(502f) Long-Term Effects of Extracellular Fluid Viscosity As a Mechanical Cue to Increase Cell Motility

Introduction: Cancer is one of the leading causes of death worldwide, with an estimated 609,360 deaths in the United States alone, in 2022 [1]. In diseases like cancer, the tumor microenvironment is a region of high viscosity, yet the short and long-term effects of the viscosity of the extracellular fluid on the signaling pathways of the cells are largely unknown. During this study, we observed that cells, counterintuitively, moved faster in fluids of elevated viscosity, similar to what has been observed in the literature [2][3][4]. Further, we uncovered that cells pre-treated in media of physiologically high viscosity form a ‘memory’, by which they sustain enhanced migration even after the removal of the viscous stimulus. We believe that our work will help in gaining insights on cell behavior under physiologically relevant conditions as well as translate in vitro studies to an in vivo setting more efficiently.

Materials and Methods: A polydimethylsiloxane (PDMS)-based microfluidic device was designed and fabricated using photolithography techniques and replica molding to create microchannels mimicking the narrow capillaries that the cancer cells travel through during metastasis. The microchannels are 3 µm in width and 10 µm in height (Fig. A). MDA-MB-231 adenocarcinoma cells are primarily used in the study. For select experiments, SUM159 human breast cancer cells, 4T1 murine breast cancer cells, and human dermal fibroblasts GM05565 are used. Methylcellulose of specified concentrations is used to increase the fluid viscosities. Cell migration was visualized and recorded with time-lapse phase-contrast microscopy. The activity or localization of proteins of interest was observed by immunofluorescence or with fluorescently tagged live reporters, such as LifeAct fused with GFP or genetically encoded calcium sensor GCaMP6s. Pharmacological inhibitors were used to decipher migration mechanisms. Images are analyzed using ImageJ (Fiji) and IMARIS (Bitplane).

Results and Discussion: MDA-MB-231 adenocarcinoma cells exhibit a viscous ‘memory’ by which cells pre-treated for 6 days at either of 3cP or 8cP media exhibit elevated migration speeds, compared to non-pretreated or naïve cells, at baseline viscosities of 0.77cP (Fig.B). The memory effects are highly persistent with enhanced motility observed for as long as 10 days after 6 days of pre-treatment in 8cP media (Fig. C).

Furthermore, we show that the ‘memory’ is mediated by the nuclear localization of Yes-associated protein (YAP), which is an effector of the Hippo signaling pathway. Immunofluorescence staining reveals that YAP nuclear entry occurs within 1hr of pre-treatment in 8cP media (Fig. D). We show that actin-branching protein ARP2/3, Na⁺/H⁺ -exchanger-1(NHE1) and mechanosensitive ion channel TRPV4 act in conjunction to translocate YAP into the nucleus (Fig.E).

Finally, we show that the enhanced motility exhibited by the pre-treated cells is independent of increased calcium activity. Rather, the increased cell volume for the entire duration of viscosity-induced cellular memory, and the coincidental cell volume decrease with the loss of memory indicates the potential involvement of ion channels (Fig. F). We are in the process of delineating the role of such ion channels and their potential crosstalk with contractility and the Hippo pathway.

Conclusion: During our investigations, we have uncovered the novel signaling pathway responsible for enhanced cell motility in extracellular media of elevated viscosity, as well as the ability of cells to form a ‘memory’ of their surrounding fluid viscosity. We have shown that this viscosity-induced memory is highly persistent across multiple cell generations, and we are in the process of elucidating the role of YAP in mediating the epigenetic changes responsible for the formation of this memory. Taken together, our work indicates that extracellular viscosity regulates both short- and long-term cellular phenotypes and behaviour with (patho)physiological relevance to cancer cell migration and metastasis.

References:

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