Abstract A016: Electrical impedance assessment of the effect of LBH589 on the cellular behavior and migratory potential of breast cancer cells

Abstract

Abstract We have previously shown histone deacetylase inhibitor, LBH589 (Panobinostat), to increase E-cadherin expression and alter mesenchymal MDA-MB-231 cell morphology to a more epithelial phenotype suggestive of the reversal of EMT (epithelial-to-mesenchymal transition). As EMT has been linked to enhanced cell motility, we examined the effects of LBH589 on breast cancer cells in vitro. Electrical Cell-substrate Impedance Sensing (ECIS) allows for real-time, noninvasive measurement of cell behavior and reproducible wounding by lethal electroporation of the cell monolayer for migration studies. Method: A 1 uA, 4kHz AC current applied across surface electrodes (250-μm diameter) provided electrical impedance monitoring capability, as MBA-MB-231 or MCF-7 breast cancer cells attached, spread, proliferate on the substrate. Measurements were reported as calculated resistance and capacitance. As cells grow on the substrate, the cell-electrode interaction is such that the current is forced to flow underneath and between cells. At 64 kHz, the capacitance is an indicator of electrode surface coverage. At 1 kHz, the resistance is an indicator of tight-junction formation between adjacent cells. As a monolayer develops, a decrease in capacitance and increase in resistance is observed. Upon visual confirmation of established cell monolayers, cells were treated with the LBH589 and cellular response was measured for 24 hrs. Afterwards, an elevated current (1 mA, 40 kHz, duration >200 ms) was applied to the surface electrode, permanently damaging cell plasma membranes directly above the electrode and creating a defined gap in the monolayer or ‘wound’. Repopulation of the ‘wound’ was then monitored for 24 hrs. Results: Cells were successfully grown and electrically monitored on the ECIS arrays. Micrographs show that proliferating MCF-7 and MDA-MB-231 cells gradually covered the electrode surface. MCF-7 cells arranged into a ‘cobblestone’ monolayer without gaps between adjacent cells, whereas MDA-MB-231 cells formed a loose monolayer with spaces between neighboring cells. At 22 hrs, the capacitance values for MCF-7 and MDA-MB-231 cells were observed to stabilize at ~1.3 nF and ~7.5 nF, respectively. The corresponding resistance values were ~40,000 Ohm and ~2,100 Ohm, respectively. LBH589 exposure did not affect MCF-7 morphology, as capacitance or resistance values remained nearly constant at ~1.5 nF and ~40,000 Ohm, respectively. However, LBH589 appears to have a gradual effect on MDA-MB-231 cell monolayers, decreasing the capacitance value to ~3.5 nF and resistance to ~3,400 Ohm. Micrograph images taken after exposure indicate morphological changes in MDA-MB-231 cells from spindle-shaped to a more epithelial phenotypes. In situ lethal electroporation introduced a ‘wound’ in the monolayer, generating a migration assay with a circular gap 250 μm in diameter. Live/dead staining performed 30 mins after electroporation indicated that cells on the electrode were dead. The corresponding resistance and capacitance values dropped to baseline (cell-free media) levels prior to recovery to control levels (non-electroporated cells). Based on measured resistance, the time to recovery for both LBH589-treated and untreated MCF-7 cells was ~7 hrs (radial migration rate of 17.9 μm/hr). Similarly, the time to recovery for treated MDA-MB-231 cells was ~2 hrs (62.5 μm/hr). Conclusion: LBH589 appears to have an effect the morphology and the migration of MDA-MB-231, but not MCF-7, cells, as monitored by electrical measurements. ECIS provides a powerful tool to measure real-time functional cellular behavior, as well as a robust and reproducible method for assessing the migration potential of cancer cells. Measurement of dynamic cellular response following drug treatment could lead to more accurate evaluation of drug treatment against cancer. Citation Format: Theresa B. Phamduy, Lyndsay V. Rhodes, Matthew E. Burow, Douglas B. Chrisey. Electrical impedance assessment of the effect of LBH589 on the cellular behavior and migratory potential of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A016.