Antibody combinations for optimized staining of macrophages in human lung tumors.

The analysis of tumor-associated macrophages (TAMs) has a high potential to predict cancer recurrence and response to immunotherapy. However, the heterogeneity of TAMs poses a challenge for quantitative and qualitative measurements. Here, we critically evaluated by immunohistochemistry and flow cytometry two commonly used pan-macrophage markers (CD14 and CD68) as well some suggested markers for tumor-promoting M2 macrophages (CD163, CD204, CD206 and CD209) in human non-small cell lung cancer (NSCLC). Tumor, non-cancerous lung tissue, and blood were investigated. For immunohistochemistry, CD68 was confirmed to be a useful pan-macrophage marker although careful selection of antibody was found to be critical. The widely used anti-CD68 antibody clone KP-1 stains both macrophages and neutrophils, which is problematic for TAM quantification because lung tumors contain many neutrophils. For TAM counting in tumor sections, we recommend combined labeling of CD68 with a cell membrane marker such as CD14, CD163, or CD206. In flow cytometry, the commonly used combination of CD14 and HLA-DR was found to not be optimal because some TAMs do not express CD14. Instead, combined staining of CD68 and HLA-DR is preferable to gate all TAMs. Concerning macrophage phenotypic markers, the scavenger receptor CD163 was found to be expressed by a substantial fraction (50-86%) of TAMs with a large patient-to-patient variation. Approximately 50% of TAMs were positive for CD206. Surprisingly, there was no clear overlap between CD163 and CD206 positivity, and three distinct TAM sub-populations were identified in NSCLC tumors: CD163(+) CD206(+) , CD163(+) CD206(-) , and CD163(-) CD206(-) . This work should help develop macrophage-based prognostic tools for cancer.