(722a) A Novel Microfluidic Platform to Study Galvanotaxis during Confined Cell Migration

Authors: 
Tuntithavornwat, S., Johns Hopkins University
Konstantopoulos, K., Johns Hopkins University
Wang, C., Johns Hopkins University
Direct current (DC) electric fields are generated in vivo as a result of the asymmetric distribution of ion fluxes and differential regulation of voltage-gated ion channels and have been shown to regulate several (patho)physiological processes including cancer metastasis. However, little is known about the exact electrical sensory system that cells employ to respond to external electrical stimulation. Moreover, past attempts at studying the influences of DC electric fields on cell migration and reorientation have been exclusively performed on conventional two-dimensional (2D) surfaces, which do not accurately recapitulate the physiological confining microenvironment that cells have to navigate in vivo. The effects of DC electric fields on cellular morphology and migratory phenotypes in confinement remains to be elucidated.

A polydimethylsiloxane-based microfluidic device was fabricated using photolithography techniques and replica molding to mimic a physiologically relevant confining microenvironment in which cancer cells disseminate from a primary tumor. Ag/AgCl electrodes embedded in agarose were used to apply DC electric fields across the microfluidic device in real time using a programmable potentiostat. The morphology and migratory behaviors of cancer cells were monitored using live-cell time-lapse phase contrast and confocal microscopy. In this system, the activity of proteins of interest was studied by overexpression of fluorescently tagged proteins as well as by FLIM FRET biosensors. Pharmacological inhibitions and lentivirus-mediated shRNA knockdown were used to assess the function of selected proteins of interest.

In the absence of the DC electric field, cancer cells migrated freely away from the central cell reservoir, which mimics the primary tumor, through adjacent microchannels with established polarization. Based on the initial direction cells are migrating in the absence of an electric field, two distinct phenomena are observed when the DC electric field was applied. Cells with parallel/matching electric field response direction and established polarization continued to migrate in the same direction. On the contrary, cells experiencing an electric field antiparallel to established polarization reversed their direction of migration. This demonstrates that our system can be used to study the effects of DC electric fields on cell migration and repolarization simultaneously. Our results also showed that responses to electrical cues are cell line dependent, as different cancer cell lines preferred to migrate towards the anode or cathode.

Previous works have claimed that electric fields provide not only directional migration cues but also kinetic cues that increase cell migration speed on conventional 2D platforms in a voltage-dependent manner. However, in our platform to study confined migration, we found that a physiological level of the electric field (0.5 V/cm) did not alter the cell migration velocity, but did control the direction cell migration and alter the phenotype by increasing the use of a bleb-based as opposed to protrusive-based migration mode Additionally, the applied electric field increased the number, but not the size of focal adhesions. Because contractility regulates bleb-based migration, we further investigated its role in migration under the influence of DC electric field. Interestingly, we found that the DC electric field is able to bypass the cell’s reliance on cellular contractility. These data suggest a different underlying mechanism of cell migration under electric field. Currently we are investigating the role of different ion channels and ion pumps in the migration of cancer cells under electric field.

Understanding how cancer cells migrate through physiologically relevant confined spaces under the influence of electric fields could potentially offer insights into the development of novel therapeutic strategies to control cell migration and ultimately inhibit cancer metastasis.