Poster S8 - A Microfluidic Model for Electrotaxis in Tumor Microenvironments: Phenotypic Modulations and Calcium Signaling

Introduction: Cancer metastasis is one of the leading causes of death among cancer patients. Therefore, an in vitro model mimicking the tumor microenvironment where cancer cells are exposed to coupled situations of both physical confinement and endogenous electric field (EF) is essential to better understand the metastatic process. "Electrotaxis," the term coined for cell movement under the influence of EF, is responsible for a variety of biological processes ranging from wound healing, angiogenesis, and stem cell polarization to cancer metastasis. To recapitulate the tumor microenvironment, we have developed a novel and versatile PDMS-based microfluidic device that can be used to study the effect of EF in confined microchannels on cancer cells. Using HT1080 human fibrosarcoma cells as a model cell line, we study changing polarity in cell migration with respect to applied EF. We also observe the change in cell phenotype from a predominantly protrusive phenotype to a non-protrusive blebbing morphology under the application of EF. We noticed a significant influx of calcium into the cells under the application of an electric field, and further, we observed that EF can bypass the RhoA-ROCK-actomyosin-based contractility pathway and can provide contractility on its own, which we showed via inhibition of this pathway

Materials and

Methods: In our experiments, we use a 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 dimensions of 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 were inside the channels, a 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 the 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 were 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 a knockdown of Myosin IIA and use a pharmacological inhibitor of ROCK (Y27632 (10µM)) in order to study the effect of RhoA-based contractility and decipher the mechanism of migration.

Results, Conclusions, and Discussions:

Results: Without any electric field, HT1080 fibrosarcoma cells migrated freely away from the central tumoral region into the microchannels (Fig. B, upper). Upon application of an electric field (0.5V/cm), we observed that the HT1080 cells, which were originally migrating towards the anode, reversed their direction of migration after the EF was switched on (reversing side), whereas the cells migrating towards the cathode maintained their direction of motion (maintaining side) (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 a significant change from a protrusive to a 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 (we know Myosin is further downstream of the RhoA/ROCK pathway). We observe that although the motility of Myosin IIA knockdown cells without EF is lower than the control 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: We have designed a novel microfluidic device that can be used to study the effect of the cue of both electric field and confinement on cancer cells. We have uncovered that EF bypasses the role of traditional RhoA-ROCK-actomyosin-based contractility. This microfluidic device can serve as a potential in vitro tool to study the directionality of various types of cancer cells, stem cells, and immune cells, and further signaling pathways can be explored for them as well

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