Quantification of Epidermal Growth Factor Receptor ExpressionLevel and Binding Kinetics on Cell Surfaces by Surface PlasmonResonance ImagingFenni Zhang,†,‡ Shaopeng Wang,*,† Linliang Yin,†,§ Yunze Yang,†,‡ Yan Guan,†,‡ Wei Wang,∥ Han Xu,⊥

and Nongjian Tao*,†,‡,∥

†Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, United States‡School of Electrical Computer and Energy Engineering, Arizona State University, Tempe, Arizona 85287, United States§College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China∥State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University,Nanjing 210093, China⊥Amgen Inc., Thousand Oaks, California 91320, United States

*S Supporting Information

ABSTRACT: Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein thathas been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification ofthe EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis andtreatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with thesurface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in thisstudy. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow thefirst-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 105 M−1 s−1 and (1.4± 0.5) × 10−4 s−1, respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-foldvariation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm2 with an estimation of 5 × 105 EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines(A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is avaluable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution.Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membraneprotein binding kinetics is important.

Molecular targeting therapy is an advanced cancer therapy,which can improve tumor-targeting specificity compared

with the traditional chemotherapy and radiotherapy. Among allthe identified targets, one promising molecular target is theepidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) in the cell membrane. EGFR plays an essentialrole in regulating normal cell signaling, and the mutation ofEGFR leads to cell proliferation, angiogenesis, invasion,metastasis, and inhibition of apoptosis, accounting for thepathogenesis and progression of cancer cells.1−5 Therefore, insitu quantification of the EGFR expression level in the cellmembrane and ligand binding kinetics and affinity are of greatimportance for cancer diagnosis and treatment.

EGFR is a transmembrane protein, which consists of threemajor functional domains: an extracellular binding domain, ahydrophobic transmembrane domain, and an intracellulartyrosine kinase domain.6−9 When an epidermal growth factor(EGF) or transforming growth factor (TGF) ligands bind tothe extracellular domain, EGFR undergoes a transition to formreceptor homodimers or heterodimers with neighboring ErbBreceptors, which activates the intrinsic receptor tyrosine kinasedomain for signal transduction. Mutations that cause EGFR

Received: July 8, 2015Accepted: September 14, 2015Published: September 14, 2015

Article

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overexpression and constant activation often lead to uncon-trolled cell dividing.10−15 Therefore, quantification of EGFRexpression density in the cell membrane is a critical step forcancer diagnosis. Currently, the most used approach forreceptor density measurement is the radiolabeling assay,16,17

which involves synthesis of a radio-ligand and requires specialtraining and safety protection. A label-free method is desired forrapid quantification of receptor density.In order to prevent the unwanted downstream effects of

EGFR signaling, two kinds of inhibitors have been proposed forcancer treatment: tyrosine kinase inhibitors targeting theintracellular domain and monoclonal antibodies targeting theextracellular domain. The binding of these inhibitors to EGFRslows down or stops tumor cell growth.18−25 Monoclonalantibodies targeting the extracellular domain of EGFR havebeen in various stages of preclinical development and haveshown good therapeutic efficacy for treatment of a number ofcancers that have up-regulated EGFR expression lev-els.1,2,22,26,27 The kinetic constants of the binding of theseantibody drugs to EGFR are the key parameters to characterizethe efficacy of these drugs.Binding kinetic constants determine how fast a drug and its

receptor associate and dissociate, providing valuable informa-tion for drug screening and optimization.28,29 A widely usedmethod to measure the binding kinetics is to isolate the targetproteins from the cell membrane for direct or indirect bindingwith antibodies for target specific drug screening, among whichthe enzyme linked immunosorbent assay (ELISA) is mostlyused in protein studies.30−34 ELISA uses an enzyme labeledantibody for signal amplification, which has high sensitivity andselectivity. However, this method involves the extraction andpurification of target proteins, which is laborious. A moreserious drawback is that the purified proteins may lose theiroriginal structures and functions after being isolated from thenative membrane environment. Other methods, such asradiolabeling and fluorescent labeling, have been used tomeasure molecular interactions of the membrane proteins intheir native membrane environment with high sensitivity,35−37

but they are end point assays and cannot provide kineticconstants required to quantify the molecular interactions.38,39 Alabel-free method for in situ measurement of binding kinetics ofmembrane proteins in intact cells is needed for rapid andaccurate drug screening.Surface plasmon resonance (SPR) is a label-free technique to

measure the kinetics of molecular interaction.40 Surfaceplasmon resonance imaging (SPRi) extends SPR measurementto microarrays33,41−45 and enables direct measurement ofmolecular binding kinetics on the surface of mammaliancells46,47 and bacteria.48 In this paper, we report quantificationof EGFR expression density and antibody binding kinetics toEGFR on the cell surface, as an effort to establish a cell basedlabel-free SPRi platform for in situ quantification of drug-targetinteractions. A monoclonal antibody, anti-EGFR, was used tostudy the binding kinetics and affinity in EGFR overexpressedcells with single cell resolution and the ability to map cell-to-cellheterogeneity. Furthermore, the binding kinetics of cell lineswith different EGFR-expressing levels were compared, whichreveals the microenvironment in the cell membranes affectingthe drug-receptor interactions.

■ MATERIALS AND METHODSMaterials. Anti-EGFR monoclonal antibody (cat. no. 05-

101) was purchased from EMD Millipore, which was dissolved

as a 1 mg/mL stock solution, and stored in frozen aliquots.Anti-EGFR solutions used in the experiments were prepared bydiluting the stock solution with phosphate buffer solution(PBS). Alexa Fluor 488 Goat Anti-Mouse IgG1 (γ1) Antibody(cat. no. A-21121) was obtained from Life Technologies. Allreagents were analytical grade, purchased from Sigma-Aldrich,except those stated. Deionized water was used to prepare all thebuffers.

SPRi Setup. The details of the SPRi setup were describedelsewhere.47 Briefly, a collimated p-polarized light from a 680nm light-emitting diode (LED) was directed through atriangular prism onto the gold-coated glass coverslip placedon the prism. The reflected beam was captured by a CCDcamera (AVT Pike F032B) at a frame rate of 11 frames persecond (fps) through a 10× variable zoom lens (Navitar) with aspatial resolution of ∼5 μm. A Flexi-Perm silicon chamber(SARSTEDT) was placed on top of the gold chip to serve as acell-culture well. A schematic diagram of the setup is shown inFigure 1.

A gravity-based multichannel drug perfusion system (SF-77B,Warner Instruments, Connecticut) was used to deliversolutions to the target cells. The typical transition timebetween different solutions was about 1−2 s.46,47

Cell Culture. A431, A549, HeLa, and HEK293 cell lineswere obtained from the American Type Culture Collection(Rockville, MD). All the cells were cultured in a humidifiedincubator at 37 °C with 5% CO2 and 70% relative humidity.A431, HeLa, and HEK293 cells were grown in DulbeccoModified Eagle Medium (DMEM; Lonza, Walkersville, MD)with 10% Fetal Bovine Serum (FBS) and 1% penicillin andstreptomycin. A549 cells were cultured in Ham’s F-12K(Kaighn’s) Medium (Lonza, Walkersville, MD) with 10%FBS and 1% penicillin and streptomycin. Cells were passagedwith 0.25% trypsin and 0.02% ethylenediaminetetraacetic acidin a Hank’s balanced salt solution (Sigma-Aldrich, St Louis,Missouri) when they were approximately 75% confluent.In order to promote cell adhesion, the gold surface of the

chips was incubated with 10 μg/mL collagen for 2 h. Thecollagen-coated chips were rinsed with PBS buffer twice priorto seeding. Each chip was seeded with 5000 cells in 300 μL ofgrowth medium and was incubated for 1 day to allow the cellsto attach and grow. The growth medium was replaced with PBSbuffer solution prior to the experiment. The viability of cells onthe chips was visually inspected prior to the experiment using atissue culture microscope. A total of more than 50 chips weretested, and all cells showed consistent high viability similar tocells in a tissue culture plate. Cells were fixed by incubating in4% paraformaldehyde solution for 10 min at room temperatureprior to measurement.

Figure 1. Schematic diagram of the SPRi setup with cells on thesurface of gold-coated glass chip.

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Immunofluorescence. The immunofluorescence imageswere obtained with an inverted fluorescence microscope at 10×magnification (Olympus IX81). A 150 W mercury lamp wasused as the light source. For EGFR staining, the fixed cells wereincubated with 10 μg/mL anti-EGFR solution for 30 min,followed by 30 min incubation with Alexa Fluor 488 Goat Anti-Mouse IgG1 (γ1) after three rinses with PBS buffer. Thefluorescence image was captured after rinsing the well twicewith PBS buffer. The following optical filters were used toobtain the fluorescence image: excitation filter, 420−480 nm,emission filter, 515 nm.

■ RESULTS AND DISCUSSIONIn Situ Quantification of anti-EGFR and EGFR

Interaction Kinetics on the Surface of A431 Cell. TheA431 cell is a model cell line (Epidermoid Carcinoma) thatoverexpresses EGFR in the cell membrane. To investigate themolecular interaction of EGFR in its native environment, wegrew and fixed A431 cells on gold-coated glass coverslips andmeasured binding kinetics and the affinity of anti-EGFR, thespecific monoclonal antibody inhibitor targeted at theextracellular domain of EGFR, using a prism based SPRisetup. When the anti-EGFR antibody binds to EGFR on thecell membrane, the mass (36 kDa for anti-EGFR) inducedrefractive index change is recorded as SPR image intensitychanges and is presented in the form of an SPR sensorgram.Three gold chips with different batches of cells were measuredto validate the repeatability of the technique. Nonspecificbinding signals were quantified in the cell-free gold area andwere subtracted from the raw signals to obtain the specificbinding signals (Figure S1).Figure 2a shows a representative SPR image of A431 cells, in

which the bright spots represent individual or clusters of cells.

A431 cells tend to aggregate. The following steps wereperformed to measure the EGFR binding kinetics. First, PBSbuffer was flowed over the cells for 300 s at a flow rate of 350μL/min to record the baseline. Next, 2 μg/mL anti-EGFR wasintroduced to the cells for 350 s to record the associationprocess. Last, PBS buffer was reintroduced to the cells for 400 sto measure the dissociation of the antibody. During theassociation process, obvious increases in SPR intensity at all cellregions were observed, as shown in the differential image(Figure 2b). The differential image maps the SPR intensitychanges in each pixel by subtracting the original SPR imageprior adding the antibody (t = 300th second) from the SPRimage at the end of the association phase (t = 650th second).Apart from the obvious intensity increase in all cell regionscompared to the gold area, the image intensity changes atdifferent cell regions were different, which indicated theheterogeneity of EGFR express level among individual cells.Quantitative binding kinetics are extracted from the SPR

image sequences and plotted as sensorgrams, with arepresentative result shown in Figure 2c. A maximum averagedincrease of 168 RU was found in the SPR sensorgram shown inFigure 2c, where the black curve represents the averagedresponse of all the cells in Figure 2a and the gray backgroundarea shows the response from individual cells or clusters. Thebinding kinetics of anti-EGFR in A431 cells were determinedby fitting the averaged response curve with a 1:1 Langmuirbinding model (the red curve). From three independentmeasurements, the corresponding association rate constant(ka), dissociation rate constant (kd), and dissociation constant(KD) were calculated and shown in Table 1 as the mean value

of three chips. These results validated the repeatability of themeasurements and indicated a fast and strong binding. The flatblue curve in Figure 2c shows that there is no response fromthe gold area without cells and confirms the specificity of theantibody binding.

Heterogeneous Binding Kinetics of Individual Cells.An important capability of imaging based measurement is toquantify the heterogeneous responses of anti-EGFR interactionfrom individual cells, which is important for identifying tumorspecificity therapy development, since tumor tissue samplesoften mixed with normal cells. Figure 2d shows the associationand dissociation processes of five individual cells selected fromdifferent regions of the chip, where the color curves are firstorder kinetic fitting to the data (gray dots). The correspondingcells are marked with corresponding colored circles in Figure2a. From the sensorgrams, we can conclude that maximumassociation varies from cell to cell. Furthermore, the kineticconstants (Table 1) obtained from the fitting exhibit up to 7-fold variations, significantly larger than the measurement error(variation from different tests). These results reveal cell-to-cellheterogeneity in the binding of drugs to the cells, which

Figure 2. (a) A typical SPR image of a few tens of A431 cells adheredon the gold-coated glass slide. (b) Differential SPR image shows themaximum SPR intensity increase due to anti-EGFR binding to thesurface of A431 cells. (c) The average SPR sensorgrams of all cells inview (black curves, average SPR sensorgram; red curve, curve fitting;gray background, cell-to-cell variation) and the surrounding regionswithout cell coverage (blue curve). (d) The SPR sensorgrams of fiveindividual cells of different regions (gray dotted curves, individual SPRsensorgram; colored curve, corresponding fitting curves). All the plotsshown are subtracted from the nonspecific binding response (in thewhite cycle region of a) in the gold area from the raw response signals.Scale bar, 50 μm.

Table 1. Measured EGFR Binding Kinetic Constants

ka (M−1 s−1) kd (s

−1) KD (nM)

cell 1 (red curve) 6.0 × 105 2.6 × 10−4 0.43cell 2 (blue curve) 6.4 × 105 1.5 × 10−4 0.23cell 3 (green curve) 6.2 × 105 4.4 × 10−4 0.70cell 4 (yellow curve) 5.5 × 105 2.7 × 10−4 0.49cell 5 (purple curve) 5.3 × 105 5.5 × 10−5 0.10mean value of 3 chips 6.7 × 105 3.3 × 10−4 0.41

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underscores the importance of measuring binding kinetics atthe single cell level.Dose Response, Global, and Equilibrium Fitting. To

measure the dose response of EGFR to the antibody drug, anti-EGFR solutions with concentrations increasing from 0.5 to 10μg/mL were sequentially introduced to A431 cells on a sensorchip to record the association and dissociation kinetics. Foreach new dose of anti-EGFR, the cell surface was regeneratedwith 100 mM NaOH for 30 s. SPR sensorgrams for differentdoses of anti-EGFR were plotted in Figure 3a (black curves).

Both the association rate constant and maximum SPR responseincrease with anti-EGFR concentration, which is expected forfirst order kinetics. The red lines are the global fitting results ofthe sensorgrams based on the 1:1 binding kinetics model. Thekinetic constants obtained from the global fitting are ka = (2.7± 0.6) × 105 M−1 s−1, kd = (1.4 ± 0.5) × 10−4 s−1, and KD =0.53 ± 0.26 nM and are consistent with the singleconcentration fitting result (mean value in Table 1).In addition to the kinetic analysis, the equilibrium-fitting

curve was plotted based on the equilibrium binding responsesobtained at seven different concentrations (Figure 3b), and theobtained dissociation constant, KD, was determined to be 5.1nM, in the range of reported value.49 The KD value obtainedfrom the equilibrium-fitting curve depends on the ligand (anti-EGFR) concentration. In general, the KD value will increase asthe concentration of the ligand increases,50 which may accountfor the difference between the global fitting result andequilibrium fitting result. As the concentrations of anti-EGFRused in our experiments are all bigger than the dissociationconstant calculated in global fitting, it is reasonable that the KDobtained from the equilibrium-fitting curve is larger than the KDobtained from kinetic response curves.Determination of EGFR Expression Level. In addition to

determination of the binding kinetics, the SPR signal can alsobe used to quantify the expression level of EGFR in the A431cell membrane. In the SPR sensorgram, the equilibriumresponse represented the saturated amount of anti-EGFRbound to A431 cells, from which we can estimate EGFRreceptor density by assuming the 1:1 binding between anti-EGFR and EGFR on the cell surface. With 10 μg/mL anti-EGFR stimulation, the equilibrium response was calculated to

be a 190 RU increase in SPR intensity, which can be used forreceptor density calculation. Assume a 1 RU SPR responsecorresponds to 1 pg/mm2 mass application, the binding of anti-EGFR induced 190 pg/mm2 in the cell surface, whichcorresponds to ∼636 receptors/μm2. Given the averagediameter of 25 μm for suspended A431 cells, the averagenumber of EGFR receptors is around 1.2 × 106 per cell. Thisnumber is consistent with the results measured by flowcytometry and fluorescent labeling.51 This result shows thatcell-based SPRi is able to quantify membrane receptor densitywithout any labeling and is potentially a valuable tool forbiomarker discovery and cancer diagnosis.

EGFR Binding Kinetics and Expression Levels inDifferent Cell Lines. To investigate how the native cellularmicroenvironment affects the drug-receptor interactions, bind-ing kinetics of anti-EGFR to EGFR in four cell lines withdifferent expression levels were studied. These cell lines includeEGFR-positive A431, HeLa and A549, and EGFR-negativeHEK293 as a negative control. Figure 4a shows the SPR imagesof different cell lines before the antibody administration.Differential SPR images in Figure 4b show the SPR intensityincrease due to anti-EGFR binding. An obvious increase wasobserved in A431 cells, while the HeLa cell and A549 cellsshowed much smaller responses. Figure 4c shows the averagedresponses curves of a few tens of cells (black lines) and thekinetic fitting curves (red lines). The gray area represents thedistribution, which measures the heterogeneity for each cellline. Typical association and dissociation curves were observedin HeLa and A549 cells with reduced responses compare withA431. No specific response from EGFR-negative HEK293 cellswas observed, confirming that the specific anti-EGFR andEGFR interactions were responsible for the SPR responses incell regions. From the equilibrium binding responses, EGFRdensity in HeLa and A549 were found to be 270 and 142receptors/μm2, respectively, corresponding to 0.53 and 0.28million per cell, much lower than A431.For cross validation, the EGFR expression levels in different

cell lines were confirmed by immunofluorescence imaging.Figure 4d shows the fluorescence image of the four cell linesstained with anti-EGFR antibody followed by a fluorescence-labeled secondary antibody. The intensity of the fluorescentsignal is proportional to the EGFR abundance. Consistent withSPRi results, the A431 cell has the highest fluorescenceintensity, while HeLa and A549 cells exhibit lower emission,and HEK293, the negative control, shows no detectablefluorescent signal.The kinetic constants in the three EGFR-positive cell lines

were determined from fitting the averaged cell responses withfirst order kinetics. Table 2 listed the EGFR density andbinding kinetics of the three cell lines we measured. The tableshows a trend of increased rate constants (ka and kd) andstronger affinity (lower KD) with lower receptor density, whichcould be caused by the steric hindrance effect. This observationshows that the membrane microenvironment of the EGFRreceptors may play an important role in drug/receptorinteractions, and this information could not be obtained bytraditional approaches that study proteins isolated from thecells.

■ CONCLUSIONSWe have quantified the binding kinetics of the monoclonal anti-EGFR antibody to its membrane bound target receptor EGFRin cultured cells with SPRi. The imaging capability of this label-

Figure 3. (a) SPR sensorgrams of a cell at different concentrations ofanti-EGFR and the corresponding global fitting (red curves). Anti-EGFR concentrations are 0.5, 1, 2, 5, and 10 μg/mL from the bottomto top curve, respectively. All the plots shown are subtracted from thenonspecific binding response in the gold area from the raw responsesignals. (b) The equilibrium-fitting curve. The anti-EGFR concen-tration (plotted in log scale) was 0.2, 0.3, 0.5, 1, 2, 5, and 10 μg/mL,respectively. The equilibrium response values at different concen-trations are obtained by fitting the association section of sensorgramswith first order kinetics. At least three independent measurementswere performed for each concentration.

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free method enables the mapping of individual cell responses.The real time detection capability provides quantitativeinformation on the membrane bound receptor density andligand interaction kinetics. While the averaged kinetic constantsare consistent with the literature data, we have observed severalfold cell-to-cell variations in kinetic constants. Furthermore, wefound a correlation between EGFR density and binding kineticsamong different EGFR positive cell lines. Our resultsdemonstrated that SPRi is able to directly quantify themembrane bound receptor expression level and ligand bindingkinetics without the need of labeling. Our findings alsounderscore the importance of an in situ study of drug/receptorinteraction in intact cells.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.5b02572.

Nonspecific binding and correction method (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*Shaopeng Wang, E-mail: shaopeng.wang@asu.edu.*Nongjian Tao, E-mail: njtao@asu.edu.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support from National Institute of Health(1R01GM107165-01A1 and 1R44GM106579-01), NationalNatural Science Foundation of China (21405080), and NaturalScience Foundation of Jiangsu Province (BK20140592) isacknowledged.

■ REFERENCES(1) Mendelsohn, J.; Baselga, J. Oncogene 2000, 19, 6550−6565.

Figure 4. Anti-EGFR interactions with EGFR-positive (A431, HeLa, and A549) and EGFR-negative (HEK 293) cell lines. (a) SPR images beforeanti-EGFR binding. (b) Differential SPR images after anti-EGFR binding. (c) The SPR sensorgrams of 2 μg/mL anti-EGFR binding. All the plotsshown are subtracted from the nonspecific binding response in the gold area from the raw response signals. (d) Immunofluorescence images bystaining EGFR.

Table 2. EGFR Densities and Binding Kinetic Constantswith anti-EGFR in Different Cell Lines

cellline

EGFR density (molecules/μm2)

ka (M−1

s−1) kd(s−1) KD(nM)

A431 636 2.7 × 105 1.4 × 10−4 0.53Hela 270 5.4 × 105 2.4 × 10−4 0.45A549 142 2.4 × 106 7.0 × 10−4 0.29

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