Fig. 3

Tumor-infiltrating immune cells produce TNFα within implanted osteosarcomas in mice. a Disaggregated unfixed 1029H-Luc subcutaneous tumor cells were phenotyped by flow cytometry. mCherry-expressing cells were designated osteosarcoma cells; these lacked markers for myeloid and NK cells. Immunophenotyping identified macrophages, dendritic cells, neutrophils and natural killer cells. No cells expressed detectable Siglec-F, CD103, CD3 or Ly6C markers. b Tumors from mice treated with saline (S) or GDC-0152 50 mg/kg (G) were harvested and disaggregated. Cells were incubated in media containing brefeldin-A, with or without 100 nM GDC-0152 (G) or 100 μg/ml LPS (LP), then incubated with a panel of antibodies recognizing cell type markers (as in panel a), fixed and then stained for TNFα. mCherry fluorescence was not detected after fixation, so unstained cells were designated as “osteosarcoma or other”. Positively identified neutrophils, macrophages and natural killer cells are grouped as “immune cells”. The percentage of cells of each type in each sample, expressing and lacking TNFα were calculated (n = 3, +/− SEM)