Long-Term Effects of Exposure to Elevated Extracellular Fluid Viscosity on Cell Motility and Metastasis

Introduction:: Cancer is one of the leading causes of death worldwide, with an estimated 609,360 deaths in the United States alone, in 2022. 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. 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 offer insights into 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 photo- and soft-lithography techniques 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. We have used shRNA and CRISPR/Cas9-based gene-editing tools, genetically encoded live-cell calcium sensors like GCaMP6s, and FLIM/FRET microscopy to evaluate the molecular signaling pathways. Pharmacological inhibitors were used to perturb the pathways for migration. We also tracked metastasis using the ex-ovo chick embryo model along with intravital confocal imaging. Images are analyzed using ImageJ (Fiji) and IMARIS (Bitplane).


Results, Conclusions, and Discussions:: MDA-MB-231 adenocarcinoma cells develop a viscous ‘memory’ after pre-treatment for 6 days at either 3cP or 8cP media, as evidenced by their enhanced migration at the baseline viscosity of 0.77cP, compared to non-pretreated (termed as naïve) cells, (Fig.B). The memory effects are passed across generations with enhanced motility observed for several days post-transfer to 0.77cP media.

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 (Fig. C). 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.D). We will also discuss the interplay of YAP nuclear translocation and actomyosin contractility on cells upon exposure to highly viscous media.

Finally, we show that the increased cell volume for the entire duration of viscosity-induced cellular memory (Fig. E), and the coincidental cell volume decrease with the loss of memory indicates the potential involvement of ion channels. Pre-treated 4T1 murine breast cancer cells, compared to naïve cells, exhibited a higher contact length with the CAM vasculature, thereby suggesting these cells can hijack the blood vessels of the surrounding non-malignant tissues more efficiently (Fig. F).

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 elucidating the role of YAP in mediating the epigenetic changes responsible for the formation of this memory. Our work also establishes media viscosity as a novel physical cue that enhances the metastatic potential of cancer cells. Taken together, our work indicates that extracellular viscosity regulates both short- and long-term cellular phenotypes and behavior with (patho)physiological relevance to cancer cell migration and metastasis.


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