Real-Time and Label-Free Detection of Cellular Response to

ARTICLE pubs.acs.org/ac

Real-Time and Label-Free Detection of Cellular Response to Signaling Mediated by Distinct Subclasses of Epidermal Growth Factor Receptors Jennifer Y. Chen, Minghong Li,§ Lynn S. Penn, and Jun Xi* Chemistry Department, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States

bS Supporting Information ABSTRACT: Epidermal growth factor receptors (EGFRs) have often shown two distinct binding affinities for epidermal growth factor. It is the high-affinity EGFR that is predominantly responsible for mediating the cell signaling that plays an indispensable role in cell growth, proliferation, motility, and differentiation. We applied the quartz crystal microbalance with dissipation monitoring (QCM-D) to track short-term cellular responses to EGFR signaling in human carcinoma A431 cells. Cellular responses to high- and low-affinity EGFR signaling were detected individually as well as simultaneously based on changes in mass and viscoelasticity of cells. These responses are associated with EGF-induced biological processes including the cytoskeleton remodeling and calcium influx. QCM-D provides a label-free sensor technology that can be exploited to investigate the role of high-affinity EGFR in cancer development and cancer prognosis.

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he epidermal growth factor receptor (EGFR) is a transmembrane receptor that regulates cell growth, proliferation, motility, and differentiation through its downstream signaling pathways.1,2 These pathways are activated by the binding of epidermal growth factor (EGF) to the extracellular domain of EGFR, followed by phosphorylation of the cytoplasmic tyrosine kinase. Ligand binding studies with radiolabeled EGF suggest that there are two subclasses of EGFR:3,4 high-affinity EGFR, which exhibits kd-values of a few nanomolar or less, and lowaffinity EGFR, which exhibits kd-values of 10 nM or above. Even though high-affinity EGFR often accounts for less than 10% of the total EGFR, it is the subclass that is most responsible for the regulation of cell growth, proliferation, motility, and differentiation.5
7 By contrast, low-affinity EGFR controls Ca2þ influx and fluid-phase pinocytosis7 but contributes little to the abovementioned regulation. It is known that an aberrant EGFR signaling can induce uncontrolled cell growth and a malignant phenotype,8 such as tumors. Because of the critical role of high-affinity EGFR in regulation of cell growth, proliferation, motility, and differentiation, the extent of cell signaling mediated by high-affinity EGFR can therefore be very informative for assessing the role of EGFR in cancer development and cancer prognosis.9 This requires a detection system that is capable of tracking cellular responses specifically to high-affinity EGFR signaling. Traditionally, cell signaling is examined biochemically based on the quantitation of the concentration of proteins or other molecules that are involved in the signaling process. Such end-point detection is often limited in providing the actual kinetic information concerning the process of cell signaling. When cellular molecules are tagged with fluorescent labels, their physical activities can be r 2011 American Chemical Society

tracked in real time to render kinetic information on cell signaling pathways. However, the presence of fluorescent labels can potentially create a non-native and physiologically irrelevant cellular environment for the molecules of interest,10
12 which may lead to ambiguous results. These problems can be circumvented with the use of real-time and label-free sensor technologies, which have previously been applied to the detection of EGF-induced cellular responses. These sensor-based detections measure changes in refractive index or electrical impedance resulting from EGF-induced changes in either mass or ion distribution in cells, respectively.13,14 However, because these changes can result from both high- and low-affinity EGFR signaling, these measurements have not shown to be specific for detecting individual subclasses of EGFR signaling. The quartz crystal microbalance with dissipation monitoring (QCM-D) is an ultrasensitive technique, consisting of a piezoelectric quartz crystal in the shape of a thin disk that is made to be part of an electrical circuit.15,16 This technique provides a labelfree and real-time measurement of changes in frequency, Δf, and energy dissipation, ΔD, of a layer attached to the quartz surface. Changes in frequency are indicative of changes in mass, while changes in dissipation are indicative of changes in the viscoelastic character of the layer. QCM-D and other versions of QCM have been successfully used in various biological analyses.17,18 They have become particularly attractive to the field of cell biology because of the capability to monitor cell
surface interactions in a dynamic, label-free, and noninvasive way.19 To our knowledge, Received: January 19, 2011 Accepted: March 5, 2011 Published: March 25, 2011 3141

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Figure 1. Real-time QCM-D measurements of the responses of A431 cells to EGF exposure. (a) ΔD-responses in the presence and absence of 10 nM EGF. Peak DM is indicated by the arrow. The lowest values of ΔD-responses are indicated by dotted line H. (b) Δf-responses in the presence and absence of 10 nM EGF. Peak L and peak fM are indicated by the arrows. Parts a and b show data from the same control and experimental pair. Part b has an expanded x-axis compared with that of part a to make the features observed more visible at an earlier time. The frequency responses remain unchanged between 60 and 120 min.

however, none of these QCM-based techniques have been applied to the study of receptor-mediated cell signaling. This report describes a novel application of QCM-D, a realtime and label-free sensor technology, for the assessment of the cell signaling mediated by high-affinity EGFR. It is known that high-affinity EGFR is associated with the cytoskeleton in A431 cells.20 When EGF binds to and activates high-affinity EGFR, cytoskeletal structures undergo drastic reorganization,21 which in turn induces cell rounding, membrane ruffling, and extension of filopodia.22 These morphological changes are closely associated with EGFR-mediated cell signaling and trafficking.23 Meanwhile, reorganization of the cytoskeleton also alters the mechanical properties,24 i.e., the viscoelastic character, of the cell. Thus, being able to monitor changes in viscoelasticity of a layer of cells attached to a surface is expected to provide an assessment of the function of high-affinity EGFR.

’ EXPERIMENTAL SECTION Reagents and Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin
EDTA, HEPES buffer, and HBSS buffer were purchased from Invitrogen. A431 cells were obtained from American Type Tissue Collection. Human epidermal growth factor was purchased from Peprotech. PD158780 and ethylene glycol tetraacetic acid (EGTA) were obtained from EMD Biosciences. EGFR monoclonal antibody mAb2 E9 was obtained from Santa Cruz Biotechnology, Inc. Cytochalasin D was purchased from Tocris Cookson. All other chemicals were obtained from Sigma-Aldrich. Cell Culture. A431 cells were cultured in T75 Corning culture flasks and maintained under a humidified atmosphere at 37 °C and 5% CO2 in DMEM containing 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The cells were usually harvested at 90% confluency. QCM-D Measurement. A quartz crystal microbalance with dissipation monitoring (E4, Q-Sense) was used to record changes in the resonant frequencies (Δf) and the energy dissipation (ΔD) as a function of time at the odd overtones (n = 3, 5, 7). All measurements were carried out on gold-deposited, polished circular (14 mm, QSX 301), AT-cut sensor crystals with a fundamental resonant frequency of 5 MHz. The sensors were prepared as follows: First the gold-coated QCM-D sensors were washed with water and ethanol and then exposed to UV-ozone for 20 min.

The sensors were then stored in the tissue culture hood under UV light. Each sensor was then placed into an individual well in a 12-well tissue culture plate along with A431 cells that were harvested from the T75 culture flask. The cells were allowed to attach and grow on the top side of gold sensor under a humidified atmosphere at 37 °C and 5% CO2. Once they reached 90% confluency, the cells on the sensors were washed with phosphate buffered saline and starved in serum free medium for 18 h. On the day of the QCM-D measurement, the sensors with the cultured cells were carefully rinsed with the assay buffer (20 mM HEPES in HBSS buffer, pH 7.2) and then the back sides of the sensors were dabbed with Kimwipe to remove residual buffer. Each sensor with cells attached was then mounted in an open module (Q-sense) and incubated in 400 μL of the assay buffer at 37 °C. Changes in frequency (Δf) and dissipation (ΔD) were then recorded simultaneously. After stable baselines were achieved for all four sensors, the assay buffer was removed from each module and EGF in 400 μL of the assay buffer that had been prewarmed to 37 °C was added. The measurements of both changes in frequency (Δf) and dissipation (ΔD) were allowed to continue at 37 °C for 3 h. For experiments involving pretreatments, the cells were incubated with the pretreatment solutions at 37 °C for a minimum of 30 min prior to the addition of EGF. All experiments were replicated a minimum of three times; variation among replicates was approximately (5 units for both frequency and dissipation measurements. Data Analysis. All curves describing dose-dependence were generated by plotting the average amplitudes ((1 std deviation) of ΔD-responses taken at dotted line H (see Figure 1a) and Δfresponses taken at peak L (see Figure 1b) as a function of EGF concentrations. (H stands for high-affinity and L stands for lowaffinity). Amplitude is the difference between the experimental value and the control value. The EC50 values were determined from the fitted curves with PSI-Plot (Poly Software International). The estimated EGFR binding affinities were computed from the relationship, kd = EC50.

’ RESULTS AND DISCUSSION In the present study, the QCM-D was used to track the shortterm, EGF-induced response of a confluent monolayer of human carcinoma A431 cells. Because of the variations that are often associated with cell assays, all the experiments described here were repeated a minimum of three times to ensure the reliability 3142

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Figure 2. Real-time QCM-D measurements of the responses of A431 cells to EGF exposure. (a) The ΔD-response of the cells pretreated with EGFR tyrosine kinase inhibitor, PD158780, showing suppression of EGF-induced response. (b) The Δf-response (peak L) of the cells pretreated with EGFR tyrosine kinase inhibitor, PD158780, showing suppression of EGF-induced response.

Figure 3. Real-time QCM-D measurements of the responses of A431 cells to EGF exposure. (a) The ΔD-response of the cells pretreated with EGFR monoclonal antibody mAb 2E9, showing no suppression of EGF-induced response. (b) The Δf-response (peak L) of the cells pretreated with mAb 2E9, showing a significant suppression of EGF-induced response.

and reproducibility of the results. Stable baselines were achieved for all experiments prior to the addition of EGF (Supporting Information, Figures S1
S4). Although the measurements were conducted at overtones n = 3, 5 and 7, the results from higher overtones echoed those from n = 3 but were much less intense, so only the results from n = 3 are presented. Figure 1a shows the effect of 10 nM EGF on the ΔD-response of the cells as a function of time. The cells treated with (EGFtreated) or without EGF (buffer-treated) show an initial sharp increase in ΔD (peak DM), which is due to mechanical perturbation of the cells by the transfer of liquid by pipetting. (DM stands for dissipation, mechanical perturbation). After an initial rise, a rapid decrease in ΔD is shown by both cell samples. However, the EGF-treated cells exhibited a much greater decrease in ΔD than the buffer-treated cells did. This difference represents the EGF-induced ΔD-response of the cells. The observed large decrease in ΔD of the EGF-treated cells indicates a decrease in the viscous character and suggests an increase in rigidity of the cell monolayer. This is consistent with the known stiffening and rounding of A431 cells in response to the EGF treatment.22 The lowest value of ΔD was reached at about 50 min (at dotted line H), which agrees well with previous observations that A431 cells develop the most extreme rounding around 45 min.25 (H stands for high-affinity). The Δf-responses of the cells as a function of time from the same experiment are shown in Figure 1b. The initial response of both the EGF-treated and buffer-treated cells was a sharp decrease (peak fM), resulting from the above-mentioned

mechanical perturbation. (fM stands for frequency, mechanical perturbation). After this, the Δf-response of the buffer-treated cells rose and leveled off at a constant value, while the Δfresponse of the EGF-treated cells continued to rise to produce peak L. (L stands for low-affinity). Thus peak L represents the EGF-induced Δf-response of the cells. The increase in Δf shown by the ascending side of peak L reflects a decrease in mass. The particular overtone (n = 3) used in our study senses the bottom portion of the cell layer as well as the shallow pockets of liquid medium residing underneath the cells.19 Thus, the mass decrease could arise from the transport of ions or liquid medium out of the shallow pockets underneath the cells into the cytoplasm19 (vide infra). Next, we determined if both the EGF-induced ΔD- and Δfresponses observed above were indeed cellular responses to EGFR-mediated cell signaling. Figure 2a,b shows the results for cells pretreated and not pretreated with PD158780, a potent inhibitor of EGFR tyrosine kinase,26 prior to the exposure to 10 nM EGF. The cells without the pretreatment showed the expected decrease in ΔD (Figure 2a) and the expected appearance of peak L (Figure 2b). The pretreatment with the inhibitor suppressed the decrease in ΔD (Figure 2a) as well as the development of peak L (Figure 2b). These inhibitor-suppressed responses were strikingly similar to the responses of the buffertreated cells in Figure 1a,b, which had not been exposed to EGF. This indicated that EGFR tyrosine kinase is responsible for the large decrease in ΔD observed in Figure 1a and also for peak L in 3143

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Figure 4. Dose-dependent, EGF-induced responses of A431 cells. (a) Dose-dependent ΔD-responses versus time. (b) Dose-dependent Δf-responses versus time. (c) The amplitudes of ΔD-responses at dotted line H, average (1 std deviation of at least three replicate experiments, as a function of EGF concentration. (d) The amplitudes of Δf-responses at peak L, average (1 std deviation of at least three replicate experiments, as a function of EGF concentration.

Figure 1b. Thus the ΔD- and Δf-responses observed in Figure 1a, b were due to EGFR-mediated cell signaling. The next logical question would be which subclass of EGFR, the low-affinity or the high-affinity, is responsible for ΔD- and Δfresponses to EGF shown in Figure 1a,b. To determine this, we compared the ΔD-response of cells exposed to 10 nM EGF with and without pretreatment with a saturating amount of EGFR monoclonal antibody mAb 2E9 (333 nM). This antibody is known to block cell signaling mediated by low-affinity EGFR but not to affect high-affinity EGFR signaling in A431 cells.7 Figure 3a shows that the cells with and without pretreatment of the antibody gave nearly identical decreases in ΔD. Because the antibody had no effect on the ΔD-response of the cells, the ΔD-response is therefore due to the cell signaling mediated by high-affinity EGFR alone. The Δf-response of the cells pretreated with the antibody shows that peak L was significantly reduced by the antibody pretreatment (Figure 3b); therefore, peak L reflects the cellular response to low-affinity EGFR signaling. To further verify the specificity of the QCM-D signal toward the individual subclass of EGFR, the dose
response of EGF was examined. As shown in Figure 4a,b, the higher the concentrations of EGF, the greater the amplitudes of the ΔD-response at dotted line H and the Δf-response at peak L. The dose
response curves were obtained from the amplitudes of ΔD and Δf-responses vs EGF concentrations, shown in Figure 4c,d. The EC50 values were determined to be 2.1 nM for high-affinity EGFR signaling from amplitudes of ΔD-responses in Figure 4c and 39 nM for lowaffinity EGFR signaling from amplitudes of Δf-responses in Figure 4d. If the EC50-values are used as approximate measures of binding affinity,27 our values for high-affinity and low-affinity EGFRs are consistent with kd-values obtained by others. Overall,

the results of our dose
response study further validate that the QCM-D signals, ΔD at dotted line H and Δf at peak L, are specific for the high-affinity and low-affinity EGFR, respectively. These signals provide a quantitative measure of the extent of EGFR signaling. There are biosensors that track changes in cellular morphology, adhesion, ion distribution, and mass distribution in response to a stimulus.28
30 It is often difficult to identify the exact cellular process or processes that are responsible for the biosensor signals. Combining the extensive information available from the literature on EGFR signaling in A431 cells with the QCMD, we sought to identify the cellular processes primarily responsible for the ΔD- and Δf-responses observed in our study. To investigate the specific cause of the ΔD-response, we examined whether the ΔD-response was due to the remodeling of the cytoskeleton, a process that is integral to the cell signaling mediated through high-affinity EGFR. Cytochalasin D (CD) is a potent, cell-permeable inhibitor of actin polymerization and is capable of attenuating the remodeling of the cytoskeleton31 and inhibiting the stiffening of cells. As shown in Figure 5a, 0.6 μM of CD substantially suppressed the ΔD-response of the cells to 10 nM of EGF, confirming that cytoskeleton remodeling is the major cause of the ΔD-response to EGF. This echoes the findings of Heitmann and co-workers’ that a change in the cytoskeleton is a major contributor to the dissipation-related QCM response of cells.19 To investigate the underlying cause of peak L in the Δfresponse, we focused our attention on the ascending side of peak L, which shows an increase in Δf, an indication of a decrease in mass within the sensing volume. We suggest that this loss of mass is caused by the transport of extracellular ions and/or liquid 3144

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Figure 5. The identification of EGF-induced cellular processes that are primarily responsible for ΔD- and Δf-responses. (a) ΔD-response was suppressed substantially for the cells pretreated with an inhibitor of actin polymerization, cytochalasin D. The Δf-response of the cells in this experiment is not shown. (b) Δf-response at peak L was almost abolished for the cells pretreated with a calcium chelator, EGTA; peak L of the EGTA treated cells is only 6 Hz while that of the control (with no EGTA pretreatment) is 42 Hz. The ΔD-response of the cells in this experiment is not shown.

medium from underneath the cell layer into the cytoplasm above. This transport can be accomplished by means of Ca2þ influx and/or fluid-phase pinocytosis; both of these processes have previously been shown to accompany the activation of lowaffinity EGFR in A431 cells.7 To verify if either of these processes is responsible for peak L, we measured the 10 nM EGF-induced response of the cells pretreated with 3 mM of ethylene glycol tetraacetic acid (EGTA). A divalent ion chelator, EGTA blocks calcium influx by trapping Ca2þ and preventing its entry into cells.32,33 The result, in Figure 5b, shows that the presence of 3 mM of EGTA substantially reduced peak L with respect to the control: peak L of the EGTA treated cells is only 6 Hz while that of the control (no EGTA pretreatment) is 42 Hz. In addition, the time scale of peak L agrees well with that of calcium influx previously detected by others using fluorescent labels.7 These results strongly support that peak L is caused by the EGFinduced Ca2þ influx. The near-complete suppression of peak L by EGTA implies that other cellular processes, such as fluidphase pinocytosis, do not contribute significantly to peak L. The descending side of peak L most probably corresponds to the restoration of the ion balance of the cells.

In recent years, mechanical properties of cells have become the subject of considerable scientific research because of the potential link between these properties and human diseases.37 With a quantifiable measure of cytoskeleton remodeling based on energy dissipation of cells, QCM-D offers a noninvasive alternative to complement the existing techniques for tracking changes in mechanical properties of the cells in a real-time and continuous manner. This may also help obtain a fundamental understanding of the correlation between cell function and cell mechanical properties.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 215-895-2648. Fax: 215-8951265. Present Addresses §

’ CONCLUSIONS QCM-D provides a novel approach for monitoring the shortterm responses of cells to EGFR-mediated signaling by real-time tracking of changes in the dissipation and frequency responses of cells attached to a surface. Our study revealed that changes in dissipation, ΔD, are associated with the remodeling of the cytoskeleton and represent the cellular response to signaling mediated by high-affinity EGFR. Changes in frequency, Δf, are associated with the calcium influx and represent the cellular response to signaling mediated by low-affinity EGFR. To our knowledge, this is the first example of tracking real-time cell signaling based on the measurement of cellular mechanical response in a label-free manner. We envision that the unique capability of QCM-D can be exploited to investigate the role of high-affinity EGFR in development of cancer and will complement the existing approach to prognosis of cancer. Because of the importance of the cytoskeleton in regulation of cell signaling and trafficking in general,34,35 QCM-D may have the potential to be applied to the study of other types of receptor-mediated cell signaling and trafficking.36

Department of Ophthalmology, University of Pennsylvania, 422 Curie Boulevard, Philadelphia, PA 19104.

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