Evaluating Inhibition of the Epidermal Growth Factor (EGF)-Induced Response of Mutant MCF10A Cells with an Acoustic Sensor

Abstract

Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

On the Measurement of Energy Dissipation of Adhered Cells with the Quartz Microbalance with Dissipation Monitoring

“…Assuming that the cell-ECM bonds are uniformly distributed over the basal region of the entire cell layer, the D -response shown in Figure 4D clearly supports a linear correlation between the D -response and the level of cell adhesion quantified with the number of cell-ECM bonds. This result corroborates our previous experimental finding of a linear correlation between the Δ D -response and the level of cell adhesion 39,40 . Surprisingly, this linear correlation remains validate across a wide dynamic range that covers the bond number from 1.0×10 6 up to approximately 1.7×10 11 , corresponding to a range of the D -response from 150×10 −6 up to approximately 500×10 −6 .…”

“…D m =5.12×10 −4 , D 0 =−3.64×10 −4 and B 0 =9.68×10 10 are fitting parameters. To verify our previous experimental finding of a linear correlation between the Δ D -response and the level of cell adhesion 39,40 , the range of the D -response in this plot that can offer a linear correlation was explored: The data points corresponding to the leveled D -response at bond numbers of 5×10 11 and higher (in Figure 4C) were removed. The remaining data points were replotted as shown in Figure 4D and fitted with a linear regression function: D = tB + D 0 , where D is the energy dissipation factor and B is the bond number.…”

“…We sought to evaluate the effectiveness of our biomechanical model in assessing the EGF-induced de-adhesion of A431 cells using the QCM-D measurement as a benchmark. Previously we used the QCM-D to examine the EGF-induced de-adhesion of MCF10A cells 39,40,70 with the aid of immunostained vinculin within the focal adhesions. A similar approach was employed in this study: We quantified the size/relative fluorescence intensity (RFU) of vinculins at various time points based on the areal density of focal adhesions of EGF-treated A431 cells shown in Figure 5A (the experimental methods are detailed in S7).…”

“…Considering our experimental finding of the linear correlation between the areal density of focal adhesions and the Δ D -response of adherent cells 39,40 , we expect that the dissipative processes that contribute to the Δ D -response should be those cellular behaviors that are capable of influencing the dynamic restructuring of focal adhesions. These cellular behaviors include those that have been overlooked by the current modeling approaches.…”

“…Thus, such a Δ f -based measurement is more of a qualitative assessment of the cell adhesion. The time-dependent change of energy dissipation (Δ D ) − has been used to complement the measurement of the Δ f -response by providing information on the intrinsic structural and mechanical properties of adhered cells. ,− However, the dissipative processes that are responsible for the measured Δ D -response of adhered cells remains controversial. ,,,, …”