Supplementary Figure S6 from Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

Supplementary Figure S6. (A and B) Representative experiment and summary results showing the effect of indicated blood and tumor FcR+ effectors on the growth of A431 tumor cell spheroids in the presence of cetuximab (1ug/ml). GFP+A431 cells were seeded at 500 cells/well in a round bottom ultra-low adhesion cell culture plate designed to form spheroids. Indicated FcR+ effectors were added at E:T ratio 50:1 into the spheroid cultures. The growth of tumor spheroids was monitored in the IncuCyte® Live Cell Analysis System for 48 hrs. Representative images of GFP+A431 tumor cell spheroids co-cultured with indicated effectors and cetuximab for 48 hrs taken from IncuCyte® Live Cell Analysis System (original magnification x10). Scale bar, 400 µm. Percentage of tumor cell growth inhibition/stimulation in the presence of anti-EGFR Abs was calculated at 48 hrs using the formula: (FI)(A431+Ab)-FI(A431+effectors+Ab)/FI(A431+Ab)x100%, FI-Integrated fluorescence intensity. Data are represented as mean ± SEM. Wilcoxon matched pairs test (n=7). (C) Representative dotplots showing the different levels of ADT mediated by indicated FcR+ effectors co-cultured with spheroids formed with PKH67-labeled A431 cells. FcR+ effectors were co-cultured at a E:T ratio 50:1 in the presence or absence of cetuximab for 12 hrs. Representative results from 1 of 3 experiments are shown. (D and E) Representative experiment and summary results showing the effect of PBN and TAN on the growth of GFP+A431 tumor cell spheroids in the presence of IgA anti-EGFR Ab (IgA) (1ug/ml). Experiments were performed as described in (A and B). Original magnification x10. Scale bar, 400 µm. Data are represented as mean ± SEM. Wilcoxon matched pairs test (n=5). (F) Representative dotplots showing the ADT mediated by PBN and TAN co-cultured with spheroids formed with PKH67-lableld A431 cells. PBN and TAN were co-cultured at a E:T ratio 50:1 in the presence or absence of IgA anti-EGFR Ab for 12 hrs. Representative results from 1 of 3 experiments are shown.