Antibody combinations for optimized staining of macrophages in human lung tumours

Scand J Immunol. 2020 Jul;92(1):e12889. doi: 10.1111/sji.12889. Epub 2020 May 10.


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.

Keywords: flow cytometry; human; immunohistochemistry; lung cancer; macrophages; tumour.

MeSH terms

  • Antigens, CD / analysis*
  • Antigens, Differentiation, Myelomonocytic / analysis*
  • Biomarkers, Tumor / analysis
  • Carcinoma, Non-Small-Cell Lung / diagnosis*
  • Carcinoma, Non-Small-Cell Lung / immunology
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Cell Adhesion Molecules / analysis
  • Flow Cytometry
  • Humans
  • Immunohistochemistry
  • Lectins, C-Type / analysis
  • Lipopolysaccharide Receptors / analysis*
  • Lung Neoplasms / diagnosis*
  • Lung Neoplasms / immunology
  • Lung Neoplasms / pathology
  • Macrophages, Alveolar / immunology*
  • Mannose-Binding Lectins / analysis
  • Prognosis
  • Receptors, Cell Surface / analysis*
  • Scavenger Receptors, Class A / analysis


  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Biomarkers, Tumor
  • CD14 protein, human
  • CD163 antigen
  • CD68 antigen, human
  • Cell Adhesion Molecules
  • DC-specific ICAM-3 grabbing nonintegrin
  • Lectins, C-Type
  • Lipopolysaccharide Receptors
  • MSR1 protein, human
  • Mannose-Binding Lectins
  • Receptors, Cell Surface
  • Scavenger Receptors, Class A
  • mannose receptor