See also Figure?S1. Considering that the marker SiglecF is frequently used to discriminate mouse eosinophils from neutrophils (Bochner, 2009; Zhang et?al., 2004) and can also be expressed by lung macrophages (Engblom et?al., 2017), we asked whether SiglecFhigh CD11b+ Ly-6G+ cells in lung tumors define bona fide neutrophils that express SiglecF or other immune cells, such as eosinophils, that upregulate Ly-6G. however, some of these cells fundamental features remain unexplored. Here, we show that tumor-infiltrating CD11b+ Ly-6G+ SiglecFhigh cells are bona fide mature neutrophils and therefore differ from other myeloid cells, including SiglecFhigh eosinophils, SiglecFhigh macrophages, and CD11b+ Ly-6G+ myeloid-derived suppressor cells. We further show that SiglecFhigh neutrophils gradually accumulate in growing tumors, where they can live for several days; this lifespan is in marked contrast to that of their SiglecFlow counterparts and neutrophils in general, which live for several hours only. Together, these findings reveal distinct attributes for tumor-promoting SiglecFhigh neutrophils and help explain their deleterious accumulation in the tumor bed. (but not a and deleted alleles of the tumor suppressor gene (Pfirschke et?al., 2016). Lung adenocarcinoma nodules growing in these mice are infiltrated by myeloid cells, including monocytes, macrophages, and neutrophils (Cortez-Retamozo et?al., 2012, 2013); this myeloid cell infiltrate is also observed in a sizable fraction of human lung adenocarcinomas (Lavin et?al., 2017; Zilionis et?al., 2019). Flow cytometry analysis of mouse lung tumor tissues identified several immune populations, including CD11b+ Ly-6GC SiglecFhigh cells, defined as eosinophils, and CD11b+ Ly-6G+ cells, defined as neutrophil-like cells (Figure?1 A). These two subsets showed distinct side-scatter and forward-scatter profiles as expected (Figures S1A and S1B). Around two-thirds of CD11b+ Ly-6G+ cells were SiglecFlow, as commonly reported for neutrophils, whereas the remaining one-third were SiglecFhigh (Figure?1A). The number of eosinophils, SiglecFlow neutrophils, and SiglecFhigh neutrophils, per milligram tissue, was greater in tumor-bearing compared to tumor-free lungs (Figure?S1C). Open in a separate window Figure?1 SiglecFhigh CD11b+ Ly-6G+ Cells Resemble Neutrophils (A) Cells obtained from lung tissue of KP1.9 tumor-bearing mice (day 29 after intravenous tumor cell injection) were stained by flow cytometry to identify eosinophils (CD11b+ Ly-6G? SiglecF+), SiglecFlow (CD11b+ Ly-6G+ SiglecFlow), and SiglecFhigh neutrophils (CD11b+ Ly-6G+ SiglecFhigh). Representative dot plots are shown (pre-gated on live cells). (B) Suspensions of CD45+ cells for single-cell RNA sequencing were prepared from murine KP1.9 lung tumors (T) (n?= 2) and lung tissue of healthy mice (H) (n?= 2; Engblom et?al., 2017; Zilionis et?al., 2019). Major cell types (neutrophils highlighted in red) were identified by a Bayesian cell classifier TNFRSF4 as reported in Zilionis et?al. (2019), and neutrophils were defined as tumor (T)-based on the expression of genes correlated to SiglecF (Engblom et?al., 2017). The heatmap shows a comparison of the 3 neutrophil subsets and of alveolar macrophages (M?4), monocytes, dendritic cells (DCs), and basophils (rows) to immune profiles defined by the Immgen consortium (columns). (C) Representative histogram (left) and quantification of geometric mean fluorescence intensity (gMFI) followed by fluorescence-minus one (FMO) signal subtraction (right) of CCR3 expression measured by flow cytometry in eosinophils and SiglecFlow and SiglecFhigh neutrophils (day 29 after intravenous tumor cell injection; n?= 4 mice/group). (D) Representative histogram (left) and quantification of delta gMFI (right) of SiglecE expression measured by flow cytometry in eosinophils and SiglecFlow and SiglecFhigh neutrophils (day 29 after intravenous tumor cell injection; n?= 4 mice/group). Data are represented KW-2478 as mean SEM. For comparisons between KW-2478 two groups, Students two-tailed t test was used. ????p?< 0.0001. See also Figure?S1. Considering that the marker SiglecF is frequently used to discriminate mouse eosinophils from neutrophils (Bochner, 2009; Zhang et?al., 2004) and can also be expressed by lung macrophages (Engblom et?al., 2017), we asked whether SiglecFhigh CD11b+ Ly-6G+ cells in lung tumors define bona fide neutrophils that express SiglecF or other immune cells, such as eosinophils, that upregulate Ly-6G. To this end, we first interrogated single-cell transcriptomic data of CD45+ cells from healthy lungs and KP1.9 lung tumors (Zilionis et?al., 2019). We used a single-cell SiglecF expression score (Engblom et?al., 2017) KW-2478 to operationally separate and cells?resembling neutrophils in tumor tissue (T-and T-cells resembling neutrophils in healthy lungs (H-cells to be neutrophils. The average likelihood for being a neutrophil or an eosinophil was 1 KW-2478 and 0, respectively (Figure?1B). As expected, both H-and T-cells also qualified as bona fide neutrophils (Figure?1B). Control myeloid cell types did not show a likelihood to be neutrophils; this included alveolar macrophages, which are referred to as M?4 in Zilionis et?al. (2019) and express SiglecF (Figure?1B). Of note, we could not identify eosinophils in our scRNA-seq datasets, even though the study was performed on total CD45+ cells. It is possible that eosinophils abundant RNase activity degrades mRNA before the reverse transcription KW-2478 reaction (H?m?l?inen et?al., 1999). Eosinophils will also be absent from additional scRNA-seq datasets (Azizi et?al., 2018; Davidson et?al., 2020; Zhang et?al., 2020), further highlighting the difficulty to detect these cells by scRNA-seq. We.