The identification of macrophage-enriched glycoproteins using glycoproteomics

Prostate cancer is a leading cause of cancer-related deaths of men in the United States. Whereas the localized disease is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Immune cells that primarily scavenge debris and promote prostate cancer angiogenesis and wound repair are M2 macrophages. They are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) and have been reported to associate with solid tumors and aide in proliferation, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. To identify novel surface glycoproteins expressed on M2 macrophages, we developed a novel method of creating homogeneous populations of human macrophages from human CD14⁺ monocytes in vitro. These homogeneous M1 macrophages secrete pro-inflammatory cytokines, and our M2 macrophages secrete antiinflammatory cytokines as well as vascular endothelial growth factor (VEGF). To identify enriched surface glycoproteins, we then performed solid-phase extraction of N-linked glycopeptides followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on our homogeneous macrophage populations. We discovered five novel peptides that are enriched exclusively on human M2 macrophages relative to human M1 macrophages and human CD14⁺ monocytes. Finally, we determined whether these surface glycoproteins, found enriched on M2 macrophages, were also expressed in human metastatic castrate-resistant prostate cancer (mCRPC) tissues. Using mCRPC tissues from rapid autopsies, we were able to determine M2 macrophage infiltration by using immunohistochemistry and flow cytometry. These findings highlight the presence of macrophage infiltration in human mCRPC but also surface glycoproteins that could be used for prognosis of localized disease and for targeting strategies.