LAUSR

The analysis of tumour‐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 as some suggested markers for tumour‐promoting M2 macrophages (CD163, CD204, CD206 and CD209) in human non–small cell lung cancer (NSCLC). Tumour, 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 tumours contain many neutrophils. For TAM counting in tumour sections, we recommend combined labelling 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 tumours: CD163+CD206+, CD163+CD206− and CD163−CD206−. This work should help develop macrophage‐based prognostic tools for cancer.