Materials and Methods: A polydimethylsiloxane (PDMS)-based microfluidic device was designed and fabricated in-house to create microchannels mimicking the narrow capillaries that the cancer cells travel through in vivo during metastasis. The microchannels are 3 µm in width and 10 µm in height. MDA-MB-231 triple-negative breast cancer cells are primarily used in the study. Methylcellulose of specified concentrations is used to increase fluid viscosities. Bulk 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, immunofluorescence, and FLIM/FRET microscopy to evaluate the molecular signaling pathways. Pharmacological inhibitors were used to perturb some of the pathways for migration. Images are analyzed using ImageJ (Fiji) and IMARIS (Bitplane).
Results and Discussion: MDA-MB-231 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. The memory effects are passed across generations with enhanced motility observed for multiple days post-transfer to 0.77cP media after extended preconditioning at either 3cP or 8cP.
Furthermore, we show that the ‘memory’ is mediated by the inhibition of the canonical ‘Hippo’ pathway, leading to the nuclear localization of the Yes-associated protein (YAP), which is an effector of the Hippo signaling pathway. To decipher the YAP-mediated transcriptional changes associated with viscous memory, we performed bulk 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 ERK-MAPK and PI3K/Akt pathways have come up as two of the most upregulated pathways between naïve (sample A) and preconditioned (sample B) or memory (sample C) cells. We are currently elucidating the roles of these pathways in the formation of viscous memory and 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 after orthotopic delivery in the mammary fat pad in mice. 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 uncovered the novel signaling pathway responsible for enhanced cell motility in extracellular media of elevated viscosity, and 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 ERK-MAPK and PI3K/Akt pathways in mediating YAP-dependent formation of this memory. Taken together, our work indicates that extracellular fluid viscosity regulates both short- and long-term cellular phenotypes with physiological relevance to cancer cell migration and metastasis.