Fig 1.
Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10A EGF-independent growth and sensitivity to gefitinib.
A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using MTS assay by measuring absorbance at 490 nm over a period of 8 days. B. The doubling times of the cell lines were calculated from the rates of proliferation. As neither MCF10A nor MCF10A-WT cell proliferated in the absence of EGF, a doubling time could not be calculated. C. Cells were treated with varying concentrations of gefitinib in the absence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (* p<0.05). D. Cells were treated with varying concentrations of gefitinib in the presence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (**** p<0.0001). 10A: MCF10A-EV, WT: MCF10A-EGFR-WT, GS: MCF10A-EGFR-GS, DEL: MCF10A-EGFR-DEL. Data from duplicate experiments.
Fig 2.
EGFR-DEL expression enhances anchorage-independent growth of MCF10A cells in the absence of EGF.
Cells were cultured in media containing growth factor-reduced Matrigel with 2% horse serum in the absence (- EGF) or presence of 5 ng/ml EGF (+ EGF). At 13 days spheroid formation assessed was assessed with phase contrast microscopy. Scale bars = 400 μm.
Fig 3.
Overexpression of wild type or mutant EGFR increases the growth of MCF10CA1a mammary fat pad xeongrafts.
Female BALB/c nude mice were injected in the mammary fat pad with 5x106 cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -EGFR-WT, GS: MCF10CA1a -EGFR-GS, DEL: MCF10CA1a -EGFR-DEL A. Representative bioluminescent images of individual mice taken on day 14 and day 43. B. Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). C. Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. D. Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).
Fig 4.
Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10CA1a EGF-independent growth but not sensitivity to gefitinib.
A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using real-time imaging on the Incucyte Zoom and measuring the increase in confluency over 4–5 days. The doubling times of the cell lines were calculated from the rates of proliferation from 36–60 hours (24 hour period). While there was no difference in proliferation between the cell lines in the presence of EGF, in the absence of EGF all three EGFR constructs reduced MCF10CA1a doubling time (*p<0.05, **** p<0.0001,, unpaired T-test, n = 6). B. Representative growth curves for cells grown in EGF-free media with measurements of confluency taken every 6 hours for up to 102 hours. C-D. Calculated IC50 values of gefitinib for inhibition of proliferation (C) or cell toxicity (D) from cells treated for 48 hours in various doses of gefitinib and monitored in real-time using the Incucyte Zoom (** p<0.01, unpaired T-test, n = 3). E-G. Calculated mean IC50 values from cells treated for seven days in various doses of gefitinib (E, n = 2), afatinib (F, n = 4) or cetuximab (G, n = 1) and cell death determined using the MTS assay. CA: MCF10CA1a-EV, WT: MCF10CA1a-EGFR-WT, GS: MCF10CA1a-EGFR-GS, DEL: MCF10CA1a-EGFR-DEL.
Fig 5.
Docetaxel/doxorubicin chemotherapy of MCF10CA1a is unaffected by EGFR mutation or in combination with afatinib.
Cells were cultured in the presence of EGF and treated with docetaxel/doxorubicin in a 5:1 ratio (DD) alone or in combination with the half IC50 dose of afatinib (DDA) for seven days. Cell survival was assessed using the MTS assay and the IC50 dose was reported using the docetaxel concentrations. CA: MCF10CA1a-EV, WT: MCF10CA1a-EGFR-WT, GS: MCF10CA1a-EGFR-GS, DEL: MCF10CA1a-EGFR-DEL.
Fig 6.
Mutant EGFR expression increases the migratory and invasive abilities of MCF10CA1a cells.
A. MCF10CA1a cells expressing empty vector (CA), wild-type EGFR (WT), G719S EGFR (GS) or E746-A750 Deletion EGFR (DEL) were seeded in serum-free media (SFM) at 40000 cells/well in 16-well xCELLigence plates and their migration to full media containing 20 ng/ml EGF monitored. The top panel is the average of the relative migration of the cells at 48 hours of three independent experiments while the bottom panel is a representative trace from one experiment of the absolute migration index over the course of the experiment (*p<0.05, unpaired T-test). B. MCF10CA1a cells expressing empty vector (CA), wild-type EGFR (WT), G719S EGFR (GS) or E746-A750 Deletion EGFR (DEL) were seeded in SFM containing Matrigel at 100000 cells/well in 16-well xCELLigence plates and their invasion to full media containing 20 ng/ml EGF monitored. The top panel is the average of the relative invasion of the cells at 48 hours of two independent experiments while the bottom panel is a representative trace from one experiment of the absolute invasion index over the course of the experiment (*p<0.05, unpaired T-test).
Fig 7.
Circos plot summarizing the gene amplification and loss differences between MCF10A and MCF10CA1a.
The outer band details chromosome number and position, middle band represents MCF10A and the inner band represents MCF10CA1a. In each case, values plotted are from copy number analysis of Illumina Human Omni 2.5M SNP array data. Red regions highlight gene amplification while green regions represent gene loss.
Table 1.
Frequency of each class of mutations in MCF10A and MCF10CA1a cells.