Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b(+) myelomonocytic cells.
It is well established that cancer cells can recruit CD11b(+) myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor-infiltrating CD11b(+) myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564-1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b(+) myeloid cells isolated from peripheral blood and from tumors, using six-color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor-derived samples concur to generate artifacts in the phenotype of tumor-infiltrating CD11b(+) subpopulations. We show that the heterogeneity of tumor-infiltrating CD11b(+) subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor-infiltrating CD11b(+) cells was particularly sensitive to these parameters compared to circulating CD11b(+) cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor-infiltrating CD11b(+) cell populations.
Kuonen
Francois
F
Division of Experimental Oncology, Centre Pluridiscipliniaire d'Oncologie (CePO), Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne (UNIL), Faculty of Biology and Medicine, Lausanne, Switzerland.
Touvrey
Cedric
C
Laurent
Julien
J
Ruegg
Curzio
C
D000818:Animals; D016477:Artifacts; D001854:Bone Marrow Cells / Q000378:metabolism / Q000473:pathology*; D018845:CD11 Antigens / Q000378:metabolism; D002449:Cell Aggregation; D016923:Cell Death; D045744:Cell Line, Tumor; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016246:Lymphocytes, Tumor-Infiltrating / Q000378:metabolism / Q000473:pathology*; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000378:metabolism / Q000473:pathology*; D011961:Receptors, Fc / Q000378:metabolism*
D018845:CD11 Antigens; D011961:Receptors, Fc