; 8: 958-969Crossref PubMed Scopus (5363) Google Scholar). Classically activated macrophages are induced by IFN-γ and have a high capacity to present antigen. Alternatively activated macrophages are induced by the cytokine IL-4, which promotes type 2 responses. As SCCs usually progress, in association with a Th2 microenvironment and low levels of IFN-γ, it is thought that the net immune response is ineffective at suppressing tumor growth. Hence, TAMs have commonly been considered alternatively activated or strongly skewed to the M2 phenotype (Biswas et al., 2006Biswas S.K. Gangi L. Paul S. et al.A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation).Blood. 2006; 107: 2112-2122Crossref PubMed Scopus (500) Google Scholar; Martinez et al., 2009Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (1865) Google Scholar; Siveen and Kuttan, 2009Siveen K.S. Kuttan G. Role of macrophages in tumour progression.Immunol Lett. 2009; 123: 97-102Crossref PubMed Scopus (261) Google Scholar; Gordon and Martinez, 2010Gordon S. Martinez F.O. Alternative activation of macrophages: mechanism and functions.Immunity. 2010; 32: 593-604Abstract Full Text Full Text PDF PubMed Scopus (2421) Google Scholar). However, there has been renewed debate over the phenotypic activation of TAMs as the physiology of these macrophages has been shown to change over time and to demonstrate remarkable plasticity (Mosser and Edwards, 2008Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat Rev Immunol. 2008; 8: 958-969Crossref PubMed Scopus (5363) Google Scholar). Given the importance of TAMs contributing to tumor growth, and the current conflicting state of the understanding of TAM activation, we set out to phenotypically characterize macrophages in SCC. Initially, we used a nonbiased genomic approach to guide our choice of markers to further evaluate TAMs. “M1” and “M2” activated macrophage gene sets (Fuentes-Duculan et al., 2010Fuentes-Duculan J. Suarez-Farinas M. Zaba L.C. et al.A subpopulation of CD163-positive macrophages is classically activated in psoriasis.J Invest Dermatol. 2010; 130: 2412-2422Crossref PubMed Scopus (185) Google Scholar) were analyzed in our SCC genomic phenotype (Haider et al., 2006Haider A.S. Peters S.B. Kaporis H. et al.Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia.J Invest Dermatol. 2006; 126: 869-881Crossref PubMed Scopus (147) Google Scholar) using gene set enrichment analysis (GSEA) (Subramanian et al., 2005Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (20148) Google Scholar; Bluth et al., 2009Bluth M.J. Zaba L.C. Moussai D. et al.Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation.J Invest Dermatol. 2009; 129: 2451-2462Crossref PubMed Scopus (50) Google Scholar). We then identified candidate genes that were expressed in the macrophage and SCC gene sets and performed immunofluorescence on SCCs versus CD163, as our constitutive macrophage marker. Previously, we have shown that in normal skin and psoriasis, CD163 is the most useful marker of dermal macrophages (Zaba et al., 2007Zaba L.C. Fuentes-Duculan J. Steinman R.M. et al.Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.J Clin Invest. 2007; 117: 2517-2525Crossref PubMed Scopus (235) Google Scholar). We expanded on that work to characterize TAMs in human SCC. We found the following: (1) compared with CD68, CD163 was a more abundant, sensitive, and accurate marker of TAMs; (2) there was an increase in the protumoral factors matrix metalloproteinases 9 and 11 (MMP9 and MMP11) in SCC, and CD163+ TAMs produced MMP9 and MMP11; (3) there was coexpression of CD163 and alternatively activated “M2” markers, CD209 and CCL18 (chemokine (C-C motif) ligand 18); (4) there was enrichment for classically activated “M1” genes in SCC, which was confirmed in situ by colocalization of CD163 and phosphorylated STAT1 (STAT1p), IL-23p19, IL-12/IL-23p40, and CD127; and (5) a subset of TAMs in SCC was bi-activated as CD163+ cells expressed markers for both M1 and M2, shown by triple-label immunofluorescence. These data support heterogeneous activation states of TAMs in SCC, and suggest that a dynamic model of macrophage activation would be more useful to characterize TAMs. Furthermore, driving TAM activation toward a more dominant anticancer phenotype might be a potential therapeutic strategy. Macrophages were quantified in SCC and normal skin (n=8–18) using CD163, which we consider a reliable marker of macrophages in normal skin and psoriasis, and CD68, the widely accepted macrophage marker (Zaba et al., 2007Zaba L.C. Fuentes-Duculan J. Steinman R.M. et al.Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.J Clin Invest. 2007; 117: 2517-2525Crossref PubMed Scopus (235) Google Scholar; Bluth et al., 2009Bluth M.J. Zaba L.C. Moussai D. et al.Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation.J Invest Dermatol. 2009; 129: 2451-2462Crossref PubMed Scopus (50) Google Scholar; Fuentes-Duculan et al., 2010Fuentes-Duculan J. Suarez-Farinas M. Zaba L.C. et al.A subpopulation of CD163-positive macrophages is classically activated in psoriasis.J Invest Dermatol. 2010; 130: 2412-2422Crossref PubMed Scopus (185) Google Scholar). Representative immunohistochemistry is shown for CD163 and CD68, and cell counts of the cases are presented (Figure 1a and b). The vast majority of CD163+ and CD68+ macrophages were surrounding, rather than infiltrating, the SCC tumor nests, and both CD163+ and CD68+ macrophages were significantly increased, ∼2-fold, in SCC compared with normal skin (P<0.001 for both). Additionally, using double-label immunofluorescence, CD163 colocalized with CD68 but there were CD163+ cells that did not coexpress CD68, suggesting that CD163 is a more robust and sensitive marker of macrophages in the skin than CD68 (Figure 1c). We also evaluated the coexpression of CD163 with the well-known dendritic cell (DC) marker, CD11c (Bluth et al., 2009Bluth M.J. Zaba L.C. Moussai D. et al.Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation.J Invest Dermatol. 2009; 129: 2451-2462Crossref PubMed Scopus (50) Google Scholar). As we have previously shown in normal skin (Zaba et al., 2007Zaba L.C. Fuentes-Duculan J. Steinman R.M. et al.Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.J Clin Invest. 2007; 117: 2517-2525Crossref PubMed Scopus (235) Google Scholar), CD163+ cells in SCC also did not colocalize with this DC marker (Figure 1d), demonstrating that these are two distinct leukocyte populations. In contrast, CD68+ cells close to SCC tumor nests did show colocalization with CD11c (Figure 1e). TAMs may produce factors that encourage tumorigenesis. We have shown that SCC TAMs produce the prolymphangiogenic factor vascular endothelial growth factor-C, which favors tumor growth and development (Moussai et al., 2011Moussai D. Mitsui H. Pettersen J.S. et al.The human cutaneous squamous cell carcinoma microenvironment is characterized by increased lymphatic density and enhanced expression of macrophage-derived VEGF-C.J Invest Dermatol. 2011; 131: 229-236Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). MMPs, the enzymes that may contribute to tumor invasion by degrading the matrix surrounding tumor nodules, may also be produced by TAMs in SCC. MMP1, MMP10, and MMP13 genes have been shown to be upregulated in SCC (Haider et al., 2006Haider A.S. Peters S.B. Kaporis H. et al.Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia.J Invest Dermatol. 2006; 126: 869-881Crossref PubMed Scopus (147) Google Scholar). MMP9 (gelatinase B) and MMP11 (stromelysin-3) proteins correlate with increased tumor aggressiveness (Pinto et al., 2003Pinto C.A. Carvalho P.E. Antonangelo L. et al.Morphometric evaluation of tumor matrix metalloproteinase 9 predicts survival after surgical resection of adenocarcinoma of the lung.Clin Cancer Res. 2003; 9: 3098-3104PubMed Google Scholar; Buergy et al., 2009Buergy D. Weber T. Maurer G.D. et al.Urokinase receptor, MMP-1 and MMP-9 are markers to differentiate prognosis, adenoma and carcinoma in thyroid malignancies.Int J Cancer. 2009; 125: 894-901Crossref PubMed Scopus (45) Google Scholar; Shah et al., 2010Shah S.A. Spinale F.G. Ikonomidis J.S. et al.Differential matrix metalloproteinase levels in adenocarcinoma and squamous cell carcinoma of the lung.J Thorac Cardiovasc Surg. 2010; 139: 984-990Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; Steidl et al., 2010Steidl C. Lee T. Shah S.P. et al.Tumor-associated macrophages and survival in classic Hodgkin's lymphoma.N Engl J Med. 2010; 362: 875-885Crossref PubMed Scopus (904) Google Scholar; Zhao et al., 2010Zhao Z.S. Chu Y.Q. Ye Z.Y. et al.Overexpression of matrix metalloproteinase 11 in human gastric carcinoma and its clinicopathologic significance.Hum Pathol. 2010; 41: 686-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Neither MMP9 nor MMP11 have been classified as products representative of either state of macrophage activation. We showed that there was increased gene expression by reverse transcriptase-PCR (RT-PCR) of MMP9 in SCC compared with adjacent nontumoral skin and normal skin (P=0.07 and 0.008, respectively, Figure 2a). Similarly, MMP11 was also increased in SCC compared with adjacent nontumoral skin and normal skin (P=0.003 and 0.025, respectively, Figure 2b). We then asked whether TAMs could be a possible source of the increased MMP9 and MMP11. There was abundant colocalization of MMP9 and MMP11 with CD163+ macrophages in SCCs compared with normal skin (Figure 2c and d). These findings suggest that TAMs secrete protumoral products in the SCC microenvironment. We evaluated expression of STAT6p based on the association of Th2 cells and the tumor microenvironment (Todaro et al., 2008Todaro M. Lombardo Y. Francipane M.G. et al.Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.Cell Death Differ. 2008; 15: 762-772Crossref PubMed Scopus (154) Google Scholar; de Oliveira et al., 2009de Oliveira M.V. Fraga C.A. Gomez R.S. et al.Immunohistochemical expression of interleukin-4, -6, -8, and -12 in inflammatory cells in surrounding invasive front of oral squamous cell carcinoma.Head Neck. 2009; 31: 1439-1446Crossref PubMed Scopus (19) Google Scholar). STAT6 has an important role in signaling pathways that lead to the differentiation of Th2 cells, and STAT6p translocates to the nucleus in IL-4-activated cells (Takeda et al., 1996Takeda K. Tanaka T. Shi W. et al.Essential role of Stat6 in IL-4 signalling.Nature. 1996; 380: 627-630Crossref PubMed Scopus (1220) Google Scholar; Forbes et al., 2010Forbes E. van Panhuys N. Min B. et al.Differential requirements for IL-4/STAT6 signalling in CD4 T-cell fate determination and Th2-immune effector responses.Immunol Cell Biol. 2010; 88: 240-243Crossref PubMed Scopus (28) Google Scholar). STAT6p colocalization with CD163 was abundant in the inflammatory infiltrate associated with SCC compared with normal skin (Figure 3a), suggesting the presence of IL-4 activation in TAMs. To further evaluate the tumor microenvironment, we used M1 and M2 gene sets to correlate with the SCC genomic phenotype using GSEA (Subramanian et al., 2005Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (20148) Google Scholar; Lamb et al., 2006Lamb J. Crawford E.D. Peck D. et al.The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3008) Google Scholar; Bluth et al., 2009Bluth M.J. Zaba L.C. Moussai D. et al.Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation.J Invest Dermatol. 2009; 129: 2451-2462Crossref PubMed Scopus (50) Google Scholar). We have used this approach previously (Bluth et al., 2009Bluth M.J. Zaba L.C. Moussai D. et al.Myeloid dendritic cells from human cutaneous squamous cell carcinoma are poor stimulators of T-cell proliferation.J Invest Dermatol. 2009; 129: 2451-2462Crossref PubMed Scopus (50) Google Scholar), and have described it thoroughly in a previous publication (Suarez-Farinas et al., 2010Suarez-Farinas M. Lowes M.A. Zaba L.C. et al.Evaluation of the psoriasis transcriptome across different studies by gene set enrichment analysis (GSEA).PLoS One. 2010; 5: e10247Crossref PubMed Scopus (115) Google Scholar). Fuentes-Duculan et al., 2010Fuentes-Duculan J. Suarez-Farinas M. Zaba L.C. et al.A subpopulation of CD163-positive macrophages is classically activated in psoriasis.J Invest Dermatol. 2010; 130: 2412-2422Crossref PubMed Scopus (185) Google Scholar) recently published these sets of genes defining “M1” macrophages, induced with IFN-γ, and “M2” macrophages, induced with IL-4, compared with control. We hypothesized that there should be greater expression of M2 macrophage genes in the SCC genomic phenotype (Martinez et al., 2009Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (1865) Google Scholar; Siveen and Kuttan, 2009Siveen K.S. Kuttan G. Role of macrophages in tumour progression.Immunol Lett. 2009; 123: 97-102Crossref PubMed Scopus (261) Google Scholar), defined by the SCC versus normal skin genes (Subramanian et al., 2007Subramanian A. Kuehn H. Gould J. et al.GSEA-P: a desktop application for gene set enrichment analysis.Bioinformatics. 2007; 23: 3251-3253Crossref PubMed Scopus (696) Google Scholar). However, the M2 gene set was not significantly enriched in SCC genomic phenotype, which may reflect that the M2 gene set is similarly expressed in both SCC and normal skin. Despite the lack of enrichment of M2 genes in the SCC transcriptome, some published M2 genes (Martinez et al., 2006Martinez F.O. Gordon S. Locati M. et al.Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression.J Immunol. 2006; 177: 7303-7311Crossref PubMed Scopus (1563) Google Scholar) were upregulated in our M2 gene set (Table 1), including CD209 (DC-SIGN) (Soilleux et al., 2002Soilleux E.J. Morris L.S. Leslie G. et al.Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro.J Leukoc Biol. 2002; 71: 445-457PubMed Google Scholar; Puig-Kroger et al., 2004Puig-Kroger A. Serrano-Gomez D. Caparros E. et al.Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages.J Biol Chem. 2004; 279: 25680-25688Crossref PubMed Scopus (83) Google Scholar), CCL17 (Bonecchi et al., 1998Bonecchi R. Sozzani S. Stine J.T. et al.Divergent effects of interleukin-4 and interferon-gamma on macrophage-derived chemokine production: an amplification circuit of polarized T helper 2 responses.Blood. 1998; 92: 2668-2671PubMed Google Scholar; Katakura et al., 2004Katakura T. Miyazaki M. Kobayashi M. et al.CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages.J Immunol. 2004; 172: 1407-1413Crossref PubMed Scopus (130) Google Scholar), and CCL18 (Kodelja et al., 1998Kodelja V. Muller C. Politz O. et al.Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern.J Immunol. 1998; 160: 1411-1418PubMed Google Scholar; Mantovani et al., 2004Mantovani A. Sica A. Sozzani S. et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol. 2004; 25: 677-686Abstract Full Text Full Text PDF PubMed Scopus (3923) Google Scholar; Martinez et al., 2006Martinez F.O. Gordon S. Locati M. et al.Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression.J Immunol. 2006; 177: 7303-7311Crossref PubMed Scopus (1563) Google Scholar; Gustafsson et al., 2008Gustafsson C. Mjosberg J. Matussek A. et al.Gene expression profiling of human decidual macrophages: evidence for immunosuppressive phenotype.PLoS One. 2008; 3: e2078Crossref PubMed Scopus (250) Google Scholar; Kwan et al., 2008Kwan W.H. Navarro-Sanchez E. Dumortier H. et al.Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth.PLoS Negl Trop Dis. 2008; 2: e311Crossref PubMed Scopus (41) Google Scholar). By double-label immunofluorescence, CD163+ cells colocalized with CD209 and CCL18 in SCC and to a lesser extent in normal skin (Figure 3b and c). In the SCC microenvironment, we have thus shown that TAMs expressed some surface markers (CD209) and chemokines (CCL18) of M2-type macrophages. In addition and consistent with previous findings (Schutyser et al., 2005Schutyser E. Richmond A. Van Damme J. Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes.J Leukoc Biol. 2005; 78: 14-26Crossref PubMed Scopus (180) Google Scholar), we showed that in normal skin, macrophages at steady state are in an alternatively activated state.Table 1Representative upregulated genes in the M1 and M2 macrophage gene sets1Fuentes-Duculan et al., 2010. and the SCC transcriptome2Haider et al., 2006.Representative genes3Genes representative of M1 and M2 macrophages were selected and their fold change in the M1 and M2 gene sets and the SCC transcriptome are listed.M1 gene setM2 gene setSCC transcriptome6SCC versus normal skin transcriptome, fold change and false discovery rate (FDR) <0.05.M1 STAT18.515Genes not differentially expressed in the M2 gene set compared with control.2.47 Mx-119.035Genes not differentially expressed in the M2 gene set compared with control.2.89 IL-23p193.105Genes not differentially expressed in the M2 gene set compared with control.2.34 IL-12/IL-23p403.365Genes not differentially expressed in the M2 gene set compared with control.1.11 CD1274.445Genes not differentially expressed in the M2 gene set compared with control.7HU95 chip did not include this probe.M2 CD2094Genes not differentially expressed in the M1 gene set compared with control.9.257HU95 chip did not include this probe. CCL184Genes not differentially expressed in the M1 gene set compared with control.3.433.558FDR >0.05. CCL174Genes not differentially expressed in the M1 gene set compared with control.2.581.43Abbreviations: CCL18, chemokine (C-C motif) ligand 18; Mx-1, myxovirus resistance 1; SCC, squamous cell carcinoma; STAT1, signal transducer and activator of transcription 1.1 Fuentes-Duculan et al., 2010Fuentes-Duculan J. Suarez-Farinas M. Zaba L.C. et al.A subpopulation of CD163-positive macrophages is classically activated in psoriasis.J Invest Dermatol. 2010; 130: 2412-2422Crossref PubMed Scopus (185) Google Scholar.2 Haider et al., 2006Haider A.S. Peters S.B. Kaporis H. et al.Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia.J Invest Dermatol. 2006; 126: 869-881Crossref PubMed Scopus (147) Google Scholar.3 Genes representative of M1 and M2 macrophages were selected and their fold change in the M1 and M2 gene sets and the SCC transcriptome are listed.4 Genes not differentially expressed in the M1 gene set compared with control.5 Genes not differentially expressed in the M2 gene set compared with control.6 SCC versus normal skin transcriptome, fold change and false discovery rate (FDR) <0.05.7 HU95 chip did not include this probe.8 FDR >0.05. Open table in a new tab Abbreviations: CCL18, chemokine (C-C motif) ligand 18; Mx-1, myxov