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

Abstract

<p>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.</p>