**2. Investigation of the dynamic mechanical response of an A431 cell monolayer in response to EGF**

#### **2.1. Mechanical behavior of an A431 cell monolayer probed with AFM**

An example of the use of the AFM to assess change in the mechanical response of cells upon exposure to a biologically active molecule is illustrated in this section. Figure 3 shows the Young's moduli, obtained at a probe speed of 5.8 µm/s and indentation depth of ~500 nm, for two hundred randomly selected A431 cells before and after the treatment with a 40-nM

EGF solution (Yang et al., 2012). The focus is on the comparison, rather than on the modulus values themselves. The two samples show a statistically significant increase (p < 0.05) in average modulus as a result of the treatment: 11.2 ± 2.8 kPa for untreated cells and 18.7 ± 2.0 kPa for treated cells.

Dynamic Mechanical Response of Epithelial Cells to Epidermal Growth Factor 177

Although both stiffness and dissipation of the cells increase simultaneously, the hysteresivity of the cells, which is determined based on the ratio of area within the hysteresis loop to area under the approach curve (Figure 1) (Collinsworth et al., 2002; Fung, 1984; Smith et al., 2005), is not constant but increases as a function of time, shown in Figure 5. According to the SGR model, upon a non-thermal stimulation (e.g., ATP depletion, or cell relaxing agent), the cell can undergo a change in mechanical ordering state either toward the glass transition as hysteresivity decreases or away from the glass transition as hysteresivity increases. So the cell can modulate its mechanical state between a more solid-like state and a more liquid-like state (Smith et al., 2005). The increase in hysteresivity in Figure 5 implies that the mechanical state of the EGF-treated cells moves away from the glass transition and possibly takes on a more fluidic behavior. This interpretation is consistent with morphological changes observed in A431 cells responding to EGF stimulation, where the cells undergo cell rounding, membrane ruffling, and filopodia extension, all of which might be facilitated by a more fluid-like

**Figure 5.** Hysteresivity versus time for exposure of A431 cells to EGF at 0, 10 nM, 20 nM, and 40 nM (Yang et al., 2012). A 30-min baseline was established for each measurement prior to the addition of EGF. Trend lines were used to connect each data point to illustrate the trend for each response curve.

An example of the use of QCM-D to assess change in the mechanical response of cells upon exposure to a biologically active molecule is shown in Figure 6. This figure shows ∆*f* and ∆*D* obtained simultaneously in response to addition of a solution of EGF. The spike at 5 min is an artifact of the manual addition of the solution with a pipette. After the initial spike, ∆*D* goes down and then slowly recovers, while ∆*f* steadily increases with time (Chen et al., 2011). Because ∆*D* was substantially more sensitive than ∆*f* to dosage of EGF, we focus the

Figure 7 shows the QCM-D measurement of ∆*D* for a monolayer of A431 cells exposed to buffer alone and to EGF. Figure 7A reveals that, after the spike caused by manual addition

**2.2. Mechanical behavior of an A431 cell monolayer probed with QCM-D** 

state of the cells (Chinkers et al., 1981).

descriptions primarily on the changes in ∆*D*.

**Figure 3.** Histograms of the distributions of the Young's modulus of two hundred randomly selected A431 cells before (A) and after (B) the treatment with a 40-nM EGF solution in buffer (Yang et al., 2012).

Figure 4 shows changes in amount of the dissipated energy of cells upon EGF stimulation. It is evident that energy dissipation of a cell, as indicated by the area of the hysteresis loop, increases after the treatment with 40 nM EGF (Figures 4A and 4B). Figure 4C summarize the distribution of such differences exhibited by one hundred randomly selected cells. A statistically significant difference (p < 0.05) in energy dissipation per cycle is shown: 3.09 ± 0.79 fJ before the treatment and 5.10 ± 0.71 fJ after the treatment.

**Figure 4.** Force-displacement curves for a single cell before (A) and after (B) the treatment with 40 nM EGF. Histograms (C) of energy dissipation for one hundred randomly selected cells before (blue) and after (red) the treatment with EGF.

The underlying meaning of a simultaneous increase in both stiffness (Young's modulus) and energy dissipation of the cells upon the EGF treatment can be better understood with the soft glassy rheology (SGR) model (Fabry et al., 2001). In this model, the cell is considered as a soft glassy material that is structurally disordered and metastable (Sollich, 1998). The hysteresivity of the cell, *η*, defined as the fraction of the elastic (input) energy that is dissipated as heat, is frequency insensitive and remains constant in the low frequency regime (Fredberg & Stamenovic, 1989).

Although both stiffness and dissipation of the cells increase simultaneously, the hysteresivity of the cells, which is determined based on the ratio of area within the hysteresis loop to area under the approach curve (Figure 1) (Collinsworth et al., 2002; Fung, 1984; Smith et al., 2005), is not constant but increases as a function of time, shown in Figure 5. According to the SGR model, upon a non-thermal stimulation (e.g., ATP depletion, or cell relaxing agent), the cell can undergo a change in mechanical ordering state either toward the glass transition as hysteresivity decreases or away from the glass transition as hysteresivity increases. So the cell can modulate its mechanical state between a more solid-like state and a more liquid-like state (Smith et al., 2005). The increase in hysteresivity in Figure 5 implies that the mechanical state of the EGF-treated cells moves away from the glass transition and possibly takes on a more fluidic behavior. This interpretation is consistent with morphological changes observed in A431 cells responding to EGF stimulation, where the cells undergo cell rounding, membrane ruffling, and filopodia extension, all of which might be facilitated by a more fluid-like state of the cells (Chinkers et al., 1981).

176 Viscoelasticity – From Theory to Biological Applications

kPa for treated cells.

EGF solution (Yang et al., 2012). The focus is on the comparison, rather than on the modulus values themselves. The two samples show a statistically significant increase (p < 0.05) in average modulus as a result of the treatment: 11.2 ± 2.8 kPa for untreated cells and 18.7 ± 2.0

**Figure 3.** Histograms of the distributions of the Young's modulus of two hundred randomly selected A431 cells before (A) and after (B) the treatment with a 40-nM EGF solution in buffer (Yang et al., 2012).

Figure 4 shows changes in amount of the dissipated energy of cells upon EGF stimulation. It is evident that energy dissipation of a cell, as indicated by the area of the hysteresis loop, increases after the treatment with 40 nM EGF (Figures 4A and 4B). Figure 4C summarize the distribution of such differences exhibited by one hundred randomly selected cells. A statistically significant difference (p < 0.05) in energy dissipation per cycle is shown: 3.09 ±

**Figure 4.** Force-displacement curves for a single cell before (A) and after (B) the treatment with 40 nM EGF. Histograms (C) of energy dissipation for one hundred randomly selected cells before (blue) and

The underlying meaning of a simultaneous increase in both stiffness (Young's modulus) and energy dissipation of the cells upon the EGF treatment can be better understood with the soft glassy rheology (SGR) model (Fabry et al., 2001). In this model, the cell is considered as a soft glassy material that is structurally disordered and metastable (Sollich, 1998). The hysteresivity of the cell, *η*, defined as the fraction of the elastic (input) energy that is dissipated as heat, is frequency insensitive and remains constant in the low frequency

0.79 fJ before the treatment and 5.10 ± 0.71 fJ after the treatment.

after (red) the treatment with EGF.

regime (Fredberg & Stamenovic, 1989).

**Figure 5.** Hysteresivity versus time for exposure of A431 cells to EGF at 0, 10 nM, 20 nM, and 40 nM (Yang et al., 2012). A 30-min baseline was established for each measurement prior to the addition of EGF. Trend lines were used to connect each data point to illustrate the trend for each response curve.

#### **2.2. Mechanical behavior of an A431 cell monolayer probed with QCM-D**

An example of the use of QCM-D to assess change in the mechanical response of cells upon exposure to a biologically active molecule is shown in Figure 6. This figure shows ∆*f* and ∆*D* obtained simultaneously in response to addition of a solution of EGF. The spike at 5 min is an artifact of the manual addition of the solution with a pipette. After the initial spike, ∆*D* goes down and then slowly recovers, while ∆*f* steadily increases with time (Chen et al., 2011). Because ∆*D* was substantially more sensitive than ∆*f* to dosage of EGF, we focus the descriptions primarily on the changes in ∆*D*.

Figure 7 shows the QCM-D measurement of ∆*D* for a monolayer of A431 cells exposed to buffer alone and to EGF. Figure 7A reveals that, after the spike caused by manual addition

of the solution, the control cells (0 nM EGF) showed a small decrease in ∆*D*, while the experimental cells (10 nM EGF) exhibited a large decrease. This difference represents the change in mechanical energy dissipation of the basal areas of the cells induced by EGF.

Dynamic Mechanical Response of Epithelial Cells to Epidermal Growth Factor 179

inhibitor, the cells showed a greatly suppressed ∆*D* response upon exposure to EGF, while the cells without pretreatment showed the expected decrease in ∆*D* (Figure 8). These results are evidence that the EGF-induced the ∆*D* response is due to EGFR-mediated cell signaling.

**Figure 8.** The ΔD response of the cells pretreated with EGFR tyrosine kinase inhibitor, PD158780, showing suppression of 10 nM EGF-induced response. This figure is adapted with permission from

The change in mechanical properties of cells has been attributed to remodeling of the cytoskeleton (Kuznetsova et al., 2007), which can be induced by EGF treatment (Rijken et al., 1995; Rijken et al., 1998). Direct evidence of remodeling of the cytoskeleton can be obtained with fluorescence imaging. For this, the cells were first treated with a 10-nM EGF solution for 60 min under the same conditions used for both AFM and QCM-D measurements. The actin filaments of the cytoskeleton were then stained with fluorescently labeled phalloidin

**Figure 9.** Remodeling of actin filaments in a monolayer of A431 cells induced by the treatment of 10 nM of EGF at 37°C. (A) and (B) show fluorescence-stained actin cortex at the top-half of the cell layer before

As shown in Figures 9A and B, the top portion of the membrane skeleton exhibited an increase in brightness after the 60-min EGF treatment, indicating an increase of the number and size of the cortical actin filaments. Considering that the cortical actin provides cells with a structural framework, the increase in cortical actin can be assumed responsible for the increased rigidity of the cells, which is manifested as the increase in Young's modulus measured by AFM. In addition, because both energy dissipation and hysteresivity were derived from the same set of force-displacement curves used for determining Young's

and after the 60-min treatment with EGF solution, respectively (Yang et al., 2012).

**2.3. Remodeling of cytoskeleton probed with fluorescence imaging** 

(Chen et al., 2011). Copyright 2011, American Chemical Society.

and imaged with an inverted fluorescence microscope.

**Figure 6.** Typical response of A431 cells to EGF. Both ∆*f* and ∆*D* are shown as a function of time.

**Figure 7.** Real-time QCM-D measurements of the Δ*D* response of a monolayer of A431 cells to exposure to EGF (Chen et al., 2011). (A) Δ*D* response of the cells in the presence (bottom) and absence (top) of 10 nM EGF. (B) Δ*D* response of the cells pretreated with an inhibitor of actin polymerization, cytochalasin D, showing suppression of the EGF-induced response. Both figures are adapted with permission from (Chen et al., 2011). Copyright 2011, American Chemical Society.

A likely cause of the reduction in ∆*D* upon exposure to EGF was the remodeling of the cytoskeleton, a process integral to the mechanical response of the cell. This remodeling can be interfered with if the polymerization of actin is inhibited. Figure 7B shows that pretreatment of the monolayer of cells with a 0.6 µM solution of cytochalasin D, a potent, cell-permeable inhibitor of actin polymerization (Schliwa, 1982), diminished the magnitude of the response of the cells to EGF. This result supports the remodeling of the cytoskeleton as the cause of the ∆*D* response, which echoes the previous finding of Heitmann and coworkers' (Heitmann et al., 2007).

It is desirable to connect the EGF-induced changes in ∆*D* to EGFR-mediated cell signaling. As already mentioned, one of the domains of EGFR, the receptor for EGF, is the intracellular tyrosine kinase domain. This domain is integral to the signaling pathway within the cell that transmits the effects of EGF to other parts of the cell, and if this domain is inhibited, the signaling pathway is disrupted. PD158780 has been shown to be a potent inhibitor of the tyrosine kinase domain for EGFR (Rewcastle et al., 1996). With a pretreatment with this inhibitor, the cells showed a greatly suppressed ∆*D* response upon exposure to EGF, while the cells without pretreatment showed the expected decrease in ∆*D* (Figure 8). These results are evidence that the EGF-induced the ∆*D* response is due to EGFR-mediated cell signaling.

178 Viscoelasticity – From Theory to Biological Applications

of the solution, the control cells (0 nM EGF) showed a small decrease in ∆*D*, while the experimental cells (10 nM EGF) exhibited a large decrease. This difference represents the change in mechanical energy dissipation of the basal areas of the cells induced by EGF.

**Figure 6.** Typical response of A431 cells to EGF. Both ∆*f* and ∆*D* are shown as a function of time.

**Figure 7.** Real-time QCM-D measurements of the Δ*D* response of a monolayer of A431 cells to exposure to EGF (Chen et al., 2011). (A) Δ*D* response of the cells in the presence (bottom) and absence (top) of 10 nM EGF. (B) Δ*D* response of the cells pretreated with an inhibitor of actin polymerization, cytochalasin D, showing suppression of the EGF-induced response. Both figures are adapted with permission from

A likely cause of the reduction in ∆*D* upon exposure to EGF was the remodeling of the cytoskeleton, a process integral to the mechanical response of the cell. This remodeling can be interfered with if the polymerization of actin is inhibited. Figure 7B shows that pretreatment of the monolayer of cells with a 0.6 µM solution of cytochalasin D, a potent, cell-permeable inhibitor of actin polymerization (Schliwa, 1982), diminished the magnitude of the response of the cells to EGF. This result supports the remodeling of the cytoskeleton as the cause of the ∆*D* response, which echoes the previous finding of Heitmann and

It is desirable to connect the EGF-induced changes in ∆*D* to EGFR-mediated cell signaling. As already mentioned, one of the domains of EGFR, the receptor for EGF, is the intracellular tyrosine kinase domain. This domain is integral to the signaling pathway within the cell that transmits the effects of EGF to other parts of the cell, and if this domain is inhibited, the signaling pathway is disrupted. PD158780 has been shown to be a potent inhibitor of the tyrosine kinase domain for EGFR (Rewcastle et al., 1996). With a pretreatment with this

(Chen et al., 2011). Copyright 2011, American Chemical Society.

coworkers' (Heitmann et al., 2007).

**Figure 8.** The ΔD response of the cells pretreated with EGFR tyrosine kinase inhibitor, PD158780, showing suppression of 10 nM EGF-induced response. This figure is adapted with permission from (Chen et al., 2011). Copyright 2011, American Chemical Society.

### **2.3. Remodeling of cytoskeleton probed with fluorescence imaging**

The change in mechanical properties of cells has been attributed to remodeling of the cytoskeleton (Kuznetsova et al., 2007), which can be induced by EGF treatment (Rijken et al., 1995; Rijken et al., 1998). Direct evidence of remodeling of the cytoskeleton can be obtained with fluorescence imaging. For this, the cells were first treated with a 10-nM EGF solution for 60 min under the same conditions used for both AFM and QCM-D measurements. The actin filaments of the cytoskeleton were then stained with fluorescently labeled phalloidin and imaged with an inverted fluorescence microscope.

**Figure 9.** Remodeling of actin filaments in a monolayer of A431 cells induced by the treatment of 10 nM of EGF at 37°C. (A) and (B) show fluorescence-stained actin cortex at the top-half of the cell layer before and after the 60-min treatment with EGF solution, respectively (Yang et al., 2012).

As shown in Figures 9A and B, the top portion of the membrane skeleton exhibited an increase in brightness after the 60-min EGF treatment, indicating an increase of the number and size of the cortical actin filaments. Considering that the cortical actin provides cells with a structural framework, the increase in cortical actin can be assumed responsible for the increased rigidity of the cells, which is manifested as the increase in Young's modulus measured by AFM. In addition, because both energy dissipation and hysteresivity were derived from the same set of force-displacement curves used for determining Young's modulus of the top region of the cells, it is reasonable to assume that the increases in dissipation and hysteresivity were also related to the increase in cortical actin filaments.

Dynamic Mechanical Response of Epithelial Cells to Epidermal Growth Factor 181

Interestingly, both hysteresivity (*η*) and the change in energy dissipation factor (∆*D*) required about approximately the same amount of time (30 to 40 min) to reach each respective maxima or minimum after the addition of 10 nM of EGF. Such a similarity suggests that both of these regional mechanical responses (∆*D* and *η*) were mediated by

In this chapter, the examples were provided to illustrate how to employ a combination of AFM and QCM-D to characterize the mechanical behavior of cells in response to exposure to EGF. This unique combination allowed the comparative assessment of the upper volume of the cell bodies as well as the basal areas of the cells. Results from both parts of the A431 cells reveal a regionally specific mechanical behavior of the cells, which can be attributed to the distinct cytoskeleton structures utilized by the cells to alter the local structure in response to EGF stimulation. The signaling pathways that mediate the remodeling of the cytoskeleton in the upper volume of the cell bodies are likely closely coupled to those in the basal areas of the cells. There is a clear correlation between the time-dependent mechanical response and

Overall, the combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during the dynamic cell signaling process than either technique alone. The use of combined techniques to track real-time cell signaling based on the measurement of cellular mechanical response in a label-free manner is a powerful approach for investigating the role of EGFR in causing abnormal cell behavior. This combined, real-time approach may have the potential to be applied to the study of other types of receptor-mediated cell signaling and trafficking. This approach should contribute to the fundamental understanding of the correlation between cell function and cell mechanical

closely coupled cell signaling pathway(s) of EGFR.

the dynamic process of EGFR signaling.

Jun Xi, Lynn S. Penn and Jennifer Y. Chen

*Drexel University, Department of Chemistry, Philadelphia, PA 19104, USA* 

*Michigan State University, Department of Electrical and Computer Engineering,* 

GM084520. We also appreciate the financial support from Drexel University.

This manuscript recapitulates some information from original works that have previously been published in *Analytical Chemistry* (Chen et al., 2011) and *Experimental Cell Research*  (Yang et al., 2012). This work was supported in part by NSF Grants IIS-0713346 and DMI-0500372, ONR Grants N00014-04-1-0799 and N00014-07-1-0935, and NIH Grant R43

**3. Conclusions** 

properties.

**Author details** 

Ning Xi and Ruiguo Yang

*East Lansing, MI 48824, USA* 

**Acknowledgement** 

The basal area of the cell monolayer, i.e., the area probed by the QCM-D, was also examined by means of fluorescence imaging. In this case, the stress fibers, which are actin filaments that reside in the bottom portion of the cells and are involved in the formation of focal adhesions attaching the cell to the substrate, were studied. As shown in Figures 10A and 10B, the cells displayed a decrease in amount and size of stress fibers after 60 min of exposure to EGF. Thus the decrease in dissipation observed by means of the QCM-D technique can be reasonably assumed to be related to a reduction in number and size of the actin stress fibers in the basal region of the cells. In addition, this reduction in stress fibers suggests a loss of adhesion and/or contact between the cells and the solid substrate, which is consistent with the EGF-induced cell rounding and retracting that has been reported previously (Chinkers et al., 1979; 1981).

**Figure 10.** Remodeling of actin filaments in a monolayer of A431 cells induced by the treatment of 10 nM of EGF at 37°C. (A) and (B) show fluorescence-stained stress fibers at the basal area of the cell layer before and after the 60-min EGF treatment, respectively.
