Journal: Cancer Research

Article Title: Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

doi: 10.1158/0008-5472.CAN-23-2135

Figure Lengend Snippet: The ability of blood and tumor myeloid cells to perform ADT and kill EGFR hi A431 cells in the presence of anti-EGFR Abs under conditions representing solid human tumors. A, The frequencies of CD45 − EpCam + cells, CD14 + CD206 + HLA-DR hi TAM, and CD11b + CD66b + CD14 − TAN were analyzed by flow cytometry in digested lung tumors. Paired t test. B and C, Representative dot plots showing the levels of trogocytosis and killing activity mediated by blood monocytes ( B ) and PBNs ( C ) cocultured with PKH67 + A431 cells at the indicated E:T ratios in the presence of cetuximab and IgA anti-EGFR Abs, respectively, for 12 hours. Nonspecific human IgG1 or IgA2 isotype control Abs were used as a control. D and E, Cumulative flow cytometry results showing the ability of indicated FcR + effectors to kill PKH67 + A431 tumor cells in the presence of cetuximab (IgG) or IgA anti-EGFR (IgA) Abs at a 2:1 E:T ratio in a 12-hour FACS-based assay. One-way ANOVA with Tukey multiple comparisons tests. Summary graphs represent the total tumoricidal activity of effectors calculated as described in Materials and Methods. F–H, Representative images of GFP + A431 tumor cells cocultured with blood monocytes (patient LC441) at different E:T ratios and cetuximab for 48 hours in the IncuCyte Live Cell Analysis System. Image of A431+Ab, where Ab is a cetuximab, is intentionally the same in both and . Scale bar, 400 μm. Representative experiments showing the kinetics of A431 tumor cell growth when cocultured with blood monocytes ( F ), PBNs ( G ), and TAMs ( H ) in the presence of anti-EGFR Abs at the indicated E:T ratios in the IncuCyte Live Cell Analysis System. I and J, Summary results showing the kinetics of GFP + A431 tumor cell growth when cocultured with FcR + effectors in the presence of cetuximab or IgA anti-EGFR Ab at 2:1 E:T ratio in the IncuCyte Live Cell System. The percentage of tumor cell growth inhibition/stimulation was calculated at 48 hours. One-way ANOVA with Tukey multiple comparisons tests. K, Representative dot plots and cumulative flow cytometry data demonstrating the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue. Paired t test. MFI, mean fluorescence intensity. L, Representative flow cytometry histograms showing the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue in comparison with A431 and A549 tumor cells. Number of patients is indicated on the graphs. All data represented as mean ± SEM. FcR + effectors were freshly isolated for all experiments.

Article Snippet: Tumor-associated neutrophils (TAN) were isolated from tumor single-cell suspensions using positive selection with CD15 or CD66b antibody–conjugated magnetic microbeads (130–046–601 and 130–111–552, Miltenyi Biotec Inc.) according to the manufacturer's protocol as previously described ( ).

Techniques: Flow Cytometry, Activity Assay, Control, Cell Analysis, Inhibition, Expressing, Fluorescence, Comparison, Isolation