Article Figures & Data
Figures
- Figure 1.
ADCC model system and the derivation of ADCC resistance. A, An in vitro NK cell–mediated ADCC model system consisting of an NK-like cell line (NK92-CD16V), an EGFR monoclonal antibody (cetuximab; red-filled circles), and EGFR-expressing A431 cells. The combination of target, effector, and antibody creates optimal conditions for ADCC. B, Schematic of the workflow of the 4 conditions of continuous exposure. Untreated control, cetuximab (1 μg/mL)-treated control, and NK cell–mediated ADCC in the absence and presence of cetuximab (1 μg/mL). C, Time course of A431 cell survival in response to ADCC exposure conditions. A431 cells were seeded and exposed to ADCC conditions for the indicated times, as described in Materials and Methods. ***, P < 0.001 by two-tailed t test across all time points as indicated on the graph. Error bars, SEM. D, Specific lysis of 20,000 ADCCR1 cells and 20,000 ADCCS1 cells by NK92-CD16V cells in the presence of cetuximab (1 μg/mL) for 4 hours at a 1:1 E:T ratio. **, P < 0.01 by two-tailed t test. Error bars, SEM. E, In vitro proliferation of ADCCS1 cells and ADCCR1 cells in the absence of ADCC conditions. ***, P < 0.001; **, P < 0.01 by two-tailed t test for days 2–6 and day 7, respectively. Error bars, SEM. F, Growth of subcutaneous tumors derived from ADCCS1 and ADCCR1 cells in Balb/c nude mice. N = 10 in each group. P value calculated by two-tailed t test as indicated on the graph. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars, SEM. G, Influence of secreted factors by ADCCR1 cells on ADCC sensitivity of ADCCS1 cells. Bar graph, ADCCR1 (R1) cells compared with mixed ADCCR1/ADCCS1 (S) cells at indicated percentages. Error bars, SEM.
- Figure 2.
EGFR and HER2 expression in ADCCS1 and ADCCR1 cells. A, A representative flow cytometry analysis of EGFR cell-surface expression in ADCCS1 (blue) cells and ADCCR1 (red) cells with isotype control expression for ADCCS1 (light blue) and ADCCR1 (light red). B, EGFR expression in ADCCS1 and ADCCR1 cells measured by mRNA, proteomic, and phosphoproteomic analysis. Measures of mRNA expression as well as proteomic and phosphoproteomic peptide counts were normalized by mean-centered scaling across sample groups (Z-score) to provide comparable distributions between assay types. Analysis was done on ADCCS1 and ADCCR1 cells post 33 challenges passaged for 3–4 times. C, Western blot for EGFR protein expression in ADCCS1 and ADCCR1 cells. ADCCS1 and ADCCR1 cells post 33 challenges passaged for 3–4 times were used. Densitometry values of expression relative to GAPDH indicated below the blot. D, Representative flow cytometry analysis of HER2 cell-surface expression in ADCCS1 (blue) and ADCCR1 (red) cells with isotype control expression for ADCCS1 (light blue) and ADCCR1 (light red). E, Specific lysis of ADCCR1(target) and ADCCS1(target) cells by NK92-CD16V(effectors) cells at a 1:1 E:T ratio in the presence of trastuzumab (5 μg/mL) for 4 hours. **, P < 0.01 by two-tailed t test. F, ADCC-induced specific lysis percentage (bars) and corresponding EGFR expression geometric mean by flow cytometry (solid line) in ADCCS1 cells and in ADCCR1 cells as a function of serial in vitro passaging (P, passage number) following the cessation of ADCC exposure.
- Figure 3.
Association of interferon response and histone gene expression with ADCC resistance. A, Heat map of gene expression assessed by whole-genome Illumina bead arrays in ADCCS1 and ADCCR1 cells. Differential gene-expression analysis was conducted for genes possessing at least 2-fold changes and an adjusted FDR of P < 0.01. The heat map is based on hierarchical clustering of both samples (columns) and probes (rows) and contains 388 total probes for 334 unique genes. Reduced (blue) and increased (red) gene expression is shown based on z-score assessment across each probe (row). B, Volcano plot of differential gene expression in ADCCR1 compared with ADCCS1 cells. Differential gene-expression analysis was conducted for genes possessing an adjusted FDR of P < 0.01. The dotted line (vertical) indicates the P value threshold 4 and –4 Log2 FC indicating significantly upregulated and downregulated genes, respectively (red). C, Western blot of JAK1, STAT1, NFκB p65, and p-NFκB p65 in ADCCS1 and ADCCR1 cells. ADCCS1 and ADCCR1 cells post 33 challenges passaged for 3–4 times were used. Densitometry values of expression relative to GAPDH indicated below the blots. D, Diagram of 300 genes found to be upregulated in ADCCR1 cells compared with ADCCS1 cells by CoGAPS analysis. Overexpressed (red) and underexpressed (green) genes in ADCCR1 compared with ADCCS1 cells. The interferon-induced and histone-associated gene clusters are identified in the bottom right portion of the diagram within hatched boxes.
- Figure 4.
Effects of pharmacologic modification of histone-associated proteins identified by CoGAPS gene-expression analysis on ADCC sensitivity. A, Western blot of PCAF (KAT2B) in ADCCS1 and ADCCR1 cells. ADCCS1 and ADCCR1 cells post 33 challenges passaged for 3–4 times were used. Densitometry values of expression relative to GAPDH indicated below. B, Specific lysis of ADCCS1 (blue) and ADCCR1 (red) measured by ADCC assay at 4 hours when pretreated with increasing concentrations of histone acetyl transferase inhibitor (HATi) C646 for 2 hours. C, Specific lysis of ADCCS1 (blue) and ADCCR1 (red) measured by ADCC assay at 4 hours when pretreated with increasing concentrations of DNMT (DNMTi), pan-HDAC (pan-HDACi), and histone demethylase inhibitors.
- Figure 5.
NK cell activation and conjugation to EGFR+ target cells under ADCC conditions. A, Representative dot plots of NK activation measured by flow cytometry analysis using CD107α (APC) and GFP+ NK92-CD16V cells at a 1:1 E:T for 2 hours as described in Materials and Methods. ADCCS1 (top row) and ADCCR1 cells (bottom row) were incubated with NK92-CD16V cells in the absence (middle) or presence of cetuximab (1 μg/mL; right) for 2 hours. B, ELISA measuring IFNγ levels in the media of ADCCS1 (blue bars) and ADCCR1 (red bars) 4 hours after exposure to ADCC conditions (cetuximab [1 μg/mL] plus NK92-CD16V cells) at E:T ratios of 0–4:1 and NK92-CD16V cells in the absence of cetuximab. C, Western blot analysis of granzyme B and perforin protein expression in target cells 2 hours after exposure to T (media control), Ab (cetuximab only at 1 μg/mL), NK (NK92-CD16V cells only at 1:1 E:T), and ADCC (NK92-CD16V cells at 1:1 E:T plus 1 μg/mL cetuximab). D, Percentage of NK cells conjugated to target was measured by a multiwell conjugation assay as described in Materials and Methods. ADCCS1 (blue) and ADCCR1 (red) were incubated with NK92-CD16V cells at a 1:1 E:T ratio in the absence (NK, checkered bar) or in the presence of cetuximab (1 μg/mL; ADCC, solid bar) for 2 hours. *, P < 0.05 by two-tailed t test.
- Figure 6.
Cell-surface screen of ADCCS1 and ADCCR1 cells. A, Dot plot comparing geometric means of ADCCS1 and ADCCR1 cell-surface molecule expression measured by the BD Lyoplate assay as described in Materials and Methods. Molecules with highest differential cell-surface expression in ADCCS1 cells are shown in the box. BD Lyoplate screen geometric means of ADCCS1 and ADCCR1 are shown in Supplementary Table S1. B, Geometric means of molecules with the highest differential expression (box in A) in ADCCS1 (blue) compared with ADCCR1 (red) cells. C, Representative histograms of selected cell-surface molecules with reduced cell-surface expression on ADCCR1 cells based on lower protein expression. Light gray histograms, negative control; dark gray histograms, expression of total protein in permeabilized ADCCR1 cells; open histograms, cell-surface expression of indicated molecule in ADCCR1 cells. D, Representative histograms of selected cell-surface molecules with reduced cell-surface expression based on reduced transport to cell surface. Light gray histograms, negative control; dark gray histograms, expression of total protein in permeabilized ADCCR1 cells; open histograms, cell-surface expression of the indicated molecule in ADCCR1 cells.
Additional Files
Supplementary Data
- Supplementary Data - S1,S2,S3,S4,S5,S6,S7, Supplemental table 1,supplemental table 2
- Supplementary Table 1 - Supplementary Table 1
- Supplementary Figure Legends - Legends for Supplemental Figures 1-7 in Supplementary Data file
Volume 7, Issue 2
