(604c) A Novel Microfluidic Device to Study the Influence of Electric Field on Cancer Cell Motility Under Confinement and Its Underlying Effect on Cellular Contractility

Introduction/Background: Cancer metastasis is one of the leading causes of death amongst cancer patients. Therefore, in vitro model mimicking the tumor microenvironment where cancer cells are exposed to coupled situation of both physical confinement and endogenous electric field (EF) is essential to better understand the metastatic process. “Galvanotaxis”, which is the term coined for cell movement under the influence of EF is responsible for a variety of biological processes ranging from wound healing [1], angiogenesis[2], stem cell polarization[3] to cancer metastasis[4]. Current literature on galvanotaxis of cancer cells mostly focusses on 2D substrates and very little is known about cancer cell motility under confinement (in arteries and capillaries). This is important because cells use different signaling pathways during migration in confined and unconfined spaces[5].In our study, we have developed a novel and versatile microfluidic device which can be used to study the effect of EF on different cell types and study separately the migration and reversal of cells under EF. For our study, we used HT1080 fibrosarcoma cells as a model cell to study influence of EF on their migration and investigate the underlying signaling pathways associated with the process.

Materials and Methods: In our experiments, we use polydimethylsiloxane (PDMS) based microfluidic device. We use photolithography to cross-link the SU-8 spin-coated on a Si wafer via a patterned mask and UV light. Then by replica molding we mix PDMS with a curing agent (1:10 ratio) and bake the mixture. The patterned PDMS is then bonded to a glass coverslip via plasma treatment. Our device has a central chamber which is the seeding area with a height of 50 µm and the set of channels on each side have dimension as 3 µm width and 10 µm height (Fig. A). Cells are seeded in the central chamber, and they freely move away into the microchannels. Once cells are inside the channels, DC electric field was applied to the device through Ag/AgCl electrodes on both sides via a programmable potentiostat. HT1080 fibrosarcoma cells were used in the study. Under applied EF (0.5V/cm), cells on the left array maintain their migration towards cathode (maintaining side) while in the right array, cells reverse their direction towards the cathode (reversing side) (Fig. B). Time-lapse phase-contrast microscopy and confocal microscopy was used for cell migration and immunofluorescence study respectively. RhoA-FRET biosensor was used to study the activity of RhoA using FLIM microscopy. We also generate knockdown of Myosin IIA and use pharmacological inhibitor of ROCK (Y27632 (10µM)) in order to study effect of RhoA based contractility and decipher the mechanism of migration.

Results and Discussion: In the absence of an electric field, HT1080 fibrosarcoma cells migrated freely away from the central reservoir into the microchannels (Fig. B, upper). In the presence of electric field, we observe that the HT1080 cells, which were originally migrating towards the anode reversed their direction of migration after the EF was switched on, whereas the cells migrating towards the cathode maintained their direction of motion (Fig. B, lower). The corresponding velocities of the cells are quantified as well (Fig. C). For both the maintaining and reversing side HT1080 cells undergo significant change from a protrusive to non-protrusive blebbing phenotype (Fig. D). Since the RhoA-ROCK-actomyosin-based pathway of contractility influences the cell phenotype, we quantified RhoA activity in cells migrating on the maintaining and reversing side using a FRET-based RhoA activity biosensor. We found that the cells reversing under EF have significantly more RhoA activity than the cells without EF (Fig. E). On the maintaining side of the device, we found that EF leads to faster cell migration than its no EF counterpart under the ROCK-inhibited migration (via inhibitor Y27632) of fibrosarcoma cells (Fig. F), in other words, EF bypasses the role of actomyosin-based contractility while maintaining the direction of cell migration under EF. This was also verified by using Myosin IIA knockdown cells (here we know Myosin is further downstream of RhoA/ROCK pathway). We observe that although motility of Myosin IIA knockdown cells without EF is lower than the control but under the influence of EF the cell velocity is restored pointing to the fact that EF provides additional contractility for the cells to move faster (Fig. G).

Conclusion: Using a novel microfluidic device, we have uncovered that in the maintaining side EF bypasses the role of RhoA-ROCK-actomyosin based contractility. Thus, these insights into pathways governing galvanotaxis can be of potential interest for developing therapeutics for cancer and other diseases. This microfluidic device can serve as a potential in vitro tool to study directionality of various types of cancer cells, stem cells, immune cells and further signaling pathways can be explored for various systems.

References:

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