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 611,720 deaths in the United States alone, in 2024. In diseases like cancer, the tumor microenvironment is highly viscous, yet the short and long-term effects of the extracellular fluid viscosity 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. Further, we uncovered that cells pre-treated in media of physiologically high viscosity form a ‘memory’, by which they sustain enhanced migration, compared to non-pretreated (hereafter termed ‘naïve’) cells, even after the removal of the viscous stimulus. We believe that our work will help in gaining insights on tumor cell behavior under circulation 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. RNA-Seq was performed to compare the transcriptomes of MDA-MB-231 cells exposed to fluids of different viscosities. Cell migration was visualized and recorded with time-lapse phase-contrast microscopy. We have used shRNA, doxycycline-inducible gene editing systems, and FLIM/FRET microscopy to evaluate the molecular signaling pathways. Pharmacological inhibitors were used to perturb the pathways for migration. Images are analyzed using ImageJ (Fiji) and IMARIS (Bitplane).

Results, Conclusions, and Discussions: MDA-MB-231 adenocarcinoma cells exhibit a viscous ‘memory’ by which cells pre-treated for 6 days at 8cP media exhibit elevated migration speeds at baseline viscosities of 0.77cP, compared to non-pretreated or naïve cells, (Fig.B). The memory effects are passed across generations with enhanced motility observed for as long as 10 days post-transfer to 0.77cP media after extended preconditioning at 8cP. (Fig. C).
Furthermore, we show that the ‘memory’ is mediated by the nuclear localization of the Yes-associated protein (YAP), which is an effector of the Hippo signaling pathway (Fig. D). To decipher the YAP-mediated transcriptional changes associated with viscous memory, we performed RNA-seq to compare the transcriptomes of MDA-MB-231 cells exposed to 0.77 cP (sample A; naïve cells) or 8 cP (sample B; preconditioned cells) or viscosity preconditioned cells that were subsequently allowed to incubate at baseline viscosity (0.77 cP) for 1 day (sample C; memory cells). We identified potential gene candidates involved in the imprinting of viscous memory (not shown), which are also upregulated in triple-negative breast cancer patient databases relative to normal controls. By subjecting the Differentially Expressed Genes (DEGs) to Reactome Pathway Analysis, the PI3K/Akt has come up as one of the most upregulated pathways between naïve (sample A) and memory (sample C) cells. We are currently elucidating the role of the PI3K/Akt pathway in driving the migration of such ‘memory’ cells.
Finally, we show that cells that have undergone pre-treatment with fluids of elevated viscosities exhibit higher (compared to naïve cells) metastasis in the lungs after tail-vein injection in mice (Fig. E). Thus, our work establishes extracellular fluid viscosity as a novel physical cue that enhances the metastatic potential of cancer cells.

Conclusion: During our investigations, 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 formation of this memory and the mechanism behind the enhanced motility observed in the cells with pre-formed memory. Taken together, our work indicates that extracellular viscosity regulates both short- and long-term cellular behavior with (patho)physiological relevance to cancer cell migration and metastasis.

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