Abstract
Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206low macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.
Highlights
Macrophages show a remarkable degree of plasticity and exert diverse functions, depending on the microenvironmental stimuli [1]
To explore whether tumor-released mesothelin could bind to macrophages, frozen cells isolated from human ascites (n = 6) or from solid tumors (n = 8) of ovarian cancer patients, as well as healthy donor monocytes (n = 12), were stained with anti-Epcam, anti-CD45, anti-CD14, anti-CD206, anti-mesothelin (K1) monoclonal antibodies (mAb) and 7-AAD
None of the healthy donor CD45+CD14+ cells expressed CD206 or bound to anti-mesothelin K1 antibody (Fig. 1A lower panels and Fig. 1B). These results were the first evidence that soluble mesothelin could bind to ascites-infiltrating CD206high macrophages from ovarian cancer patients and to some tumor-infiltrating CD206low macrophages
Summary
Macrophages show a remarkable degree of plasticity and exert diverse functions, depending on the microenvironmental stimuli [1]. Tumor polarization of macrophages represents an essential immune escape mechanism that results in a hampered innate immune response leading to a poor adaptive immunity [3,4]. TAMs constitute a predominant cell population of the tumor microenvironment and are correlated with poor clinical outcome [9]. Mouse studies suggest a critical role for CSF-1 in attracting monocytes at the tumor site [1], while cytokine imbalance in favour of IL-10 and TGF-b in the microenvironment could foster immunosuppression and polarize macrophages to elicit pro-tumoral functions [10]. Hagemann and colleagues have proposed that macrophage differentiation towards TAMs involves a ‘‘chemical conversation’’ via exchange of soluble extracellular mediators between ovarian tumor cells and macrophages [11,12]
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