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

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posted on 2024-04-01, 07:21 authored by Sunil Singhal, Abhishek S. Rao, Jason Stadanlick, Kyle Bruns, Neil T. Sullivan, Andres Bermudez, Adam Honig-Frand, Ryan Krouse, Sachinthani Arambepola, Emily Guo, Edmund K. Moon, George Georgiou, Thomas Valerius, Steven M. Albelda, Evgeniy B. Eruslanov

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.

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National Cancer Institute (NCI)

United States Department of Health and Human Services

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Janssen Pharmaceutica (Janssen)

National Institutes of Health (NIH)

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ARTICLE ABSTRACT

The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)–expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells “nibbled off” tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.

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