Supplementary Figure S8. (A and B) Representative experiment and summary results showing the effect of blood and tumor FcR+ effectors on the growth of GFP+A549 tumor cell spheroids in the presence of cetuximab (1ug/ml). Experiments were performed as described in supplemental figure 5 (A and B). Original magnification x10. Scale bar, 400 µm. Data are represented as mean ± SEM. Wilcoxon matched pairs test (n=6). Indicated FcR+ effectors were freshly isolated for all experiments. (C) Flow cytometry histogram showing the expression of EGFR on the surface of H1975 tumor cell line in comparison with A431 and A549 tumor cells. (D and E) Cumulative flow cytometry results showing the different levels of trogocytosis and killing activity mediated by blood and tumor FcR+ effectors co-cultured with PKH67+ H1975 tumor cells at a 50:1 E:T ratio in the presence of cetuximab or isotype control Abs for 12 hrs. All comparisons used one-way ANOVA with Turkey’s multiple comparisons tests. All data represented as mean ± SEM. FcR+ effectors were freshly isolated for all experiments. IC-isotype control Ab.
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.
