Haynes (Duke University). of the PI3K pathway. In human melanoma tissues, tumor-infiltrating macrophages expressing CD7 are present. These melanomas, with CD7-positive inflammatory cell infiltrations, frequently highly express SECTM1, including an N-terminal, soluble form, which can be detected in the sera of metastatic melanoma patients but not in normal sera. Taken together, our data demonstrate that CD7 is present on monocytes and tumor macrophages, and that its ligand, SECTM1, is frequently expressed in corresponding melanoma tissues, possibly acting as a chemoattractant for monocytes to modulate the melanoma microenvironment. Introduction Tumor-associated macrophages (TAMs) are a major component of tumor stroma, and they modulate the tumor microenvironment by increasing tumor initiation and growth, remodeling the extracellular matrix, promoting angiogenesis and suppressing anti-tumor immunity. High numbers of macrophages are associated with a poor prognosis in a variety of cancers, including breast cancer and colon cancer (Qian and Pollard, 2010; Solinas system to differentiate monocytes to macrophages using melanoma-conditioned media (Wang em et al Kif2c /em ., 2012a). Because it has been shown that myeloid-derived suppressor cells (MDSC) express many markers similar to macrophages and share many similar functions with tumor-associated macrophages in human tumors (Nagaraj and Gabrilovich, 2010), we further characterized MCMI/M? to exclude the possibility of MDSC contamination. We found that MCMI/M? express CD16 and HLA-DR, two markers that are negative for MDSC (Figure 1a). MCMI/M? also express a late-stage macrophage marker (Figure 1b). Together, AX20017 these AX20017 data and our previous work indicate that MCMI/? are highly similar to the tumor-associated macrophages. Open in a separate window Figure 1 Expression of CD7 by monocytes and macrophagesExpression of CD16, HLA DR (a) and the late stage maker (b) was analyzed by flow cytometry. 1205Lu-M?) were stained with the fluorescence-conjugated anti-CD16, HLA DR and late stage macrophage marker. (c) Real-time PCR was used to analyze the expression of CD7 in monocytes, GM-CSF, M-CSF differentiated macrophages, and C8161 (“type”:”entrez-nucleotide”,”attrs”:”text”:”C68161″,”term_id”:”2427091″,”term_text”:”C68161″C68161-M?) and 1205Lu (1205Lu-M?) melanoma conditioned media differentiated M?. Samples were normalized to GAPDH. Monocytes (d) M-CSF/M?, M-CSF/M(|), C8161/M?) and 1205Lu/M?) (e) were stained with the anti-human CD7 monoclonal antibody, AX20017 3A1, following by FITC-conjugated anti-mouse IgG staining for FACS analysis. Filled: Isotype control, black line: anti-CD7. (f) Western blot analysis expression of CD7 in monocytes, GM-CSF/M?), M-CSF/M?, and C8161/M? and 1205Lu/M?. RAb11 was used as a loading control. To confirm the expression of CD7 in monocytes and macrophages, we performed real-time PCR for CD7 on monocytes, MCMI/M?, M-CSF/M? and GM-CSF/M? (Hume and MacDonald, 2012). We found that mRNA levels for CD7 were expressed at a high level in monocytes, while expression of CD7 was expressed at a lower level in M-CSF/M? and in MCMI/M?. A much lower level of CD7 expression was detected in GM-CSF/M? (Figure 1c). Next, we examined the expression of CD7 at the protein level in monocytes, M-CSF/M?, GM-CSF/M? and in MCMI-M? by flow cytometric analysis with the anti-CD7 antibody, 3A1, which also was used to stain for cell surface expression of CD7 in T cells. Corresponding to the RNA expression studies, CD7 was also expressed in monocytes (Figure 1d), M-CSF/M?, C8161/M? and 1205Lu/M?, but not in GM-CSF/M? (Figure 1e). Finally, we conducted western blot analysis with a novel rabbit anti-human CD7 monoclonal antibody, which recognizes the C-terminal 25 amino acids of the CD7 molecule. This peptide was used to produce the antibody because it contains no homologous sequence in other human molecules, and staining of cells known to be negative for CD7 expression by RT-PCR supports the specificity of this antibody (data not shown). Consistent with the flow AX20017 cytometric analysis results, western blot analysis of purified monocytes and macrophages with this antibody revealed an anticipated 40 kDa band, with the highest level of CD7 expression in monocytes and with lowest levels in GM-CSF/M? (Figure 1e). Despite the widespread use of anti-CD7 antibodies, there has not been a definitive study demonstrating CD7 expression on monocytes and macrophages. In order to better understand this, we used several other commercially available CD7 monoclonal antibodies to look for the expression of CD7 on these cell types and, contrary to our results described above, these antibodies did not detect the expression of CD7 in monocytes. It is possible that those antibodies recognize different epitopes or have a lower affinity than the CD7 antibodies used in this study. However, similar differences in results with different anti-CD7 antibodies were also seen in studies to detect the expression of CD7 in T cells (Haynes, 1981). It is also possible that different epitopes of CD7 are present in monocytes compared to T cells due to modification(s) or clustering with other molecules. For example, an anti-CD7 monoclonal antibody, clone 3D9, does not recognize intestinal intraepithelial lymphocytes (IEL), whereas most other anti-CD7 antibodies recognize them (Russell.