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. 2016 Aug;15(8):1879-89.
doi: 10.1158/1535-7163.MCT-15-0335. Epub 2016 May 25.

Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells

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Free PMC article

Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells

Ramraj Velmurugan et al. Mol Cancer Ther. .
Free PMC article

Abstract

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is protumorigenic. In the current study, we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. Mol Cancer Ther; 15(8); 1879-89. ©2016 AACR.

Conflict of interest statement

Conflicts of interest: No potential conflicts of interest declared.

Figures

Figure 1
Figure 1
Macrophages reduce breast cancer cell viability in the presence of trastuzumab. J774A.1 (A) or RAW264.7 (B) macrophages were plated in 48 well plates with MDA-MB-453 or SK-BR-3 breast cancer cells at a 4:1 effector:target cell ratio (2.5×104:6.25×103 cells) and 1 μg/ml trastuzumab (Ab) or PBS vehicle (-ve) was added 24 hours later. Following 72 hours, cells were harvested and the remaining number of cancer cells quantitated by flow cytometry. The number of live cancer cells in each sample is shown as a fraction of the corresponding vehicle control. C, cell numbers following incubation of cancer cells as in A,B but without macrophages. Error bars represent standard errors. Student's t-test was performed to indicate statistical significance (denoted by *; p < 0.05).
Figure 2
Figure 2
J774A.1 and RAW264.7 macrophages exhibit different phagocytic activities. A, MDA-MB-453 cells were harvested and opsonized by incubation with 10 μg/ml Alexa 555-labeled trastuzumab at room temperature for ten minutes followed by washing. The opsonized cancer cells (2.5×104 cells/imaging dish) were added to adhered, IFNγ-activated J774A.1 macrophages (4×104 cells) for 30 minutes and the samples fixed and stained. Trogosomes and a completely engulfed cancer cell are indicated by white and yellow arrows, respectively. B, J774A.1 macrophages and MDA-MB-453 cells were incubated as in A, fixed, permeabilized and mouse CD45 detected using FITC-labeled mouse CD45-specific antibody. C, representative flow cytometry plots to show the identification of the whole cell phagocytosis (WCP) population. Macrophages were plated for 18 hours, followed by addition of EdU-treated cancer cells at a 10:1 effector:target cell ratio in the presence of 1 μg/ml trastuzumab or PBS vehicle for 6 hours. The samples were then harvested and stained for mouse CD45 (macrophages) and cancer cells accessible to the medium were detected using labeled pertuzumab. The following cell populations can be identified: macrophage only (1); cancer cell only (2); macrophage:cancer cell conjugate (3); macrophage that has performed WCP (4). D, fluorescence microscopy images of cells representative of the populations numbered 1, 2, 3 and 4 in panel C. E, time-course of WCP using J774A.1 macrophages and MDA-MB-453 cancer cells. F, comparison of WCP activity using different macrophage cells with MDA-MB-453 cells after co-incubation for 6 hours. G, plot of percentage WCP against percentage HER2 reduction for the data shown in Fig. 2F. The percentage of HER2 reduction from the cell surface was calculated from the ratio of the surface pertuzumab (MFI) remaining in the non-phagocytosed cancer cell population to surface pertuzumab (MFI) in samples without antibody treatment. H,I, comparison of WCP activity using J774A.1 (H) or RAW264.7 (I) macrophages with different breast cancer cell lines after co-incubation for 6 hours. Control in panels C, E-I, represent co-cultures incubated without trastuzumab. Error bars represent standard errors. Student's t-test was performed to indicate statistical significance (denoted by *; p < 0.05). n.s., no significant difference (p>0.05). For panels A, B and D, scale bars = 5 μm.
Figure 3
Figure 3
RAW264.7, J774A.1 and human monocyte-derived macrophages induce similar levels of apoptosis in opsonized cancer cells. Macrophages and cancer cells were plated at an effector:target cell ratio of 4:1 in a T25 culture flask (3×106:7.5×105 cells), 1 μg/ml trastuzumab was added 18-24 hours later and the cells were imaged. A, individual frames showing an SK-BR-3 cell undergoing cell death in a long-term light microscopy imaging experiment for a co-culture of SK-BR-3 and RAW264.7 cells in the presence of 1 μg/ml trastuzumab. Times of acquisition of each image are indicated. B,C,D, MDA-MB-453 cells were plated alone or co-incubated with RAW264.7, J774A.1 or human monocyte-derived macrophages at a 4:1 effector:target cell ratio in the presence of 1 μg/ml trastuzumab (Ab) or PBS vehicle (-ve) for 36 hours. B, representative dot-plots for pertuzumab fluorescence vs. PI fluoresence for cancer cells from co-cultures of RAW264.7 or human macrophages with cancer cells in the presence (trastuzumab) and absence of antibody (control). C,D, fraction of annexin V, PI double-positive cancer cells in co-cultures determined by flow cytometry. E,F, samples shown in C and D, respectively, were stained with fluorescently labeled pertuzumab after harvesting and the mean fluorescent intensity (MFI) of pertuzumab on the cancer cell populations determined. Error bars represent standard errors. Student's t-test was performed to indicate statistical significance (denoted by *; p < 0.05).
Figure 4
Figure 4
Fluorescence microscopy analyses using MUM reveals that trogocytosis involves tubular extensions of the cancer cells. A, individual frames from a live imaging experiment of MDA-MB-453: RAW264.7 cell conjugates. The cancer cells were opsonized by incubation with 10 μg/ml Alexa 555-labeled trastuzumab at room temperature for ten minutes followed by washing. These cells were added to IFN-γ-activated RAW264.7 macrophages expressing MEM-GFP (to label the plasma membrane; pseudocolored green) plated in dishes 30 minutes prior to imaging at a 1:1 effector:target cell ratio (2.5×104 cells). Grayscale images represent individual frames of the trastuzumab signal for the boxed region in the pseudo-colored image showing trastuzumab (red) and MEM-GFP (green) signals. Yellow arrows indicate punctate structures accumulating inside the macrophage. B, schematic diagram of the MUM configuration and representative images from multiple focal planes of a macrophage:cancer cell conjugate formed between IFN-γ-activated MEM-GFP expressing RAW264.7 macrophages and MDA-MB-453 cancer cells opsonized with Alexa 555-labeled trastuzumab as above. C, individual frames showing the trastuzumab signal (black and white panels) of live cell imaging from two focal planes displaying a trogocytic event involving tubulation. Yellow arrows indicate intermediates in the tubulation process. Numbers in the lower part of each panel represent the acquisition time (seconds) for each image pair. The left panel shows an overlay of the MEM-GFP (pseudocolored green) and trastuzumab (pseudocolored red) at 1020 sec in the 1.8 μm focal plane. The boxed region is expanded in the grayscale images. D, E, flow cytometry analyses of pertuzumab and CFSE staining levels in MDA-MB-453 cells co-incubated with RAW264.7 macrophages as described in Fig. 1B. Scale bars = 5 μm.
Figure 5
Figure 5
Antibodies with enhanced affinity for activating FcγRs have increased trogocytic and cell-killing activity. A, thioglycollate-elicited macrophages isolated from C57BL/6 mice transgenically expressing human FcγRs (hFcγR macrophages; 26) were plated with MDA-MB-453 cells at an effector:target ratio of 0.4:1 (4×104:1×105 cells). 18 hours later, 1 μg/ml wild type (WT) trastuzumab or engineered trastuzumab variants with enhanced affinity for FcγRIIa and FcγRIIIa (AE and ADE), combined with 0.5 μg/ml Alexa 488-labeled Fab fragments derived from pertuzumab, were added to the co-cultures for 60 minutes. As controls, cells were also co-cultured without trastuzumab (-ve). The mean fluorescence intensity (MFI) values for pertuzumab Fab staining in the macrophage population are shown. B, WCP assay was performed as before using hFcγR macrophages plated for 18 hours, followed by addition of 10-fold lower numbers of cancer cells and 10 μg/ml WT, AE or ADE trastuzumab variants for 6 hours. Trogocytosis (C) and WCP (D) assays were performed as in A,B, but in the presence of 10 mg/ml IVIG. Under the conditions of the trogocytosis assays (60 minutes incubation), phagocytosis of cancer cells was at background, control levels (data not shown). E, hFcγR macrophages were plated with cancer cells at an effector:target cell ratio of 2:1 (5×104:2.5×104 cells) or cancer cells alone for 24 hours, followed by addition of 10 mg/ml IVIG and 10 μg/ml WT, AE or ADE variants of trastuzumab. The medium was replaced by new medium containing the same additions after 3 days. Cells were harvested after 5 days and the remaining numbers of cancer cells quantitated. Error bars represent standard errors. For A-E, one-way ANOVA analyses were carried out followed by a Tukey's multiple comparisons test between all sample pairs with a confidence interval of 95%. Horizontal lines indicate significant differences between sample pairs.

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